Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014...

13
Research Article An Immunomodulatory Protein (Ling Zhi-8) from a Ganoderma lucidum Induced Acceleration of Wound Healing in Rat Liver Tissues after Monopolar Electrosurgery Hao-Jan Lin, 1,2 Yushan-Sophie Chang, 3,4 Li-Hsiang Lin, 1,4,5 Chiung-Fang Haung, 4,6 Chia-Yu Wu, 1,7 and Keng-Liang Ou 2,4,8,9 1 School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei 110, Taiwan 2 Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan 3 Division of Oral Rehabilitation and Center of Pediatric Dentistry, Department of Dentistry, Taipei Medical University Hospital, Taipei 110, Taiwan 4 Research Center for Biomedical Devices and Prototyping Production, Taipei Medical University, Taipei 110, Taiwan 5 Department of Dentistry, Sijhih Cathay General Hospital, Taipei 221, Taiwan 6 School of Dental Technology, College of Oral Medicine, Taipei Medical University, Taipei 110, Taiwan 7 Division of Oral and Maxillofacial Surgery, Department of Dentistry, Taipei Medical University Hospital, Taipei, Taiwan 8 Graduate Institute of Biomedical Materials and Tissue Engineering, College of Oral Medicine, Taipei Medical University, Taipei 110, Taiwan 9 Department of Dentistry, Taipei Medical University-Shuang Ho Hospital, Taipei 235, Taiwan Correspondence should be addressed to Chia-Yu Wu; [email protected] and Keng-Liang Ou; [email protected] Received 13 November 2013; Revised 4 January 2014; Accepted 4 March 2014; Published 5 May 2014 Academic Editor: Kuo-Pao Lai Copyright © 2014 Hao-Jan Lin et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e purpose of this study was to investigate the effect of an immunomodulatory protein (Ling Zhi-8, LZ-8) on wound healing in rat liver tissues aſter monopolar electrosurgery. Animals were sacrificed for evaluations at 0, 3, 7, and 28 days postoperatively. It was found that the wound with the LZ-8 treatment significantly increases wound healing. Western blot analysis clearly indicated that the expression of NF-B was decreased at 3, 7, and 28 days when liver tissues were treated with LZ-8. Moreover, caspase-3 activity of the liver tissue also significantly decreases at 7 and 28 days, respectively. DAPI staining and TUNEL assays revealed that only a minimal dispersion of NF-B was found on the liver tissue treated with LZ-8 at day 7 as compared with day 3 and tissues without LZ-8 treatment. Similarly, apoptosis was decreased on liver tissues treated with LZ-8 at 7 days when compared to the control (monopolar electrosurgery) tissues. erefore, the analytical results demonstrated that LZ-8 induced acceleration of wound healing in rat liver tissues aſter monopolar electrosurgery. 1. Introduction Conventional electrosurgery has been clinically available for more than half a century and widely used as an alternative to the scalpel to reduce blood during surgery. e mech- anism of electrosurgical cutting is well defined in all sur- gical operations areas [1, 2]. However, more attention is focused on the surrounding tissue damage during incision by heat dissipation. e bipolar electrosurgery as an exam- ple allows more direct current application but can only be used for hemostasis. e monopolar electrosurgery has been well documented as alternative modalities in oral and maxillofacial surgery, although steel scalpel is considered to be the instrument of choice for surgical incisions [2]. Both monopolar and bipolar surgeries can achieve incision and coagulation of tissue. However, the monopolar mode is more effective than the bipolar for incision or cutting action and possesses distinct advantages over the bipolar mode for this purpose [3]. ere are wide varieties of electrode shapes available for monopolar incision than the bipolar. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2014, Article ID 916531, 12 pages http://dx.doi.org/10.1155/2014/916531

Transcript of Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014...

Page 1: Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014 › 916531.pdfResearch Article An Immunomodulatory Protein (Ling Zhi-8) from a

Research ArticleAn Immunomodulatory Protein (Ling Zhi-8) froma Ganoderma lucidum Induced Acceleration of Wound Healingin Rat Liver Tissues after Monopolar Electrosurgery

Hao-Jan Lin12 Yushan-Sophie Chang34 Li-Hsiang Lin145 Chiung-Fang Haung46

Chia-Yu Wu17 and Keng-Liang Ou2489

1 School of Dentistry College of Oral Medicine Taipei Medical University Taipei 110 Taiwan2 Research Center for Biomedical Implants and Microsurgery Devices Taipei Medical University Taipei 110 Taiwan3Division of Oral Rehabilitation and Center of Pediatric Dentistry Department of DentistryTaipei Medical University Hospital Taipei 110 Taiwan

4Research Center for Biomedical Devices and Prototyping Production Taipei Medical University Taipei 110 Taiwan5Department of Dentistry Sijhih Cathay General Hospital Taipei 221 Taiwan6 School of Dental Technology College of Oral Medicine Taipei Medical University Taipei 110 Taiwan7Division of Oral and Maxillofacial Surgery Department of Dentistry Taipei Medical University Hospital Taipei Taiwan8Graduate Institute of Biomedical Materials and Tissue Engineering College of Oral Medicine Taipei Medical UniversityTaipei 110 Taiwan

9Department of Dentistry Taipei Medical University-Shuang Ho Hospital Taipei 235 Taiwan

Correspondence should be addressed to Chia-Yu Wu borgiawugmailcom and Keng-Liang Ou kloutmuedutw

Received 13 November 2013 Revised 4 January 2014 Accepted 4 March 2014 Published 5 May 2014

Academic Editor Kuo-Pao Lai

Copyright copy 2014 Hao-Jan Lin et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The purpose of this study was to investigate the effect of an immunomodulatory protein (Ling Zhi-8 LZ-8) on wound healing inrat liver tissues after monopolar electrosurgery Animals were sacrificed for evaluations at 0 3 7 and 28 days postoperatively Itwas found that the wound with the LZ-8 treatment significantly increases wound healing Western blot analysis clearly indicatedthat the expression of NF-120581B was decreased at 3 7 and 28 days when liver tissues were treated with LZ-8 Moreover caspase-3activity of the liver tissue also significantly decreases at 7 and 28 days respectively DAPI staining and TUNEL assays revealed thatonly a minimal dispersion of NF-120581B was found on the liver tissue treated with LZ-8 at day 7 as compared with day 3 and tissueswithout LZ-8 treatment Similarly apoptosis was decreased on liver tissues treatedwith LZ-8 at 7 days when compared to the control(monopolar electrosurgery) tissuesTherefore the analytical results demonstrated that LZ-8 induced acceleration of wound healingin rat liver tissues after monopolar electrosurgery

1 Introduction

Conventional electrosurgery has been clinically available formore than half a century and widely used as an alternativeto the scalpel to reduce blood during surgery The mech-anism of electrosurgical cutting is well defined in all sur-gical operations areas [1 2] However more attention isfocused on the surrounding tissue damage during incisionby heat dissipation The bipolar electrosurgery as an exam-ple allows more direct current application but can only

be used for hemostasis The monopolar electrosurgery hasbeen well documented as alternative modalities in oral andmaxillofacial surgery although steel scalpel is consideredto be the instrument of choice for surgical incisions [2]Both monopolar and bipolar surgeries can achieve incisionand coagulation of tissue However the monopolar mode ismore effective than the bipolar for incision or cutting actionand possesses distinct advantages over the bipolar modefor this purpose [3] There are wide varieties of electrodeshapes available for monopolar incision than the bipolar

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 916531 12 pageshttpdxdoiorg1011552014916531

2 Evidence-Based Complementary and Alternative Medicine

Although incision can be accomplished by both but thebipolarmode ismore inefficient and restrictive in applicationBipolar coagulation is slower than monopolar activity [3]Their slow action is considered a disadvantage for surgicalprocedures but proves to be of significant benefit for moreprecise microsurgical procedures due to their easy usagewide availability incision accuracy and minimal damageto adjacent tissues The use of steel scalpel in monopolarelectrosurgery remains an imperative need for electrosurgeryoperating system

The effects of electrosurgery (ES) on histoarchitecturevary depending on the power output [4] and frequency [5]of ES unit waveform selection [6] and shape and size ofactive electrode [7] It is well observed that wound healing forelectrosurgery delays through comparative studies for elec-trosurgical and scalpel wounds [8] Electrosurgical woundshave more inflammatory responses and tissue destructionbut the viability of osteoblasts in both categories shows thesame in both kinds of wounds with no increase in osteoclastswhich could indicate no bone resorption occurrences Onthe contrary [9] it could be found out that there will be nodifferences on wound healing between ES and periodontalknives when the gingival resection is shallow Howeverthere could be intense inflammatory and bone loss in heightresulting from bone necrosis during deep resection Otherresearch study [10] shows that although there is loss oftissue soon after ES 70 to 100 of the lost tissue is regainedover a period of months When teeth with cervical amalgamrestoration get contact with active electrode tip to simulatefrom clinical application report [11] there is no evidenceof extensive damage or necrosis of pulp [12] Macroscopicobservation and histological examination of our latest studyclearly show that commercial stainless steel needles producelarger injury area than CrN-needles [13] As reported slowwound healing found as a result of operations while usingboth mono- and bipolar electrosurgery devices an extratherapy is highly recommended to enhance this woundhealing after surgery which this paper clearly reports

ldquoLing Zhirdquo is a Chinese term representing combinationof spiritual potency and essence of immortality and widelyregarded as the ldquoherb of spiritual potencyrdquo symbolizingsuccess well-being divine power and longevity Amongcultivated mushrooms G lucidum is unique in that itspharmaceutical rather than nutritional value is paramountA variety of commercial G lucidum products are available invarious forms such as powders dietary supplements and teaThese are produced from different parts of the mushroomincluding mycelia spores and fruit body Ling Zhi-8 (LZ-8)an immunomodulatory protein isolated from mycelia of Glucidum could be considered as one of the major bioactivesubstances of its kind [14] They also contain excellent ther-mal and acidity stability together with moderate resistivity toalkalinity and dehydration [15] LZ-8 consists of 110 aminoacids and a molecular mass of 124 kDa which is similarto that of the variable region of the immunoglobulin heavychain [16] LZ-8 exerts mitogenic activities in vitro and hasalso shown in vivo immunomodulatory activities [17] Ling-Zhi had been widely recommended for specific applicationsand attributed health benefits for treatment of migraine

hypertension arthritis bronchitis asthma anorexia gas-tritis hemorrhoids and diabetes and for the control ofblood glucose levels modulation of the immune systemhepatoprotection bacteriostasis and more

A recent study haswell demonstrated that LZ-8 effectivelypromotes the activation and maturation of immature humandendritic cells via regulating NF-120581B and MAPKs path ways[18] Hitherto several studies have focused on wound healingactions in different tissues after electrosurgery inducinginflammatory reaction and reepithelialization and stimu-lating angiogenesis However there is relatively little infor-mation pertaining to wound healing activity and possiblemechanism on liver tissues Hence the present study wasdesigned to have a three pronged approach (a) to examinethe wound size injury fibrosis and apoptosis in rat modelfor the liver tissues after monopolar electrosurgery (b) toascertain whether LZ-8 protein enhances wound healing forthe liver tissues created by surgery and (c) to elucidate thepossiblemechanismof LZ-8 onwoundhealing viaNF-120581Bandcaspase-3 expressions

2 Materials and Methods

21 Materials LZ-8 was provided by Yeastern Biotech CoLtd Taiwan Anti-NF-120581B (MAB3026) caspase-3 (AB1899)120572-tubulin mAb (MAB1501) monoclonal antibodies (mAbs)horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG) and sheep anti-mouse IgGwere all from Chemicon Millipore Temecula CA Thenitrocellulose membrane (HybondTM-C Extra AmershamBiosciences Corp Hong Kong China) enhanced chemilu-minescence (ECL) Western blotting detection reagent andanalysis system (WBLUC0100) were also purchased fromMillipore Temecula CA LZ-8 was dissolved in phosphatebuffered saline (PBS) and stored at 4∘C until the time ofusageapplication used

22 Animals and Surgical Device Sixteen healthy male Spra-gue-Dawley rats weighing 200 to 300 grams (g) eachprocured from BioLASCO (Taiwan) were used for theexperiments These Sprague-Dawley rats were prepared andmaintained under the guidelines for care and usage oflaboratory animals at 25∘C and exposed to 12 h of dark and12 h of lightTheywere fed a standard balanced pellet diet andwater ad libitum The protocols for animal experiments werereviewed and approved by the Institutional Animal Care andUse Committee of Taipei Medical University (LAC-99-0037)Rats were marked to permit individual identification andwere kept in their cages for 14 days prior to experimentationto allow for acclimatization to the laboratory conditions Noreusable commercial electrosurgical monopolar tip (ERBEnumber 20191-377 USA needle type stainless steel 304) wasused

23 Surgical Procedure General anesthesia was inducedwith inhaled isoflurane for the animals The operative sitewas cleansed and draped in a sterile fashion Conven-tional stainless steel (SS) needle electrodes were used with

Evidence-Based Complementary and Alternative Medicine 3

SS

0 7 28Days

3

SS (stainless steel)

500 120583m 500 120583m 500 120583m 500 120583m

(a)

SS +

LZ-

8

0 7 28Days

3

LZ-8 (Ling Zhi-8)

500 120583m 500 120583m 500 120583m 500 120583m

(b)

Figure 1 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for different healing days (HampE staining)(a) SS group and (b) SS + LZ-8 group

an electrosurgical unit The liver was exposed through mid-line laparotomywith a retractor to avoid injury to the liver Anunmodified SS-needle was inserted to the liver lobe (3mm indepth) to create an anterior lesion using a fixed power setting(20W) with a fixed activation time (3 seconds) The powersetting and activation time were commonly used parametersand were based on previous study settings For LZ-8 proteingroup 10 120583L of 1mgmL LZ-8 protein solution was pipettedand dropped on the wound surface For control group andexperiment group 10120583L of normal saline was pipetted anddropped on the wound surface

24 Weight of Sticking Tissue The weights of each SS-needlewere recorded perioperatively and the weight of stickingtissue was calculated as the weight of needle after operationminus the weight of needle before operation

25Thermography The effects of the SS-needles on tempera-tures across the live lobe surface were determined from ther-mographs obtained with a thermal-imaging infrared camera(Advanced Thermo TVS-500EX NEC Avio TechnologiesTokyo Japan) The highest temperature was recorded forevery test After recording the thermograph the surroundingsoft tissues were carefully repaired and the surgical woundwas closed in layers No anti-inflammatory regimen was usedafter surgery

26 Histological Examination to Detect Injury Area Histo-pathological studies were performed to provide structuralevidence ofmediated tissue injury and protective effect of LZ-8 These animals were sacrificed on days 0 3 7 and 28 afteroperation respectively Tissues were then cut into pieces ofdesired size and fixed in Bouinrsquos fluid fixative immediatelyafter autopsy Fixation was carried out at room temperaturefor 24 hrs after which these fix tissues were transferred to70 alcohol Several changes of 70 alcohol were given untilthe yellow colour disappeared from the tissues The tissueswere then dehydrated by passing through ascending grades ofalcohol (30 50 70 90 and 100) cleared in methylsalicylate and infiltrated with wax at 57∘C The clear tissueswere embedded in the paraffin Conventional techniques ofparaffinwax sectioningwere used for histological studies [19]Paraffin wax sections of 6 to 8 120583m thickness were cut using arotary microtome (Leica Germany) The sections were fur-ther stained with hematoxylin and eosin (HampE) andMassonrsquostrichrome staining and then washed in 90 alcohol for fewseconds Subsequently the stained sections were dehydratedin 100 alcohol cleared in xylene and mounted in DPXmountant The stained slides were observed in a Carl Zeiss(Germany) Axio 2 Plus research microscope Images werecaptured through a CCD camera (BX51 Olympus Japan)in a computer and processed using Carl Zeiss Axiovisionsoftware The total injury areas caused by the SS-needleare measured using image analysis software (SPOT basic

4 Evidence-Based Complementary and Alternative Medicine

(a)

TA Thermal injury area IA Injury area (trauma)

TA

IA

TA

IA

TA

IA

TAIA

TA

TA

IA

IA

SSSS

+ L

Z-8

SS SS + LZ-8

SS SS + LZ-80

10

20

30

40

50

60

70

80

90

100

Are

a (

)

Thermal injury areaInjury area

Wound area (TA + IA)

(c)(b)

500 120583m

100 120583m

100 120583m

500 120583m

Figure 2 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 0 days (Massonrsquos trichrome staining)(a) wound area (b) the site of thermal injury area and injury area and (c) percentage of each area Data are expressed as the means plusmn SEM(119899 = 5) No significant difference in the thermal injury area injury area and wound area at day 0

software SPOT imaging solutions MI USA) In additiondifferent histological features were observed on the injurysite Therefore the injured area was further divided intodifferent classifications to demonstrate the injury caused bythermal spread and the area of every portion was measured

27 Western Blot Analysis The retrieved liver tissues werehomogenized and lysed in an ice-cold protein lysis buffer andcentrifuged at 13000 rpm for 15 minutes at 4∘C Protein con-centrations of the lysates were determined using a Bio-Radprotein assay (Bio-Rad Laboratories Hercules CA USA)The lysates (20120583glane) were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in10 gels and electroblotted onto a nitrocellulose membrane(HybondTM-C Extra Amersham Biosciences Corp HongKong China) The membrane was blocked for 30 minutesat room temperature in 3 nonfat milk Tris-buffered salinewith 005 Tween 20 and then incubated overnight at4∘C with the indicated primary antibodies against NF-120581B(1 500 MAB3026 Chemicon Millipore Temecula CA) orcaspase-3 (1 200 AB1899 Millipore Temecula CA) andthen incubated with horseradish peroxidase-conjugated sec-ondary antibodies for 2 hours at room temperature After

washing the membrane was developed using chemilumi-nescent substrates (WBLUC0100 Millipore Temecula CA)Densitometric analysis of the gels was performed using Gel-Pro analyzer software and the results were expressed relativeto the density of 120573-actin (1 500 MAB1501 ChemiconMillipore Temecula CA)

28 Immunofluorescence Staining and TUNEL Staining Forimmunofluorescence staining liver tissues were embeddedin Cryomatrix (Thermo Shandon Pittsburgh PA USA) andstored at minus80∘C Cryostat sections (5ndash7mm thickness) werefixed in a mixture of methanol and acetone (1 1) at minus20∘Cfor 5 minutes and incubate in 1 Triton X-100 for another5 minutes Nonspecific activity was blocked by incubatingthe sections with 10 horse serum in a buffer containing002 Tween 20 in phosphate-buffered saline for 30 minutesSections were then incubated overnight with primary anti-Caspase-3 at 4∘C Antigen localization was indicated bya Cy5 dye (red fluorescence 111-176-003 Jackson ImmuneResearch West Grove PA) Finally the samples were stainedwith 410158406-diamidino-2-phenyl-indole (DAPI sc-3598 SantaCruz Biotechnology CA) for 7 minutes and examined usingfluorescent microscope Normal liver tissues obtained from

Evidence-Based Complementary and Alternative Medicine 5

NA

TA

IA

IA

TA

NA

NA Normal area TA Thermal injury area IA Injury area (trauma)

SS SS + LZ-80

10

20

30

40

50

60

70

80

Are

a (

)

Thermal injury areaInjury area

Wound area (TA + IA)

SSSS

+ L

Z-8

(a)

(c) (b)

100 120583m

100 120583m

lowastlowast

lowast

lowastlowastlowast

SS SS + LZ-8

500 120583m 500 120583m

Figure 3 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 3 days (Massonrsquos trichrome staining)(a) wound area (b) the site of thermal injury area injury area and normal area and (c) percentage of each area Data are expressed as themeans plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

healthy rats undergo identical procedures Negative controlswere processed identically except that the primary antibodieswere replaced with IgG An in situ cell death detection kit(terminal deoxynucleotidyl transferase dUTP nick end label-ing TUNEL 11684795910 RocheMannheim Germany) wasused to detect apoptotic cells and the apoptotic cells wereindicated by a green fluorescent FITC fluorochrome

29 Statistical Analysis Data were expressed as mean plusmnstandard error of the mean (SEM) Data were analyzed usinganalysis of variance (ANOVA) All statistical analyses wereperformed using SPSS version 120 (SPSS Inc Chicago IL)Values of 119875 lt 005 were considered significant

3 Results

31 Stimulatory Effects of LZ-8 on Wound Healing in RatLiver Tissues after Monopolar Electrosurgery In generallybleeding and healing could easily be reduced when tissuesare subjected to electrosurgery To determine whether LZ-8could enhance wound healing on liver tissues after surgerywe compared the wound size after monopolar electrosurgeryand the operated tissues that had been treatedwith LZ-8 for 03 7 and 28 daysThe observed results of HampE stained section

of liver tissues show that a significant reduced of wound sizetreated with LZ-8 (3 7 and 28 days) dependently relative tothe normal (untreated) rats as shown in Figure 1 Moreoverthe mean wound sizes for these liver tissues were moreprominently reduced in tissues that were treated with LZ-8 at28 days when compared with untreated groups and with day3 and day 7 The results clearly indicated that treatment withLZ-8 enhances wounds healing faster after surgery in rat livertissues

32 Effects of LZ-8 on the Site of Thermal Damage Injuryand Wounds of Liver Tissues after Surgery Figure 2 showshistological sections of monopolar SS on the operated livertissues and tissues subjected to LZ-8 treatment (SS + LZ-8)for 0 days (Massonrsquos trichrome staining) Clearly there wasno significant difference in the total injury areas caused by theSS and SS + LZ-8 treatments Moreover the injury area andthermal injury area in SS group were also similar to those ofSS + LZ-8 group at day 0 However the results demonstratedthat damage of liver tissues in the form of injury thermalinjury and wound was negligible in SS group for 3 days(Figure 3) when compared to 0 days (Figure 2) Interestinglythe effect of injury thermal injury and wound of liver tissueswas significantly reduced in SS + LZ-8 group for 3 days It

6 Evidence-Based Complementary and Alternative Medicine

NA Normal area FA Fibrosis area AA Apoptosis area

NA

FAFAAA

AA

AAFA

FA

NA

SSSS

+ L

Z-8

(b)

50 120583m

50 120583m

50 120583m

50 120583m

SS SS + LZ-80

10

20

30

40

50

60

70

80

90

Are

a (

)

Fibrosis area Injury areaApoptosis area Wound area (FA + IA + AA)

(c)

lowast

lowastlowast

lowastlowastlowast

(a)

SS SS + LZ-8

500 120583m500 120583m

Figure 4 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 7 days (Massonrsquos trichrome staining)(a) wound area (b) the site of apoptosis area fibrosis area and normal area and (c) percentage of each area Data are expressed as themeans plusmnSEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

was found that the injury area (119875 lt 005) thermal injuryarea (119875 lt 001) and wound area (119875 lt 0001) of SS + LZ-8groupwere reducedmore significantly than those of SS groupat day 3 Furthermore Massonrsquos trichrome staining was usedto examine the formation of fibrotic tissue No fibrotic tissuewas evident for either group at day 0 or day 3

33 Effects of LZ-8 on the Areas of Fibrosis Injury ApoptosisandWounds of Liver Tissues after 7 Days of Surgery A furtherstudy was performed to determine whether LZ-8 couldenhance the areas of fibrosis reduce injury and wounds andinhibit apoptosis of monopolar SS induced liver incision interms of withdrawing the above observed injuriesThe resultsclearly confirmed that injury apoptosis and wound of livertissues in SS operated groups that had been treated withLZ-8 for 7 days were significantly alleviated when comparedwith SS operated groups without LZ-8 treatment (Figure 4)Nevertheless the areas of fibrosis and numbers of cell deathin liver tissues for treated rats with LZ-8 were comparativelyhigher than those of SS operated rats Thus the resultsindicate that LZ-8 treatment could not affect the reduction ofliver fibrosis andmore effective on the inhibition of apoptosiswhich are caused by surgery

34 Effects of LZ-8 on the Areas of Fibrosis Injury Apoptosisand Wounds of Liver Tissues after 28 Days of Surgery Sincedifferential observations were found on the areas of fibrosisand apoptosis in liver tissues of LZ-8 supplemented rats thathave already been operated with SS for 7 days we furtherextended the duration of treatment up to 28 days (Figure 5)to confirm the hypothesis Our analysis obviously exhibitsfurther evidence that the effect of LZ-8 was similar to thatof 7-days treatment

35 Does LZ-8 Enhance Wound Healing in SS Operated LiverTissues via Inhibiting the Expression of NF-120581B and Caspase-3It is well known that the angiogenesis is essential in woundhealing Various studies have demonstrated the inhibition ofangiogenesis by the blockage of NF-120581B A recent study hasalso suggested that caspase-3 plays an important role duringskin wound healing [20] Thus we performed this study todeterminewhether thewound healing action of LZ-8was dueto the effect on inhibiting of NF-120581B and caspase-3 expressionthe tissue levels of their protein were examined by Westernblot analysis As shown in Figure 6(a) liver tissues exposedto electrosurgery with SS for 3 days could slightly inducethe expression of NF-120581B and it gradually increased up to

Evidence-Based Complementary and Alternative Medicine 7

SS0

5

10

15

20

25

30

Are

a (

)

Fibrosis area Apoptosis area

Wound area (FA + AA)

(a)

(c) (b)

FA Fibrosis area AA Apoptosis area PA Proliferate area

SSSS

+ L

Z-8

SS SS + LZ-8

SS + LZ-8

AA

FA

PA

AA

FA

AA

PA

lowast

lowast

lowastlowast

500 120583m500 120583m

50 120583m

50 120583m

Figure 5The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 28 days (Massonrsquos trichrome staining)(a) wound area (b) the site of apoptosis area fibrosis area and proliferate area and (c) percentage of each area Data are expressed as themeans plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

28 days Interestingly liver tissues further treated with LZ-8 significantly reduced the ratio of NF-120581B expression in aduration dependent manner (3 to 28 days) A similar resultwas observed on the expression of caspase-3 Therefore theresults can suggest that treatment of liver tissues after surgerywith LZ-8 was found to lower the mean NF-120581B and caspase-3 expressions so that it approaches wound healing propertypresumably by limiting NF-120581B and caspase-3 expression inliver tissuesThese results were further confirmed and shownas in Figure 6(b)

36 LZ-8 Inhibits NF-120581BNuclear Translocation in SS OperatedLiver Tissues The ability of LZ-8 to inhibit NF-120581B dispersionwas determined by red fluorescence (Cy5) intensity analysisof immunolabelled antibodies directed against NF-120581B andHoechst reagent to simultaneously identify nuclei (Figure 7)The intensity values of NF-120581B red fluorescence in the nucleusof SS operated tissues that were treated with LZ-8 for 3 dayswere found to be lesser as compared to SS operated livertissues without LZ-8 treatment Interestingly the presence ofLZ-8 in SS treated liver tissues for 7 days reached amaximuminhibition of nuclear fluorescence of NF-120581B than that oftreated tissues with SS alone These results can obviouslyindicate that LZ-8 plays a major role in inhibiting of NF-120581B

nuclear translocation in liver tissues during the process ofwound healing after electrosurgery

37 LZ-8 Reduces Apoptotic Cell Death in Liver Tissues duringWoundHealing after Electrosurgery Extensive fragmentationof nuclear DNA that generates a large number of DNAdouble-strand breaks is one of the most characteristic eventsof apoptosis An assay that relies on detection of DNA strandbreaks (DSBs) in situ by labeling them with fluorochromeshas been developed to identify and quantify apoptotic cellsby fluorescence microscopy or cytometry The assay is com-monly called TUNEL the acronym of Terminal deoxynu-cleotidyl transferase-mediated d-UTP Nick End Labeling Inthis study a very clear TUNEL-positive cells were observedin rats hepatic cells after 7 days of electrosurgery (Figure 8)However the number or TUNEL expression continued todecrease more prominently in LZ-8 treated hepatic cells thathad been exposed with monopolar electrosurgery for 7 daysThe results obtained from this study demonstrate that a slightapoptosis occurs in hepatic cells after electrosurgery whichwas as a result of LZ-8 inhibition and provide evidencefor high level of angiogenesis arises during wound healingprocess

8 Evidence-Based Complementary and Alternative Medicine

SS

NF-120581B

NF-120581B

SS + LZ-8

0 3 7 2800

05

10

15

20

25

Relat

ive e

xpre

ssio

n of

NF-120581

B

Treatment duration (day)

SSSS + LZ-8

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowast

lowastlowast

(a)

0 3 7 2800

05

10

15

20

Relat

ive e

xpre

ssio

n of

casp

ase-

3

Treatment duration (day)

SSSS + LZ-8

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Caspase-3SS

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowastlowast

lowastlowastlowast

(b)

Figure 6TheWestern blot assay results from rats liver treated with SS group and SS + LZ-8 group after different healing days (a) expressionsof NF-120581B and (b) expressions of caspase-3 Data are expressed as the means plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 comparedwith SS group

4 Discussion

The purpose of this study was to determine the effect ofthe LZ-8 an immunomodulatory protein derived from Glucidum when it is used for wound healing on hepatictissues and to compare this with monopolar electrosurgeryoperated liver tissues on the wounds size fibrosis thermalinjury and apoptosis The results of this study show thatLZ-8 enhances wound healing actions in hepatic tissuesafter electrosurgery Importantly this study has identifiedthat NF-120581B and caspase-3 activation are associated with LZ-8 on enhancing wound healing after surgery Purpose ofelectrical energy or coagulation of bleeding vessels onto tissueis frequently used in surgery Coagulation techniques suchas monopolar and laser techniques are commonly used indifferent surgical disciplines [21 22]

Monopolar and bipolar coagulation effects on brain tissuewere reported and the results of experimental trials were

compared [23 24] They observed less trauma after bipolarcoagulation compared with monopolar technique A studyhas demonstrated that bipolar mode of stimulation claimedto be another paradigm of a potential hazard comparedwith monopolar stimulation Temel et al [25] observed braindamage after bipolar but not monopolar and stimulationin rats However monophasic pulses were used whichmight have created larger lesions in a bipolar configurationMonopolar electrosurgery causes sparking current spreadand thermal damage in the tissues because of excessivegeneration of heat These problems will induce deleteriouseffects to the tissue in proximity to the operative site Inaddition when comparing monopolar electrosurgery andthe scalpel incision electrosurgery produces more tissuealteration and histological thermal damage when a lateralheat was increased by the low frequency radio wave of05 to 29MHz [26] Consequently the resultant effect ofmonopolar systems is excessive tissue damage and delayed

Evidence-Based Complementary and Alternative Medicine 9

Cy 5 DAPI Merge

Control

SS

SS + LZ-8

Day 3

SS

SS + LZ-8

Day 7

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

Figure 7 The immunofluorescence staining results from rats liver treated with SS group and SS + LZ-8 group after different healing days

healing Therefore additional drug supplements or therapyneed to be required to accelerate the wound healing actionon tissues after electrosurgery

A new immunomodulatory protein known as LZ-8 wasisolated from the mycelia of G lucidum and characterizedby chromatographicelectrophoretic techniques [17] Bothin vivo immunomodulation activity and in vitro mitogenicactivity of LZ-8 have been reported by Kino et al [14] Pre-vious studies have also demonstrated that topical applicationof oils improves some parameters of wound healing such asstimulation of connective tissue fiber [27] reduction in thethickness of the necrotic cell layer edge around the wound[28] and acceleration of wound closure [29 30] Due toexcellent thermal and acid stability together with a moderateresistance to alkali and dehydration LZ-8 is appropriate toapply tissues after surgery even with a mild thermal heatIn this study this is the first and novel results that LZ-8accelerates the reduction of the wound size thermal injuryand increase fibrosis area in rat liver after surgery and it

indicates that LZ-8 has potent ability to enhance the effect ofwound healing In a latest study we have also demonstratedthat the total areas of liver injury lateral thermal injury andfibrotic tissues were significantly higher in SS treated hepatictissues than that of CrN-coated needles that had been usedfor surgery [13]

NF-120581B activation plays a role in the regulation of morethan 400 gene product expressions associatedwith inflamma-tion cell survival proliferation invasion and angiogenesis[31 32] NF-120581B normally binds to I120581B120572 which impedes NF-120581B nuclear translocation from the cytoplasm to the nucleusNF-120581B is reported to be a vital link between hepatic injuryfibrosis and even hepatocellular carcinoma [33] NF-120581Benters the nucleus with the degradation of I120581B and activatesthe expression of specific genes that induce the apoptosiscascade [34] Once the cells are exposed to inflammatorystimuli including LPS and TNF-120572 I120581B120572 is phosphorylatedleading to I120581B120572 degradation and nuclear translocation of NF-120581B In our latest study it was noticed that liver tissues that had

10 Evidence-Based Complementary and Alternative Medicine

TUNEL DAPI Merge

SSSS

+ L

Z-8

250 120583m 250 120583m 250 120583m

250 120583m 250 120583m 250 120583m

Figure 8 The TUNEL staining assay results from rats liver treated with SS group and SS + LZ-8 group after 7 days of healing

been operated with SS-needles secreted higher levels of NF-120581B expression and its nuclear translocation than that of CrNoperated tissues [13] Fibrosis is an important component ofadvanced chronic inflammatory liver disease Although thechanges in hepatic structures that occur in liver cirrhosis havebeen described in detail the pathogenesis of fibrosis in duringelectrosurgery process is largely unknown Our latest reportindicates that monopolar elecrtrosurgery-induced hepaticfibrosis after repeated liver injury and the mechanisms aremainly based on the upregulation of NF-120581B and caspase-3expression in the liver tissues [13]

In this study we thus evaluated whether LZ-8 had anyeffect on nuclear translocation of NF-120581B during monopolarelectrosurgery and the results found for the first time thatLZ-8 inhibits nuclear translocation of NF-120581B in hepaticcells after 3 and 7 days of monopolar electrosurgery Yatesand Rayner [35] studied the effects of fatty acid supple-mentation on activation of NF-120581B and AP-1 after a skininjury since these transcription factors are highly associ-ated with wound healing They have also reported a novelfinding which indicates that wounding of normal humankeratinocytes in culture causes NF-120581B translocation Therelevance of NF-120581B activation to wound healing is sug-gested by the results that HeNe laser treatment a processshown to accelerate healing in a model of wounding [36]promoted the translocation of NF-120581B A previous studyalso proved that a ganoderma immunomodulatory proteincloned from Ganoderma microsporum holds anti-invasiveand anti-inflammatory activities in TNF-120572 induced humanalveolar epithelial A549 cells via inhibition of the I120581B120572 andNF-120581B pathway [37]

Apoptosis in wound healing is a promising issue Therehas been some evaluation of chronic diabetic wounds in

mice that suggests that there is increased cellular apoptosislikely due to hyperglycemia which produces reactive oxygenspecies that mediate mitochondrial release of cytochrome cand activate caspase-3 [37 38] Apoptosis is also involved inthe regulation of collagen synthesis and degradation withinwounds through regulation of fibroblast numbers and colla-genase activity [39] Recent work in another mouse modelsuggests that hypertrophic scars demonstrate decreased ratesof apoptosis with decreased expression of cleaved caspase-3Over expression of various caspase family members inducesapoptosis in cultured mammalian cells [40] Among thecysteine proteases caspase-3 is believed to be one of themost commonly involved in the execution of apoptosisin various cell types [41] The assay of TUNEL stainingcan identify the apoptotic cells via DNA fragmentation bylabeling the terminal ends of nucleic acids [42 43] Fromour latest study of TUNEL assay it can be clearly foundthat the apoptosis event was more significant in SS-needleoperated liver tissues [13] In this study TUNEL stainingprovides direct evidence of apoptosis in SS operated livertissues after 7 days Both the activation of caspase-3 and theinduction of apoptotic cell death could be suppressed by theaddition of antioxidants in the cultures [44] It has also beendemonstrated that LZ-8 is able to effectively promote theactivation and maturation of immature dendritic cells via thenuclear factor-kappaB and mitogen-activated protein kinasepathways [18] Moreover the present study observed theeffects of (i) inhibiting expression and nuclear translocationof NF-120581B and (ii) inhibiting expression of caspase-3 andreduction of apoptotic cell death by LZ-8 in liver tissuesafter electrosurgery which indicates that the wound healingproperty of LZ-8 may possibly occur through inhibition oftranscription factor NF-120581B and apoptotic factor caspase-3

Evidence-Based Complementary and Alternative Medicine 11

5 Conclusion

Under the same conditions LZ-8 treatment potentiates onreducing wound size and thermal injury An increasing fibro-sis and reducing apoptosis were found in LZ-8 treated livertissues during electrosurgery LZ-8 treatment significantlyinhibits NF-120581B and caspase-3 expressions and also reducesNF-120581B nuclear translocation and apoptotic cell death Theresults generated from this study suggest that LZ-8 has potenteffects on increasing wound healing action against the elec-trosurgical induced liver injury via the possible mechanismof inhibiting NF-120581B and caspase-3 expressions

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors would like to thank the Department of HealthExecutive Yuan Taiwan (Contract no MOHW103-TDU-N-211-133001) and TaipeiMedical University and TaipeiMedicalHospital (Contract no 100TMU-TMUH-05) for financiallysupporting this research Dr Hao-Jan Lin and Dr Yushan-Sophie Chang are considered the first coauthors of the paper

References

[1] J Liboon W Funkhouser and D J Terris ldquoA comparison ofmucosal incisions made by scalpel CO

2

laser electrocauteryand constant-voltage electrocauteryrdquo OtolaryngologymdashHeadand Neck Surgery vol 116 no 3 pp 379ndash385 1997

[2] J F Carew R F Ward A LaBruna P A Torzilli and W SSchley ldquoEffects of scalpel electrocautery and CO

2

and KTPlasers on wound healing in rat tonguesrdquoThe Laryngoscope vol108 no 3 pp 373ndash380 1998

[3] J A Pearce Electrosurgery John Wiley amp Sons New York NYUSA 1986

[4] W L Maness F W Rober R E Clark E Cataldo andA W Haddad ldquoTissue damage from electrosurgical poweroutput variations in hamster tonguesrdquoThe Journal of ProstheticDentistry vol 42 no 4 pp 456ndash460 1979

[5] W L Maness F W Roeber R E Clark E Cataldo D Riisand A W Haddad ldquoHistologic evaluation of electrosurgerywith varying frequency andwaveformrdquoThe Journal of ProstheticDentistry vol 40 no 3 pp 304ndash308 1978

[6] J Friedman J Margolin and S Piliero ldquoA preliminary study ofthe histological effects of three different types of electrosurgicalcurrentsrdquoThe New York State Dental Journal vol 40 no 6 pp349ndash353 1974

[7] W H Noble K D McClatchey and G D Douglass ldquoAhistologic comparison of effects of electrosurgical resectionusing different electrodesrdquo The Journal of Prosthetic Dentistryvol 35 no 5 pp 575ndash579 1976

[8] K C Nixon K F Adkins and D W Keys ldquoHistologicalevaluation of effects produced in alveolar bone followinggingival incision with an electrosurgical scalpelrdquo Journal ofPeriodontology vol 46 no 1 pp 40ndash44 1975

[9] I Glickman and L R Imber ldquoComparison of gingival resec-tion with electrosurgery and periodontal knivesmdasha biometric

and histologic studyrdquo Journal of Periodontology vol 41 no 3pp 142ndash148 1970

[10] D H Coelho J Cavallaro and E A Rothschild ldquoGingivalrecession with electrosurgery for impression makingrdquo TheJournal of Prosthetic Dentistry vol 33 no 4 pp 422ndash426 1975

[11] P B Robertson B Luscher L S Spangberg and B M LevyldquoPulpal and periodontal effects of electrosurgery involvingcervical metallic restorationsrdquo Oral Surgery Oral Medicine andOral Pathology vol 46 no 5 pp 702ndash710 1978

[12] R DrsquoSouza ldquoPulpal and periapical immune response to electro-surgical contact of cervical metallic restorations in monkeysrdquoQuintessence International vol 17 no 12 pp 803ndash808 1986

[13] K-L Ou J-S Chu H Hosseinkhani J-F Chiou and C-HYu ldquoBiomedical nanostructured coating for minimally invasivesurgery devices applications characterization cell cytotoxicityevaluation and an animal study in ratrdquo Surgical Endoscopy 2014

[14] K Kino A Yamashita K Yamaoka et al ldquoIsolation and char-acterization of a new immunomodulatory protein Ling Zhi-8(LZ-8) from Ganoderma lucidiumrdquo The Journal of BiologicalChemistry vol 264 no 1 pp 472ndash478 1989

[15] AMurasugi S Tanaka N Komiyama et al ldquoMolecular cloningof a cDNAand a gene encoding an immunomodulatory proteinLing Zhi-8 from a fungusGanoderma lucidiumrdquoThe Journal ofBiological Chemistry vol 266 no 4 pp 2486ndash2493 1991

[16] M-H Tong P-J Chien H-H Chang M-J Tsai and F SheuldquoHigh processing tolerances of immunomodulatory proteins inEnoki and Reishi mushroomsrdquo Journal of Agricultural and FoodChemistry vol 56 no 9 pp 3160ndash3166 2008

[17] L Huang F Sun C Liang et al ldquoCrystal structure of LZ-8 fromthemedicinal fungusGanoderma lucidiumrdquo Proteins StructureFunction and Bioinformatics vol 75 no 2 pp 524ndash527 2009

[18] Y-L Lin Y-C Liang Y-S Tseng et al ldquoAn immunomodulatoryprotein Ling Zhi-8 induced activation and maturation ofhuman monocyte-derived dendritic cells by the NF-120581B andMAPK pathwaysrdquo Journal of Leukocyte Biology vol 86 no 4pp 877ndash889 2009

[19] R A D Drury and E A Wallington Carletonrsquos HistologicalTechnique 1981

[20] Y Du D-W Guan and R Zhao ldquoAn immunohistochemicalstudy on the expression of caspase-3 in the skin wound healingrdquoFa Yi Xue Za Zhi vol 20 no 1 pp 1ndash3 2004

[21] K E Grund T Straub and G Farin ldquoNew haemostatictechniques argon plasma coagulationrdquo Baillierersquos Best Practiceand Research in Clinical Gastroenterology vol 13 no 1 pp 67ndash84 1999

[22] D Hellwig R Haag V Bartel et al ldquoApplication of newelectrosurgical devices and probes in endoscopic neurosurgeryrdquoNeurological Research vol 21 no 1 pp 67ndash72 1999

[23] B Chehrazi and W F Collins Jr ldquoA comparison of effects ofbipolar and monopolar electrocoagulation in brainrdquo Journal ofNeurosurgery vol 54 no 2 pp 197ndash203 1981

[24] J Kerl and J Staubesand ldquoAn ultrastructural report on thechoice between monopolar and bipolar electrocoagulationrdquoActa Neurochirurgica vol 92 no 1ndash4 pp 123ndash131 1988

[25] Y Temel V Visser-Vandewalle M van derWolf et al ldquoMonop-olar versus bipolar high frequency stimulation in the rat subtha-lamic nucleus differences in histological damagerdquoNeuroscienceLetters vol 367 no 1 pp 92ndash96 2004

[26] L I Malis ldquoNew trends in microsurgery and applied technol-ogyrdquo in Advanced Technology in Neurosurgery F Pluchino andG Broggi Eds pp 1ndash16 Springer Berlin Germany 1988

12 Evidence-Based Complementary and Alternative Medicine

[27] C R B Cardoso M A Souza E A V Ferro S Favoreto Jrand J D O Pena ldquoInfluence of topical administration of n-3and n-6 essential and n-9 nonessential fatty acids on the healingof cutaneous woundsrdquoWound Repair and Regeneration vol 12no 2 pp 235ndash243 2004

[28] L M Pereira E Hatanaka E F Martins et al ldquoEffect of oleicand linoleic acids on the inflammatory phase of wound healingin ratsrdquo Cell Biochemistry and Function vol 26 no 2 pp 197ndash204 2008

[29] N-Y ParkGValacchi andY Lim ldquoEffect of dietary conjugatedlinoleic acid supplementation on early inflammatory responsesduring cutaneous wound healingrdquo Mediators of Inflammationvol 2010 Article ID 342328 8 pages 2010

[30] M Otranto A P do Nascimento and A Monte-Alto-CostaldquoEffects of supplementation with different edible oils on cuta-neous wound healingrdquoWound Repair and Regeneration vol 18no 6 pp 629ndash636 2010

[31] X Dolcet D Llobet J Pallares and X Matias-Guiu ldquoNF-kBin development and progression of human cancerrdquo VirchowsArchiv vol 446 no 5 pp 475ndash482 2005

[32] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[33] T Luedde and R F Schwabe ldquoNF-120581B in the livermdashlinkinginjury fibrosis and hepatocellular carcinomardquo Nature ReviewsGastroenterology amp Hepatology vol 8 no 2 pp 108ndash118 2011

[34] J A Schmid and A Birbach ldquoIkappaB kinase beta (IKKbetaIKK2IKBKB)mdasha keymolecule in signaling to the transcriptionfactor NF-kappaBrdquo Cytokine amp Growth Factor Reviews vol 19no 2 pp 157ndash165 2008

[35] S Yates and T E Rayner ldquoTranscription factor activation inresponse to cutaneous injury role of AP-1 in reepithelializa-tionrdquo Wound Repair and Regeneration vol 10 no 1 pp 5ndash152002

[36] A F Haas R R Isseroff R G Wheeland P A Rood and PJ Graves ldquoLow-energy helium-neon laser irradiation increasesthe motility of cultured human keratinocytesrdquo The Journal ofInvestigative Dermatology vol 94 no 6 pp 822ndash826 1990

[37] C-H Lin Y-M Hsiao C-C Ou et al ldquoGMI a Ganodermaimmunomodulatory protein down-regulates tumor necrosisfactor 120572-induced expression of matrix metalloproteinase 9 viaNF-120581Bpathway in human alveolar epithelial A549 cellsrdquo Journalof agricultural and food chemistry vol 58 no 22 pp 12014ndash12021 2010

[38] L Cai W Li G Wang L Guo Y Jiang and Y J KangldquoHyperglycemia-induced apoptosis in mouse myocardiummitochondrial cytochrome c-mediated caspase-3 activationpathwayrdquo Diabetes vol 51 no 6 pp 1938ndash1948 2002

[39] N K Rai K Tripathi D Sharma andV K Shukla ldquoApoptosis abasic physiologic process in wound healingrdquo The InternationalJournal of Lower Extremity Wounds vol 4 no 3 pp 138ndash1442005

[40] J Bian and Y Sun ldquoTranscriptional activation by p53 of thehuman type IV collagenase (gelatinase A or matrix metallopro-teinase 2) promoterrdquoMolecular and Cellular Biology vol 17 no11 pp 6330ndash6338 1997

[41] S Wang MMiura Y-K Jung et al ldquoIdentification and charac-terization of ich-3 a member of the interleukin-1120573 convertingenzyme (ICE)Ced-3 family and an upstream regulator of ICErdquoThe Journal of Biological Chemistry vol 271 no 34 pp 20580ndash20587 1996

[42] E S Alnemri D J Livingston D W Nicholson et al ldquoHumanICECED-3 protease nomenclaturerdquo Cell vol 87 no 2 article171 1996

[43] T D Hewitson T Bisucci and I A Darby ldquoHistochemicallocalization of apoptosis with in situ labeling of fragmentedDNArdquo Methods in Molecular Biology vol 326 pp 227ndash2342006

[44] D T Loo ldquoIn situ detection of apoptosis by the TUNEL assayan overview of techniquesrdquo in DNA Damage Detection In SituEx Vivo and In Vivo vol 682 ofMethods in Molecular Biologypp 3ndash13 Humana Press New Jersey NJ USA 2011

Submit your manuscripts athttpwwwhindawicom

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Behavioural Neurology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 2: Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014 › 916531.pdfResearch Article An Immunomodulatory Protein (Ling Zhi-8) from a

2 Evidence-Based Complementary and Alternative Medicine

Although incision can be accomplished by both but thebipolarmode ismore inefficient and restrictive in applicationBipolar coagulation is slower than monopolar activity [3]Their slow action is considered a disadvantage for surgicalprocedures but proves to be of significant benefit for moreprecise microsurgical procedures due to their easy usagewide availability incision accuracy and minimal damageto adjacent tissues The use of steel scalpel in monopolarelectrosurgery remains an imperative need for electrosurgeryoperating system

The effects of electrosurgery (ES) on histoarchitecturevary depending on the power output [4] and frequency [5]of ES unit waveform selection [6] and shape and size ofactive electrode [7] It is well observed that wound healing forelectrosurgery delays through comparative studies for elec-trosurgical and scalpel wounds [8] Electrosurgical woundshave more inflammatory responses and tissue destructionbut the viability of osteoblasts in both categories shows thesame in both kinds of wounds with no increase in osteoclastswhich could indicate no bone resorption occurrences Onthe contrary [9] it could be found out that there will be nodifferences on wound healing between ES and periodontalknives when the gingival resection is shallow Howeverthere could be intense inflammatory and bone loss in heightresulting from bone necrosis during deep resection Otherresearch study [10] shows that although there is loss oftissue soon after ES 70 to 100 of the lost tissue is regainedover a period of months When teeth with cervical amalgamrestoration get contact with active electrode tip to simulatefrom clinical application report [11] there is no evidenceof extensive damage or necrosis of pulp [12] Macroscopicobservation and histological examination of our latest studyclearly show that commercial stainless steel needles producelarger injury area than CrN-needles [13] As reported slowwound healing found as a result of operations while usingboth mono- and bipolar electrosurgery devices an extratherapy is highly recommended to enhance this woundhealing after surgery which this paper clearly reports

ldquoLing Zhirdquo is a Chinese term representing combinationof spiritual potency and essence of immortality and widelyregarded as the ldquoherb of spiritual potencyrdquo symbolizingsuccess well-being divine power and longevity Amongcultivated mushrooms G lucidum is unique in that itspharmaceutical rather than nutritional value is paramountA variety of commercial G lucidum products are available invarious forms such as powders dietary supplements and teaThese are produced from different parts of the mushroomincluding mycelia spores and fruit body Ling Zhi-8 (LZ-8)an immunomodulatory protein isolated from mycelia of Glucidum could be considered as one of the major bioactivesubstances of its kind [14] They also contain excellent ther-mal and acidity stability together with moderate resistivity toalkalinity and dehydration [15] LZ-8 consists of 110 aminoacids and a molecular mass of 124 kDa which is similarto that of the variable region of the immunoglobulin heavychain [16] LZ-8 exerts mitogenic activities in vitro and hasalso shown in vivo immunomodulatory activities [17] Ling-Zhi had been widely recommended for specific applicationsand attributed health benefits for treatment of migraine

hypertension arthritis bronchitis asthma anorexia gas-tritis hemorrhoids and diabetes and for the control ofblood glucose levels modulation of the immune systemhepatoprotection bacteriostasis and more

A recent study haswell demonstrated that LZ-8 effectivelypromotes the activation and maturation of immature humandendritic cells via regulating NF-120581B and MAPKs path ways[18] Hitherto several studies have focused on wound healingactions in different tissues after electrosurgery inducinginflammatory reaction and reepithelialization and stimu-lating angiogenesis However there is relatively little infor-mation pertaining to wound healing activity and possiblemechanism on liver tissues Hence the present study wasdesigned to have a three pronged approach (a) to examinethe wound size injury fibrosis and apoptosis in rat modelfor the liver tissues after monopolar electrosurgery (b) toascertain whether LZ-8 protein enhances wound healing forthe liver tissues created by surgery and (c) to elucidate thepossiblemechanismof LZ-8 onwoundhealing viaNF-120581Bandcaspase-3 expressions

2 Materials and Methods

21 Materials LZ-8 was provided by Yeastern Biotech CoLtd Taiwan Anti-NF-120581B (MAB3026) caspase-3 (AB1899)120572-tubulin mAb (MAB1501) monoclonal antibodies (mAbs)horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG) and sheep anti-mouse IgGwere all from Chemicon Millipore Temecula CA Thenitrocellulose membrane (HybondTM-C Extra AmershamBiosciences Corp Hong Kong China) enhanced chemilu-minescence (ECL) Western blotting detection reagent andanalysis system (WBLUC0100) were also purchased fromMillipore Temecula CA LZ-8 was dissolved in phosphatebuffered saline (PBS) and stored at 4∘C until the time ofusageapplication used

22 Animals and Surgical Device Sixteen healthy male Spra-gue-Dawley rats weighing 200 to 300 grams (g) eachprocured from BioLASCO (Taiwan) were used for theexperiments These Sprague-Dawley rats were prepared andmaintained under the guidelines for care and usage oflaboratory animals at 25∘C and exposed to 12 h of dark and12 h of lightTheywere fed a standard balanced pellet diet andwater ad libitum The protocols for animal experiments werereviewed and approved by the Institutional Animal Care andUse Committee of Taipei Medical University (LAC-99-0037)Rats were marked to permit individual identification andwere kept in their cages for 14 days prior to experimentationto allow for acclimatization to the laboratory conditions Noreusable commercial electrosurgical monopolar tip (ERBEnumber 20191-377 USA needle type stainless steel 304) wasused

23 Surgical Procedure General anesthesia was inducedwith inhaled isoflurane for the animals The operative sitewas cleansed and draped in a sterile fashion Conven-tional stainless steel (SS) needle electrodes were used with

Evidence-Based Complementary and Alternative Medicine 3

SS

0 7 28Days

3

SS (stainless steel)

500 120583m 500 120583m 500 120583m 500 120583m

(a)

SS +

LZ-

8

0 7 28Days

3

LZ-8 (Ling Zhi-8)

500 120583m 500 120583m 500 120583m 500 120583m

(b)

Figure 1 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for different healing days (HampE staining)(a) SS group and (b) SS + LZ-8 group

an electrosurgical unit The liver was exposed through mid-line laparotomywith a retractor to avoid injury to the liver Anunmodified SS-needle was inserted to the liver lobe (3mm indepth) to create an anterior lesion using a fixed power setting(20W) with a fixed activation time (3 seconds) The powersetting and activation time were commonly used parametersand were based on previous study settings For LZ-8 proteingroup 10 120583L of 1mgmL LZ-8 protein solution was pipettedand dropped on the wound surface For control group andexperiment group 10120583L of normal saline was pipetted anddropped on the wound surface

24 Weight of Sticking Tissue The weights of each SS-needlewere recorded perioperatively and the weight of stickingtissue was calculated as the weight of needle after operationminus the weight of needle before operation

25Thermography The effects of the SS-needles on tempera-tures across the live lobe surface were determined from ther-mographs obtained with a thermal-imaging infrared camera(Advanced Thermo TVS-500EX NEC Avio TechnologiesTokyo Japan) The highest temperature was recorded forevery test After recording the thermograph the surroundingsoft tissues were carefully repaired and the surgical woundwas closed in layers No anti-inflammatory regimen was usedafter surgery

26 Histological Examination to Detect Injury Area Histo-pathological studies were performed to provide structuralevidence ofmediated tissue injury and protective effect of LZ-8 These animals were sacrificed on days 0 3 7 and 28 afteroperation respectively Tissues were then cut into pieces ofdesired size and fixed in Bouinrsquos fluid fixative immediatelyafter autopsy Fixation was carried out at room temperaturefor 24 hrs after which these fix tissues were transferred to70 alcohol Several changes of 70 alcohol were given untilthe yellow colour disappeared from the tissues The tissueswere then dehydrated by passing through ascending grades ofalcohol (30 50 70 90 and 100) cleared in methylsalicylate and infiltrated with wax at 57∘C The clear tissueswere embedded in the paraffin Conventional techniques ofparaffinwax sectioningwere used for histological studies [19]Paraffin wax sections of 6 to 8 120583m thickness were cut using arotary microtome (Leica Germany) The sections were fur-ther stained with hematoxylin and eosin (HampE) andMassonrsquostrichrome staining and then washed in 90 alcohol for fewseconds Subsequently the stained sections were dehydratedin 100 alcohol cleared in xylene and mounted in DPXmountant The stained slides were observed in a Carl Zeiss(Germany) Axio 2 Plus research microscope Images werecaptured through a CCD camera (BX51 Olympus Japan)in a computer and processed using Carl Zeiss Axiovisionsoftware The total injury areas caused by the SS-needleare measured using image analysis software (SPOT basic

4 Evidence-Based Complementary and Alternative Medicine

(a)

TA Thermal injury area IA Injury area (trauma)

TA

IA

TA

IA

TA

IA

TAIA

TA

TA

IA

IA

SSSS

+ L

Z-8

SS SS + LZ-8

SS SS + LZ-80

10

20

30

40

50

60

70

80

90

100

Are

a (

)

Thermal injury areaInjury area

Wound area (TA + IA)

(c)(b)

500 120583m

100 120583m

100 120583m

500 120583m

Figure 2 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 0 days (Massonrsquos trichrome staining)(a) wound area (b) the site of thermal injury area and injury area and (c) percentage of each area Data are expressed as the means plusmn SEM(119899 = 5) No significant difference in the thermal injury area injury area and wound area at day 0

software SPOT imaging solutions MI USA) In additiondifferent histological features were observed on the injurysite Therefore the injured area was further divided intodifferent classifications to demonstrate the injury caused bythermal spread and the area of every portion was measured

27 Western Blot Analysis The retrieved liver tissues werehomogenized and lysed in an ice-cold protein lysis buffer andcentrifuged at 13000 rpm for 15 minutes at 4∘C Protein con-centrations of the lysates were determined using a Bio-Radprotein assay (Bio-Rad Laboratories Hercules CA USA)The lysates (20120583glane) were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in10 gels and electroblotted onto a nitrocellulose membrane(HybondTM-C Extra Amersham Biosciences Corp HongKong China) The membrane was blocked for 30 minutesat room temperature in 3 nonfat milk Tris-buffered salinewith 005 Tween 20 and then incubated overnight at4∘C with the indicated primary antibodies against NF-120581B(1 500 MAB3026 Chemicon Millipore Temecula CA) orcaspase-3 (1 200 AB1899 Millipore Temecula CA) andthen incubated with horseradish peroxidase-conjugated sec-ondary antibodies for 2 hours at room temperature After

washing the membrane was developed using chemilumi-nescent substrates (WBLUC0100 Millipore Temecula CA)Densitometric analysis of the gels was performed using Gel-Pro analyzer software and the results were expressed relativeto the density of 120573-actin (1 500 MAB1501 ChemiconMillipore Temecula CA)

28 Immunofluorescence Staining and TUNEL Staining Forimmunofluorescence staining liver tissues were embeddedin Cryomatrix (Thermo Shandon Pittsburgh PA USA) andstored at minus80∘C Cryostat sections (5ndash7mm thickness) werefixed in a mixture of methanol and acetone (1 1) at minus20∘Cfor 5 minutes and incubate in 1 Triton X-100 for another5 minutes Nonspecific activity was blocked by incubatingthe sections with 10 horse serum in a buffer containing002 Tween 20 in phosphate-buffered saline for 30 minutesSections were then incubated overnight with primary anti-Caspase-3 at 4∘C Antigen localization was indicated bya Cy5 dye (red fluorescence 111-176-003 Jackson ImmuneResearch West Grove PA) Finally the samples were stainedwith 410158406-diamidino-2-phenyl-indole (DAPI sc-3598 SantaCruz Biotechnology CA) for 7 minutes and examined usingfluorescent microscope Normal liver tissues obtained from

Evidence-Based Complementary and Alternative Medicine 5

NA

TA

IA

IA

TA

NA

NA Normal area TA Thermal injury area IA Injury area (trauma)

SS SS + LZ-80

10

20

30

40

50

60

70

80

Are

a (

)

Thermal injury areaInjury area

Wound area (TA + IA)

SSSS

+ L

Z-8

(a)

(c) (b)

100 120583m

100 120583m

lowastlowast

lowast

lowastlowastlowast

SS SS + LZ-8

500 120583m 500 120583m

Figure 3 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 3 days (Massonrsquos trichrome staining)(a) wound area (b) the site of thermal injury area injury area and normal area and (c) percentage of each area Data are expressed as themeans plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

healthy rats undergo identical procedures Negative controlswere processed identically except that the primary antibodieswere replaced with IgG An in situ cell death detection kit(terminal deoxynucleotidyl transferase dUTP nick end label-ing TUNEL 11684795910 RocheMannheim Germany) wasused to detect apoptotic cells and the apoptotic cells wereindicated by a green fluorescent FITC fluorochrome

29 Statistical Analysis Data were expressed as mean plusmnstandard error of the mean (SEM) Data were analyzed usinganalysis of variance (ANOVA) All statistical analyses wereperformed using SPSS version 120 (SPSS Inc Chicago IL)Values of 119875 lt 005 were considered significant

3 Results

31 Stimulatory Effects of LZ-8 on Wound Healing in RatLiver Tissues after Monopolar Electrosurgery In generallybleeding and healing could easily be reduced when tissuesare subjected to electrosurgery To determine whether LZ-8could enhance wound healing on liver tissues after surgerywe compared the wound size after monopolar electrosurgeryand the operated tissues that had been treatedwith LZ-8 for 03 7 and 28 daysThe observed results of HampE stained section

of liver tissues show that a significant reduced of wound sizetreated with LZ-8 (3 7 and 28 days) dependently relative tothe normal (untreated) rats as shown in Figure 1 Moreoverthe mean wound sizes for these liver tissues were moreprominently reduced in tissues that were treated with LZ-8 at28 days when compared with untreated groups and with day3 and day 7 The results clearly indicated that treatment withLZ-8 enhances wounds healing faster after surgery in rat livertissues

32 Effects of LZ-8 on the Site of Thermal Damage Injuryand Wounds of Liver Tissues after Surgery Figure 2 showshistological sections of monopolar SS on the operated livertissues and tissues subjected to LZ-8 treatment (SS + LZ-8)for 0 days (Massonrsquos trichrome staining) Clearly there wasno significant difference in the total injury areas caused by theSS and SS + LZ-8 treatments Moreover the injury area andthermal injury area in SS group were also similar to those ofSS + LZ-8 group at day 0 However the results demonstratedthat damage of liver tissues in the form of injury thermalinjury and wound was negligible in SS group for 3 days(Figure 3) when compared to 0 days (Figure 2) Interestinglythe effect of injury thermal injury and wound of liver tissueswas significantly reduced in SS + LZ-8 group for 3 days It

6 Evidence-Based Complementary and Alternative Medicine

NA Normal area FA Fibrosis area AA Apoptosis area

NA

FAFAAA

AA

AAFA

FA

NA

SSSS

+ L

Z-8

(b)

50 120583m

50 120583m

50 120583m

50 120583m

SS SS + LZ-80

10

20

30

40

50

60

70

80

90

Are

a (

)

Fibrosis area Injury areaApoptosis area Wound area (FA + IA + AA)

(c)

lowast

lowastlowast

lowastlowastlowast

(a)

SS SS + LZ-8

500 120583m500 120583m

Figure 4 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 7 days (Massonrsquos trichrome staining)(a) wound area (b) the site of apoptosis area fibrosis area and normal area and (c) percentage of each area Data are expressed as themeans plusmnSEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

was found that the injury area (119875 lt 005) thermal injuryarea (119875 lt 001) and wound area (119875 lt 0001) of SS + LZ-8groupwere reducedmore significantly than those of SS groupat day 3 Furthermore Massonrsquos trichrome staining was usedto examine the formation of fibrotic tissue No fibrotic tissuewas evident for either group at day 0 or day 3

33 Effects of LZ-8 on the Areas of Fibrosis Injury ApoptosisandWounds of Liver Tissues after 7 Days of Surgery A furtherstudy was performed to determine whether LZ-8 couldenhance the areas of fibrosis reduce injury and wounds andinhibit apoptosis of monopolar SS induced liver incision interms of withdrawing the above observed injuriesThe resultsclearly confirmed that injury apoptosis and wound of livertissues in SS operated groups that had been treated withLZ-8 for 7 days were significantly alleviated when comparedwith SS operated groups without LZ-8 treatment (Figure 4)Nevertheless the areas of fibrosis and numbers of cell deathin liver tissues for treated rats with LZ-8 were comparativelyhigher than those of SS operated rats Thus the resultsindicate that LZ-8 treatment could not affect the reduction ofliver fibrosis andmore effective on the inhibition of apoptosiswhich are caused by surgery

34 Effects of LZ-8 on the Areas of Fibrosis Injury Apoptosisand Wounds of Liver Tissues after 28 Days of Surgery Sincedifferential observations were found on the areas of fibrosisand apoptosis in liver tissues of LZ-8 supplemented rats thathave already been operated with SS for 7 days we furtherextended the duration of treatment up to 28 days (Figure 5)to confirm the hypothesis Our analysis obviously exhibitsfurther evidence that the effect of LZ-8 was similar to thatof 7-days treatment

35 Does LZ-8 Enhance Wound Healing in SS Operated LiverTissues via Inhibiting the Expression of NF-120581B and Caspase-3It is well known that the angiogenesis is essential in woundhealing Various studies have demonstrated the inhibition ofangiogenesis by the blockage of NF-120581B A recent study hasalso suggested that caspase-3 plays an important role duringskin wound healing [20] Thus we performed this study todeterminewhether thewound healing action of LZ-8was dueto the effect on inhibiting of NF-120581B and caspase-3 expressionthe tissue levels of their protein were examined by Westernblot analysis As shown in Figure 6(a) liver tissues exposedto electrosurgery with SS for 3 days could slightly inducethe expression of NF-120581B and it gradually increased up to

Evidence-Based Complementary and Alternative Medicine 7

SS0

5

10

15

20

25

30

Are

a (

)

Fibrosis area Apoptosis area

Wound area (FA + AA)

(a)

(c) (b)

FA Fibrosis area AA Apoptosis area PA Proliferate area

SSSS

+ L

Z-8

SS SS + LZ-8

SS + LZ-8

AA

FA

PA

AA

FA

AA

PA

lowast

lowast

lowastlowast

500 120583m500 120583m

50 120583m

50 120583m

Figure 5The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 28 days (Massonrsquos trichrome staining)(a) wound area (b) the site of apoptosis area fibrosis area and proliferate area and (c) percentage of each area Data are expressed as themeans plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

28 days Interestingly liver tissues further treated with LZ-8 significantly reduced the ratio of NF-120581B expression in aduration dependent manner (3 to 28 days) A similar resultwas observed on the expression of caspase-3 Therefore theresults can suggest that treatment of liver tissues after surgerywith LZ-8 was found to lower the mean NF-120581B and caspase-3 expressions so that it approaches wound healing propertypresumably by limiting NF-120581B and caspase-3 expression inliver tissuesThese results were further confirmed and shownas in Figure 6(b)

36 LZ-8 Inhibits NF-120581BNuclear Translocation in SS OperatedLiver Tissues The ability of LZ-8 to inhibit NF-120581B dispersionwas determined by red fluorescence (Cy5) intensity analysisof immunolabelled antibodies directed against NF-120581B andHoechst reagent to simultaneously identify nuclei (Figure 7)The intensity values of NF-120581B red fluorescence in the nucleusof SS operated tissues that were treated with LZ-8 for 3 dayswere found to be lesser as compared to SS operated livertissues without LZ-8 treatment Interestingly the presence ofLZ-8 in SS treated liver tissues for 7 days reached amaximuminhibition of nuclear fluorescence of NF-120581B than that oftreated tissues with SS alone These results can obviouslyindicate that LZ-8 plays a major role in inhibiting of NF-120581B

nuclear translocation in liver tissues during the process ofwound healing after electrosurgery

37 LZ-8 Reduces Apoptotic Cell Death in Liver Tissues duringWoundHealing after Electrosurgery Extensive fragmentationof nuclear DNA that generates a large number of DNAdouble-strand breaks is one of the most characteristic eventsof apoptosis An assay that relies on detection of DNA strandbreaks (DSBs) in situ by labeling them with fluorochromeshas been developed to identify and quantify apoptotic cellsby fluorescence microscopy or cytometry The assay is com-monly called TUNEL the acronym of Terminal deoxynu-cleotidyl transferase-mediated d-UTP Nick End Labeling Inthis study a very clear TUNEL-positive cells were observedin rats hepatic cells after 7 days of electrosurgery (Figure 8)However the number or TUNEL expression continued todecrease more prominently in LZ-8 treated hepatic cells thathad been exposed with monopolar electrosurgery for 7 daysThe results obtained from this study demonstrate that a slightapoptosis occurs in hepatic cells after electrosurgery whichwas as a result of LZ-8 inhibition and provide evidencefor high level of angiogenesis arises during wound healingprocess

8 Evidence-Based Complementary and Alternative Medicine

SS

NF-120581B

NF-120581B

SS + LZ-8

0 3 7 2800

05

10

15

20

25

Relat

ive e

xpre

ssio

n of

NF-120581

B

Treatment duration (day)

SSSS + LZ-8

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowast

lowastlowast

(a)

0 3 7 2800

05

10

15

20

Relat

ive e

xpre

ssio

n of

casp

ase-

3

Treatment duration (day)

SSSS + LZ-8

Caspase-3SS + LZ-8

Caspase-3SS

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowastlowast

lowastlowastlowast

(b)

Figure 6TheWestern blot assay results from rats liver treated with SS group and SS + LZ-8 group after different healing days (a) expressionsof NF-120581B and (b) expressions of caspase-3 Data are expressed as the means plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 comparedwith SS group

4 Discussion

The purpose of this study was to determine the effect ofthe LZ-8 an immunomodulatory protein derived from Glucidum when it is used for wound healing on hepatictissues and to compare this with monopolar electrosurgeryoperated liver tissues on the wounds size fibrosis thermalinjury and apoptosis The results of this study show thatLZ-8 enhances wound healing actions in hepatic tissuesafter electrosurgery Importantly this study has identifiedthat NF-120581B and caspase-3 activation are associated with LZ-8 on enhancing wound healing after surgery Purpose ofelectrical energy or coagulation of bleeding vessels onto tissueis frequently used in surgery Coagulation techniques suchas monopolar and laser techniques are commonly used indifferent surgical disciplines [21 22]

Monopolar and bipolar coagulation effects on brain tissuewere reported and the results of experimental trials were

compared [23 24] They observed less trauma after bipolarcoagulation compared with monopolar technique A studyhas demonstrated that bipolar mode of stimulation claimedto be another paradigm of a potential hazard comparedwith monopolar stimulation Temel et al [25] observed braindamage after bipolar but not monopolar and stimulationin rats However monophasic pulses were used whichmight have created larger lesions in a bipolar configurationMonopolar electrosurgery causes sparking current spreadand thermal damage in the tissues because of excessivegeneration of heat These problems will induce deleteriouseffects to the tissue in proximity to the operative site Inaddition when comparing monopolar electrosurgery andthe scalpel incision electrosurgery produces more tissuealteration and histological thermal damage when a lateralheat was increased by the low frequency radio wave of05 to 29MHz [26] Consequently the resultant effect ofmonopolar systems is excessive tissue damage and delayed

Evidence-Based Complementary and Alternative Medicine 9

Cy 5 DAPI Merge

Control

SS

SS + LZ-8

Day 3

SS

SS + LZ-8

Day 7

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

Figure 7 The immunofluorescence staining results from rats liver treated with SS group and SS + LZ-8 group after different healing days

healing Therefore additional drug supplements or therapyneed to be required to accelerate the wound healing actionon tissues after electrosurgery

A new immunomodulatory protein known as LZ-8 wasisolated from the mycelia of G lucidum and characterizedby chromatographicelectrophoretic techniques [17] Bothin vivo immunomodulation activity and in vitro mitogenicactivity of LZ-8 have been reported by Kino et al [14] Pre-vious studies have also demonstrated that topical applicationof oils improves some parameters of wound healing such asstimulation of connective tissue fiber [27] reduction in thethickness of the necrotic cell layer edge around the wound[28] and acceleration of wound closure [29 30] Due toexcellent thermal and acid stability together with a moderateresistance to alkali and dehydration LZ-8 is appropriate toapply tissues after surgery even with a mild thermal heatIn this study this is the first and novel results that LZ-8accelerates the reduction of the wound size thermal injuryand increase fibrosis area in rat liver after surgery and it

indicates that LZ-8 has potent ability to enhance the effect ofwound healing In a latest study we have also demonstratedthat the total areas of liver injury lateral thermal injury andfibrotic tissues were significantly higher in SS treated hepatictissues than that of CrN-coated needles that had been usedfor surgery [13]

NF-120581B activation plays a role in the regulation of morethan 400 gene product expressions associatedwith inflamma-tion cell survival proliferation invasion and angiogenesis[31 32] NF-120581B normally binds to I120581B120572 which impedes NF-120581B nuclear translocation from the cytoplasm to the nucleusNF-120581B is reported to be a vital link between hepatic injuryfibrosis and even hepatocellular carcinoma [33] NF-120581Benters the nucleus with the degradation of I120581B and activatesthe expression of specific genes that induce the apoptosiscascade [34] Once the cells are exposed to inflammatorystimuli including LPS and TNF-120572 I120581B120572 is phosphorylatedleading to I120581B120572 degradation and nuclear translocation of NF-120581B In our latest study it was noticed that liver tissues that had

10 Evidence-Based Complementary and Alternative Medicine

TUNEL DAPI Merge

SSSS

+ L

Z-8

250 120583m 250 120583m 250 120583m

250 120583m 250 120583m 250 120583m

Figure 8 The TUNEL staining assay results from rats liver treated with SS group and SS + LZ-8 group after 7 days of healing

been operated with SS-needles secreted higher levels of NF-120581B expression and its nuclear translocation than that of CrNoperated tissues [13] Fibrosis is an important component ofadvanced chronic inflammatory liver disease Although thechanges in hepatic structures that occur in liver cirrhosis havebeen described in detail the pathogenesis of fibrosis in duringelectrosurgery process is largely unknown Our latest reportindicates that monopolar elecrtrosurgery-induced hepaticfibrosis after repeated liver injury and the mechanisms aremainly based on the upregulation of NF-120581B and caspase-3expression in the liver tissues [13]

In this study we thus evaluated whether LZ-8 had anyeffect on nuclear translocation of NF-120581B during monopolarelectrosurgery and the results found for the first time thatLZ-8 inhibits nuclear translocation of NF-120581B in hepaticcells after 3 and 7 days of monopolar electrosurgery Yatesand Rayner [35] studied the effects of fatty acid supple-mentation on activation of NF-120581B and AP-1 after a skininjury since these transcription factors are highly associ-ated with wound healing They have also reported a novelfinding which indicates that wounding of normal humankeratinocytes in culture causes NF-120581B translocation Therelevance of NF-120581B activation to wound healing is sug-gested by the results that HeNe laser treatment a processshown to accelerate healing in a model of wounding [36]promoted the translocation of NF-120581B A previous studyalso proved that a ganoderma immunomodulatory proteincloned from Ganoderma microsporum holds anti-invasiveand anti-inflammatory activities in TNF-120572 induced humanalveolar epithelial A549 cells via inhibition of the I120581B120572 andNF-120581B pathway [37]

Apoptosis in wound healing is a promising issue Therehas been some evaluation of chronic diabetic wounds in

mice that suggests that there is increased cellular apoptosislikely due to hyperglycemia which produces reactive oxygenspecies that mediate mitochondrial release of cytochrome cand activate caspase-3 [37 38] Apoptosis is also involved inthe regulation of collagen synthesis and degradation withinwounds through regulation of fibroblast numbers and colla-genase activity [39] Recent work in another mouse modelsuggests that hypertrophic scars demonstrate decreased ratesof apoptosis with decreased expression of cleaved caspase-3Over expression of various caspase family members inducesapoptosis in cultured mammalian cells [40] Among thecysteine proteases caspase-3 is believed to be one of themost commonly involved in the execution of apoptosisin various cell types [41] The assay of TUNEL stainingcan identify the apoptotic cells via DNA fragmentation bylabeling the terminal ends of nucleic acids [42 43] Fromour latest study of TUNEL assay it can be clearly foundthat the apoptosis event was more significant in SS-needleoperated liver tissues [13] In this study TUNEL stainingprovides direct evidence of apoptosis in SS operated livertissues after 7 days Both the activation of caspase-3 and theinduction of apoptotic cell death could be suppressed by theaddition of antioxidants in the cultures [44] It has also beendemonstrated that LZ-8 is able to effectively promote theactivation and maturation of immature dendritic cells via thenuclear factor-kappaB and mitogen-activated protein kinasepathways [18] Moreover the present study observed theeffects of (i) inhibiting expression and nuclear translocationof NF-120581B and (ii) inhibiting expression of caspase-3 andreduction of apoptotic cell death by LZ-8 in liver tissuesafter electrosurgery which indicates that the wound healingproperty of LZ-8 may possibly occur through inhibition oftranscription factor NF-120581B and apoptotic factor caspase-3

Evidence-Based Complementary and Alternative Medicine 11

5 Conclusion

Under the same conditions LZ-8 treatment potentiates onreducing wound size and thermal injury An increasing fibro-sis and reducing apoptosis were found in LZ-8 treated livertissues during electrosurgery LZ-8 treatment significantlyinhibits NF-120581B and caspase-3 expressions and also reducesNF-120581B nuclear translocation and apoptotic cell death Theresults generated from this study suggest that LZ-8 has potenteffects on increasing wound healing action against the elec-trosurgical induced liver injury via the possible mechanismof inhibiting NF-120581B and caspase-3 expressions

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors would like to thank the Department of HealthExecutive Yuan Taiwan (Contract no MOHW103-TDU-N-211-133001) and TaipeiMedical University and TaipeiMedicalHospital (Contract no 100TMU-TMUH-05) for financiallysupporting this research Dr Hao-Jan Lin and Dr Yushan-Sophie Chang are considered the first coauthors of the paper

References

[1] J Liboon W Funkhouser and D J Terris ldquoA comparison ofmucosal incisions made by scalpel CO

2

laser electrocauteryand constant-voltage electrocauteryrdquo OtolaryngologymdashHeadand Neck Surgery vol 116 no 3 pp 379ndash385 1997

[2] J F Carew R F Ward A LaBruna P A Torzilli and W SSchley ldquoEffects of scalpel electrocautery and CO

2

and KTPlasers on wound healing in rat tonguesrdquoThe Laryngoscope vol108 no 3 pp 373ndash380 1998

[3] J A Pearce Electrosurgery John Wiley amp Sons New York NYUSA 1986

[4] W L Maness F W Rober R E Clark E Cataldo andA W Haddad ldquoTissue damage from electrosurgical poweroutput variations in hamster tonguesrdquoThe Journal of ProstheticDentistry vol 42 no 4 pp 456ndash460 1979

[5] W L Maness F W Roeber R E Clark E Cataldo D Riisand A W Haddad ldquoHistologic evaluation of electrosurgerywith varying frequency andwaveformrdquoThe Journal of ProstheticDentistry vol 40 no 3 pp 304ndash308 1978

[6] J Friedman J Margolin and S Piliero ldquoA preliminary study ofthe histological effects of three different types of electrosurgicalcurrentsrdquoThe New York State Dental Journal vol 40 no 6 pp349ndash353 1974

[7] W H Noble K D McClatchey and G D Douglass ldquoAhistologic comparison of effects of electrosurgical resectionusing different electrodesrdquo The Journal of Prosthetic Dentistryvol 35 no 5 pp 575ndash579 1976

[8] K C Nixon K F Adkins and D W Keys ldquoHistologicalevaluation of effects produced in alveolar bone followinggingival incision with an electrosurgical scalpelrdquo Journal ofPeriodontology vol 46 no 1 pp 40ndash44 1975

[9] I Glickman and L R Imber ldquoComparison of gingival resec-tion with electrosurgery and periodontal knivesmdasha biometric

and histologic studyrdquo Journal of Periodontology vol 41 no 3pp 142ndash148 1970

[10] D H Coelho J Cavallaro and E A Rothschild ldquoGingivalrecession with electrosurgery for impression makingrdquo TheJournal of Prosthetic Dentistry vol 33 no 4 pp 422ndash426 1975

[11] P B Robertson B Luscher L S Spangberg and B M LevyldquoPulpal and periodontal effects of electrosurgery involvingcervical metallic restorationsrdquo Oral Surgery Oral Medicine andOral Pathology vol 46 no 5 pp 702ndash710 1978

[12] R DrsquoSouza ldquoPulpal and periapical immune response to electro-surgical contact of cervical metallic restorations in monkeysrdquoQuintessence International vol 17 no 12 pp 803ndash808 1986

[13] K-L Ou J-S Chu H Hosseinkhani J-F Chiou and C-HYu ldquoBiomedical nanostructured coating for minimally invasivesurgery devices applications characterization cell cytotoxicityevaluation and an animal study in ratrdquo Surgical Endoscopy 2014

[14] K Kino A Yamashita K Yamaoka et al ldquoIsolation and char-acterization of a new immunomodulatory protein Ling Zhi-8(LZ-8) from Ganoderma lucidiumrdquo The Journal of BiologicalChemistry vol 264 no 1 pp 472ndash478 1989

[15] AMurasugi S Tanaka N Komiyama et al ldquoMolecular cloningof a cDNAand a gene encoding an immunomodulatory proteinLing Zhi-8 from a fungusGanoderma lucidiumrdquoThe Journal ofBiological Chemistry vol 266 no 4 pp 2486ndash2493 1991

[16] M-H Tong P-J Chien H-H Chang M-J Tsai and F SheuldquoHigh processing tolerances of immunomodulatory proteins inEnoki and Reishi mushroomsrdquo Journal of Agricultural and FoodChemistry vol 56 no 9 pp 3160ndash3166 2008

[17] L Huang F Sun C Liang et al ldquoCrystal structure of LZ-8 fromthemedicinal fungusGanoderma lucidiumrdquo Proteins StructureFunction and Bioinformatics vol 75 no 2 pp 524ndash527 2009

[18] Y-L Lin Y-C Liang Y-S Tseng et al ldquoAn immunomodulatoryprotein Ling Zhi-8 induced activation and maturation ofhuman monocyte-derived dendritic cells by the NF-120581B andMAPK pathwaysrdquo Journal of Leukocyte Biology vol 86 no 4pp 877ndash889 2009

[19] R A D Drury and E A Wallington Carletonrsquos HistologicalTechnique 1981

[20] Y Du D-W Guan and R Zhao ldquoAn immunohistochemicalstudy on the expression of caspase-3 in the skin wound healingrdquoFa Yi Xue Za Zhi vol 20 no 1 pp 1ndash3 2004

[21] K E Grund T Straub and G Farin ldquoNew haemostatictechniques argon plasma coagulationrdquo Baillierersquos Best Practiceand Research in Clinical Gastroenterology vol 13 no 1 pp 67ndash84 1999

[22] D Hellwig R Haag V Bartel et al ldquoApplication of newelectrosurgical devices and probes in endoscopic neurosurgeryrdquoNeurological Research vol 21 no 1 pp 67ndash72 1999

[23] B Chehrazi and W F Collins Jr ldquoA comparison of effects ofbipolar and monopolar electrocoagulation in brainrdquo Journal ofNeurosurgery vol 54 no 2 pp 197ndash203 1981

[24] J Kerl and J Staubesand ldquoAn ultrastructural report on thechoice between monopolar and bipolar electrocoagulationrdquoActa Neurochirurgica vol 92 no 1ndash4 pp 123ndash131 1988

[25] Y Temel V Visser-Vandewalle M van derWolf et al ldquoMonop-olar versus bipolar high frequency stimulation in the rat subtha-lamic nucleus differences in histological damagerdquoNeuroscienceLetters vol 367 no 1 pp 92ndash96 2004

[26] L I Malis ldquoNew trends in microsurgery and applied technol-ogyrdquo in Advanced Technology in Neurosurgery F Pluchino andG Broggi Eds pp 1ndash16 Springer Berlin Germany 1988

12 Evidence-Based Complementary and Alternative Medicine

[27] C R B Cardoso M A Souza E A V Ferro S Favoreto Jrand J D O Pena ldquoInfluence of topical administration of n-3and n-6 essential and n-9 nonessential fatty acids on the healingof cutaneous woundsrdquoWound Repair and Regeneration vol 12no 2 pp 235ndash243 2004

[28] L M Pereira E Hatanaka E F Martins et al ldquoEffect of oleicand linoleic acids on the inflammatory phase of wound healingin ratsrdquo Cell Biochemistry and Function vol 26 no 2 pp 197ndash204 2008

[29] N-Y ParkGValacchi andY Lim ldquoEffect of dietary conjugatedlinoleic acid supplementation on early inflammatory responsesduring cutaneous wound healingrdquo Mediators of Inflammationvol 2010 Article ID 342328 8 pages 2010

[30] M Otranto A P do Nascimento and A Monte-Alto-CostaldquoEffects of supplementation with different edible oils on cuta-neous wound healingrdquoWound Repair and Regeneration vol 18no 6 pp 629ndash636 2010

[31] X Dolcet D Llobet J Pallares and X Matias-Guiu ldquoNF-kBin development and progression of human cancerrdquo VirchowsArchiv vol 446 no 5 pp 475ndash482 2005

[32] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[33] T Luedde and R F Schwabe ldquoNF-120581B in the livermdashlinkinginjury fibrosis and hepatocellular carcinomardquo Nature ReviewsGastroenterology amp Hepatology vol 8 no 2 pp 108ndash118 2011

[34] J A Schmid and A Birbach ldquoIkappaB kinase beta (IKKbetaIKK2IKBKB)mdasha keymolecule in signaling to the transcriptionfactor NF-kappaBrdquo Cytokine amp Growth Factor Reviews vol 19no 2 pp 157ndash165 2008

[35] S Yates and T E Rayner ldquoTranscription factor activation inresponse to cutaneous injury role of AP-1 in reepithelializa-tionrdquo Wound Repair and Regeneration vol 10 no 1 pp 5ndash152002

[36] A F Haas R R Isseroff R G Wheeland P A Rood and PJ Graves ldquoLow-energy helium-neon laser irradiation increasesthe motility of cultured human keratinocytesrdquo The Journal ofInvestigative Dermatology vol 94 no 6 pp 822ndash826 1990

[37] C-H Lin Y-M Hsiao C-C Ou et al ldquoGMI a Ganodermaimmunomodulatory protein down-regulates tumor necrosisfactor 120572-induced expression of matrix metalloproteinase 9 viaNF-120581Bpathway in human alveolar epithelial A549 cellsrdquo Journalof agricultural and food chemistry vol 58 no 22 pp 12014ndash12021 2010

[38] L Cai W Li G Wang L Guo Y Jiang and Y J KangldquoHyperglycemia-induced apoptosis in mouse myocardiummitochondrial cytochrome c-mediated caspase-3 activationpathwayrdquo Diabetes vol 51 no 6 pp 1938ndash1948 2002

[39] N K Rai K Tripathi D Sharma andV K Shukla ldquoApoptosis abasic physiologic process in wound healingrdquo The InternationalJournal of Lower Extremity Wounds vol 4 no 3 pp 138ndash1442005

[40] J Bian and Y Sun ldquoTranscriptional activation by p53 of thehuman type IV collagenase (gelatinase A or matrix metallopro-teinase 2) promoterrdquoMolecular and Cellular Biology vol 17 no11 pp 6330ndash6338 1997

[41] S Wang MMiura Y-K Jung et al ldquoIdentification and charac-terization of ich-3 a member of the interleukin-1120573 convertingenzyme (ICE)Ced-3 family and an upstream regulator of ICErdquoThe Journal of Biological Chemistry vol 271 no 34 pp 20580ndash20587 1996

[42] E S Alnemri D J Livingston D W Nicholson et al ldquoHumanICECED-3 protease nomenclaturerdquo Cell vol 87 no 2 article171 1996

[43] T D Hewitson T Bisucci and I A Darby ldquoHistochemicallocalization of apoptosis with in situ labeling of fragmentedDNArdquo Methods in Molecular Biology vol 326 pp 227ndash2342006

[44] D T Loo ldquoIn situ detection of apoptosis by the TUNEL assayan overview of techniquesrdquo in DNA Damage Detection In SituEx Vivo and In Vivo vol 682 ofMethods in Molecular Biologypp 3ndash13 Humana Press New Jersey NJ USA 2011

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 3: Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014 › 916531.pdfResearch Article An Immunomodulatory Protein (Ling Zhi-8) from a

Evidence-Based Complementary and Alternative Medicine 3

SS

0 7 28Days

3

SS (stainless steel)

500 120583m 500 120583m 500 120583m 500 120583m

(a)

SS +

LZ-

8

0 7 28Days

3

LZ-8 (Ling Zhi-8)

500 120583m 500 120583m 500 120583m 500 120583m

(b)

Figure 1 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for different healing days (HampE staining)(a) SS group and (b) SS + LZ-8 group

an electrosurgical unit The liver was exposed through mid-line laparotomywith a retractor to avoid injury to the liver Anunmodified SS-needle was inserted to the liver lobe (3mm indepth) to create an anterior lesion using a fixed power setting(20W) with a fixed activation time (3 seconds) The powersetting and activation time were commonly used parametersand were based on previous study settings For LZ-8 proteingroup 10 120583L of 1mgmL LZ-8 protein solution was pipettedand dropped on the wound surface For control group andexperiment group 10120583L of normal saline was pipetted anddropped on the wound surface

24 Weight of Sticking Tissue The weights of each SS-needlewere recorded perioperatively and the weight of stickingtissue was calculated as the weight of needle after operationminus the weight of needle before operation

25Thermography The effects of the SS-needles on tempera-tures across the live lobe surface were determined from ther-mographs obtained with a thermal-imaging infrared camera(Advanced Thermo TVS-500EX NEC Avio TechnologiesTokyo Japan) The highest temperature was recorded forevery test After recording the thermograph the surroundingsoft tissues were carefully repaired and the surgical woundwas closed in layers No anti-inflammatory regimen was usedafter surgery

26 Histological Examination to Detect Injury Area Histo-pathological studies were performed to provide structuralevidence ofmediated tissue injury and protective effect of LZ-8 These animals were sacrificed on days 0 3 7 and 28 afteroperation respectively Tissues were then cut into pieces ofdesired size and fixed in Bouinrsquos fluid fixative immediatelyafter autopsy Fixation was carried out at room temperaturefor 24 hrs after which these fix tissues were transferred to70 alcohol Several changes of 70 alcohol were given untilthe yellow colour disappeared from the tissues The tissueswere then dehydrated by passing through ascending grades ofalcohol (30 50 70 90 and 100) cleared in methylsalicylate and infiltrated with wax at 57∘C The clear tissueswere embedded in the paraffin Conventional techniques ofparaffinwax sectioningwere used for histological studies [19]Paraffin wax sections of 6 to 8 120583m thickness were cut using arotary microtome (Leica Germany) The sections were fur-ther stained with hematoxylin and eosin (HampE) andMassonrsquostrichrome staining and then washed in 90 alcohol for fewseconds Subsequently the stained sections were dehydratedin 100 alcohol cleared in xylene and mounted in DPXmountant The stained slides were observed in a Carl Zeiss(Germany) Axio 2 Plus research microscope Images werecaptured through a CCD camera (BX51 Olympus Japan)in a computer and processed using Carl Zeiss Axiovisionsoftware The total injury areas caused by the SS-needleare measured using image analysis software (SPOT basic

4 Evidence-Based Complementary and Alternative Medicine

(a)

TA Thermal injury area IA Injury area (trauma)

TA

IA

TA

IA

TA

IA

TAIA

TA

TA

IA

IA

SSSS

+ L

Z-8

SS SS + LZ-8

SS SS + LZ-80

10

20

30

40

50

60

70

80

90

100

Are

a (

)

Thermal injury areaInjury area

Wound area (TA + IA)

(c)(b)

500 120583m

100 120583m

100 120583m

500 120583m

Figure 2 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 0 days (Massonrsquos trichrome staining)(a) wound area (b) the site of thermal injury area and injury area and (c) percentage of each area Data are expressed as the means plusmn SEM(119899 = 5) No significant difference in the thermal injury area injury area and wound area at day 0

software SPOT imaging solutions MI USA) In additiondifferent histological features were observed on the injurysite Therefore the injured area was further divided intodifferent classifications to demonstrate the injury caused bythermal spread and the area of every portion was measured

27 Western Blot Analysis The retrieved liver tissues werehomogenized and lysed in an ice-cold protein lysis buffer andcentrifuged at 13000 rpm for 15 minutes at 4∘C Protein con-centrations of the lysates were determined using a Bio-Radprotein assay (Bio-Rad Laboratories Hercules CA USA)The lysates (20120583glane) were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in10 gels and electroblotted onto a nitrocellulose membrane(HybondTM-C Extra Amersham Biosciences Corp HongKong China) The membrane was blocked for 30 minutesat room temperature in 3 nonfat milk Tris-buffered salinewith 005 Tween 20 and then incubated overnight at4∘C with the indicated primary antibodies against NF-120581B(1 500 MAB3026 Chemicon Millipore Temecula CA) orcaspase-3 (1 200 AB1899 Millipore Temecula CA) andthen incubated with horseradish peroxidase-conjugated sec-ondary antibodies for 2 hours at room temperature After

washing the membrane was developed using chemilumi-nescent substrates (WBLUC0100 Millipore Temecula CA)Densitometric analysis of the gels was performed using Gel-Pro analyzer software and the results were expressed relativeto the density of 120573-actin (1 500 MAB1501 ChemiconMillipore Temecula CA)

28 Immunofluorescence Staining and TUNEL Staining Forimmunofluorescence staining liver tissues were embeddedin Cryomatrix (Thermo Shandon Pittsburgh PA USA) andstored at minus80∘C Cryostat sections (5ndash7mm thickness) werefixed in a mixture of methanol and acetone (1 1) at minus20∘Cfor 5 minutes and incubate in 1 Triton X-100 for another5 minutes Nonspecific activity was blocked by incubatingthe sections with 10 horse serum in a buffer containing002 Tween 20 in phosphate-buffered saline for 30 minutesSections were then incubated overnight with primary anti-Caspase-3 at 4∘C Antigen localization was indicated bya Cy5 dye (red fluorescence 111-176-003 Jackson ImmuneResearch West Grove PA) Finally the samples were stainedwith 410158406-diamidino-2-phenyl-indole (DAPI sc-3598 SantaCruz Biotechnology CA) for 7 minutes and examined usingfluorescent microscope Normal liver tissues obtained from

Evidence-Based Complementary and Alternative Medicine 5

NA

TA

IA

IA

TA

NA

NA Normal area TA Thermal injury area IA Injury area (trauma)

SS SS + LZ-80

10

20

30

40

50

60

70

80

Are

a (

)

Thermal injury areaInjury area

Wound area (TA + IA)

SSSS

+ L

Z-8

(a)

(c) (b)

100 120583m

100 120583m

lowastlowast

lowast

lowastlowastlowast

SS SS + LZ-8

500 120583m 500 120583m

Figure 3 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 3 days (Massonrsquos trichrome staining)(a) wound area (b) the site of thermal injury area injury area and normal area and (c) percentage of each area Data are expressed as themeans plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

healthy rats undergo identical procedures Negative controlswere processed identically except that the primary antibodieswere replaced with IgG An in situ cell death detection kit(terminal deoxynucleotidyl transferase dUTP nick end label-ing TUNEL 11684795910 RocheMannheim Germany) wasused to detect apoptotic cells and the apoptotic cells wereindicated by a green fluorescent FITC fluorochrome

29 Statistical Analysis Data were expressed as mean plusmnstandard error of the mean (SEM) Data were analyzed usinganalysis of variance (ANOVA) All statistical analyses wereperformed using SPSS version 120 (SPSS Inc Chicago IL)Values of 119875 lt 005 were considered significant

3 Results

31 Stimulatory Effects of LZ-8 on Wound Healing in RatLiver Tissues after Monopolar Electrosurgery In generallybleeding and healing could easily be reduced when tissuesare subjected to electrosurgery To determine whether LZ-8could enhance wound healing on liver tissues after surgerywe compared the wound size after monopolar electrosurgeryand the operated tissues that had been treatedwith LZ-8 for 03 7 and 28 daysThe observed results of HampE stained section

of liver tissues show that a significant reduced of wound sizetreated with LZ-8 (3 7 and 28 days) dependently relative tothe normal (untreated) rats as shown in Figure 1 Moreoverthe mean wound sizes for these liver tissues were moreprominently reduced in tissues that were treated with LZ-8 at28 days when compared with untreated groups and with day3 and day 7 The results clearly indicated that treatment withLZ-8 enhances wounds healing faster after surgery in rat livertissues

32 Effects of LZ-8 on the Site of Thermal Damage Injuryand Wounds of Liver Tissues after Surgery Figure 2 showshistological sections of monopolar SS on the operated livertissues and tissues subjected to LZ-8 treatment (SS + LZ-8)for 0 days (Massonrsquos trichrome staining) Clearly there wasno significant difference in the total injury areas caused by theSS and SS + LZ-8 treatments Moreover the injury area andthermal injury area in SS group were also similar to those ofSS + LZ-8 group at day 0 However the results demonstratedthat damage of liver tissues in the form of injury thermalinjury and wound was negligible in SS group for 3 days(Figure 3) when compared to 0 days (Figure 2) Interestinglythe effect of injury thermal injury and wound of liver tissueswas significantly reduced in SS + LZ-8 group for 3 days It

6 Evidence-Based Complementary and Alternative Medicine

NA Normal area FA Fibrosis area AA Apoptosis area

NA

FAFAAA

AA

AAFA

FA

NA

SSSS

+ L

Z-8

(b)

50 120583m

50 120583m

50 120583m

50 120583m

SS SS + LZ-80

10

20

30

40

50

60

70

80

90

Are

a (

)

Fibrosis area Injury areaApoptosis area Wound area (FA + IA + AA)

(c)

lowast

lowastlowast

lowastlowastlowast

(a)

SS SS + LZ-8

500 120583m500 120583m

Figure 4 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 7 days (Massonrsquos trichrome staining)(a) wound area (b) the site of apoptosis area fibrosis area and normal area and (c) percentage of each area Data are expressed as themeans plusmnSEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

was found that the injury area (119875 lt 005) thermal injuryarea (119875 lt 001) and wound area (119875 lt 0001) of SS + LZ-8groupwere reducedmore significantly than those of SS groupat day 3 Furthermore Massonrsquos trichrome staining was usedto examine the formation of fibrotic tissue No fibrotic tissuewas evident for either group at day 0 or day 3

33 Effects of LZ-8 on the Areas of Fibrosis Injury ApoptosisandWounds of Liver Tissues after 7 Days of Surgery A furtherstudy was performed to determine whether LZ-8 couldenhance the areas of fibrosis reduce injury and wounds andinhibit apoptosis of monopolar SS induced liver incision interms of withdrawing the above observed injuriesThe resultsclearly confirmed that injury apoptosis and wound of livertissues in SS operated groups that had been treated withLZ-8 for 7 days were significantly alleviated when comparedwith SS operated groups without LZ-8 treatment (Figure 4)Nevertheless the areas of fibrosis and numbers of cell deathin liver tissues for treated rats with LZ-8 were comparativelyhigher than those of SS operated rats Thus the resultsindicate that LZ-8 treatment could not affect the reduction ofliver fibrosis andmore effective on the inhibition of apoptosiswhich are caused by surgery

34 Effects of LZ-8 on the Areas of Fibrosis Injury Apoptosisand Wounds of Liver Tissues after 28 Days of Surgery Sincedifferential observations were found on the areas of fibrosisand apoptosis in liver tissues of LZ-8 supplemented rats thathave already been operated with SS for 7 days we furtherextended the duration of treatment up to 28 days (Figure 5)to confirm the hypothesis Our analysis obviously exhibitsfurther evidence that the effect of LZ-8 was similar to thatof 7-days treatment

35 Does LZ-8 Enhance Wound Healing in SS Operated LiverTissues via Inhibiting the Expression of NF-120581B and Caspase-3It is well known that the angiogenesis is essential in woundhealing Various studies have demonstrated the inhibition ofangiogenesis by the blockage of NF-120581B A recent study hasalso suggested that caspase-3 plays an important role duringskin wound healing [20] Thus we performed this study todeterminewhether thewound healing action of LZ-8was dueto the effect on inhibiting of NF-120581B and caspase-3 expressionthe tissue levels of their protein were examined by Westernblot analysis As shown in Figure 6(a) liver tissues exposedto electrosurgery with SS for 3 days could slightly inducethe expression of NF-120581B and it gradually increased up to

Evidence-Based Complementary and Alternative Medicine 7

SS0

5

10

15

20

25

30

Are

a (

)

Fibrosis area Apoptosis area

Wound area (FA + AA)

(a)

(c) (b)

FA Fibrosis area AA Apoptosis area PA Proliferate area

SSSS

+ L

Z-8

SS SS + LZ-8

SS + LZ-8

AA

FA

PA

AA

FA

AA

PA

lowast

lowast

lowastlowast

500 120583m500 120583m

50 120583m

50 120583m

Figure 5The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 28 days (Massonrsquos trichrome staining)(a) wound area (b) the site of apoptosis area fibrosis area and proliferate area and (c) percentage of each area Data are expressed as themeans plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

28 days Interestingly liver tissues further treated with LZ-8 significantly reduced the ratio of NF-120581B expression in aduration dependent manner (3 to 28 days) A similar resultwas observed on the expression of caspase-3 Therefore theresults can suggest that treatment of liver tissues after surgerywith LZ-8 was found to lower the mean NF-120581B and caspase-3 expressions so that it approaches wound healing propertypresumably by limiting NF-120581B and caspase-3 expression inliver tissuesThese results were further confirmed and shownas in Figure 6(b)

36 LZ-8 Inhibits NF-120581BNuclear Translocation in SS OperatedLiver Tissues The ability of LZ-8 to inhibit NF-120581B dispersionwas determined by red fluorescence (Cy5) intensity analysisof immunolabelled antibodies directed against NF-120581B andHoechst reagent to simultaneously identify nuclei (Figure 7)The intensity values of NF-120581B red fluorescence in the nucleusof SS operated tissues that were treated with LZ-8 for 3 dayswere found to be lesser as compared to SS operated livertissues without LZ-8 treatment Interestingly the presence ofLZ-8 in SS treated liver tissues for 7 days reached amaximuminhibition of nuclear fluorescence of NF-120581B than that oftreated tissues with SS alone These results can obviouslyindicate that LZ-8 plays a major role in inhibiting of NF-120581B

nuclear translocation in liver tissues during the process ofwound healing after electrosurgery

37 LZ-8 Reduces Apoptotic Cell Death in Liver Tissues duringWoundHealing after Electrosurgery Extensive fragmentationof nuclear DNA that generates a large number of DNAdouble-strand breaks is one of the most characteristic eventsof apoptosis An assay that relies on detection of DNA strandbreaks (DSBs) in situ by labeling them with fluorochromeshas been developed to identify and quantify apoptotic cellsby fluorescence microscopy or cytometry The assay is com-monly called TUNEL the acronym of Terminal deoxynu-cleotidyl transferase-mediated d-UTP Nick End Labeling Inthis study a very clear TUNEL-positive cells were observedin rats hepatic cells after 7 days of electrosurgery (Figure 8)However the number or TUNEL expression continued todecrease more prominently in LZ-8 treated hepatic cells thathad been exposed with monopolar electrosurgery for 7 daysThe results obtained from this study demonstrate that a slightapoptosis occurs in hepatic cells after electrosurgery whichwas as a result of LZ-8 inhibition and provide evidencefor high level of angiogenesis arises during wound healingprocess

8 Evidence-Based Complementary and Alternative Medicine

SS

NF-120581B

NF-120581B

SS + LZ-8

0 3 7 2800

05

10

15

20

25

Relat

ive e

xpre

ssio

n of

NF-120581

B

Treatment duration (day)

SSSS + LZ-8

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowast

lowastlowast

(a)

0 3 7 2800

05

10

15

20

Relat

ive e

xpre

ssio

n of

casp

ase-

3

Treatment duration (day)

SSSS + LZ-8

Caspase-3SS + LZ-8

Caspase-3SS

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowastlowast

lowastlowastlowast

(b)

Figure 6TheWestern blot assay results from rats liver treated with SS group and SS + LZ-8 group after different healing days (a) expressionsof NF-120581B and (b) expressions of caspase-3 Data are expressed as the means plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 comparedwith SS group

4 Discussion

The purpose of this study was to determine the effect ofthe LZ-8 an immunomodulatory protein derived from Glucidum when it is used for wound healing on hepatictissues and to compare this with monopolar electrosurgeryoperated liver tissues on the wounds size fibrosis thermalinjury and apoptosis The results of this study show thatLZ-8 enhances wound healing actions in hepatic tissuesafter electrosurgery Importantly this study has identifiedthat NF-120581B and caspase-3 activation are associated with LZ-8 on enhancing wound healing after surgery Purpose ofelectrical energy or coagulation of bleeding vessels onto tissueis frequently used in surgery Coagulation techniques suchas monopolar and laser techniques are commonly used indifferent surgical disciplines [21 22]

Monopolar and bipolar coagulation effects on brain tissuewere reported and the results of experimental trials were

compared [23 24] They observed less trauma after bipolarcoagulation compared with monopolar technique A studyhas demonstrated that bipolar mode of stimulation claimedto be another paradigm of a potential hazard comparedwith monopolar stimulation Temel et al [25] observed braindamage after bipolar but not monopolar and stimulationin rats However monophasic pulses were used whichmight have created larger lesions in a bipolar configurationMonopolar electrosurgery causes sparking current spreadand thermal damage in the tissues because of excessivegeneration of heat These problems will induce deleteriouseffects to the tissue in proximity to the operative site Inaddition when comparing monopolar electrosurgery andthe scalpel incision electrosurgery produces more tissuealteration and histological thermal damage when a lateralheat was increased by the low frequency radio wave of05 to 29MHz [26] Consequently the resultant effect ofmonopolar systems is excessive tissue damage and delayed

Evidence-Based Complementary and Alternative Medicine 9

Cy 5 DAPI Merge

Control

SS

SS + LZ-8

Day 3

SS

SS + LZ-8

Day 7

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

Figure 7 The immunofluorescence staining results from rats liver treated with SS group and SS + LZ-8 group after different healing days

healing Therefore additional drug supplements or therapyneed to be required to accelerate the wound healing actionon tissues after electrosurgery

A new immunomodulatory protein known as LZ-8 wasisolated from the mycelia of G lucidum and characterizedby chromatographicelectrophoretic techniques [17] Bothin vivo immunomodulation activity and in vitro mitogenicactivity of LZ-8 have been reported by Kino et al [14] Pre-vious studies have also demonstrated that topical applicationof oils improves some parameters of wound healing such asstimulation of connective tissue fiber [27] reduction in thethickness of the necrotic cell layer edge around the wound[28] and acceleration of wound closure [29 30] Due toexcellent thermal and acid stability together with a moderateresistance to alkali and dehydration LZ-8 is appropriate toapply tissues after surgery even with a mild thermal heatIn this study this is the first and novel results that LZ-8accelerates the reduction of the wound size thermal injuryand increase fibrosis area in rat liver after surgery and it

indicates that LZ-8 has potent ability to enhance the effect ofwound healing In a latest study we have also demonstratedthat the total areas of liver injury lateral thermal injury andfibrotic tissues were significantly higher in SS treated hepatictissues than that of CrN-coated needles that had been usedfor surgery [13]

NF-120581B activation plays a role in the regulation of morethan 400 gene product expressions associatedwith inflamma-tion cell survival proliferation invasion and angiogenesis[31 32] NF-120581B normally binds to I120581B120572 which impedes NF-120581B nuclear translocation from the cytoplasm to the nucleusNF-120581B is reported to be a vital link between hepatic injuryfibrosis and even hepatocellular carcinoma [33] NF-120581Benters the nucleus with the degradation of I120581B and activatesthe expression of specific genes that induce the apoptosiscascade [34] Once the cells are exposed to inflammatorystimuli including LPS and TNF-120572 I120581B120572 is phosphorylatedleading to I120581B120572 degradation and nuclear translocation of NF-120581B In our latest study it was noticed that liver tissues that had

10 Evidence-Based Complementary and Alternative Medicine

TUNEL DAPI Merge

SSSS

+ L

Z-8

250 120583m 250 120583m 250 120583m

250 120583m 250 120583m 250 120583m

Figure 8 The TUNEL staining assay results from rats liver treated with SS group and SS + LZ-8 group after 7 days of healing

been operated with SS-needles secreted higher levels of NF-120581B expression and its nuclear translocation than that of CrNoperated tissues [13] Fibrosis is an important component ofadvanced chronic inflammatory liver disease Although thechanges in hepatic structures that occur in liver cirrhosis havebeen described in detail the pathogenesis of fibrosis in duringelectrosurgery process is largely unknown Our latest reportindicates that monopolar elecrtrosurgery-induced hepaticfibrosis after repeated liver injury and the mechanisms aremainly based on the upregulation of NF-120581B and caspase-3expression in the liver tissues [13]

In this study we thus evaluated whether LZ-8 had anyeffect on nuclear translocation of NF-120581B during monopolarelectrosurgery and the results found for the first time thatLZ-8 inhibits nuclear translocation of NF-120581B in hepaticcells after 3 and 7 days of monopolar electrosurgery Yatesand Rayner [35] studied the effects of fatty acid supple-mentation on activation of NF-120581B and AP-1 after a skininjury since these transcription factors are highly associ-ated with wound healing They have also reported a novelfinding which indicates that wounding of normal humankeratinocytes in culture causes NF-120581B translocation Therelevance of NF-120581B activation to wound healing is sug-gested by the results that HeNe laser treatment a processshown to accelerate healing in a model of wounding [36]promoted the translocation of NF-120581B A previous studyalso proved that a ganoderma immunomodulatory proteincloned from Ganoderma microsporum holds anti-invasiveand anti-inflammatory activities in TNF-120572 induced humanalveolar epithelial A549 cells via inhibition of the I120581B120572 andNF-120581B pathway [37]

Apoptosis in wound healing is a promising issue Therehas been some evaluation of chronic diabetic wounds in

mice that suggests that there is increased cellular apoptosislikely due to hyperglycemia which produces reactive oxygenspecies that mediate mitochondrial release of cytochrome cand activate caspase-3 [37 38] Apoptosis is also involved inthe regulation of collagen synthesis and degradation withinwounds through regulation of fibroblast numbers and colla-genase activity [39] Recent work in another mouse modelsuggests that hypertrophic scars demonstrate decreased ratesof apoptosis with decreased expression of cleaved caspase-3Over expression of various caspase family members inducesapoptosis in cultured mammalian cells [40] Among thecysteine proteases caspase-3 is believed to be one of themost commonly involved in the execution of apoptosisin various cell types [41] The assay of TUNEL stainingcan identify the apoptotic cells via DNA fragmentation bylabeling the terminal ends of nucleic acids [42 43] Fromour latest study of TUNEL assay it can be clearly foundthat the apoptosis event was more significant in SS-needleoperated liver tissues [13] In this study TUNEL stainingprovides direct evidence of apoptosis in SS operated livertissues after 7 days Both the activation of caspase-3 and theinduction of apoptotic cell death could be suppressed by theaddition of antioxidants in the cultures [44] It has also beendemonstrated that LZ-8 is able to effectively promote theactivation and maturation of immature dendritic cells via thenuclear factor-kappaB and mitogen-activated protein kinasepathways [18] Moreover the present study observed theeffects of (i) inhibiting expression and nuclear translocationof NF-120581B and (ii) inhibiting expression of caspase-3 andreduction of apoptotic cell death by LZ-8 in liver tissuesafter electrosurgery which indicates that the wound healingproperty of LZ-8 may possibly occur through inhibition oftranscription factor NF-120581B and apoptotic factor caspase-3

Evidence-Based Complementary and Alternative Medicine 11

5 Conclusion

Under the same conditions LZ-8 treatment potentiates onreducing wound size and thermal injury An increasing fibro-sis and reducing apoptosis were found in LZ-8 treated livertissues during electrosurgery LZ-8 treatment significantlyinhibits NF-120581B and caspase-3 expressions and also reducesNF-120581B nuclear translocation and apoptotic cell death Theresults generated from this study suggest that LZ-8 has potenteffects on increasing wound healing action against the elec-trosurgical induced liver injury via the possible mechanismof inhibiting NF-120581B and caspase-3 expressions

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors would like to thank the Department of HealthExecutive Yuan Taiwan (Contract no MOHW103-TDU-N-211-133001) and TaipeiMedical University and TaipeiMedicalHospital (Contract no 100TMU-TMUH-05) for financiallysupporting this research Dr Hao-Jan Lin and Dr Yushan-Sophie Chang are considered the first coauthors of the paper

References

[1] J Liboon W Funkhouser and D J Terris ldquoA comparison ofmucosal incisions made by scalpel CO

2

laser electrocauteryand constant-voltage electrocauteryrdquo OtolaryngologymdashHeadand Neck Surgery vol 116 no 3 pp 379ndash385 1997

[2] J F Carew R F Ward A LaBruna P A Torzilli and W SSchley ldquoEffects of scalpel electrocautery and CO

2

and KTPlasers on wound healing in rat tonguesrdquoThe Laryngoscope vol108 no 3 pp 373ndash380 1998

[3] J A Pearce Electrosurgery John Wiley amp Sons New York NYUSA 1986

[4] W L Maness F W Rober R E Clark E Cataldo andA W Haddad ldquoTissue damage from electrosurgical poweroutput variations in hamster tonguesrdquoThe Journal of ProstheticDentistry vol 42 no 4 pp 456ndash460 1979

[5] W L Maness F W Roeber R E Clark E Cataldo D Riisand A W Haddad ldquoHistologic evaluation of electrosurgerywith varying frequency andwaveformrdquoThe Journal of ProstheticDentistry vol 40 no 3 pp 304ndash308 1978

[6] J Friedman J Margolin and S Piliero ldquoA preliminary study ofthe histological effects of three different types of electrosurgicalcurrentsrdquoThe New York State Dental Journal vol 40 no 6 pp349ndash353 1974

[7] W H Noble K D McClatchey and G D Douglass ldquoAhistologic comparison of effects of electrosurgical resectionusing different electrodesrdquo The Journal of Prosthetic Dentistryvol 35 no 5 pp 575ndash579 1976

[8] K C Nixon K F Adkins and D W Keys ldquoHistologicalevaluation of effects produced in alveolar bone followinggingival incision with an electrosurgical scalpelrdquo Journal ofPeriodontology vol 46 no 1 pp 40ndash44 1975

[9] I Glickman and L R Imber ldquoComparison of gingival resec-tion with electrosurgery and periodontal knivesmdasha biometric

and histologic studyrdquo Journal of Periodontology vol 41 no 3pp 142ndash148 1970

[10] D H Coelho J Cavallaro and E A Rothschild ldquoGingivalrecession with electrosurgery for impression makingrdquo TheJournal of Prosthetic Dentistry vol 33 no 4 pp 422ndash426 1975

[11] P B Robertson B Luscher L S Spangberg and B M LevyldquoPulpal and periodontal effects of electrosurgery involvingcervical metallic restorationsrdquo Oral Surgery Oral Medicine andOral Pathology vol 46 no 5 pp 702ndash710 1978

[12] R DrsquoSouza ldquoPulpal and periapical immune response to electro-surgical contact of cervical metallic restorations in monkeysrdquoQuintessence International vol 17 no 12 pp 803ndash808 1986

[13] K-L Ou J-S Chu H Hosseinkhani J-F Chiou and C-HYu ldquoBiomedical nanostructured coating for minimally invasivesurgery devices applications characterization cell cytotoxicityevaluation and an animal study in ratrdquo Surgical Endoscopy 2014

[14] K Kino A Yamashita K Yamaoka et al ldquoIsolation and char-acterization of a new immunomodulatory protein Ling Zhi-8(LZ-8) from Ganoderma lucidiumrdquo The Journal of BiologicalChemistry vol 264 no 1 pp 472ndash478 1989

[15] AMurasugi S Tanaka N Komiyama et al ldquoMolecular cloningof a cDNAand a gene encoding an immunomodulatory proteinLing Zhi-8 from a fungusGanoderma lucidiumrdquoThe Journal ofBiological Chemistry vol 266 no 4 pp 2486ndash2493 1991

[16] M-H Tong P-J Chien H-H Chang M-J Tsai and F SheuldquoHigh processing tolerances of immunomodulatory proteins inEnoki and Reishi mushroomsrdquo Journal of Agricultural and FoodChemistry vol 56 no 9 pp 3160ndash3166 2008

[17] L Huang F Sun C Liang et al ldquoCrystal structure of LZ-8 fromthemedicinal fungusGanoderma lucidiumrdquo Proteins StructureFunction and Bioinformatics vol 75 no 2 pp 524ndash527 2009

[18] Y-L Lin Y-C Liang Y-S Tseng et al ldquoAn immunomodulatoryprotein Ling Zhi-8 induced activation and maturation ofhuman monocyte-derived dendritic cells by the NF-120581B andMAPK pathwaysrdquo Journal of Leukocyte Biology vol 86 no 4pp 877ndash889 2009

[19] R A D Drury and E A Wallington Carletonrsquos HistologicalTechnique 1981

[20] Y Du D-W Guan and R Zhao ldquoAn immunohistochemicalstudy on the expression of caspase-3 in the skin wound healingrdquoFa Yi Xue Za Zhi vol 20 no 1 pp 1ndash3 2004

[21] K E Grund T Straub and G Farin ldquoNew haemostatictechniques argon plasma coagulationrdquo Baillierersquos Best Practiceand Research in Clinical Gastroenterology vol 13 no 1 pp 67ndash84 1999

[22] D Hellwig R Haag V Bartel et al ldquoApplication of newelectrosurgical devices and probes in endoscopic neurosurgeryrdquoNeurological Research vol 21 no 1 pp 67ndash72 1999

[23] B Chehrazi and W F Collins Jr ldquoA comparison of effects ofbipolar and monopolar electrocoagulation in brainrdquo Journal ofNeurosurgery vol 54 no 2 pp 197ndash203 1981

[24] J Kerl and J Staubesand ldquoAn ultrastructural report on thechoice between monopolar and bipolar electrocoagulationrdquoActa Neurochirurgica vol 92 no 1ndash4 pp 123ndash131 1988

[25] Y Temel V Visser-Vandewalle M van derWolf et al ldquoMonop-olar versus bipolar high frequency stimulation in the rat subtha-lamic nucleus differences in histological damagerdquoNeuroscienceLetters vol 367 no 1 pp 92ndash96 2004

[26] L I Malis ldquoNew trends in microsurgery and applied technol-ogyrdquo in Advanced Technology in Neurosurgery F Pluchino andG Broggi Eds pp 1ndash16 Springer Berlin Germany 1988

12 Evidence-Based Complementary and Alternative Medicine

[27] C R B Cardoso M A Souza E A V Ferro S Favoreto Jrand J D O Pena ldquoInfluence of topical administration of n-3and n-6 essential and n-9 nonessential fatty acids on the healingof cutaneous woundsrdquoWound Repair and Regeneration vol 12no 2 pp 235ndash243 2004

[28] L M Pereira E Hatanaka E F Martins et al ldquoEffect of oleicand linoleic acids on the inflammatory phase of wound healingin ratsrdquo Cell Biochemistry and Function vol 26 no 2 pp 197ndash204 2008

[29] N-Y ParkGValacchi andY Lim ldquoEffect of dietary conjugatedlinoleic acid supplementation on early inflammatory responsesduring cutaneous wound healingrdquo Mediators of Inflammationvol 2010 Article ID 342328 8 pages 2010

[30] M Otranto A P do Nascimento and A Monte-Alto-CostaldquoEffects of supplementation with different edible oils on cuta-neous wound healingrdquoWound Repair and Regeneration vol 18no 6 pp 629ndash636 2010

[31] X Dolcet D Llobet J Pallares and X Matias-Guiu ldquoNF-kBin development and progression of human cancerrdquo VirchowsArchiv vol 446 no 5 pp 475ndash482 2005

[32] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[33] T Luedde and R F Schwabe ldquoNF-120581B in the livermdashlinkinginjury fibrosis and hepatocellular carcinomardquo Nature ReviewsGastroenterology amp Hepatology vol 8 no 2 pp 108ndash118 2011

[34] J A Schmid and A Birbach ldquoIkappaB kinase beta (IKKbetaIKK2IKBKB)mdasha keymolecule in signaling to the transcriptionfactor NF-kappaBrdquo Cytokine amp Growth Factor Reviews vol 19no 2 pp 157ndash165 2008

[35] S Yates and T E Rayner ldquoTranscription factor activation inresponse to cutaneous injury role of AP-1 in reepithelializa-tionrdquo Wound Repair and Regeneration vol 10 no 1 pp 5ndash152002

[36] A F Haas R R Isseroff R G Wheeland P A Rood and PJ Graves ldquoLow-energy helium-neon laser irradiation increasesthe motility of cultured human keratinocytesrdquo The Journal ofInvestigative Dermatology vol 94 no 6 pp 822ndash826 1990

[37] C-H Lin Y-M Hsiao C-C Ou et al ldquoGMI a Ganodermaimmunomodulatory protein down-regulates tumor necrosisfactor 120572-induced expression of matrix metalloproteinase 9 viaNF-120581Bpathway in human alveolar epithelial A549 cellsrdquo Journalof agricultural and food chemistry vol 58 no 22 pp 12014ndash12021 2010

[38] L Cai W Li G Wang L Guo Y Jiang and Y J KangldquoHyperglycemia-induced apoptosis in mouse myocardiummitochondrial cytochrome c-mediated caspase-3 activationpathwayrdquo Diabetes vol 51 no 6 pp 1938ndash1948 2002

[39] N K Rai K Tripathi D Sharma andV K Shukla ldquoApoptosis abasic physiologic process in wound healingrdquo The InternationalJournal of Lower Extremity Wounds vol 4 no 3 pp 138ndash1442005

[40] J Bian and Y Sun ldquoTranscriptional activation by p53 of thehuman type IV collagenase (gelatinase A or matrix metallopro-teinase 2) promoterrdquoMolecular and Cellular Biology vol 17 no11 pp 6330ndash6338 1997

[41] S Wang MMiura Y-K Jung et al ldquoIdentification and charac-terization of ich-3 a member of the interleukin-1120573 convertingenzyme (ICE)Ced-3 family and an upstream regulator of ICErdquoThe Journal of Biological Chemistry vol 271 no 34 pp 20580ndash20587 1996

[42] E S Alnemri D J Livingston D W Nicholson et al ldquoHumanICECED-3 protease nomenclaturerdquo Cell vol 87 no 2 article171 1996

[43] T D Hewitson T Bisucci and I A Darby ldquoHistochemicallocalization of apoptosis with in situ labeling of fragmentedDNArdquo Methods in Molecular Biology vol 326 pp 227ndash2342006

[44] D T Loo ldquoIn situ detection of apoptosis by the TUNEL assayan overview of techniquesrdquo in DNA Damage Detection In SituEx Vivo and In Vivo vol 682 ofMethods in Molecular Biologypp 3ndash13 Humana Press New Jersey NJ USA 2011

Submit your manuscripts athttpwwwhindawicom

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Disease Markers

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 4: Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014 › 916531.pdfResearch Article An Immunomodulatory Protein (Ling Zhi-8) from a

4 Evidence-Based Complementary and Alternative Medicine

(a)

TA Thermal injury area IA Injury area (trauma)

TA

IA

TA

IA

TA

IA

TAIA

TA

TA

IA

IA

SSSS

+ L

Z-8

SS SS + LZ-8

SS SS + LZ-80

10

20

30

40

50

60

70

80

90

100

Are

a (

)

Thermal injury areaInjury area

Wound area (TA + IA)

(c)(b)

500 120583m

100 120583m

100 120583m

500 120583m

Figure 2 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 0 days (Massonrsquos trichrome staining)(a) wound area (b) the site of thermal injury area and injury area and (c) percentage of each area Data are expressed as the means plusmn SEM(119899 = 5) No significant difference in the thermal injury area injury area and wound area at day 0

software SPOT imaging solutions MI USA) In additiondifferent histological features were observed on the injurysite Therefore the injured area was further divided intodifferent classifications to demonstrate the injury caused bythermal spread and the area of every portion was measured

27 Western Blot Analysis The retrieved liver tissues werehomogenized and lysed in an ice-cold protein lysis buffer andcentrifuged at 13000 rpm for 15 minutes at 4∘C Protein con-centrations of the lysates were determined using a Bio-Radprotein assay (Bio-Rad Laboratories Hercules CA USA)The lysates (20120583glane) were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in10 gels and electroblotted onto a nitrocellulose membrane(HybondTM-C Extra Amersham Biosciences Corp HongKong China) The membrane was blocked for 30 minutesat room temperature in 3 nonfat milk Tris-buffered salinewith 005 Tween 20 and then incubated overnight at4∘C with the indicated primary antibodies against NF-120581B(1 500 MAB3026 Chemicon Millipore Temecula CA) orcaspase-3 (1 200 AB1899 Millipore Temecula CA) andthen incubated with horseradish peroxidase-conjugated sec-ondary antibodies for 2 hours at room temperature After

washing the membrane was developed using chemilumi-nescent substrates (WBLUC0100 Millipore Temecula CA)Densitometric analysis of the gels was performed using Gel-Pro analyzer software and the results were expressed relativeto the density of 120573-actin (1 500 MAB1501 ChemiconMillipore Temecula CA)

28 Immunofluorescence Staining and TUNEL Staining Forimmunofluorescence staining liver tissues were embeddedin Cryomatrix (Thermo Shandon Pittsburgh PA USA) andstored at minus80∘C Cryostat sections (5ndash7mm thickness) werefixed in a mixture of methanol and acetone (1 1) at minus20∘Cfor 5 minutes and incubate in 1 Triton X-100 for another5 minutes Nonspecific activity was blocked by incubatingthe sections with 10 horse serum in a buffer containing002 Tween 20 in phosphate-buffered saline for 30 minutesSections were then incubated overnight with primary anti-Caspase-3 at 4∘C Antigen localization was indicated bya Cy5 dye (red fluorescence 111-176-003 Jackson ImmuneResearch West Grove PA) Finally the samples were stainedwith 410158406-diamidino-2-phenyl-indole (DAPI sc-3598 SantaCruz Biotechnology CA) for 7 minutes and examined usingfluorescent microscope Normal liver tissues obtained from

Evidence-Based Complementary and Alternative Medicine 5

NA

TA

IA

IA

TA

NA

NA Normal area TA Thermal injury area IA Injury area (trauma)

SS SS + LZ-80

10

20

30

40

50

60

70

80

Are

a (

)

Thermal injury areaInjury area

Wound area (TA + IA)

SSSS

+ L

Z-8

(a)

(c) (b)

100 120583m

100 120583m

lowastlowast

lowast

lowastlowastlowast

SS SS + LZ-8

500 120583m 500 120583m

Figure 3 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 3 days (Massonrsquos trichrome staining)(a) wound area (b) the site of thermal injury area injury area and normal area and (c) percentage of each area Data are expressed as themeans plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

healthy rats undergo identical procedures Negative controlswere processed identically except that the primary antibodieswere replaced with IgG An in situ cell death detection kit(terminal deoxynucleotidyl transferase dUTP nick end label-ing TUNEL 11684795910 RocheMannheim Germany) wasused to detect apoptotic cells and the apoptotic cells wereindicated by a green fluorescent FITC fluorochrome

29 Statistical Analysis Data were expressed as mean plusmnstandard error of the mean (SEM) Data were analyzed usinganalysis of variance (ANOVA) All statistical analyses wereperformed using SPSS version 120 (SPSS Inc Chicago IL)Values of 119875 lt 005 were considered significant

3 Results

31 Stimulatory Effects of LZ-8 on Wound Healing in RatLiver Tissues after Monopolar Electrosurgery In generallybleeding and healing could easily be reduced when tissuesare subjected to electrosurgery To determine whether LZ-8could enhance wound healing on liver tissues after surgerywe compared the wound size after monopolar electrosurgeryand the operated tissues that had been treatedwith LZ-8 for 03 7 and 28 daysThe observed results of HampE stained section

of liver tissues show that a significant reduced of wound sizetreated with LZ-8 (3 7 and 28 days) dependently relative tothe normal (untreated) rats as shown in Figure 1 Moreoverthe mean wound sizes for these liver tissues were moreprominently reduced in tissues that were treated with LZ-8 at28 days when compared with untreated groups and with day3 and day 7 The results clearly indicated that treatment withLZ-8 enhances wounds healing faster after surgery in rat livertissues

32 Effects of LZ-8 on the Site of Thermal Damage Injuryand Wounds of Liver Tissues after Surgery Figure 2 showshistological sections of monopolar SS on the operated livertissues and tissues subjected to LZ-8 treatment (SS + LZ-8)for 0 days (Massonrsquos trichrome staining) Clearly there wasno significant difference in the total injury areas caused by theSS and SS + LZ-8 treatments Moreover the injury area andthermal injury area in SS group were also similar to those ofSS + LZ-8 group at day 0 However the results demonstratedthat damage of liver tissues in the form of injury thermalinjury and wound was negligible in SS group for 3 days(Figure 3) when compared to 0 days (Figure 2) Interestinglythe effect of injury thermal injury and wound of liver tissueswas significantly reduced in SS + LZ-8 group for 3 days It

6 Evidence-Based Complementary and Alternative Medicine

NA Normal area FA Fibrosis area AA Apoptosis area

NA

FAFAAA

AA

AAFA

FA

NA

SSSS

+ L

Z-8

(b)

50 120583m

50 120583m

50 120583m

50 120583m

SS SS + LZ-80

10

20

30

40

50

60

70

80

90

Are

a (

)

Fibrosis area Injury areaApoptosis area Wound area (FA + IA + AA)

(c)

lowast

lowastlowast

lowastlowastlowast

(a)

SS SS + LZ-8

500 120583m500 120583m

Figure 4 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 7 days (Massonrsquos trichrome staining)(a) wound area (b) the site of apoptosis area fibrosis area and normal area and (c) percentage of each area Data are expressed as themeans plusmnSEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

was found that the injury area (119875 lt 005) thermal injuryarea (119875 lt 001) and wound area (119875 lt 0001) of SS + LZ-8groupwere reducedmore significantly than those of SS groupat day 3 Furthermore Massonrsquos trichrome staining was usedto examine the formation of fibrotic tissue No fibrotic tissuewas evident for either group at day 0 or day 3

33 Effects of LZ-8 on the Areas of Fibrosis Injury ApoptosisandWounds of Liver Tissues after 7 Days of Surgery A furtherstudy was performed to determine whether LZ-8 couldenhance the areas of fibrosis reduce injury and wounds andinhibit apoptosis of monopolar SS induced liver incision interms of withdrawing the above observed injuriesThe resultsclearly confirmed that injury apoptosis and wound of livertissues in SS operated groups that had been treated withLZ-8 for 7 days were significantly alleviated when comparedwith SS operated groups without LZ-8 treatment (Figure 4)Nevertheless the areas of fibrosis and numbers of cell deathin liver tissues for treated rats with LZ-8 were comparativelyhigher than those of SS operated rats Thus the resultsindicate that LZ-8 treatment could not affect the reduction ofliver fibrosis andmore effective on the inhibition of apoptosiswhich are caused by surgery

34 Effects of LZ-8 on the Areas of Fibrosis Injury Apoptosisand Wounds of Liver Tissues after 28 Days of Surgery Sincedifferential observations were found on the areas of fibrosisand apoptosis in liver tissues of LZ-8 supplemented rats thathave already been operated with SS for 7 days we furtherextended the duration of treatment up to 28 days (Figure 5)to confirm the hypothesis Our analysis obviously exhibitsfurther evidence that the effect of LZ-8 was similar to thatof 7-days treatment

35 Does LZ-8 Enhance Wound Healing in SS Operated LiverTissues via Inhibiting the Expression of NF-120581B and Caspase-3It is well known that the angiogenesis is essential in woundhealing Various studies have demonstrated the inhibition ofangiogenesis by the blockage of NF-120581B A recent study hasalso suggested that caspase-3 plays an important role duringskin wound healing [20] Thus we performed this study todeterminewhether thewound healing action of LZ-8was dueto the effect on inhibiting of NF-120581B and caspase-3 expressionthe tissue levels of their protein were examined by Westernblot analysis As shown in Figure 6(a) liver tissues exposedto electrosurgery with SS for 3 days could slightly inducethe expression of NF-120581B and it gradually increased up to

Evidence-Based Complementary and Alternative Medicine 7

SS0

5

10

15

20

25

30

Are

a (

)

Fibrosis area Apoptosis area

Wound area (FA + AA)

(a)

(c) (b)

FA Fibrosis area AA Apoptosis area PA Proliferate area

SSSS

+ L

Z-8

SS SS + LZ-8

SS + LZ-8

AA

FA

PA

AA

FA

AA

PA

lowast

lowast

lowastlowast

500 120583m500 120583m

50 120583m

50 120583m

Figure 5The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 28 days (Massonrsquos trichrome staining)(a) wound area (b) the site of apoptosis area fibrosis area and proliferate area and (c) percentage of each area Data are expressed as themeans plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

28 days Interestingly liver tissues further treated with LZ-8 significantly reduced the ratio of NF-120581B expression in aduration dependent manner (3 to 28 days) A similar resultwas observed on the expression of caspase-3 Therefore theresults can suggest that treatment of liver tissues after surgerywith LZ-8 was found to lower the mean NF-120581B and caspase-3 expressions so that it approaches wound healing propertypresumably by limiting NF-120581B and caspase-3 expression inliver tissuesThese results were further confirmed and shownas in Figure 6(b)

36 LZ-8 Inhibits NF-120581BNuclear Translocation in SS OperatedLiver Tissues The ability of LZ-8 to inhibit NF-120581B dispersionwas determined by red fluorescence (Cy5) intensity analysisof immunolabelled antibodies directed against NF-120581B andHoechst reagent to simultaneously identify nuclei (Figure 7)The intensity values of NF-120581B red fluorescence in the nucleusof SS operated tissues that were treated with LZ-8 for 3 dayswere found to be lesser as compared to SS operated livertissues without LZ-8 treatment Interestingly the presence ofLZ-8 in SS treated liver tissues for 7 days reached amaximuminhibition of nuclear fluorescence of NF-120581B than that oftreated tissues with SS alone These results can obviouslyindicate that LZ-8 plays a major role in inhibiting of NF-120581B

nuclear translocation in liver tissues during the process ofwound healing after electrosurgery

37 LZ-8 Reduces Apoptotic Cell Death in Liver Tissues duringWoundHealing after Electrosurgery Extensive fragmentationof nuclear DNA that generates a large number of DNAdouble-strand breaks is one of the most characteristic eventsof apoptosis An assay that relies on detection of DNA strandbreaks (DSBs) in situ by labeling them with fluorochromeshas been developed to identify and quantify apoptotic cellsby fluorescence microscopy or cytometry The assay is com-monly called TUNEL the acronym of Terminal deoxynu-cleotidyl transferase-mediated d-UTP Nick End Labeling Inthis study a very clear TUNEL-positive cells were observedin rats hepatic cells after 7 days of electrosurgery (Figure 8)However the number or TUNEL expression continued todecrease more prominently in LZ-8 treated hepatic cells thathad been exposed with monopolar electrosurgery for 7 daysThe results obtained from this study demonstrate that a slightapoptosis occurs in hepatic cells after electrosurgery whichwas as a result of LZ-8 inhibition and provide evidencefor high level of angiogenesis arises during wound healingprocess

8 Evidence-Based Complementary and Alternative Medicine

SS

NF-120581B

NF-120581B

SS + LZ-8

0 3 7 2800

05

10

15

20

25

Relat

ive e

xpre

ssio

n of

NF-120581

B

Treatment duration (day)

SSSS + LZ-8

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowast

lowastlowast

(a)

0 3 7 2800

05

10

15

20

Relat

ive e

xpre

ssio

n of

casp

ase-

3

Treatment duration (day)

SSSS + LZ-8

Caspase-3SS + LZ-8

Caspase-3SS

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowastlowast

lowastlowastlowast

(b)

Figure 6TheWestern blot assay results from rats liver treated with SS group and SS + LZ-8 group after different healing days (a) expressionsof NF-120581B and (b) expressions of caspase-3 Data are expressed as the means plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 comparedwith SS group

4 Discussion

The purpose of this study was to determine the effect ofthe LZ-8 an immunomodulatory protein derived from Glucidum when it is used for wound healing on hepatictissues and to compare this with monopolar electrosurgeryoperated liver tissues on the wounds size fibrosis thermalinjury and apoptosis The results of this study show thatLZ-8 enhances wound healing actions in hepatic tissuesafter electrosurgery Importantly this study has identifiedthat NF-120581B and caspase-3 activation are associated with LZ-8 on enhancing wound healing after surgery Purpose ofelectrical energy or coagulation of bleeding vessels onto tissueis frequently used in surgery Coagulation techniques suchas monopolar and laser techniques are commonly used indifferent surgical disciplines [21 22]

Monopolar and bipolar coagulation effects on brain tissuewere reported and the results of experimental trials were

compared [23 24] They observed less trauma after bipolarcoagulation compared with monopolar technique A studyhas demonstrated that bipolar mode of stimulation claimedto be another paradigm of a potential hazard comparedwith monopolar stimulation Temel et al [25] observed braindamage after bipolar but not monopolar and stimulationin rats However monophasic pulses were used whichmight have created larger lesions in a bipolar configurationMonopolar electrosurgery causes sparking current spreadand thermal damage in the tissues because of excessivegeneration of heat These problems will induce deleteriouseffects to the tissue in proximity to the operative site Inaddition when comparing monopolar electrosurgery andthe scalpel incision electrosurgery produces more tissuealteration and histological thermal damage when a lateralheat was increased by the low frequency radio wave of05 to 29MHz [26] Consequently the resultant effect ofmonopolar systems is excessive tissue damage and delayed

Evidence-Based Complementary and Alternative Medicine 9

Cy 5 DAPI Merge

Control

SS

SS + LZ-8

Day 3

SS

SS + LZ-8

Day 7

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

Figure 7 The immunofluorescence staining results from rats liver treated with SS group and SS + LZ-8 group after different healing days

healing Therefore additional drug supplements or therapyneed to be required to accelerate the wound healing actionon tissues after electrosurgery

A new immunomodulatory protein known as LZ-8 wasisolated from the mycelia of G lucidum and characterizedby chromatographicelectrophoretic techniques [17] Bothin vivo immunomodulation activity and in vitro mitogenicactivity of LZ-8 have been reported by Kino et al [14] Pre-vious studies have also demonstrated that topical applicationof oils improves some parameters of wound healing such asstimulation of connective tissue fiber [27] reduction in thethickness of the necrotic cell layer edge around the wound[28] and acceleration of wound closure [29 30] Due toexcellent thermal and acid stability together with a moderateresistance to alkali and dehydration LZ-8 is appropriate toapply tissues after surgery even with a mild thermal heatIn this study this is the first and novel results that LZ-8accelerates the reduction of the wound size thermal injuryand increase fibrosis area in rat liver after surgery and it

indicates that LZ-8 has potent ability to enhance the effect ofwound healing In a latest study we have also demonstratedthat the total areas of liver injury lateral thermal injury andfibrotic tissues were significantly higher in SS treated hepatictissues than that of CrN-coated needles that had been usedfor surgery [13]

NF-120581B activation plays a role in the regulation of morethan 400 gene product expressions associatedwith inflamma-tion cell survival proliferation invasion and angiogenesis[31 32] NF-120581B normally binds to I120581B120572 which impedes NF-120581B nuclear translocation from the cytoplasm to the nucleusNF-120581B is reported to be a vital link between hepatic injuryfibrosis and even hepatocellular carcinoma [33] NF-120581Benters the nucleus with the degradation of I120581B and activatesthe expression of specific genes that induce the apoptosiscascade [34] Once the cells are exposed to inflammatorystimuli including LPS and TNF-120572 I120581B120572 is phosphorylatedleading to I120581B120572 degradation and nuclear translocation of NF-120581B In our latest study it was noticed that liver tissues that had

10 Evidence-Based Complementary and Alternative Medicine

TUNEL DAPI Merge

SSSS

+ L

Z-8

250 120583m 250 120583m 250 120583m

250 120583m 250 120583m 250 120583m

Figure 8 The TUNEL staining assay results from rats liver treated with SS group and SS + LZ-8 group after 7 days of healing

been operated with SS-needles secreted higher levels of NF-120581B expression and its nuclear translocation than that of CrNoperated tissues [13] Fibrosis is an important component ofadvanced chronic inflammatory liver disease Although thechanges in hepatic structures that occur in liver cirrhosis havebeen described in detail the pathogenesis of fibrosis in duringelectrosurgery process is largely unknown Our latest reportindicates that monopolar elecrtrosurgery-induced hepaticfibrosis after repeated liver injury and the mechanisms aremainly based on the upregulation of NF-120581B and caspase-3expression in the liver tissues [13]

In this study we thus evaluated whether LZ-8 had anyeffect on nuclear translocation of NF-120581B during monopolarelectrosurgery and the results found for the first time thatLZ-8 inhibits nuclear translocation of NF-120581B in hepaticcells after 3 and 7 days of monopolar electrosurgery Yatesand Rayner [35] studied the effects of fatty acid supple-mentation on activation of NF-120581B and AP-1 after a skininjury since these transcription factors are highly associ-ated with wound healing They have also reported a novelfinding which indicates that wounding of normal humankeratinocytes in culture causes NF-120581B translocation Therelevance of NF-120581B activation to wound healing is sug-gested by the results that HeNe laser treatment a processshown to accelerate healing in a model of wounding [36]promoted the translocation of NF-120581B A previous studyalso proved that a ganoderma immunomodulatory proteincloned from Ganoderma microsporum holds anti-invasiveand anti-inflammatory activities in TNF-120572 induced humanalveolar epithelial A549 cells via inhibition of the I120581B120572 andNF-120581B pathway [37]

Apoptosis in wound healing is a promising issue Therehas been some evaluation of chronic diabetic wounds in

mice that suggests that there is increased cellular apoptosislikely due to hyperglycemia which produces reactive oxygenspecies that mediate mitochondrial release of cytochrome cand activate caspase-3 [37 38] Apoptosis is also involved inthe regulation of collagen synthesis and degradation withinwounds through regulation of fibroblast numbers and colla-genase activity [39] Recent work in another mouse modelsuggests that hypertrophic scars demonstrate decreased ratesof apoptosis with decreased expression of cleaved caspase-3Over expression of various caspase family members inducesapoptosis in cultured mammalian cells [40] Among thecysteine proteases caspase-3 is believed to be one of themost commonly involved in the execution of apoptosisin various cell types [41] The assay of TUNEL stainingcan identify the apoptotic cells via DNA fragmentation bylabeling the terminal ends of nucleic acids [42 43] Fromour latest study of TUNEL assay it can be clearly foundthat the apoptosis event was more significant in SS-needleoperated liver tissues [13] In this study TUNEL stainingprovides direct evidence of apoptosis in SS operated livertissues after 7 days Both the activation of caspase-3 and theinduction of apoptotic cell death could be suppressed by theaddition of antioxidants in the cultures [44] It has also beendemonstrated that LZ-8 is able to effectively promote theactivation and maturation of immature dendritic cells via thenuclear factor-kappaB and mitogen-activated protein kinasepathways [18] Moreover the present study observed theeffects of (i) inhibiting expression and nuclear translocationof NF-120581B and (ii) inhibiting expression of caspase-3 andreduction of apoptotic cell death by LZ-8 in liver tissuesafter electrosurgery which indicates that the wound healingproperty of LZ-8 may possibly occur through inhibition oftranscription factor NF-120581B and apoptotic factor caspase-3

Evidence-Based Complementary and Alternative Medicine 11

5 Conclusion

Under the same conditions LZ-8 treatment potentiates onreducing wound size and thermal injury An increasing fibro-sis and reducing apoptosis were found in LZ-8 treated livertissues during electrosurgery LZ-8 treatment significantlyinhibits NF-120581B and caspase-3 expressions and also reducesNF-120581B nuclear translocation and apoptotic cell death Theresults generated from this study suggest that LZ-8 has potenteffects on increasing wound healing action against the elec-trosurgical induced liver injury via the possible mechanismof inhibiting NF-120581B and caspase-3 expressions

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors would like to thank the Department of HealthExecutive Yuan Taiwan (Contract no MOHW103-TDU-N-211-133001) and TaipeiMedical University and TaipeiMedicalHospital (Contract no 100TMU-TMUH-05) for financiallysupporting this research Dr Hao-Jan Lin and Dr Yushan-Sophie Chang are considered the first coauthors of the paper

References

[1] J Liboon W Funkhouser and D J Terris ldquoA comparison ofmucosal incisions made by scalpel CO

2

laser electrocauteryand constant-voltage electrocauteryrdquo OtolaryngologymdashHeadand Neck Surgery vol 116 no 3 pp 379ndash385 1997

[2] J F Carew R F Ward A LaBruna P A Torzilli and W SSchley ldquoEffects of scalpel electrocautery and CO

2

and KTPlasers on wound healing in rat tonguesrdquoThe Laryngoscope vol108 no 3 pp 373ndash380 1998

[3] J A Pearce Electrosurgery John Wiley amp Sons New York NYUSA 1986

[4] W L Maness F W Rober R E Clark E Cataldo andA W Haddad ldquoTissue damage from electrosurgical poweroutput variations in hamster tonguesrdquoThe Journal of ProstheticDentistry vol 42 no 4 pp 456ndash460 1979

[5] W L Maness F W Roeber R E Clark E Cataldo D Riisand A W Haddad ldquoHistologic evaluation of electrosurgerywith varying frequency andwaveformrdquoThe Journal of ProstheticDentistry vol 40 no 3 pp 304ndash308 1978

[6] J Friedman J Margolin and S Piliero ldquoA preliminary study ofthe histological effects of three different types of electrosurgicalcurrentsrdquoThe New York State Dental Journal vol 40 no 6 pp349ndash353 1974

[7] W H Noble K D McClatchey and G D Douglass ldquoAhistologic comparison of effects of electrosurgical resectionusing different electrodesrdquo The Journal of Prosthetic Dentistryvol 35 no 5 pp 575ndash579 1976

[8] K C Nixon K F Adkins and D W Keys ldquoHistologicalevaluation of effects produced in alveolar bone followinggingival incision with an electrosurgical scalpelrdquo Journal ofPeriodontology vol 46 no 1 pp 40ndash44 1975

[9] I Glickman and L R Imber ldquoComparison of gingival resec-tion with electrosurgery and periodontal knivesmdasha biometric

and histologic studyrdquo Journal of Periodontology vol 41 no 3pp 142ndash148 1970

[10] D H Coelho J Cavallaro and E A Rothschild ldquoGingivalrecession with electrosurgery for impression makingrdquo TheJournal of Prosthetic Dentistry vol 33 no 4 pp 422ndash426 1975

[11] P B Robertson B Luscher L S Spangberg and B M LevyldquoPulpal and periodontal effects of electrosurgery involvingcervical metallic restorationsrdquo Oral Surgery Oral Medicine andOral Pathology vol 46 no 5 pp 702ndash710 1978

[12] R DrsquoSouza ldquoPulpal and periapical immune response to electro-surgical contact of cervical metallic restorations in monkeysrdquoQuintessence International vol 17 no 12 pp 803ndash808 1986

[13] K-L Ou J-S Chu H Hosseinkhani J-F Chiou and C-HYu ldquoBiomedical nanostructured coating for minimally invasivesurgery devices applications characterization cell cytotoxicityevaluation and an animal study in ratrdquo Surgical Endoscopy 2014

[14] K Kino A Yamashita K Yamaoka et al ldquoIsolation and char-acterization of a new immunomodulatory protein Ling Zhi-8(LZ-8) from Ganoderma lucidiumrdquo The Journal of BiologicalChemistry vol 264 no 1 pp 472ndash478 1989

[15] AMurasugi S Tanaka N Komiyama et al ldquoMolecular cloningof a cDNAand a gene encoding an immunomodulatory proteinLing Zhi-8 from a fungusGanoderma lucidiumrdquoThe Journal ofBiological Chemistry vol 266 no 4 pp 2486ndash2493 1991

[16] M-H Tong P-J Chien H-H Chang M-J Tsai and F SheuldquoHigh processing tolerances of immunomodulatory proteins inEnoki and Reishi mushroomsrdquo Journal of Agricultural and FoodChemistry vol 56 no 9 pp 3160ndash3166 2008

[17] L Huang F Sun C Liang et al ldquoCrystal structure of LZ-8 fromthemedicinal fungusGanoderma lucidiumrdquo Proteins StructureFunction and Bioinformatics vol 75 no 2 pp 524ndash527 2009

[18] Y-L Lin Y-C Liang Y-S Tseng et al ldquoAn immunomodulatoryprotein Ling Zhi-8 induced activation and maturation ofhuman monocyte-derived dendritic cells by the NF-120581B andMAPK pathwaysrdquo Journal of Leukocyte Biology vol 86 no 4pp 877ndash889 2009

[19] R A D Drury and E A Wallington Carletonrsquos HistologicalTechnique 1981

[20] Y Du D-W Guan and R Zhao ldquoAn immunohistochemicalstudy on the expression of caspase-3 in the skin wound healingrdquoFa Yi Xue Za Zhi vol 20 no 1 pp 1ndash3 2004

[21] K E Grund T Straub and G Farin ldquoNew haemostatictechniques argon plasma coagulationrdquo Baillierersquos Best Practiceand Research in Clinical Gastroenterology vol 13 no 1 pp 67ndash84 1999

[22] D Hellwig R Haag V Bartel et al ldquoApplication of newelectrosurgical devices and probes in endoscopic neurosurgeryrdquoNeurological Research vol 21 no 1 pp 67ndash72 1999

[23] B Chehrazi and W F Collins Jr ldquoA comparison of effects ofbipolar and monopolar electrocoagulation in brainrdquo Journal ofNeurosurgery vol 54 no 2 pp 197ndash203 1981

[24] J Kerl and J Staubesand ldquoAn ultrastructural report on thechoice between monopolar and bipolar electrocoagulationrdquoActa Neurochirurgica vol 92 no 1ndash4 pp 123ndash131 1988

[25] Y Temel V Visser-Vandewalle M van derWolf et al ldquoMonop-olar versus bipolar high frequency stimulation in the rat subtha-lamic nucleus differences in histological damagerdquoNeuroscienceLetters vol 367 no 1 pp 92ndash96 2004

[26] L I Malis ldquoNew trends in microsurgery and applied technol-ogyrdquo in Advanced Technology in Neurosurgery F Pluchino andG Broggi Eds pp 1ndash16 Springer Berlin Germany 1988

12 Evidence-Based Complementary and Alternative Medicine

[27] C R B Cardoso M A Souza E A V Ferro S Favoreto Jrand J D O Pena ldquoInfluence of topical administration of n-3and n-6 essential and n-9 nonessential fatty acids on the healingof cutaneous woundsrdquoWound Repair and Regeneration vol 12no 2 pp 235ndash243 2004

[28] L M Pereira E Hatanaka E F Martins et al ldquoEffect of oleicand linoleic acids on the inflammatory phase of wound healingin ratsrdquo Cell Biochemistry and Function vol 26 no 2 pp 197ndash204 2008

[29] N-Y ParkGValacchi andY Lim ldquoEffect of dietary conjugatedlinoleic acid supplementation on early inflammatory responsesduring cutaneous wound healingrdquo Mediators of Inflammationvol 2010 Article ID 342328 8 pages 2010

[30] M Otranto A P do Nascimento and A Monte-Alto-CostaldquoEffects of supplementation with different edible oils on cuta-neous wound healingrdquoWound Repair and Regeneration vol 18no 6 pp 629ndash636 2010

[31] X Dolcet D Llobet J Pallares and X Matias-Guiu ldquoNF-kBin development and progression of human cancerrdquo VirchowsArchiv vol 446 no 5 pp 475ndash482 2005

[32] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[33] T Luedde and R F Schwabe ldquoNF-120581B in the livermdashlinkinginjury fibrosis and hepatocellular carcinomardquo Nature ReviewsGastroenterology amp Hepatology vol 8 no 2 pp 108ndash118 2011

[34] J A Schmid and A Birbach ldquoIkappaB kinase beta (IKKbetaIKK2IKBKB)mdasha keymolecule in signaling to the transcriptionfactor NF-kappaBrdquo Cytokine amp Growth Factor Reviews vol 19no 2 pp 157ndash165 2008

[35] S Yates and T E Rayner ldquoTranscription factor activation inresponse to cutaneous injury role of AP-1 in reepithelializa-tionrdquo Wound Repair and Regeneration vol 10 no 1 pp 5ndash152002

[36] A F Haas R R Isseroff R G Wheeland P A Rood and PJ Graves ldquoLow-energy helium-neon laser irradiation increasesthe motility of cultured human keratinocytesrdquo The Journal ofInvestigative Dermatology vol 94 no 6 pp 822ndash826 1990

[37] C-H Lin Y-M Hsiao C-C Ou et al ldquoGMI a Ganodermaimmunomodulatory protein down-regulates tumor necrosisfactor 120572-induced expression of matrix metalloproteinase 9 viaNF-120581Bpathway in human alveolar epithelial A549 cellsrdquo Journalof agricultural and food chemistry vol 58 no 22 pp 12014ndash12021 2010

[38] L Cai W Li G Wang L Guo Y Jiang and Y J KangldquoHyperglycemia-induced apoptosis in mouse myocardiummitochondrial cytochrome c-mediated caspase-3 activationpathwayrdquo Diabetes vol 51 no 6 pp 1938ndash1948 2002

[39] N K Rai K Tripathi D Sharma andV K Shukla ldquoApoptosis abasic physiologic process in wound healingrdquo The InternationalJournal of Lower Extremity Wounds vol 4 no 3 pp 138ndash1442005

[40] J Bian and Y Sun ldquoTranscriptional activation by p53 of thehuman type IV collagenase (gelatinase A or matrix metallopro-teinase 2) promoterrdquoMolecular and Cellular Biology vol 17 no11 pp 6330ndash6338 1997

[41] S Wang MMiura Y-K Jung et al ldquoIdentification and charac-terization of ich-3 a member of the interleukin-1120573 convertingenzyme (ICE)Ced-3 family and an upstream regulator of ICErdquoThe Journal of Biological Chemistry vol 271 no 34 pp 20580ndash20587 1996

[42] E S Alnemri D J Livingston D W Nicholson et al ldquoHumanICECED-3 protease nomenclaturerdquo Cell vol 87 no 2 article171 1996

[43] T D Hewitson T Bisucci and I A Darby ldquoHistochemicallocalization of apoptosis with in situ labeling of fragmentedDNArdquo Methods in Molecular Biology vol 326 pp 227ndash2342006

[44] D T Loo ldquoIn situ detection of apoptosis by the TUNEL assayan overview of techniquesrdquo in DNA Damage Detection In SituEx Vivo and In Vivo vol 682 ofMethods in Molecular Biologypp 3ndash13 Humana Press New Jersey NJ USA 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 5: Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014 › 916531.pdfResearch Article An Immunomodulatory Protein (Ling Zhi-8) from a

Evidence-Based Complementary and Alternative Medicine 5

NA

TA

IA

IA

TA

NA

NA Normal area TA Thermal injury area IA Injury area (trauma)

SS SS + LZ-80

10

20

30

40

50

60

70

80

Are

a (

)

Thermal injury areaInjury area

Wound area (TA + IA)

SSSS

+ L

Z-8

(a)

(c) (b)

100 120583m

100 120583m

lowastlowast

lowast

lowastlowastlowast

SS SS + LZ-8

500 120583m 500 120583m

Figure 3 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 3 days (Massonrsquos trichrome staining)(a) wound area (b) the site of thermal injury area injury area and normal area and (c) percentage of each area Data are expressed as themeans plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

healthy rats undergo identical procedures Negative controlswere processed identically except that the primary antibodieswere replaced with IgG An in situ cell death detection kit(terminal deoxynucleotidyl transferase dUTP nick end label-ing TUNEL 11684795910 RocheMannheim Germany) wasused to detect apoptotic cells and the apoptotic cells wereindicated by a green fluorescent FITC fluorochrome

29 Statistical Analysis Data were expressed as mean plusmnstandard error of the mean (SEM) Data were analyzed usinganalysis of variance (ANOVA) All statistical analyses wereperformed using SPSS version 120 (SPSS Inc Chicago IL)Values of 119875 lt 005 were considered significant

3 Results

31 Stimulatory Effects of LZ-8 on Wound Healing in RatLiver Tissues after Monopolar Electrosurgery In generallybleeding and healing could easily be reduced when tissuesare subjected to electrosurgery To determine whether LZ-8could enhance wound healing on liver tissues after surgerywe compared the wound size after monopolar electrosurgeryand the operated tissues that had been treatedwith LZ-8 for 03 7 and 28 daysThe observed results of HampE stained section

of liver tissues show that a significant reduced of wound sizetreated with LZ-8 (3 7 and 28 days) dependently relative tothe normal (untreated) rats as shown in Figure 1 Moreoverthe mean wound sizes for these liver tissues were moreprominently reduced in tissues that were treated with LZ-8 at28 days when compared with untreated groups and with day3 and day 7 The results clearly indicated that treatment withLZ-8 enhances wounds healing faster after surgery in rat livertissues

32 Effects of LZ-8 on the Site of Thermal Damage Injuryand Wounds of Liver Tissues after Surgery Figure 2 showshistological sections of monopolar SS on the operated livertissues and tissues subjected to LZ-8 treatment (SS + LZ-8)for 0 days (Massonrsquos trichrome staining) Clearly there wasno significant difference in the total injury areas caused by theSS and SS + LZ-8 treatments Moreover the injury area andthermal injury area in SS group were also similar to those ofSS + LZ-8 group at day 0 However the results demonstratedthat damage of liver tissues in the form of injury thermalinjury and wound was negligible in SS group for 3 days(Figure 3) when compared to 0 days (Figure 2) Interestinglythe effect of injury thermal injury and wound of liver tissueswas significantly reduced in SS + LZ-8 group for 3 days It

6 Evidence-Based Complementary and Alternative Medicine

NA Normal area FA Fibrosis area AA Apoptosis area

NA

FAFAAA

AA

AAFA

FA

NA

SSSS

+ L

Z-8

(b)

50 120583m

50 120583m

50 120583m

50 120583m

SS SS + LZ-80

10

20

30

40

50

60

70

80

90

Are

a (

)

Fibrosis area Injury areaApoptosis area Wound area (FA + IA + AA)

(c)

lowast

lowastlowast

lowastlowastlowast

(a)

SS SS + LZ-8

500 120583m500 120583m

Figure 4 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 7 days (Massonrsquos trichrome staining)(a) wound area (b) the site of apoptosis area fibrosis area and normal area and (c) percentage of each area Data are expressed as themeans plusmnSEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

was found that the injury area (119875 lt 005) thermal injuryarea (119875 lt 001) and wound area (119875 lt 0001) of SS + LZ-8groupwere reducedmore significantly than those of SS groupat day 3 Furthermore Massonrsquos trichrome staining was usedto examine the formation of fibrotic tissue No fibrotic tissuewas evident for either group at day 0 or day 3

33 Effects of LZ-8 on the Areas of Fibrosis Injury ApoptosisandWounds of Liver Tissues after 7 Days of Surgery A furtherstudy was performed to determine whether LZ-8 couldenhance the areas of fibrosis reduce injury and wounds andinhibit apoptosis of monopolar SS induced liver incision interms of withdrawing the above observed injuriesThe resultsclearly confirmed that injury apoptosis and wound of livertissues in SS operated groups that had been treated withLZ-8 for 7 days were significantly alleviated when comparedwith SS operated groups without LZ-8 treatment (Figure 4)Nevertheless the areas of fibrosis and numbers of cell deathin liver tissues for treated rats with LZ-8 were comparativelyhigher than those of SS operated rats Thus the resultsindicate that LZ-8 treatment could not affect the reduction ofliver fibrosis andmore effective on the inhibition of apoptosiswhich are caused by surgery

34 Effects of LZ-8 on the Areas of Fibrosis Injury Apoptosisand Wounds of Liver Tissues after 28 Days of Surgery Sincedifferential observations were found on the areas of fibrosisand apoptosis in liver tissues of LZ-8 supplemented rats thathave already been operated with SS for 7 days we furtherextended the duration of treatment up to 28 days (Figure 5)to confirm the hypothesis Our analysis obviously exhibitsfurther evidence that the effect of LZ-8 was similar to thatof 7-days treatment

35 Does LZ-8 Enhance Wound Healing in SS Operated LiverTissues via Inhibiting the Expression of NF-120581B and Caspase-3It is well known that the angiogenesis is essential in woundhealing Various studies have demonstrated the inhibition ofangiogenesis by the blockage of NF-120581B A recent study hasalso suggested that caspase-3 plays an important role duringskin wound healing [20] Thus we performed this study todeterminewhether thewound healing action of LZ-8was dueto the effect on inhibiting of NF-120581B and caspase-3 expressionthe tissue levels of their protein were examined by Westernblot analysis As shown in Figure 6(a) liver tissues exposedto electrosurgery with SS for 3 days could slightly inducethe expression of NF-120581B and it gradually increased up to

Evidence-Based Complementary and Alternative Medicine 7

SS0

5

10

15

20

25

30

Are

a (

)

Fibrosis area Apoptosis area

Wound area (FA + AA)

(a)

(c) (b)

FA Fibrosis area AA Apoptosis area PA Proliferate area

SSSS

+ L

Z-8

SS SS + LZ-8

SS + LZ-8

AA

FA

PA

AA

FA

AA

PA

lowast

lowast

lowastlowast

500 120583m500 120583m

50 120583m

50 120583m

Figure 5The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 28 days (Massonrsquos trichrome staining)(a) wound area (b) the site of apoptosis area fibrosis area and proliferate area and (c) percentage of each area Data are expressed as themeans plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

28 days Interestingly liver tissues further treated with LZ-8 significantly reduced the ratio of NF-120581B expression in aduration dependent manner (3 to 28 days) A similar resultwas observed on the expression of caspase-3 Therefore theresults can suggest that treatment of liver tissues after surgerywith LZ-8 was found to lower the mean NF-120581B and caspase-3 expressions so that it approaches wound healing propertypresumably by limiting NF-120581B and caspase-3 expression inliver tissuesThese results were further confirmed and shownas in Figure 6(b)

36 LZ-8 Inhibits NF-120581BNuclear Translocation in SS OperatedLiver Tissues The ability of LZ-8 to inhibit NF-120581B dispersionwas determined by red fluorescence (Cy5) intensity analysisof immunolabelled antibodies directed against NF-120581B andHoechst reagent to simultaneously identify nuclei (Figure 7)The intensity values of NF-120581B red fluorescence in the nucleusof SS operated tissues that were treated with LZ-8 for 3 dayswere found to be lesser as compared to SS operated livertissues without LZ-8 treatment Interestingly the presence ofLZ-8 in SS treated liver tissues for 7 days reached amaximuminhibition of nuclear fluorescence of NF-120581B than that oftreated tissues with SS alone These results can obviouslyindicate that LZ-8 plays a major role in inhibiting of NF-120581B

nuclear translocation in liver tissues during the process ofwound healing after electrosurgery

37 LZ-8 Reduces Apoptotic Cell Death in Liver Tissues duringWoundHealing after Electrosurgery Extensive fragmentationof nuclear DNA that generates a large number of DNAdouble-strand breaks is one of the most characteristic eventsof apoptosis An assay that relies on detection of DNA strandbreaks (DSBs) in situ by labeling them with fluorochromeshas been developed to identify and quantify apoptotic cellsby fluorescence microscopy or cytometry The assay is com-monly called TUNEL the acronym of Terminal deoxynu-cleotidyl transferase-mediated d-UTP Nick End Labeling Inthis study a very clear TUNEL-positive cells were observedin rats hepatic cells after 7 days of electrosurgery (Figure 8)However the number or TUNEL expression continued todecrease more prominently in LZ-8 treated hepatic cells thathad been exposed with monopolar electrosurgery for 7 daysThe results obtained from this study demonstrate that a slightapoptosis occurs in hepatic cells after electrosurgery whichwas as a result of LZ-8 inhibition and provide evidencefor high level of angiogenesis arises during wound healingprocess

8 Evidence-Based Complementary and Alternative Medicine

SS

NF-120581B

NF-120581B

SS + LZ-8

0 3 7 2800

05

10

15

20

25

Relat

ive e

xpre

ssio

n of

NF-120581

B

Treatment duration (day)

SSSS + LZ-8

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowast

lowastlowast

(a)

0 3 7 2800

05

10

15

20

Relat

ive e

xpre

ssio

n of

casp

ase-

3

Treatment duration (day)

SSSS + LZ-8

Caspase-3SS + LZ-8

Caspase-3SS

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowastlowast

lowastlowastlowast

(b)

Figure 6TheWestern blot assay results from rats liver treated with SS group and SS + LZ-8 group after different healing days (a) expressionsof NF-120581B and (b) expressions of caspase-3 Data are expressed as the means plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 comparedwith SS group

4 Discussion

The purpose of this study was to determine the effect ofthe LZ-8 an immunomodulatory protein derived from Glucidum when it is used for wound healing on hepatictissues and to compare this with monopolar electrosurgeryoperated liver tissues on the wounds size fibrosis thermalinjury and apoptosis The results of this study show thatLZ-8 enhances wound healing actions in hepatic tissuesafter electrosurgery Importantly this study has identifiedthat NF-120581B and caspase-3 activation are associated with LZ-8 on enhancing wound healing after surgery Purpose ofelectrical energy or coagulation of bleeding vessels onto tissueis frequently used in surgery Coagulation techniques suchas monopolar and laser techniques are commonly used indifferent surgical disciplines [21 22]

Monopolar and bipolar coagulation effects on brain tissuewere reported and the results of experimental trials were

compared [23 24] They observed less trauma after bipolarcoagulation compared with monopolar technique A studyhas demonstrated that bipolar mode of stimulation claimedto be another paradigm of a potential hazard comparedwith monopolar stimulation Temel et al [25] observed braindamage after bipolar but not monopolar and stimulationin rats However monophasic pulses were used whichmight have created larger lesions in a bipolar configurationMonopolar electrosurgery causes sparking current spreadand thermal damage in the tissues because of excessivegeneration of heat These problems will induce deleteriouseffects to the tissue in proximity to the operative site Inaddition when comparing monopolar electrosurgery andthe scalpel incision electrosurgery produces more tissuealteration and histological thermal damage when a lateralheat was increased by the low frequency radio wave of05 to 29MHz [26] Consequently the resultant effect ofmonopolar systems is excessive tissue damage and delayed

Evidence-Based Complementary and Alternative Medicine 9

Cy 5 DAPI Merge

Control

SS

SS + LZ-8

Day 3

SS

SS + LZ-8

Day 7

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

Figure 7 The immunofluorescence staining results from rats liver treated with SS group and SS + LZ-8 group after different healing days

healing Therefore additional drug supplements or therapyneed to be required to accelerate the wound healing actionon tissues after electrosurgery

A new immunomodulatory protein known as LZ-8 wasisolated from the mycelia of G lucidum and characterizedby chromatographicelectrophoretic techniques [17] Bothin vivo immunomodulation activity and in vitro mitogenicactivity of LZ-8 have been reported by Kino et al [14] Pre-vious studies have also demonstrated that topical applicationof oils improves some parameters of wound healing such asstimulation of connective tissue fiber [27] reduction in thethickness of the necrotic cell layer edge around the wound[28] and acceleration of wound closure [29 30] Due toexcellent thermal and acid stability together with a moderateresistance to alkali and dehydration LZ-8 is appropriate toapply tissues after surgery even with a mild thermal heatIn this study this is the first and novel results that LZ-8accelerates the reduction of the wound size thermal injuryand increase fibrosis area in rat liver after surgery and it

indicates that LZ-8 has potent ability to enhance the effect ofwound healing In a latest study we have also demonstratedthat the total areas of liver injury lateral thermal injury andfibrotic tissues were significantly higher in SS treated hepatictissues than that of CrN-coated needles that had been usedfor surgery [13]

NF-120581B activation plays a role in the regulation of morethan 400 gene product expressions associatedwith inflamma-tion cell survival proliferation invasion and angiogenesis[31 32] NF-120581B normally binds to I120581B120572 which impedes NF-120581B nuclear translocation from the cytoplasm to the nucleusNF-120581B is reported to be a vital link between hepatic injuryfibrosis and even hepatocellular carcinoma [33] NF-120581Benters the nucleus with the degradation of I120581B and activatesthe expression of specific genes that induce the apoptosiscascade [34] Once the cells are exposed to inflammatorystimuli including LPS and TNF-120572 I120581B120572 is phosphorylatedleading to I120581B120572 degradation and nuclear translocation of NF-120581B In our latest study it was noticed that liver tissues that had

10 Evidence-Based Complementary and Alternative Medicine

TUNEL DAPI Merge

SSSS

+ L

Z-8

250 120583m 250 120583m 250 120583m

250 120583m 250 120583m 250 120583m

Figure 8 The TUNEL staining assay results from rats liver treated with SS group and SS + LZ-8 group after 7 days of healing

been operated with SS-needles secreted higher levels of NF-120581B expression and its nuclear translocation than that of CrNoperated tissues [13] Fibrosis is an important component ofadvanced chronic inflammatory liver disease Although thechanges in hepatic structures that occur in liver cirrhosis havebeen described in detail the pathogenesis of fibrosis in duringelectrosurgery process is largely unknown Our latest reportindicates that monopolar elecrtrosurgery-induced hepaticfibrosis after repeated liver injury and the mechanisms aremainly based on the upregulation of NF-120581B and caspase-3expression in the liver tissues [13]

In this study we thus evaluated whether LZ-8 had anyeffect on nuclear translocation of NF-120581B during monopolarelectrosurgery and the results found for the first time thatLZ-8 inhibits nuclear translocation of NF-120581B in hepaticcells after 3 and 7 days of monopolar electrosurgery Yatesand Rayner [35] studied the effects of fatty acid supple-mentation on activation of NF-120581B and AP-1 after a skininjury since these transcription factors are highly associ-ated with wound healing They have also reported a novelfinding which indicates that wounding of normal humankeratinocytes in culture causes NF-120581B translocation Therelevance of NF-120581B activation to wound healing is sug-gested by the results that HeNe laser treatment a processshown to accelerate healing in a model of wounding [36]promoted the translocation of NF-120581B A previous studyalso proved that a ganoderma immunomodulatory proteincloned from Ganoderma microsporum holds anti-invasiveand anti-inflammatory activities in TNF-120572 induced humanalveolar epithelial A549 cells via inhibition of the I120581B120572 andNF-120581B pathway [37]

Apoptosis in wound healing is a promising issue Therehas been some evaluation of chronic diabetic wounds in

mice that suggests that there is increased cellular apoptosislikely due to hyperglycemia which produces reactive oxygenspecies that mediate mitochondrial release of cytochrome cand activate caspase-3 [37 38] Apoptosis is also involved inthe regulation of collagen synthesis and degradation withinwounds through regulation of fibroblast numbers and colla-genase activity [39] Recent work in another mouse modelsuggests that hypertrophic scars demonstrate decreased ratesof apoptosis with decreased expression of cleaved caspase-3Over expression of various caspase family members inducesapoptosis in cultured mammalian cells [40] Among thecysteine proteases caspase-3 is believed to be one of themost commonly involved in the execution of apoptosisin various cell types [41] The assay of TUNEL stainingcan identify the apoptotic cells via DNA fragmentation bylabeling the terminal ends of nucleic acids [42 43] Fromour latest study of TUNEL assay it can be clearly foundthat the apoptosis event was more significant in SS-needleoperated liver tissues [13] In this study TUNEL stainingprovides direct evidence of apoptosis in SS operated livertissues after 7 days Both the activation of caspase-3 and theinduction of apoptotic cell death could be suppressed by theaddition of antioxidants in the cultures [44] It has also beendemonstrated that LZ-8 is able to effectively promote theactivation and maturation of immature dendritic cells via thenuclear factor-kappaB and mitogen-activated protein kinasepathways [18] Moreover the present study observed theeffects of (i) inhibiting expression and nuclear translocationof NF-120581B and (ii) inhibiting expression of caspase-3 andreduction of apoptotic cell death by LZ-8 in liver tissuesafter electrosurgery which indicates that the wound healingproperty of LZ-8 may possibly occur through inhibition oftranscription factor NF-120581B and apoptotic factor caspase-3

Evidence-Based Complementary and Alternative Medicine 11

5 Conclusion

Under the same conditions LZ-8 treatment potentiates onreducing wound size and thermal injury An increasing fibro-sis and reducing apoptosis were found in LZ-8 treated livertissues during electrosurgery LZ-8 treatment significantlyinhibits NF-120581B and caspase-3 expressions and also reducesNF-120581B nuclear translocation and apoptotic cell death Theresults generated from this study suggest that LZ-8 has potenteffects on increasing wound healing action against the elec-trosurgical induced liver injury via the possible mechanismof inhibiting NF-120581B and caspase-3 expressions

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors would like to thank the Department of HealthExecutive Yuan Taiwan (Contract no MOHW103-TDU-N-211-133001) and TaipeiMedical University and TaipeiMedicalHospital (Contract no 100TMU-TMUH-05) for financiallysupporting this research Dr Hao-Jan Lin and Dr Yushan-Sophie Chang are considered the first coauthors of the paper

References

[1] J Liboon W Funkhouser and D J Terris ldquoA comparison ofmucosal incisions made by scalpel CO

2

laser electrocauteryand constant-voltage electrocauteryrdquo OtolaryngologymdashHeadand Neck Surgery vol 116 no 3 pp 379ndash385 1997

[2] J F Carew R F Ward A LaBruna P A Torzilli and W SSchley ldquoEffects of scalpel electrocautery and CO

2

and KTPlasers on wound healing in rat tonguesrdquoThe Laryngoscope vol108 no 3 pp 373ndash380 1998

[3] J A Pearce Electrosurgery John Wiley amp Sons New York NYUSA 1986

[4] W L Maness F W Rober R E Clark E Cataldo andA W Haddad ldquoTissue damage from electrosurgical poweroutput variations in hamster tonguesrdquoThe Journal of ProstheticDentistry vol 42 no 4 pp 456ndash460 1979

[5] W L Maness F W Roeber R E Clark E Cataldo D Riisand A W Haddad ldquoHistologic evaluation of electrosurgerywith varying frequency andwaveformrdquoThe Journal of ProstheticDentistry vol 40 no 3 pp 304ndash308 1978

[6] J Friedman J Margolin and S Piliero ldquoA preliminary study ofthe histological effects of three different types of electrosurgicalcurrentsrdquoThe New York State Dental Journal vol 40 no 6 pp349ndash353 1974

[7] W H Noble K D McClatchey and G D Douglass ldquoAhistologic comparison of effects of electrosurgical resectionusing different electrodesrdquo The Journal of Prosthetic Dentistryvol 35 no 5 pp 575ndash579 1976

[8] K C Nixon K F Adkins and D W Keys ldquoHistologicalevaluation of effects produced in alveolar bone followinggingival incision with an electrosurgical scalpelrdquo Journal ofPeriodontology vol 46 no 1 pp 40ndash44 1975

[9] I Glickman and L R Imber ldquoComparison of gingival resec-tion with electrosurgery and periodontal knivesmdasha biometric

and histologic studyrdquo Journal of Periodontology vol 41 no 3pp 142ndash148 1970

[10] D H Coelho J Cavallaro and E A Rothschild ldquoGingivalrecession with electrosurgery for impression makingrdquo TheJournal of Prosthetic Dentistry vol 33 no 4 pp 422ndash426 1975

[11] P B Robertson B Luscher L S Spangberg and B M LevyldquoPulpal and periodontal effects of electrosurgery involvingcervical metallic restorationsrdquo Oral Surgery Oral Medicine andOral Pathology vol 46 no 5 pp 702ndash710 1978

[12] R DrsquoSouza ldquoPulpal and periapical immune response to electro-surgical contact of cervical metallic restorations in monkeysrdquoQuintessence International vol 17 no 12 pp 803ndash808 1986

[13] K-L Ou J-S Chu H Hosseinkhani J-F Chiou and C-HYu ldquoBiomedical nanostructured coating for minimally invasivesurgery devices applications characterization cell cytotoxicityevaluation and an animal study in ratrdquo Surgical Endoscopy 2014

[14] K Kino A Yamashita K Yamaoka et al ldquoIsolation and char-acterization of a new immunomodulatory protein Ling Zhi-8(LZ-8) from Ganoderma lucidiumrdquo The Journal of BiologicalChemistry vol 264 no 1 pp 472ndash478 1989

[15] AMurasugi S Tanaka N Komiyama et al ldquoMolecular cloningof a cDNAand a gene encoding an immunomodulatory proteinLing Zhi-8 from a fungusGanoderma lucidiumrdquoThe Journal ofBiological Chemistry vol 266 no 4 pp 2486ndash2493 1991

[16] M-H Tong P-J Chien H-H Chang M-J Tsai and F SheuldquoHigh processing tolerances of immunomodulatory proteins inEnoki and Reishi mushroomsrdquo Journal of Agricultural and FoodChemistry vol 56 no 9 pp 3160ndash3166 2008

[17] L Huang F Sun C Liang et al ldquoCrystal structure of LZ-8 fromthemedicinal fungusGanoderma lucidiumrdquo Proteins StructureFunction and Bioinformatics vol 75 no 2 pp 524ndash527 2009

[18] Y-L Lin Y-C Liang Y-S Tseng et al ldquoAn immunomodulatoryprotein Ling Zhi-8 induced activation and maturation ofhuman monocyte-derived dendritic cells by the NF-120581B andMAPK pathwaysrdquo Journal of Leukocyte Biology vol 86 no 4pp 877ndash889 2009

[19] R A D Drury and E A Wallington Carletonrsquos HistologicalTechnique 1981

[20] Y Du D-W Guan and R Zhao ldquoAn immunohistochemicalstudy on the expression of caspase-3 in the skin wound healingrdquoFa Yi Xue Za Zhi vol 20 no 1 pp 1ndash3 2004

[21] K E Grund T Straub and G Farin ldquoNew haemostatictechniques argon plasma coagulationrdquo Baillierersquos Best Practiceand Research in Clinical Gastroenterology vol 13 no 1 pp 67ndash84 1999

[22] D Hellwig R Haag V Bartel et al ldquoApplication of newelectrosurgical devices and probes in endoscopic neurosurgeryrdquoNeurological Research vol 21 no 1 pp 67ndash72 1999

[23] B Chehrazi and W F Collins Jr ldquoA comparison of effects ofbipolar and monopolar electrocoagulation in brainrdquo Journal ofNeurosurgery vol 54 no 2 pp 197ndash203 1981

[24] J Kerl and J Staubesand ldquoAn ultrastructural report on thechoice between monopolar and bipolar electrocoagulationrdquoActa Neurochirurgica vol 92 no 1ndash4 pp 123ndash131 1988

[25] Y Temel V Visser-Vandewalle M van derWolf et al ldquoMonop-olar versus bipolar high frequency stimulation in the rat subtha-lamic nucleus differences in histological damagerdquoNeuroscienceLetters vol 367 no 1 pp 92ndash96 2004

[26] L I Malis ldquoNew trends in microsurgery and applied technol-ogyrdquo in Advanced Technology in Neurosurgery F Pluchino andG Broggi Eds pp 1ndash16 Springer Berlin Germany 1988

12 Evidence-Based Complementary and Alternative Medicine

[27] C R B Cardoso M A Souza E A V Ferro S Favoreto Jrand J D O Pena ldquoInfluence of topical administration of n-3and n-6 essential and n-9 nonessential fatty acids on the healingof cutaneous woundsrdquoWound Repair and Regeneration vol 12no 2 pp 235ndash243 2004

[28] L M Pereira E Hatanaka E F Martins et al ldquoEffect of oleicand linoleic acids on the inflammatory phase of wound healingin ratsrdquo Cell Biochemistry and Function vol 26 no 2 pp 197ndash204 2008

[29] N-Y ParkGValacchi andY Lim ldquoEffect of dietary conjugatedlinoleic acid supplementation on early inflammatory responsesduring cutaneous wound healingrdquo Mediators of Inflammationvol 2010 Article ID 342328 8 pages 2010

[30] M Otranto A P do Nascimento and A Monte-Alto-CostaldquoEffects of supplementation with different edible oils on cuta-neous wound healingrdquoWound Repair and Regeneration vol 18no 6 pp 629ndash636 2010

[31] X Dolcet D Llobet J Pallares and X Matias-Guiu ldquoNF-kBin development and progression of human cancerrdquo VirchowsArchiv vol 446 no 5 pp 475ndash482 2005

[32] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[33] T Luedde and R F Schwabe ldquoNF-120581B in the livermdashlinkinginjury fibrosis and hepatocellular carcinomardquo Nature ReviewsGastroenterology amp Hepatology vol 8 no 2 pp 108ndash118 2011

[34] J A Schmid and A Birbach ldquoIkappaB kinase beta (IKKbetaIKK2IKBKB)mdasha keymolecule in signaling to the transcriptionfactor NF-kappaBrdquo Cytokine amp Growth Factor Reviews vol 19no 2 pp 157ndash165 2008

[35] S Yates and T E Rayner ldquoTranscription factor activation inresponse to cutaneous injury role of AP-1 in reepithelializa-tionrdquo Wound Repair and Regeneration vol 10 no 1 pp 5ndash152002

[36] A F Haas R R Isseroff R G Wheeland P A Rood and PJ Graves ldquoLow-energy helium-neon laser irradiation increasesthe motility of cultured human keratinocytesrdquo The Journal ofInvestigative Dermatology vol 94 no 6 pp 822ndash826 1990

[37] C-H Lin Y-M Hsiao C-C Ou et al ldquoGMI a Ganodermaimmunomodulatory protein down-regulates tumor necrosisfactor 120572-induced expression of matrix metalloproteinase 9 viaNF-120581Bpathway in human alveolar epithelial A549 cellsrdquo Journalof agricultural and food chemistry vol 58 no 22 pp 12014ndash12021 2010

[38] L Cai W Li G Wang L Guo Y Jiang and Y J KangldquoHyperglycemia-induced apoptosis in mouse myocardiummitochondrial cytochrome c-mediated caspase-3 activationpathwayrdquo Diabetes vol 51 no 6 pp 1938ndash1948 2002

[39] N K Rai K Tripathi D Sharma andV K Shukla ldquoApoptosis abasic physiologic process in wound healingrdquo The InternationalJournal of Lower Extremity Wounds vol 4 no 3 pp 138ndash1442005

[40] J Bian and Y Sun ldquoTranscriptional activation by p53 of thehuman type IV collagenase (gelatinase A or matrix metallopro-teinase 2) promoterrdquoMolecular and Cellular Biology vol 17 no11 pp 6330ndash6338 1997

[41] S Wang MMiura Y-K Jung et al ldquoIdentification and charac-terization of ich-3 a member of the interleukin-1120573 convertingenzyme (ICE)Ced-3 family and an upstream regulator of ICErdquoThe Journal of Biological Chemistry vol 271 no 34 pp 20580ndash20587 1996

[42] E S Alnemri D J Livingston D W Nicholson et al ldquoHumanICECED-3 protease nomenclaturerdquo Cell vol 87 no 2 article171 1996

[43] T D Hewitson T Bisucci and I A Darby ldquoHistochemicallocalization of apoptosis with in situ labeling of fragmentedDNArdquo Methods in Molecular Biology vol 326 pp 227ndash2342006

[44] D T Loo ldquoIn situ detection of apoptosis by the TUNEL assayan overview of techniquesrdquo in DNA Damage Detection In SituEx Vivo and In Vivo vol 682 ofMethods in Molecular Biologypp 3ndash13 Humana Press New Jersey NJ USA 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 6: Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014 › 916531.pdfResearch Article An Immunomodulatory Protein (Ling Zhi-8) from a

6 Evidence-Based Complementary and Alternative Medicine

NA Normal area FA Fibrosis area AA Apoptosis area

NA

FAFAAA

AA

AAFA

FA

NA

SSSS

+ L

Z-8

(b)

50 120583m

50 120583m

50 120583m

50 120583m

SS SS + LZ-80

10

20

30

40

50

60

70

80

90

Are

a (

)

Fibrosis area Injury areaApoptosis area Wound area (FA + IA + AA)

(c)

lowast

lowastlowast

lowastlowastlowast

(a)

SS SS + LZ-8

500 120583m500 120583m

Figure 4 The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 7 days (Massonrsquos trichrome staining)(a) wound area (b) the site of apoptosis area fibrosis area and normal area and (c) percentage of each area Data are expressed as themeans plusmnSEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

was found that the injury area (119875 lt 005) thermal injuryarea (119875 lt 001) and wound area (119875 lt 0001) of SS + LZ-8groupwere reducedmore significantly than those of SS groupat day 3 Furthermore Massonrsquos trichrome staining was usedto examine the formation of fibrotic tissue No fibrotic tissuewas evident for either group at day 0 or day 3

33 Effects of LZ-8 on the Areas of Fibrosis Injury ApoptosisandWounds of Liver Tissues after 7 Days of Surgery A furtherstudy was performed to determine whether LZ-8 couldenhance the areas of fibrosis reduce injury and wounds andinhibit apoptosis of monopolar SS induced liver incision interms of withdrawing the above observed injuriesThe resultsclearly confirmed that injury apoptosis and wound of livertissues in SS operated groups that had been treated withLZ-8 for 7 days were significantly alleviated when comparedwith SS operated groups without LZ-8 treatment (Figure 4)Nevertheless the areas of fibrosis and numbers of cell deathin liver tissues for treated rats with LZ-8 were comparativelyhigher than those of SS operated rats Thus the resultsindicate that LZ-8 treatment could not affect the reduction ofliver fibrosis andmore effective on the inhibition of apoptosiswhich are caused by surgery

34 Effects of LZ-8 on the Areas of Fibrosis Injury Apoptosisand Wounds of Liver Tissues after 28 Days of Surgery Sincedifferential observations were found on the areas of fibrosisand apoptosis in liver tissues of LZ-8 supplemented rats thathave already been operated with SS for 7 days we furtherextended the duration of treatment up to 28 days (Figure 5)to confirm the hypothesis Our analysis obviously exhibitsfurther evidence that the effect of LZ-8 was similar to thatof 7-days treatment

35 Does LZ-8 Enhance Wound Healing in SS Operated LiverTissues via Inhibiting the Expression of NF-120581B and Caspase-3It is well known that the angiogenesis is essential in woundhealing Various studies have demonstrated the inhibition ofangiogenesis by the blockage of NF-120581B A recent study hasalso suggested that caspase-3 plays an important role duringskin wound healing [20] Thus we performed this study todeterminewhether thewound healing action of LZ-8was dueto the effect on inhibiting of NF-120581B and caspase-3 expressionthe tissue levels of their protein were examined by Westernblot analysis As shown in Figure 6(a) liver tissues exposedto electrosurgery with SS for 3 days could slightly inducethe expression of NF-120581B and it gradually increased up to

Evidence-Based Complementary and Alternative Medicine 7

SS0

5

10

15

20

25

30

Are

a (

)

Fibrosis area Apoptosis area

Wound area (FA + AA)

(a)

(c) (b)

FA Fibrosis area AA Apoptosis area PA Proliferate area

SSSS

+ L

Z-8

SS SS + LZ-8

SS + LZ-8

AA

FA

PA

AA

FA

AA

PA

lowast

lowast

lowastlowast

500 120583m500 120583m

50 120583m

50 120583m

Figure 5The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 28 days (Massonrsquos trichrome staining)(a) wound area (b) the site of apoptosis area fibrosis area and proliferate area and (c) percentage of each area Data are expressed as themeans plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

28 days Interestingly liver tissues further treated with LZ-8 significantly reduced the ratio of NF-120581B expression in aduration dependent manner (3 to 28 days) A similar resultwas observed on the expression of caspase-3 Therefore theresults can suggest that treatment of liver tissues after surgerywith LZ-8 was found to lower the mean NF-120581B and caspase-3 expressions so that it approaches wound healing propertypresumably by limiting NF-120581B and caspase-3 expression inliver tissuesThese results were further confirmed and shownas in Figure 6(b)

36 LZ-8 Inhibits NF-120581BNuclear Translocation in SS OperatedLiver Tissues The ability of LZ-8 to inhibit NF-120581B dispersionwas determined by red fluorescence (Cy5) intensity analysisof immunolabelled antibodies directed against NF-120581B andHoechst reagent to simultaneously identify nuclei (Figure 7)The intensity values of NF-120581B red fluorescence in the nucleusof SS operated tissues that were treated with LZ-8 for 3 dayswere found to be lesser as compared to SS operated livertissues without LZ-8 treatment Interestingly the presence ofLZ-8 in SS treated liver tissues for 7 days reached amaximuminhibition of nuclear fluorescence of NF-120581B than that oftreated tissues with SS alone These results can obviouslyindicate that LZ-8 plays a major role in inhibiting of NF-120581B

nuclear translocation in liver tissues during the process ofwound healing after electrosurgery

37 LZ-8 Reduces Apoptotic Cell Death in Liver Tissues duringWoundHealing after Electrosurgery Extensive fragmentationof nuclear DNA that generates a large number of DNAdouble-strand breaks is one of the most characteristic eventsof apoptosis An assay that relies on detection of DNA strandbreaks (DSBs) in situ by labeling them with fluorochromeshas been developed to identify and quantify apoptotic cellsby fluorescence microscopy or cytometry The assay is com-monly called TUNEL the acronym of Terminal deoxynu-cleotidyl transferase-mediated d-UTP Nick End Labeling Inthis study a very clear TUNEL-positive cells were observedin rats hepatic cells after 7 days of electrosurgery (Figure 8)However the number or TUNEL expression continued todecrease more prominently in LZ-8 treated hepatic cells thathad been exposed with monopolar electrosurgery for 7 daysThe results obtained from this study demonstrate that a slightapoptosis occurs in hepatic cells after electrosurgery whichwas as a result of LZ-8 inhibition and provide evidencefor high level of angiogenesis arises during wound healingprocess

8 Evidence-Based Complementary and Alternative Medicine

SS

NF-120581B

NF-120581B

SS + LZ-8

0 3 7 2800

05

10

15

20

25

Relat

ive e

xpre

ssio

n of

NF-120581

B

Treatment duration (day)

SSSS + LZ-8

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowast

lowastlowast

(a)

0 3 7 2800

05

10

15

20

Relat

ive e

xpre

ssio

n of

casp

ase-

3

Treatment duration (day)

SSSS + LZ-8

Caspase-3SS + LZ-8

Caspase-3SS

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowastlowast

lowastlowastlowast

(b)

Figure 6TheWestern blot assay results from rats liver treated with SS group and SS + LZ-8 group after different healing days (a) expressionsof NF-120581B and (b) expressions of caspase-3 Data are expressed as the means plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 comparedwith SS group

4 Discussion

The purpose of this study was to determine the effect ofthe LZ-8 an immunomodulatory protein derived from Glucidum when it is used for wound healing on hepatictissues and to compare this with monopolar electrosurgeryoperated liver tissues on the wounds size fibrosis thermalinjury and apoptosis The results of this study show thatLZ-8 enhances wound healing actions in hepatic tissuesafter electrosurgery Importantly this study has identifiedthat NF-120581B and caspase-3 activation are associated with LZ-8 on enhancing wound healing after surgery Purpose ofelectrical energy or coagulation of bleeding vessels onto tissueis frequently used in surgery Coagulation techniques suchas monopolar and laser techniques are commonly used indifferent surgical disciplines [21 22]

Monopolar and bipolar coagulation effects on brain tissuewere reported and the results of experimental trials were

compared [23 24] They observed less trauma after bipolarcoagulation compared with monopolar technique A studyhas demonstrated that bipolar mode of stimulation claimedto be another paradigm of a potential hazard comparedwith monopolar stimulation Temel et al [25] observed braindamage after bipolar but not monopolar and stimulationin rats However monophasic pulses were used whichmight have created larger lesions in a bipolar configurationMonopolar electrosurgery causes sparking current spreadand thermal damage in the tissues because of excessivegeneration of heat These problems will induce deleteriouseffects to the tissue in proximity to the operative site Inaddition when comparing monopolar electrosurgery andthe scalpel incision electrosurgery produces more tissuealteration and histological thermal damage when a lateralheat was increased by the low frequency radio wave of05 to 29MHz [26] Consequently the resultant effect ofmonopolar systems is excessive tissue damage and delayed

Evidence-Based Complementary and Alternative Medicine 9

Cy 5 DAPI Merge

Control

SS

SS + LZ-8

Day 3

SS

SS + LZ-8

Day 7

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

Figure 7 The immunofluorescence staining results from rats liver treated with SS group and SS + LZ-8 group after different healing days

healing Therefore additional drug supplements or therapyneed to be required to accelerate the wound healing actionon tissues after electrosurgery

A new immunomodulatory protein known as LZ-8 wasisolated from the mycelia of G lucidum and characterizedby chromatographicelectrophoretic techniques [17] Bothin vivo immunomodulation activity and in vitro mitogenicactivity of LZ-8 have been reported by Kino et al [14] Pre-vious studies have also demonstrated that topical applicationof oils improves some parameters of wound healing such asstimulation of connective tissue fiber [27] reduction in thethickness of the necrotic cell layer edge around the wound[28] and acceleration of wound closure [29 30] Due toexcellent thermal and acid stability together with a moderateresistance to alkali and dehydration LZ-8 is appropriate toapply tissues after surgery even with a mild thermal heatIn this study this is the first and novel results that LZ-8accelerates the reduction of the wound size thermal injuryand increase fibrosis area in rat liver after surgery and it

indicates that LZ-8 has potent ability to enhance the effect ofwound healing In a latest study we have also demonstratedthat the total areas of liver injury lateral thermal injury andfibrotic tissues were significantly higher in SS treated hepatictissues than that of CrN-coated needles that had been usedfor surgery [13]

NF-120581B activation plays a role in the regulation of morethan 400 gene product expressions associatedwith inflamma-tion cell survival proliferation invasion and angiogenesis[31 32] NF-120581B normally binds to I120581B120572 which impedes NF-120581B nuclear translocation from the cytoplasm to the nucleusNF-120581B is reported to be a vital link between hepatic injuryfibrosis and even hepatocellular carcinoma [33] NF-120581Benters the nucleus with the degradation of I120581B and activatesthe expression of specific genes that induce the apoptosiscascade [34] Once the cells are exposed to inflammatorystimuli including LPS and TNF-120572 I120581B120572 is phosphorylatedleading to I120581B120572 degradation and nuclear translocation of NF-120581B In our latest study it was noticed that liver tissues that had

10 Evidence-Based Complementary and Alternative Medicine

TUNEL DAPI Merge

SSSS

+ L

Z-8

250 120583m 250 120583m 250 120583m

250 120583m 250 120583m 250 120583m

Figure 8 The TUNEL staining assay results from rats liver treated with SS group and SS + LZ-8 group after 7 days of healing

been operated with SS-needles secreted higher levels of NF-120581B expression and its nuclear translocation than that of CrNoperated tissues [13] Fibrosis is an important component ofadvanced chronic inflammatory liver disease Although thechanges in hepatic structures that occur in liver cirrhosis havebeen described in detail the pathogenesis of fibrosis in duringelectrosurgery process is largely unknown Our latest reportindicates that monopolar elecrtrosurgery-induced hepaticfibrosis after repeated liver injury and the mechanisms aremainly based on the upregulation of NF-120581B and caspase-3expression in the liver tissues [13]

In this study we thus evaluated whether LZ-8 had anyeffect on nuclear translocation of NF-120581B during monopolarelectrosurgery and the results found for the first time thatLZ-8 inhibits nuclear translocation of NF-120581B in hepaticcells after 3 and 7 days of monopolar electrosurgery Yatesand Rayner [35] studied the effects of fatty acid supple-mentation on activation of NF-120581B and AP-1 after a skininjury since these transcription factors are highly associ-ated with wound healing They have also reported a novelfinding which indicates that wounding of normal humankeratinocytes in culture causes NF-120581B translocation Therelevance of NF-120581B activation to wound healing is sug-gested by the results that HeNe laser treatment a processshown to accelerate healing in a model of wounding [36]promoted the translocation of NF-120581B A previous studyalso proved that a ganoderma immunomodulatory proteincloned from Ganoderma microsporum holds anti-invasiveand anti-inflammatory activities in TNF-120572 induced humanalveolar epithelial A549 cells via inhibition of the I120581B120572 andNF-120581B pathway [37]

Apoptosis in wound healing is a promising issue Therehas been some evaluation of chronic diabetic wounds in

mice that suggests that there is increased cellular apoptosislikely due to hyperglycemia which produces reactive oxygenspecies that mediate mitochondrial release of cytochrome cand activate caspase-3 [37 38] Apoptosis is also involved inthe regulation of collagen synthesis and degradation withinwounds through regulation of fibroblast numbers and colla-genase activity [39] Recent work in another mouse modelsuggests that hypertrophic scars demonstrate decreased ratesof apoptosis with decreased expression of cleaved caspase-3Over expression of various caspase family members inducesapoptosis in cultured mammalian cells [40] Among thecysteine proteases caspase-3 is believed to be one of themost commonly involved in the execution of apoptosisin various cell types [41] The assay of TUNEL stainingcan identify the apoptotic cells via DNA fragmentation bylabeling the terminal ends of nucleic acids [42 43] Fromour latest study of TUNEL assay it can be clearly foundthat the apoptosis event was more significant in SS-needleoperated liver tissues [13] In this study TUNEL stainingprovides direct evidence of apoptosis in SS operated livertissues after 7 days Both the activation of caspase-3 and theinduction of apoptotic cell death could be suppressed by theaddition of antioxidants in the cultures [44] It has also beendemonstrated that LZ-8 is able to effectively promote theactivation and maturation of immature dendritic cells via thenuclear factor-kappaB and mitogen-activated protein kinasepathways [18] Moreover the present study observed theeffects of (i) inhibiting expression and nuclear translocationof NF-120581B and (ii) inhibiting expression of caspase-3 andreduction of apoptotic cell death by LZ-8 in liver tissuesafter electrosurgery which indicates that the wound healingproperty of LZ-8 may possibly occur through inhibition oftranscription factor NF-120581B and apoptotic factor caspase-3

Evidence-Based Complementary and Alternative Medicine 11

5 Conclusion

Under the same conditions LZ-8 treatment potentiates onreducing wound size and thermal injury An increasing fibro-sis and reducing apoptosis were found in LZ-8 treated livertissues during electrosurgery LZ-8 treatment significantlyinhibits NF-120581B and caspase-3 expressions and also reducesNF-120581B nuclear translocation and apoptotic cell death Theresults generated from this study suggest that LZ-8 has potenteffects on increasing wound healing action against the elec-trosurgical induced liver injury via the possible mechanismof inhibiting NF-120581B and caspase-3 expressions

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors would like to thank the Department of HealthExecutive Yuan Taiwan (Contract no MOHW103-TDU-N-211-133001) and TaipeiMedical University and TaipeiMedicalHospital (Contract no 100TMU-TMUH-05) for financiallysupporting this research Dr Hao-Jan Lin and Dr Yushan-Sophie Chang are considered the first coauthors of the paper

References

[1] J Liboon W Funkhouser and D J Terris ldquoA comparison ofmucosal incisions made by scalpel CO

2

laser electrocauteryand constant-voltage electrocauteryrdquo OtolaryngologymdashHeadand Neck Surgery vol 116 no 3 pp 379ndash385 1997

[2] J F Carew R F Ward A LaBruna P A Torzilli and W SSchley ldquoEffects of scalpel electrocautery and CO

2

and KTPlasers on wound healing in rat tonguesrdquoThe Laryngoscope vol108 no 3 pp 373ndash380 1998

[3] J A Pearce Electrosurgery John Wiley amp Sons New York NYUSA 1986

[4] W L Maness F W Rober R E Clark E Cataldo andA W Haddad ldquoTissue damage from electrosurgical poweroutput variations in hamster tonguesrdquoThe Journal of ProstheticDentistry vol 42 no 4 pp 456ndash460 1979

[5] W L Maness F W Roeber R E Clark E Cataldo D Riisand A W Haddad ldquoHistologic evaluation of electrosurgerywith varying frequency andwaveformrdquoThe Journal of ProstheticDentistry vol 40 no 3 pp 304ndash308 1978

[6] J Friedman J Margolin and S Piliero ldquoA preliminary study ofthe histological effects of three different types of electrosurgicalcurrentsrdquoThe New York State Dental Journal vol 40 no 6 pp349ndash353 1974

[7] W H Noble K D McClatchey and G D Douglass ldquoAhistologic comparison of effects of electrosurgical resectionusing different electrodesrdquo The Journal of Prosthetic Dentistryvol 35 no 5 pp 575ndash579 1976

[8] K C Nixon K F Adkins and D W Keys ldquoHistologicalevaluation of effects produced in alveolar bone followinggingival incision with an electrosurgical scalpelrdquo Journal ofPeriodontology vol 46 no 1 pp 40ndash44 1975

[9] I Glickman and L R Imber ldquoComparison of gingival resec-tion with electrosurgery and periodontal knivesmdasha biometric

and histologic studyrdquo Journal of Periodontology vol 41 no 3pp 142ndash148 1970

[10] D H Coelho J Cavallaro and E A Rothschild ldquoGingivalrecession with electrosurgery for impression makingrdquo TheJournal of Prosthetic Dentistry vol 33 no 4 pp 422ndash426 1975

[11] P B Robertson B Luscher L S Spangberg and B M LevyldquoPulpal and periodontal effects of electrosurgery involvingcervical metallic restorationsrdquo Oral Surgery Oral Medicine andOral Pathology vol 46 no 5 pp 702ndash710 1978

[12] R DrsquoSouza ldquoPulpal and periapical immune response to electro-surgical contact of cervical metallic restorations in monkeysrdquoQuintessence International vol 17 no 12 pp 803ndash808 1986

[13] K-L Ou J-S Chu H Hosseinkhani J-F Chiou and C-HYu ldquoBiomedical nanostructured coating for minimally invasivesurgery devices applications characterization cell cytotoxicityevaluation and an animal study in ratrdquo Surgical Endoscopy 2014

[14] K Kino A Yamashita K Yamaoka et al ldquoIsolation and char-acterization of a new immunomodulatory protein Ling Zhi-8(LZ-8) from Ganoderma lucidiumrdquo The Journal of BiologicalChemistry vol 264 no 1 pp 472ndash478 1989

[15] AMurasugi S Tanaka N Komiyama et al ldquoMolecular cloningof a cDNAand a gene encoding an immunomodulatory proteinLing Zhi-8 from a fungusGanoderma lucidiumrdquoThe Journal ofBiological Chemistry vol 266 no 4 pp 2486ndash2493 1991

[16] M-H Tong P-J Chien H-H Chang M-J Tsai and F SheuldquoHigh processing tolerances of immunomodulatory proteins inEnoki and Reishi mushroomsrdquo Journal of Agricultural and FoodChemistry vol 56 no 9 pp 3160ndash3166 2008

[17] L Huang F Sun C Liang et al ldquoCrystal structure of LZ-8 fromthemedicinal fungusGanoderma lucidiumrdquo Proteins StructureFunction and Bioinformatics vol 75 no 2 pp 524ndash527 2009

[18] Y-L Lin Y-C Liang Y-S Tseng et al ldquoAn immunomodulatoryprotein Ling Zhi-8 induced activation and maturation ofhuman monocyte-derived dendritic cells by the NF-120581B andMAPK pathwaysrdquo Journal of Leukocyte Biology vol 86 no 4pp 877ndash889 2009

[19] R A D Drury and E A Wallington Carletonrsquos HistologicalTechnique 1981

[20] Y Du D-W Guan and R Zhao ldquoAn immunohistochemicalstudy on the expression of caspase-3 in the skin wound healingrdquoFa Yi Xue Za Zhi vol 20 no 1 pp 1ndash3 2004

[21] K E Grund T Straub and G Farin ldquoNew haemostatictechniques argon plasma coagulationrdquo Baillierersquos Best Practiceand Research in Clinical Gastroenterology vol 13 no 1 pp 67ndash84 1999

[22] D Hellwig R Haag V Bartel et al ldquoApplication of newelectrosurgical devices and probes in endoscopic neurosurgeryrdquoNeurological Research vol 21 no 1 pp 67ndash72 1999

[23] B Chehrazi and W F Collins Jr ldquoA comparison of effects ofbipolar and monopolar electrocoagulation in brainrdquo Journal ofNeurosurgery vol 54 no 2 pp 197ndash203 1981

[24] J Kerl and J Staubesand ldquoAn ultrastructural report on thechoice between monopolar and bipolar electrocoagulationrdquoActa Neurochirurgica vol 92 no 1ndash4 pp 123ndash131 1988

[25] Y Temel V Visser-Vandewalle M van derWolf et al ldquoMonop-olar versus bipolar high frequency stimulation in the rat subtha-lamic nucleus differences in histological damagerdquoNeuroscienceLetters vol 367 no 1 pp 92ndash96 2004

[26] L I Malis ldquoNew trends in microsurgery and applied technol-ogyrdquo in Advanced Technology in Neurosurgery F Pluchino andG Broggi Eds pp 1ndash16 Springer Berlin Germany 1988

12 Evidence-Based Complementary and Alternative Medicine

[27] C R B Cardoso M A Souza E A V Ferro S Favoreto Jrand J D O Pena ldquoInfluence of topical administration of n-3and n-6 essential and n-9 nonessential fatty acids on the healingof cutaneous woundsrdquoWound Repair and Regeneration vol 12no 2 pp 235ndash243 2004

[28] L M Pereira E Hatanaka E F Martins et al ldquoEffect of oleicand linoleic acids on the inflammatory phase of wound healingin ratsrdquo Cell Biochemistry and Function vol 26 no 2 pp 197ndash204 2008

[29] N-Y ParkGValacchi andY Lim ldquoEffect of dietary conjugatedlinoleic acid supplementation on early inflammatory responsesduring cutaneous wound healingrdquo Mediators of Inflammationvol 2010 Article ID 342328 8 pages 2010

[30] M Otranto A P do Nascimento and A Monte-Alto-CostaldquoEffects of supplementation with different edible oils on cuta-neous wound healingrdquoWound Repair and Regeneration vol 18no 6 pp 629ndash636 2010

[31] X Dolcet D Llobet J Pallares and X Matias-Guiu ldquoNF-kBin development and progression of human cancerrdquo VirchowsArchiv vol 446 no 5 pp 475ndash482 2005

[32] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[33] T Luedde and R F Schwabe ldquoNF-120581B in the livermdashlinkinginjury fibrosis and hepatocellular carcinomardquo Nature ReviewsGastroenterology amp Hepatology vol 8 no 2 pp 108ndash118 2011

[34] J A Schmid and A Birbach ldquoIkappaB kinase beta (IKKbetaIKK2IKBKB)mdasha keymolecule in signaling to the transcriptionfactor NF-kappaBrdquo Cytokine amp Growth Factor Reviews vol 19no 2 pp 157ndash165 2008

[35] S Yates and T E Rayner ldquoTranscription factor activation inresponse to cutaneous injury role of AP-1 in reepithelializa-tionrdquo Wound Repair and Regeneration vol 10 no 1 pp 5ndash152002

[36] A F Haas R R Isseroff R G Wheeland P A Rood and PJ Graves ldquoLow-energy helium-neon laser irradiation increasesthe motility of cultured human keratinocytesrdquo The Journal ofInvestigative Dermatology vol 94 no 6 pp 822ndash826 1990

[37] C-H Lin Y-M Hsiao C-C Ou et al ldquoGMI a Ganodermaimmunomodulatory protein down-regulates tumor necrosisfactor 120572-induced expression of matrix metalloproteinase 9 viaNF-120581Bpathway in human alveolar epithelial A549 cellsrdquo Journalof agricultural and food chemistry vol 58 no 22 pp 12014ndash12021 2010

[38] L Cai W Li G Wang L Guo Y Jiang and Y J KangldquoHyperglycemia-induced apoptosis in mouse myocardiummitochondrial cytochrome c-mediated caspase-3 activationpathwayrdquo Diabetes vol 51 no 6 pp 1938ndash1948 2002

[39] N K Rai K Tripathi D Sharma andV K Shukla ldquoApoptosis abasic physiologic process in wound healingrdquo The InternationalJournal of Lower Extremity Wounds vol 4 no 3 pp 138ndash1442005

[40] J Bian and Y Sun ldquoTranscriptional activation by p53 of thehuman type IV collagenase (gelatinase A or matrix metallopro-teinase 2) promoterrdquoMolecular and Cellular Biology vol 17 no11 pp 6330ndash6338 1997

[41] S Wang MMiura Y-K Jung et al ldquoIdentification and charac-terization of ich-3 a member of the interleukin-1120573 convertingenzyme (ICE)Ced-3 family and an upstream regulator of ICErdquoThe Journal of Biological Chemistry vol 271 no 34 pp 20580ndash20587 1996

[42] E S Alnemri D J Livingston D W Nicholson et al ldquoHumanICECED-3 protease nomenclaturerdquo Cell vol 87 no 2 article171 1996

[43] T D Hewitson T Bisucci and I A Darby ldquoHistochemicallocalization of apoptosis with in situ labeling of fragmentedDNArdquo Methods in Molecular Biology vol 326 pp 227ndash2342006

[44] D T Loo ldquoIn situ detection of apoptosis by the TUNEL assayan overview of techniquesrdquo in DNA Damage Detection In SituEx Vivo and In Vivo vol 682 ofMethods in Molecular Biologypp 3ndash13 Humana Press New Jersey NJ USA 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 7: Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014 › 916531.pdfResearch Article An Immunomodulatory Protein (Ling Zhi-8) from a

Evidence-Based Complementary and Alternative Medicine 7

SS0

5

10

15

20

25

30

Are

a (

)

Fibrosis area Apoptosis area

Wound area (FA + AA)

(a)

(c) (b)

FA Fibrosis area AA Apoptosis area PA Proliferate area

SSSS

+ L

Z-8

SS SS + LZ-8

SS + LZ-8

AA

FA

PA

AA

FA

AA

PA

lowast

lowast

lowastlowast

500 120583m500 120583m

50 120583m

50 120583m

Figure 5The wound healing effects on wounds in rat liver tissues after monopolar electrosurgery for 28 days (Massonrsquos trichrome staining)(a) wound area (b) the site of apoptosis area fibrosis area and proliferate area and (c) percentage of each area Data are expressed as themeans plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with SS group

28 days Interestingly liver tissues further treated with LZ-8 significantly reduced the ratio of NF-120581B expression in aduration dependent manner (3 to 28 days) A similar resultwas observed on the expression of caspase-3 Therefore theresults can suggest that treatment of liver tissues after surgerywith LZ-8 was found to lower the mean NF-120581B and caspase-3 expressions so that it approaches wound healing propertypresumably by limiting NF-120581B and caspase-3 expression inliver tissuesThese results were further confirmed and shownas in Figure 6(b)

36 LZ-8 Inhibits NF-120581BNuclear Translocation in SS OperatedLiver Tissues The ability of LZ-8 to inhibit NF-120581B dispersionwas determined by red fluorescence (Cy5) intensity analysisof immunolabelled antibodies directed against NF-120581B andHoechst reagent to simultaneously identify nuclei (Figure 7)The intensity values of NF-120581B red fluorescence in the nucleusof SS operated tissues that were treated with LZ-8 for 3 dayswere found to be lesser as compared to SS operated livertissues without LZ-8 treatment Interestingly the presence ofLZ-8 in SS treated liver tissues for 7 days reached amaximuminhibition of nuclear fluorescence of NF-120581B than that oftreated tissues with SS alone These results can obviouslyindicate that LZ-8 plays a major role in inhibiting of NF-120581B

nuclear translocation in liver tissues during the process ofwound healing after electrosurgery

37 LZ-8 Reduces Apoptotic Cell Death in Liver Tissues duringWoundHealing after Electrosurgery Extensive fragmentationof nuclear DNA that generates a large number of DNAdouble-strand breaks is one of the most characteristic eventsof apoptosis An assay that relies on detection of DNA strandbreaks (DSBs) in situ by labeling them with fluorochromeshas been developed to identify and quantify apoptotic cellsby fluorescence microscopy or cytometry The assay is com-monly called TUNEL the acronym of Terminal deoxynu-cleotidyl transferase-mediated d-UTP Nick End Labeling Inthis study a very clear TUNEL-positive cells were observedin rats hepatic cells after 7 days of electrosurgery (Figure 8)However the number or TUNEL expression continued todecrease more prominently in LZ-8 treated hepatic cells thathad been exposed with monopolar electrosurgery for 7 daysThe results obtained from this study demonstrate that a slightapoptosis occurs in hepatic cells after electrosurgery whichwas as a result of LZ-8 inhibition and provide evidencefor high level of angiogenesis arises during wound healingprocess

8 Evidence-Based Complementary and Alternative Medicine

SS

NF-120581B

NF-120581B

SS + LZ-8

0 3 7 2800

05

10

15

20

25

Relat

ive e

xpre

ssio

n of

NF-120581

B

Treatment duration (day)

SSSS + LZ-8

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowast

lowastlowast

(a)

0 3 7 2800

05

10

15

20

Relat

ive e

xpre

ssio

n of

casp

ase-

3

Treatment duration (day)

SSSS + LZ-8

Caspase-3SS + LZ-8

Caspase-3SS

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowastlowast

lowastlowastlowast

(b)

Figure 6TheWestern blot assay results from rats liver treated with SS group and SS + LZ-8 group after different healing days (a) expressionsof NF-120581B and (b) expressions of caspase-3 Data are expressed as the means plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 comparedwith SS group

4 Discussion

The purpose of this study was to determine the effect ofthe LZ-8 an immunomodulatory protein derived from Glucidum when it is used for wound healing on hepatictissues and to compare this with monopolar electrosurgeryoperated liver tissues on the wounds size fibrosis thermalinjury and apoptosis The results of this study show thatLZ-8 enhances wound healing actions in hepatic tissuesafter electrosurgery Importantly this study has identifiedthat NF-120581B and caspase-3 activation are associated with LZ-8 on enhancing wound healing after surgery Purpose ofelectrical energy or coagulation of bleeding vessels onto tissueis frequently used in surgery Coagulation techniques suchas monopolar and laser techniques are commonly used indifferent surgical disciplines [21 22]

Monopolar and bipolar coagulation effects on brain tissuewere reported and the results of experimental trials were

compared [23 24] They observed less trauma after bipolarcoagulation compared with monopolar technique A studyhas demonstrated that bipolar mode of stimulation claimedto be another paradigm of a potential hazard comparedwith monopolar stimulation Temel et al [25] observed braindamage after bipolar but not monopolar and stimulationin rats However monophasic pulses were used whichmight have created larger lesions in a bipolar configurationMonopolar electrosurgery causes sparking current spreadand thermal damage in the tissues because of excessivegeneration of heat These problems will induce deleteriouseffects to the tissue in proximity to the operative site Inaddition when comparing monopolar electrosurgery andthe scalpel incision electrosurgery produces more tissuealteration and histological thermal damage when a lateralheat was increased by the low frequency radio wave of05 to 29MHz [26] Consequently the resultant effect ofmonopolar systems is excessive tissue damage and delayed

Evidence-Based Complementary and Alternative Medicine 9

Cy 5 DAPI Merge

Control

SS

SS + LZ-8

Day 3

SS

SS + LZ-8

Day 7

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

Figure 7 The immunofluorescence staining results from rats liver treated with SS group and SS + LZ-8 group after different healing days

healing Therefore additional drug supplements or therapyneed to be required to accelerate the wound healing actionon tissues after electrosurgery

A new immunomodulatory protein known as LZ-8 wasisolated from the mycelia of G lucidum and characterizedby chromatographicelectrophoretic techniques [17] Bothin vivo immunomodulation activity and in vitro mitogenicactivity of LZ-8 have been reported by Kino et al [14] Pre-vious studies have also demonstrated that topical applicationof oils improves some parameters of wound healing such asstimulation of connective tissue fiber [27] reduction in thethickness of the necrotic cell layer edge around the wound[28] and acceleration of wound closure [29 30] Due toexcellent thermal and acid stability together with a moderateresistance to alkali and dehydration LZ-8 is appropriate toapply tissues after surgery even with a mild thermal heatIn this study this is the first and novel results that LZ-8accelerates the reduction of the wound size thermal injuryand increase fibrosis area in rat liver after surgery and it

indicates that LZ-8 has potent ability to enhance the effect ofwound healing In a latest study we have also demonstratedthat the total areas of liver injury lateral thermal injury andfibrotic tissues were significantly higher in SS treated hepatictissues than that of CrN-coated needles that had been usedfor surgery [13]

NF-120581B activation plays a role in the regulation of morethan 400 gene product expressions associatedwith inflamma-tion cell survival proliferation invasion and angiogenesis[31 32] NF-120581B normally binds to I120581B120572 which impedes NF-120581B nuclear translocation from the cytoplasm to the nucleusNF-120581B is reported to be a vital link between hepatic injuryfibrosis and even hepatocellular carcinoma [33] NF-120581Benters the nucleus with the degradation of I120581B and activatesthe expression of specific genes that induce the apoptosiscascade [34] Once the cells are exposed to inflammatorystimuli including LPS and TNF-120572 I120581B120572 is phosphorylatedleading to I120581B120572 degradation and nuclear translocation of NF-120581B In our latest study it was noticed that liver tissues that had

10 Evidence-Based Complementary and Alternative Medicine

TUNEL DAPI Merge

SSSS

+ L

Z-8

250 120583m 250 120583m 250 120583m

250 120583m 250 120583m 250 120583m

Figure 8 The TUNEL staining assay results from rats liver treated with SS group and SS + LZ-8 group after 7 days of healing

been operated with SS-needles secreted higher levels of NF-120581B expression and its nuclear translocation than that of CrNoperated tissues [13] Fibrosis is an important component ofadvanced chronic inflammatory liver disease Although thechanges in hepatic structures that occur in liver cirrhosis havebeen described in detail the pathogenesis of fibrosis in duringelectrosurgery process is largely unknown Our latest reportindicates that monopolar elecrtrosurgery-induced hepaticfibrosis after repeated liver injury and the mechanisms aremainly based on the upregulation of NF-120581B and caspase-3expression in the liver tissues [13]

In this study we thus evaluated whether LZ-8 had anyeffect on nuclear translocation of NF-120581B during monopolarelectrosurgery and the results found for the first time thatLZ-8 inhibits nuclear translocation of NF-120581B in hepaticcells after 3 and 7 days of monopolar electrosurgery Yatesand Rayner [35] studied the effects of fatty acid supple-mentation on activation of NF-120581B and AP-1 after a skininjury since these transcription factors are highly associ-ated with wound healing They have also reported a novelfinding which indicates that wounding of normal humankeratinocytes in culture causes NF-120581B translocation Therelevance of NF-120581B activation to wound healing is sug-gested by the results that HeNe laser treatment a processshown to accelerate healing in a model of wounding [36]promoted the translocation of NF-120581B A previous studyalso proved that a ganoderma immunomodulatory proteincloned from Ganoderma microsporum holds anti-invasiveand anti-inflammatory activities in TNF-120572 induced humanalveolar epithelial A549 cells via inhibition of the I120581B120572 andNF-120581B pathway [37]

Apoptosis in wound healing is a promising issue Therehas been some evaluation of chronic diabetic wounds in

mice that suggests that there is increased cellular apoptosislikely due to hyperglycemia which produces reactive oxygenspecies that mediate mitochondrial release of cytochrome cand activate caspase-3 [37 38] Apoptosis is also involved inthe regulation of collagen synthesis and degradation withinwounds through regulation of fibroblast numbers and colla-genase activity [39] Recent work in another mouse modelsuggests that hypertrophic scars demonstrate decreased ratesof apoptosis with decreased expression of cleaved caspase-3Over expression of various caspase family members inducesapoptosis in cultured mammalian cells [40] Among thecysteine proteases caspase-3 is believed to be one of themost commonly involved in the execution of apoptosisin various cell types [41] The assay of TUNEL stainingcan identify the apoptotic cells via DNA fragmentation bylabeling the terminal ends of nucleic acids [42 43] Fromour latest study of TUNEL assay it can be clearly foundthat the apoptosis event was more significant in SS-needleoperated liver tissues [13] In this study TUNEL stainingprovides direct evidence of apoptosis in SS operated livertissues after 7 days Both the activation of caspase-3 and theinduction of apoptotic cell death could be suppressed by theaddition of antioxidants in the cultures [44] It has also beendemonstrated that LZ-8 is able to effectively promote theactivation and maturation of immature dendritic cells via thenuclear factor-kappaB and mitogen-activated protein kinasepathways [18] Moreover the present study observed theeffects of (i) inhibiting expression and nuclear translocationof NF-120581B and (ii) inhibiting expression of caspase-3 andreduction of apoptotic cell death by LZ-8 in liver tissuesafter electrosurgery which indicates that the wound healingproperty of LZ-8 may possibly occur through inhibition oftranscription factor NF-120581B and apoptotic factor caspase-3

Evidence-Based Complementary and Alternative Medicine 11

5 Conclusion

Under the same conditions LZ-8 treatment potentiates onreducing wound size and thermal injury An increasing fibro-sis and reducing apoptosis were found in LZ-8 treated livertissues during electrosurgery LZ-8 treatment significantlyinhibits NF-120581B and caspase-3 expressions and also reducesNF-120581B nuclear translocation and apoptotic cell death Theresults generated from this study suggest that LZ-8 has potenteffects on increasing wound healing action against the elec-trosurgical induced liver injury via the possible mechanismof inhibiting NF-120581B and caspase-3 expressions

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors would like to thank the Department of HealthExecutive Yuan Taiwan (Contract no MOHW103-TDU-N-211-133001) and TaipeiMedical University and TaipeiMedicalHospital (Contract no 100TMU-TMUH-05) for financiallysupporting this research Dr Hao-Jan Lin and Dr Yushan-Sophie Chang are considered the first coauthors of the paper

References

[1] J Liboon W Funkhouser and D J Terris ldquoA comparison ofmucosal incisions made by scalpel CO

2

laser electrocauteryand constant-voltage electrocauteryrdquo OtolaryngologymdashHeadand Neck Surgery vol 116 no 3 pp 379ndash385 1997

[2] J F Carew R F Ward A LaBruna P A Torzilli and W SSchley ldquoEffects of scalpel electrocautery and CO

2

and KTPlasers on wound healing in rat tonguesrdquoThe Laryngoscope vol108 no 3 pp 373ndash380 1998

[3] J A Pearce Electrosurgery John Wiley amp Sons New York NYUSA 1986

[4] W L Maness F W Rober R E Clark E Cataldo andA W Haddad ldquoTissue damage from electrosurgical poweroutput variations in hamster tonguesrdquoThe Journal of ProstheticDentistry vol 42 no 4 pp 456ndash460 1979

[5] W L Maness F W Roeber R E Clark E Cataldo D Riisand A W Haddad ldquoHistologic evaluation of electrosurgerywith varying frequency andwaveformrdquoThe Journal of ProstheticDentistry vol 40 no 3 pp 304ndash308 1978

[6] J Friedman J Margolin and S Piliero ldquoA preliminary study ofthe histological effects of three different types of electrosurgicalcurrentsrdquoThe New York State Dental Journal vol 40 no 6 pp349ndash353 1974

[7] W H Noble K D McClatchey and G D Douglass ldquoAhistologic comparison of effects of electrosurgical resectionusing different electrodesrdquo The Journal of Prosthetic Dentistryvol 35 no 5 pp 575ndash579 1976

[8] K C Nixon K F Adkins and D W Keys ldquoHistologicalevaluation of effects produced in alveolar bone followinggingival incision with an electrosurgical scalpelrdquo Journal ofPeriodontology vol 46 no 1 pp 40ndash44 1975

[9] I Glickman and L R Imber ldquoComparison of gingival resec-tion with electrosurgery and periodontal knivesmdasha biometric

and histologic studyrdquo Journal of Periodontology vol 41 no 3pp 142ndash148 1970

[10] D H Coelho J Cavallaro and E A Rothschild ldquoGingivalrecession with electrosurgery for impression makingrdquo TheJournal of Prosthetic Dentistry vol 33 no 4 pp 422ndash426 1975

[11] P B Robertson B Luscher L S Spangberg and B M LevyldquoPulpal and periodontal effects of electrosurgery involvingcervical metallic restorationsrdquo Oral Surgery Oral Medicine andOral Pathology vol 46 no 5 pp 702ndash710 1978

[12] R DrsquoSouza ldquoPulpal and periapical immune response to electro-surgical contact of cervical metallic restorations in monkeysrdquoQuintessence International vol 17 no 12 pp 803ndash808 1986

[13] K-L Ou J-S Chu H Hosseinkhani J-F Chiou and C-HYu ldquoBiomedical nanostructured coating for minimally invasivesurgery devices applications characterization cell cytotoxicityevaluation and an animal study in ratrdquo Surgical Endoscopy 2014

[14] K Kino A Yamashita K Yamaoka et al ldquoIsolation and char-acterization of a new immunomodulatory protein Ling Zhi-8(LZ-8) from Ganoderma lucidiumrdquo The Journal of BiologicalChemistry vol 264 no 1 pp 472ndash478 1989

[15] AMurasugi S Tanaka N Komiyama et al ldquoMolecular cloningof a cDNAand a gene encoding an immunomodulatory proteinLing Zhi-8 from a fungusGanoderma lucidiumrdquoThe Journal ofBiological Chemistry vol 266 no 4 pp 2486ndash2493 1991

[16] M-H Tong P-J Chien H-H Chang M-J Tsai and F SheuldquoHigh processing tolerances of immunomodulatory proteins inEnoki and Reishi mushroomsrdquo Journal of Agricultural and FoodChemistry vol 56 no 9 pp 3160ndash3166 2008

[17] L Huang F Sun C Liang et al ldquoCrystal structure of LZ-8 fromthemedicinal fungusGanoderma lucidiumrdquo Proteins StructureFunction and Bioinformatics vol 75 no 2 pp 524ndash527 2009

[18] Y-L Lin Y-C Liang Y-S Tseng et al ldquoAn immunomodulatoryprotein Ling Zhi-8 induced activation and maturation ofhuman monocyte-derived dendritic cells by the NF-120581B andMAPK pathwaysrdquo Journal of Leukocyte Biology vol 86 no 4pp 877ndash889 2009

[19] R A D Drury and E A Wallington Carletonrsquos HistologicalTechnique 1981

[20] Y Du D-W Guan and R Zhao ldquoAn immunohistochemicalstudy on the expression of caspase-3 in the skin wound healingrdquoFa Yi Xue Za Zhi vol 20 no 1 pp 1ndash3 2004

[21] K E Grund T Straub and G Farin ldquoNew haemostatictechniques argon plasma coagulationrdquo Baillierersquos Best Practiceand Research in Clinical Gastroenterology vol 13 no 1 pp 67ndash84 1999

[22] D Hellwig R Haag V Bartel et al ldquoApplication of newelectrosurgical devices and probes in endoscopic neurosurgeryrdquoNeurological Research vol 21 no 1 pp 67ndash72 1999

[23] B Chehrazi and W F Collins Jr ldquoA comparison of effects ofbipolar and monopolar electrocoagulation in brainrdquo Journal ofNeurosurgery vol 54 no 2 pp 197ndash203 1981

[24] J Kerl and J Staubesand ldquoAn ultrastructural report on thechoice between monopolar and bipolar electrocoagulationrdquoActa Neurochirurgica vol 92 no 1ndash4 pp 123ndash131 1988

[25] Y Temel V Visser-Vandewalle M van derWolf et al ldquoMonop-olar versus bipolar high frequency stimulation in the rat subtha-lamic nucleus differences in histological damagerdquoNeuroscienceLetters vol 367 no 1 pp 92ndash96 2004

[26] L I Malis ldquoNew trends in microsurgery and applied technol-ogyrdquo in Advanced Technology in Neurosurgery F Pluchino andG Broggi Eds pp 1ndash16 Springer Berlin Germany 1988

12 Evidence-Based Complementary and Alternative Medicine

[27] C R B Cardoso M A Souza E A V Ferro S Favoreto Jrand J D O Pena ldquoInfluence of topical administration of n-3and n-6 essential and n-9 nonessential fatty acids on the healingof cutaneous woundsrdquoWound Repair and Regeneration vol 12no 2 pp 235ndash243 2004

[28] L M Pereira E Hatanaka E F Martins et al ldquoEffect of oleicand linoleic acids on the inflammatory phase of wound healingin ratsrdquo Cell Biochemistry and Function vol 26 no 2 pp 197ndash204 2008

[29] N-Y ParkGValacchi andY Lim ldquoEffect of dietary conjugatedlinoleic acid supplementation on early inflammatory responsesduring cutaneous wound healingrdquo Mediators of Inflammationvol 2010 Article ID 342328 8 pages 2010

[30] M Otranto A P do Nascimento and A Monte-Alto-CostaldquoEffects of supplementation with different edible oils on cuta-neous wound healingrdquoWound Repair and Regeneration vol 18no 6 pp 629ndash636 2010

[31] X Dolcet D Llobet J Pallares and X Matias-Guiu ldquoNF-kBin development and progression of human cancerrdquo VirchowsArchiv vol 446 no 5 pp 475ndash482 2005

[32] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[33] T Luedde and R F Schwabe ldquoNF-120581B in the livermdashlinkinginjury fibrosis and hepatocellular carcinomardquo Nature ReviewsGastroenterology amp Hepatology vol 8 no 2 pp 108ndash118 2011

[34] J A Schmid and A Birbach ldquoIkappaB kinase beta (IKKbetaIKK2IKBKB)mdasha keymolecule in signaling to the transcriptionfactor NF-kappaBrdquo Cytokine amp Growth Factor Reviews vol 19no 2 pp 157ndash165 2008

[35] S Yates and T E Rayner ldquoTranscription factor activation inresponse to cutaneous injury role of AP-1 in reepithelializa-tionrdquo Wound Repair and Regeneration vol 10 no 1 pp 5ndash152002

[36] A F Haas R R Isseroff R G Wheeland P A Rood and PJ Graves ldquoLow-energy helium-neon laser irradiation increasesthe motility of cultured human keratinocytesrdquo The Journal ofInvestigative Dermatology vol 94 no 6 pp 822ndash826 1990

[37] C-H Lin Y-M Hsiao C-C Ou et al ldquoGMI a Ganodermaimmunomodulatory protein down-regulates tumor necrosisfactor 120572-induced expression of matrix metalloproteinase 9 viaNF-120581Bpathway in human alveolar epithelial A549 cellsrdquo Journalof agricultural and food chemistry vol 58 no 22 pp 12014ndash12021 2010

[38] L Cai W Li G Wang L Guo Y Jiang and Y J KangldquoHyperglycemia-induced apoptosis in mouse myocardiummitochondrial cytochrome c-mediated caspase-3 activationpathwayrdquo Diabetes vol 51 no 6 pp 1938ndash1948 2002

[39] N K Rai K Tripathi D Sharma andV K Shukla ldquoApoptosis abasic physiologic process in wound healingrdquo The InternationalJournal of Lower Extremity Wounds vol 4 no 3 pp 138ndash1442005

[40] J Bian and Y Sun ldquoTranscriptional activation by p53 of thehuman type IV collagenase (gelatinase A or matrix metallopro-teinase 2) promoterrdquoMolecular and Cellular Biology vol 17 no11 pp 6330ndash6338 1997

[41] S Wang MMiura Y-K Jung et al ldquoIdentification and charac-terization of ich-3 a member of the interleukin-1120573 convertingenzyme (ICE)Ced-3 family and an upstream regulator of ICErdquoThe Journal of Biological Chemistry vol 271 no 34 pp 20580ndash20587 1996

[42] E S Alnemri D J Livingston D W Nicholson et al ldquoHumanICECED-3 protease nomenclaturerdquo Cell vol 87 no 2 article171 1996

[43] T D Hewitson T Bisucci and I A Darby ldquoHistochemicallocalization of apoptosis with in situ labeling of fragmentedDNArdquo Methods in Molecular Biology vol 326 pp 227ndash2342006

[44] D T Loo ldquoIn situ detection of apoptosis by the TUNEL assayan overview of techniquesrdquo in DNA Damage Detection In SituEx Vivo and In Vivo vol 682 ofMethods in Molecular Biologypp 3ndash13 Humana Press New Jersey NJ USA 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 8: Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014 › 916531.pdfResearch Article An Immunomodulatory Protein (Ling Zhi-8) from a

8 Evidence-Based Complementary and Alternative Medicine

SS

NF-120581B

NF-120581B

SS + LZ-8

0 3 7 2800

05

10

15

20

25

Relat

ive e

xpre

ssio

n of

NF-120581

B

Treatment duration (day)

SSSS + LZ-8

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowast

lowastlowast

(a)

0 3 7 2800

05

10

15

20

Relat

ive e

xpre

ssio

n of

casp

ase-

3

Treatment duration (day)

SSSS + LZ-8

Caspase-3SS + LZ-8

Caspase-3SS

0 day 3rd day 7th day 28th day

120573-Actin

120573-Actin

lowast

lowastlowast

lowastlowastlowast

(b)

Figure 6TheWestern blot assay results from rats liver treated with SS group and SS + LZ-8 group after different healing days (a) expressionsof NF-120581B and (b) expressions of caspase-3 Data are expressed as the means plusmn SEM (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 comparedwith SS group

4 Discussion

The purpose of this study was to determine the effect ofthe LZ-8 an immunomodulatory protein derived from Glucidum when it is used for wound healing on hepatictissues and to compare this with monopolar electrosurgeryoperated liver tissues on the wounds size fibrosis thermalinjury and apoptosis The results of this study show thatLZ-8 enhances wound healing actions in hepatic tissuesafter electrosurgery Importantly this study has identifiedthat NF-120581B and caspase-3 activation are associated with LZ-8 on enhancing wound healing after surgery Purpose ofelectrical energy or coagulation of bleeding vessels onto tissueis frequently used in surgery Coagulation techniques suchas monopolar and laser techniques are commonly used indifferent surgical disciplines [21 22]

Monopolar and bipolar coagulation effects on brain tissuewere reported and the results of experimental trials were

compared [23 24] They observed less trauma after bipolarcoagulation compared with monopolar technique A studyhas demonstrated that bipolar mode of stimulation claimedto be another paradigm of a potential hazard comparedwith monopolar stimulation Temel et al [25] observed braindamage after bipolar but not monopolar and stimulationin rats However monophasic pulses were used whichmight have created larger lesions in a bipolar configurationMonopolar electrosurgery causes sparking current spreadand thermal damage in the tissues because of excessivegeneration of heat These problems will induce deleteriouseffects to the tissue in proximity to the operative site Inaddition when comparing monopolar electrosurgery andthe scalpel incision electrosurgery produces more tissuealteration and histological thermal damage when a lateralheat was increased by the low frequency radio wave of05 to 29MHz [26] Consequently the resultant effect ofmonopolar systems is excessive tissue damage and delayed

Evidence-Based Complementary and Alternative Medicine 9

Cy 5 DAPI Merge

Control

SS

SS + LZ-8

Day 3

SS

SS + LZ-8

Day 7

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

Figure 7 The immunofluorescence staining results from rats liver treated with SS group and SS + LZ-8 group after different healing days

healing Therefore additional drug supplements or therapyneed to be required to accelerate the wound healing actionon tissues after electrosurgery

A new immunomodulatory protein known as LZ-8 wasisolated from the mycelia of G lucidum and characterizedby chromatographicelectrophoretic techniques [17] Bothin vivo immunomodulation activity and in vitro mitogenicactivity of LZ-8 have been reported by Kino et al [14] Pre-vious studies have also demonstrated that topical applicationof oils improves some parameters of wound healing such asstimulation of connective tissue fiber [27] reduction in thethickness of the necrotic cell layer edge around the wound[28] and acceleration of wound closure [29 30] Due toexcellent thermal and acid stability together with a moderateresistance to alkali and dehydration LZ-8 is appropriate toapply tissues after surgery even with a mild thermal heatIn this study this is the first and novel results that LZ-8accelerates the reduction of the wound size thermal injuryand increase fibrosis area in rat liver after surgery and it

indicates that LZ-8 has potent ability to enhance the effect ofwound healing In a latest study we have also demonstratedthat the total areas of liver injury lateral thermal injury andfibrotic tissues were significantly higher in SS treated hepatictissues than that of CrN-coated needles that had been usedfor surgery [13]

NF-120581B activation plays a role in the regulation of morethan 400 gene product expressions associatedwith inflamma-tion cell survival proliferation invasion and angiogenesis[31 32] NF-120581B normally binds to I120581B120572 which impedes NF-120581B nuclear translocation from the cytoplasm to the nucleusNF-120581B is reported to be a vital link between hepatic injuryfibrosis and even hepatocellular carcinoma [33] NF-120581Benters the nucleus with the degradation of I120581B and activatesthe expression of specific genes that induce the apoptosiscascade [34] Once the cells are exposed to inflammatorystimuli including LPS and TNF-120572 I120581B120572 is phosphorylatedleading to I120581B120572 degradation and nuclear translocation of NF-120581B In our latest study it was noticed that liver tissues that had

10 Evidence-Based Complementary and Alternative Medicine

TUNEL DAPI Merge

SSSS

+ L

Z-8

250 120583m 250 120583m 250 120583m

250 120583m 250 120583m 250 120583m

Figure 8 The TUNEL staining assay results from rats liver treated with SS group and SS + LZ-8 group after 7 days of healing

been operated with SS-needles secreted higher levels of NF-120581B expression and its nuclear translocation than that of CrNoperated tissues [13] Fibrosis is an important component ofadvanced chronic inflammatory liver disease Although thechanges in hepatic structures that occur in liver cirrhosis havebeen described in detail the pathogenesis of fibrosis in duringelectrosurgery process is largely unknown Our latest reportindicates that monopolar elecrtrosurgery-induced hepaticfibrosis after repeated liver injury and the mechanisms aremainly based on the upregulation of NF-120581B and caspase-3expression in the liver tissues [13]

In this study we thus evaluated whether LZ-8 had anyeffect on nuclear translocation of NF-120581B during monopolarelectrosurgery and the results found for the first time thatLZ-8 inhibits nuclear translocation of NF-120581B in hepaticcells after 3 and 7 days of monopolar electrosurgery Yatesand Rayner [35] studied the effects of fatty acid supple-mentation on activation of NF-120581B and AP-1 after a skininjury since these transcription factors are highly associ-ated with wound healing They have also reported a novelfinding which indicates that wounding of normal humankeratinocytes in culture causes NF-120581B translocation Therelevance of NF-120581B activation to wound healing is sug-gested by the results that HeNe laser treatment a processshown to accelerate healing in a model of wounding [36]promoted the translocation of NF-120581B A previous studyalso proved that a ganoderma immunomodulatory proteincloned from Ganoderma microsporum holds anti-invasiveand anti-inflammatory activities in TNF-120572 induced humanalveolar epithelial A549 cells via inhibition of the I120581B120572 andNF-120581B pathway [37]

Apoptosis in wound healing is a promising issue Therehas been some evaluation of chronic diabetic wounds in

mice that suggests that there is increased cellular apoptosislikely due to hyperglycemia which produces reactive oxygenspecies that mediate mitochondrial release of cytochrome cand activate caspase-3 [37 38] Apoptosis is also involved inthe regulation of collagen synthesis and degradation withinwounds through regulation of fibroblast numbers and colla-genase activity [39] Recent work in another mouse modelsuggests that hypertrophic scars demonstrate decreased ratesof apoptosis with decreased expression of cleaved caspase-3Over expression of various caspase family members inducesapoptosis in cultured mammalian cells [40] Among thecysteine proteases caspase-3 is believed to be one of themost commonly involved in the execution of apoptosisin various cell types [41] The assay of TUNEL stainingcan identify the apoptotic cells via DNA fragmentation bylabeling the terminal ends of nucleic acids [42 43] Fromour latest study of TUNEL assay it can be clearly foundthat the apoptosis event was more significant in SS-needleoperated liver tissues [13] In this study TUNEL stainingprovides direct evidence of apoptosis in SS operated livertissues after 7 days Both the activation of caspase-3 and theinduction of apoptotic cell death could be suppressed by theaddition of antioxidants in the cultures [44] It has also beendemonstrated that LZ-8 is able to effectively promote theactivation and maturation of immature dendritic cells via thenuclear factor-kappaB and mitogen-activated protein kinasepathways [18] Moreover the present study observed theeffects of (i) inhibiting expression and nuclear translocationof NF-120581B and (ii) inhibiting expression of caspase-3 andreduction of apoptotic cell death by LZ-8 in liver tissuesafter electrosurgery which indicates that the wound healingproperty of LZ-8 may possibly occur through inhibition oftranscription factor NF-120581B and apoptotic factor caspase-3

Evidence-Based Complementary and Alternative Medicine 11

5 Conclusion

Under the same conditions LZ-8 treatment potentiates onreducing wound size and thermal injury An increasing fibro-sis and reducing apoptosis were found in LZ-8 treated livertissues during electrosurgery LZ-8 treatment significantlyinhibits NF-120581B and caspase-3 expressions and also reducesNF-120581B nuclear translocation and apoptotic cell death Theresults generated from this study suggest that LZ-8 has potenteffects on increasing wound healing action against the elec-trosurgical induced liver injury via the possible mechanismof inhibiting NF-120581B and caspase-3 expressions

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors would like to thank the Department of HealthExecutive Yuan Taiwan (Contract no MOHW103-TDU-N-211-133001) and TaipeiMedical University and TaipeiMedicalHospital (Contract no 100TMU-TMUH-05) for financiallysupporting this research Dr Hao-Jan Lin and Dr Yushan-Sophie Chang are considered the first coauthors of the paper

References

[1] J Liboon W Funkhouser and D J Terris ldquoA comparison ofmucosal incisions made by scalpel CO

2

laser electrocauteryand constant-voltage electrocauteryrdquo OtolaryngologymdashHeadand Neck Surgery vol 116 no 3 pp 379ndash385 1997

[2] J F Carew R F Ward A LaBruna P A Torzilli and W SSchley ldquoEffects of scalpel electrocautery and CO

2

and KTPlasers on wound healing in rat tonguesrdquoThe Laryngoscope vol108 no 3 pp 373ndash380 1998

[3] J A Pearce Electrosurgery John Wiley amp Sons New York NYUSA 1986

[4] W L Maness F W Rober R E Clark E Cataldo andA W Haddad ldquoTissue damage from electrosurgical poweroutput variations in hamster tonguesrdquoThe Journal of ProstheticDentistry vol 42 no 4 pp 456ndash460 1979

[5] W L Maness F W Roeber R E Clark E Cataldo D Riisand A W Haddad ldquoHistologic evaluation of electrosurgerywith varying frequency andwaveformrdquoThe Journal of ProstheticDentistry vol 40 no 3 pp 304ndash308 1978

[6] J Friedman J Margolin and S Piliero ldquoA preliminary study ofthe histological effects of three different types of electrosurgicalcurrentsrdquoThe New York State Dental Journal vol 40 no 6 pp349ndash353 1974

[7] W H Noble K D McClatchey and G D Douglass ldquoAhistologic comparison of effects of electrosurgical resectionusing different electrodesrdquo The Journal of Prosthetic Dentistryvol 35 no 5 pp 575ndash579 1976

[8] K C Nixon K F Adkins and D W Keys ldquoHistologicalevaluation of effects produced in alveolar bone followinggingival incision with an electrosurgical scalpelrdquo Journal ofPeriodontology vol 46 no 1 pp 40ndash44 1975

[9] I Glickman and L R Imber ldquoComparison of gingival resec-tion with electrosurgery and periodontal knivesmdasha biometric

and histologic studyrdquo Journal of Periodontology vol 41 no 3pp 142ndash148 1970

[10] D H Coelho J Cavallaro and E A Rothschild ldquoGingivalrecession with electrosurgery for impression makingrdquo TheJournal of Prosthetic Dentistry vol 33 no 4 pp 422ndash426 1975

[11] P B Robertson B Luscher L S Spangberg and B M LevyldquoPulpal and periodontal effects of electrosurgery involvingcervical metallic restorationsrdquo Oral Surgery Oral Medicine andOral Pathology vol 46 no 5 pp 702ndash710 1978

[12] R DrsquoSouza ldquoPulpal and periapical immune response to electro-surgical contact of cervical metallic restorations in monkeysrdquoQuintessence International vol 17 no 12 pp 803ndash808 1986

[13] K-L Ou J-S Chu H Hosseinkhani J-F Chiou and C-HYu ldquoBiomedical nanostructured coating for minimally invasivesurgery devices applications characterization cell cytotoxicityevaluation and an animal study in ratrdquo Surgical Endoscopy 2014

[14] K Kino A Yamashita K Yamaoka et al ldquoIsolation and char-acterization of a new immunomodulatory protein Ling Zhi-8(LZ-8) from Ganoderma lucidiumrdquo The Journal of BiologicalChemistry vol 264 no 1 pp 472ndash478 1989

[15] AMurasugi S Tanaka N Komiyama et al ldquoMolecular cloningof a cDNAand a gene encoding an immunomodulatory proteinLing Zhi-8 from a fungusGanoderma lucidiumrdquoThe Journal ofBiological Chemistry vol 266 no 4 pp 2486ndash2493 1991

[16] M-H Tong P-J Chien H-H Chang M-J Tsai and F SheuldquoHigh processing tolerances of immunomodulatory proteins inEnoki and Reishi mushroomsrdquo Journal of Agricultural and FoodChemistry vol 56 no 9 pp 3160ndash3166 2008

[17] L Huang F Sun C Liang et al ldquoCrystal structure of LZ-8 fromthemedicinal fungusGanoderma lucidiumrdquo Proteins StructureFunction and Bioinformatics vol 75 no 2 pp 524ndash527 2009

[18] Y-L Lin Y-C Liang Y-S Tseng et al ldquoAn immunomodulatoryprotein Ling Zhi-8 induced activation and maturation ofhuman monocyte-derived dendritic cells by the NF-120581B andMAPK pathwaysrdquo Journal of Leukocyte Biology vol 86 no 4pp 877ndash889 2009

[19] R A D Drury and E A Wallington Carletonrsquos HistologicalTechnique 1981

[20] Y Du D-W Guan and R Zhao ldquoAn immunohistochemicalstudy on the expression of caspase-3 in the skin wound healingrdquoFa Yi Xue Za Zhi vol 20 no 1 pp 1ndash3 2004

[21] K E Grund T Straub and G Farin ldquoNew haemostatictechniques argon plasma coagulationrdquo Baillierersquos Best Practiceand Research in Clinical Gastroenterology vol 13 no 1 pp 67ndash84 1999

[22] D Hellwig R Haag V Bartel et al ldquoApplication of newelectrosurgical devices and probes in endoscopic neurosurgeryrdquoNeurological Research vol 21 no 1 pp 67ndash72 1999

[23] B Chehrazi and W F Collins Jr ldquoA comparison of effects ofbipolar and monopolar electrocoagulation in brainrdquo Journal ofNeurosurgery vol 54 no 2 pp 197ndash203 1981

[24] J Kerl and J Staubesand ldquoAn ultrastructural report on thechoice between monopolar and bipolar electrocoagulationrdquoActa Neurochirurgica vol 92 no 1ndash4 pp 123ndash131 1988

[25] Y Temel V Visser-Vandewalle M van derWolf et al ldquoMonop-olar versus bipolar high frequency stimulation in the rat subtha-lamic nucleus differences in histological damagerdquoNeuroscienceLetters vol 367 no 1 pp 92ndash96 2004

[26] L I Malis ldquoNew trends in microsurgery and applied technol-ogyrdquo in Advanced Technology in Neurosurgery F Pluchino andG Broggi Eds pp 1ndash16 Springer Berlin Germany 1988

12 Evidence-Based Complementary and Alternative Medicine

[27] C R B Cardoso M A Souza E A V Ferro S Favoreto Jrand J D O Pena ldquoInfluence of topical administration of n-3and n-6 essential and n-9 nonessential fatty acids on the healingof cutaneous woundsrdquoWound Repair and Regeneration vol 12no 2 pp 235ndash243 2004

[28] L M Pereira E Hatanaka E F Martins et al ldquoEffect of oleicand linoleic acids on the inflammatory phase of wound healingin ratsrdquo Cell Biochemistry and Function vol 26 no 2 pp 197ndash204 2008

[29] N-Y ParkGValacchi andY Lim ldquoEffect of dietary conjugatedlinoleic acid supplementation on early inflammatory responsesduring cutaneous wound healingrdquo Mediators of Inflammationvol 2010 Article ID 342328 8 pages 2010

[30] M Otranto A P do Nascimento and A Monte-Alto-CostaldquoEffects of supplementation with different edible oils on cuta-neous wound healingrdquoWound Repair and Regeneration vol 18no 6 pp 629ndash636 2010

[31] X Dolcet D Llobet J Pallares and X Matias-Guiu ldquoNF-kBin development and progression of human cancerrdquo VirchowsArchiv vol 446 no 5 pp 475ndash482 2005

[32] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[33] T Luedde and R F Schwabe ldquoNF-120581B in the livermdashlinkinginjury fibrosis and hepatocellular carcinomardquo Nature ReviewsGastroenterology amp Hepatology vol 8 no 2 pp 108ndash118 2011

[34] J A Schmid and A Birbach ldquoIkappaB kinase beta (IKKbetaIKK2IKBKB)mdasha keymolecule in signaling to the transcriptionfactor NF-kappaBrdquo Cytokine amp Growth Factor Reviews vol 19no 2 pp 157ndash165 2008

[35] S Yates and T E Rayner ldquoTranscription factor activation inresponse to cutaneous injury role of AP-1 in reepithelializa-tionrdquo Wound Repair and Regeneration vol 10 no 1 pp 5ndash152002

[36] A F Haas R R Isseroff R G Wheeland P A Rood and PJ Graves ldquoLow-energy helium-neon laser irradiation increasesthe motility of cultured human keratinocytesrdquo The Journal ofInvestigative Dermatology vol 94 no 6 pp 822ndash826 1990

[37] C-H Lin Y-M Hsiao C-C Ou et al ldquoGMI a Ganodermaimmunomodulatory protein down-regulates tumor necrosisfactor 120572-induced expression of matrix metalloproteinase 9 viaNF-120581Bpathway in human alveolar epithelial A549 cellsrdquo Journalof agricultural and food chemistry vol 58 no 22 pp 12014ndash12021 2010

[38] L Cai W Li G Wang L Guo Y Jiang and Y J KangldquoHyperglycemia-induced apoptosis in mouse myocardiummitochondrial cytochrome c-mediated caspase-3 activationpathwayrdquo Diabetes vol 51 no 6 pp 1938ndash1948 2002

[39] N K Rai K Tripathi D Sharma andV K Shukla ldquoApoptosis abasic physiologic process in wound healingrdquo The InternationalJournal of Lower Extremity Wounds vol 4 no 3 pp 138ndash1442005

[40] J Bian and Y Sun ldquoTranscriptional activation by p53 of thehuman type IV collagenase (gelatinase A or matrix metallopro-teinase 2) promoterrdquoMolecular and Cellular Biology vol 17 no11 pp 6330ndash6338 1997

[41] S Wang MMiura Y-K Jung et al ldquoIdentification and charac-terization of ich-3 a member of the interleukin-1120573 convertingenzyme (ICE)Ced-3 family and an upstream regulator of ICErdquoThe Journal of Biological Chemistry vol 271 no 34 pp 20580ndash20587 1996

[42] E S Alnemri D J Livingston D W Nicholson et al ldquoHumanICECED-3 protease nomenclaturerdquo Cell vol 87 no 2 article171 1996

[43] T D Hewitson T Bisucci and I A Darby ldquoHistochemicallocalization of apoptosis with in situ labeling of fragmentedDNArdquo Methods in Molecular Biology vol 326 pp 227ndash2342006

[44] D T Loo ldquoIn situ detection of apoptosis by the TUNEL assayan overview of techniquesrdquo in DNA Damage Detection In SituEx Vivo and In Vivo vol 682 ofMethods in Molecular Biologypp 3ndash13 Humana Press New Jersey NJ USA 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 9: Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014 › 916531.pdfResearch Article An Immunomodulatory Protein (Ling Zhi-8) from a

Evidence-Based Complementary and Alternative Medicine 9

Cy 5 DAPI Merge

Control

SS

SS + LZ-8

Day 3

SS

SS + LZ-8

Day 7

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

250 120583m

Figure 7 The immunofluorescence staining results from rats liver treated with SS group and SS + LZ-8 group after different healing days

healing Therefore additional drug supplements or therapyneed to be required to accelerate the wound healing actionon tissues after electrosurgery

A new immunomodulatory protein known as LZ-8 wasisolated from the mycelia of G lucidum and characterizedby chromatographicelectrophoretic techniques [17] Bothin vivo immunomodulation activity and in vitro mitogenicactivity of LZ-8 have been reported by Kino et al [14] Pre-vious studies have also demonstrated that topical applicationof oils improves some parameters of wound healing such asstimulation of connective tissue fiber [27] reduction in thethickness of the necrotic cell layer edge around the wound[28] and acceleration of wound closure [29 30] Due toexcellent thermal and acid stability together with a moderateresistance to alkali and dehydration LZ-8 is appropriate toapply tissues after surgery even with a mild thermal heatIn this study this is the first and novel results that LZ-8accelerates the reduction of the wound size thermal injuryand increase fibrosis area in rat liver after surgery and it

indicates that LZ-8 has potent ability to enhance the effect ofwound healing In a latest study we have also demonstratedthat the total areas of liver injury lateral thermal injury andfibrotic tissues were significantly higher in SS treated hepatictissues than that of CrN-coated needles that had been usedfor surgery [13]

NF-120581B activation plays a role in the regulation of morethan 400 gene product expressions associatedwith inflamma-tion cell survival proliferation invasion and angiogenesis[31 32] NF-120581B normally binds to I120581B120572 which impedes NF-120581B nuclear translocation from the cytoplasm to the nucleusNF-120581B is reported to be a vital link between hepatic injuryfibrosis and even hepatocellular carcinoma [33] NF-120581Benters the nucleus with the degradation of I120581B and activatesthe expression of specific genes that induce the apoptosiscascade [34] Once the cells are exposed to inflammatorystimuli including LPS and TNF-120572 I120581B120572 is phosphorylatedleading to I120581B120572 degradation and nuclear translocation of NF-120581B In our latest study it was noticed that liver tissues that had

10 Evidence-Based Complementary and Alternative Medicine

TUNEL DAPI Merge

SSSS

+ L

Z-8

250 120583m 250 120583m 250 120583m

250 120583m 250 120583m 250 120583m

Figure 8 The TUNEL staining assay results from rats liver treated with SS group and SS + LZ-8 group after 7 days of healing

been operated with SS-needles secreted higher levels of NF-120581B expression and its nuclear translocation than that of CrNoperated tissues [13] Fibrosis is an important component ofadvanced chronic inflammatory liver disease Although thechanges in hepatic structures that occur in liver cirrhosis havebeen described in detail the pathogenesis of fibrosis in duringelectrosurgery process is largely unknown Our latest reportindicates that monopolar elecrtrosurgery-induced hepaticfibrosis after repeated liver injury and the mechanisms aremainly based on the upregulation of NF-120581B and caspase-3expression in the liver tissues [13]

In this study we thus evaluated whether LZ-8 had anyeffect on nuclear translocation of NF-120581B during monopolarelectrosurgery and the results found for the first time thatLZ-8 inhibits nuclear translocation of NF-120581B in hepaticcells after 3 and 7 days of monopolar electrosurgery Yatesand Rayner [35] studied the effects of fatty acid supple-mentation on activation of NF-120581B and AP-1 after a skininjury since these transcription factors are highly associ-ated with wound healing They have also reported a novelfinding which indicates that wounding of normal humankeratinocytes in culture causes NF-120581B translocation Therelevance of NF-120581B activation to wound healing is sug-gested by the results that HeNe laser treatment a processshown to accelerate healing in a model of wounding [36]promoted the translocation of NF-120581B A previous studyalso proved that a ganoderma immunomodulatory proteincloned from Ganoderma microsporum holds anti-invasiveand anti-inflammatory activities in TNF-120572 induced humanalveolar epithelial A549 cells via inhibition of the I120581B120572 andNF-120581B pathway [37]

Apoptosis in wound healing is a promising issue Therehas been some evaluation of chronic diabetic wounds in

mice that suggests that there is increased cellular apoptosislikely due to hyperglycemia which produces reactive oxygenspecies that mediate mitochondrial release of cytochrome cand activate caspase-3 [37 38] Apoptosis is also involved inthe regulation of collagen synthesis and degradation withinwounds through regulation of fibroblast numbers and colla-genase activity [39] Recent work in another mouse modelsuggests that hypertrophic scars demonstrate decreased ratesof apoptosis with decreased expression of cleaved caspase-3Over expression of various caspase family members inducesapoptosis in cultured mammalian cells [40] Among thecysteine proteases caspase-3 is believed to be one of themost commonly involved in the execution of apoptosisin various cell types [41] The assay of TUNEL stainingcan identify the apoptotic cells via DNA fragmentation bylabeling the terminal ends of nucleic acids [42 43] Fromour latest study of TUNEL assay it can be clearly foundthat the apoptosis event was more significant in SS-needleoperated liver tissues [13] In this study TUNEL stainingprovides direct evidence of apoptosis in SS operated livertissues after 7 days Both the activation of caspase-3 and theinduction of apoptotic cell death could be suppressed by theaddition of antioxidants in the cultures [44] It has also beendemonstrated that LZ-8 is able to effectively promote theactivation and maturation of immature dendritic cells via thenuclear factor-kappaB and mitogen-activated protein kinasepathways [18] Moreover the present study observed theeffects of (i) inhibiting expression and nuclear translocationof NF-120581B and (ii) inhibiting expression of caspase-3 andreduction of apoptotic cell death by LZ-8 in liver tissuesafter electrosurgery which indicates that the wound healingproperty of LZ-8 may possibly occur through inhibition oftranscription factor NF-120581B and apoptotic factor caspase-3

Evidence-Based Complementary and Alternative Medicine 11

5 Conclusion

Under the same conditions LZ-8 treatment potentiates onreducing wound size and thermal injury An increasing fibro-sis and reducing apoptosis were found in LZ-8 treated livertissues during electrosurgery LZ-8 treatment significantlyinhibits NF-120581B and caspase-3 expressions and also reducesNF-120581B nuclear translocation and apoptotic cell death Theresults generated from this study suggest that LZ-8 has potenteffects on increasing wound healing action against the elec-trosurgical induced liver injury via the possible mechanismof inhibiting NF-120581B and caspase-3 expressions

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors would like to thank the Department of HealthExecutive Yuan Taiwan (Contract no MOHW103-TDU-N-211-133001) and TaipeiMedical University and TaipeiMedicalHospital (Contract no 100TMU-TMUH-05) for financiallysupporting this research Dr Hao-Jan Lin and Dr Yushan-Sophie Chang are considered the first coauthors of the paper

References

[1] J Liboon W Funkhouser and D J Terris ldquoA comparison ofmucosal incisions made by scalpel CO

2

laser electrocauteryand constant-voltage electrocauteryrdquo OtolaryngologymdashHeadand Neck Surgery vol 116 no 3 pp 379ndash385 1997

[2] J F Carew R F Ward A LaBruna P A Torzilli and W SSchley ldquoEffects of scalpel electrocautery and CO

2

and KTPlasers on wound healing in rat tonguesrdquoThe Laryngoscope vol108 no 3 pp 373ndash380 1998

[3] J A Pearce Electrosurgery John Wiley amp Sons New York NYUSA 1986

[4] W L Maness F W Rober R E Clark E Cataldo andA W Haddad ldquoTissue damage from electrosurgical poweroutput variations in hamster tonguesrdquoThe Journal of ProstheticDentistry vol 42 no 4 pp 456ndash460 1979

[5] W L Maness F W Roeber R E Clark E Cataldo D Riisand A W Haddad ldquoHistologic evaluation of electrosurgerywith varying frequency andwaveformrdquoThe Journal of ProstheticDentistry vol 40 no 3 pp 304ndash308 1978

[6] J Friedman J Margolin and S Piliero ldquoA preliminary study ofthe histological effects of three different types of electrosurgicalcurrentsrdquoThe New York State Dental Journal vol 40 no 6 pp349ndash353 1974

[7] W H Noble K D McClatchey and G D Douglass ldquoAhistologic comparison of effects of electrosurgical resectionusing different electrodesrdquo The Journal of Prosthetic Dentistryvol 35 no 5 pp 575ndash579 1976

[8] K C Nixon K F Adkins and D W Keys ldquoHistologicalevaluation of effects produced in alveolar bone followinggingival incision with an electrosurgical scalpelrdquo Journal ofPeriodontology vol 46 no 1 pp 40ndash44 1975

[9] I Glickman and L R Imber ldquoComparison of gingival resec-tion with electrosurgery and periodontal knivesmdasha biometric

and histologic studyrdquo Journal of Periodontology vol 41 no 3pp 142ndash148 1970

[10] D H Coelho J Cavallaro and E A Rothschild ldquoGingivalrecession with electrosurgery for impression makingrdquo TheJournal of Prosthetic Dentistry vol 33 no 4 pp 422ndash426 1975

[11] P B Robertson B Luscher L S Spangberg and B M LevyldquoPulpal and periodontal effects of electrosurgery involvingcervical metallic restorationsrdquo Oral Surgery Oral Medicine andOral Pathology vol 46 no 5 pp 702ndash710 1978

[12] R DrsquoSouza ldquoPulpal and periapical immune response to electro-surgical contact of cervical metallic restorations in monkeysrdquoQuintessence International vol 17 no 12 pp 803ndash808 1986

[13] K-L Ou J-S Chu H Hosseinkhani J-F Chiou and C-HYu ldquoBiomedical nanostructured coating for minimally invasivesurgery devices applications characterization cell cytotoxicityevaluation and an animal study in ratrdquo Surgical Endoscopy 2014

[14] K Kino A Yamashita K Yamaoka et al ldquoIsolation and char-acterization of a new immunomodulatory protein Ling Zhi-8(LZ-8) from Ganoderma lucidiumrdquo The Journal of BiologicalChemistry vol 264 no 1 pp 472ndash478 1989

[15] AMurasugi S Tanaka N Komiyama et al ldquoMolecular cloningof a cDNAand a gene encoding an immunomodulatory proteinLing Zhi-8 from a fungusGanoderma lucidiumrdquoThe Journal ofBiological Chemistry vol 266 no 4 pp 2486ndash2493 1991

[16] M-H Tong P-J Chien H-H Chang M-J Tsai and F SheuldquoHigh processing tolerances of immunomodulatory proteins inEnoki and Reishi mushroomsrdquo Journal of Agricultural and FoodChemistry vol 56 no 9 pp 3160ndash3166 2008

[17] L Huang F Sun C Liang et al ldquoCrystal structure of LZ-8 fromthemedicinal fungusGanoderma lucidiumrdquo Proteins StructureFunction and Bioinformatics vol 75 no 2 pp 524ndash527 2009

[18] Y-L Lin Y-C Liang Y-S Tseng et al ldquoAn immunomodulatoryprotein Ling Zhi-8 induced activation and maturation ofhuman monocyte-derived dendritic cells by the NF-120581B andMAPK pathwaysrdquo Journal of Leukocyte Biology vol 86 no 4pp 877ndash889 2009

[19] R A D Drury and E A Wallington Carletonrsquos HistologicalTechnique 1981

[20] Y Du D-W Guan and R Zhao ldquoAn immunohistochemicalstudy on the expression of caspase-3 in the skin wound healingrdquoFa Yi Xue Za Zhi vol 20 no 1 pp 1ndash3 2004

[21] K E Grund T Straub and G Farin ldquoNew haemostatictechniques argon plasma coagulationrdquo Baillierersquos Best Practiceand Research in Clinical Gastroenterology vol 13 no 1 pp 67ndash84 1999

[22] D Hellwig R Haag V Bartel et al ldquoApplication of newelectrosurgical devices and probes in endoscopic neurosurgeryrdquoNeurological Research vol 21 no 1 pp 67ndash72 1999

[23] B Chehrazi and W F Collins Jr ldquoA comparison of effects ofbipolar and monopolar electrocoagulation in brainrdquo Journal ofNeurosurgery vol 54 no 2 pp 197ndash203 1981

[24] J Kerl and J Staubesand ldquoAn ultrastructural report on thechoice between monopolar and bipolar electrocoagulationrdquoActa Neurochirurgica vol 92 no 1ndash4 pp 123ndash131 1988

[25] Y Temel V Visser-Vandewalle M van derWolf et al ldquoMonop-olar versus bipolar high frequency stimulation in the rat subtha-lamic nucleus differences in histological damagerdquoNeuroscienceLetters vol 367 no 1 pp 92ndash96 2004

[26] L I Malis ldquoNew trends in microsurgery and applied technol-ogyrdquo in Advanced Technology in Neurosurgery F Pluchino andG Broggi Eds pp 1ndash16 Springer Berlin Germany 1988

12 Evidence-Based Complementary and Alternative Medicine

[27] C R B Cardoso M A Souza E A V Ferro S Favoreto Jrand J D O Pena ldquoInfluence of topical administration of n-3and n-6 essential and n-9 nonessential fatty acids on the healingof cutaneous woundsrdquoWound Repair and Regeneration vol 12no 2 pp 235ndash243 2004

[28] L M Pereira E Hatanaka E F Martins et al ldquoEffect of oleicand linoleic acids on the inflammatory phase of wound healingin ratsrdquo Cell Biochemistry and Function vol 26 no 2 pp 197ndash204 2008

[29] N-Y ParkGValacchi andY Lim ldquoEffect of dietary conjugatedlinoleic acid supplementation on early inflammatory responsesduring cutaneous wound healingrdquo Mediators of Inflammationvol 2010 Article ID 342328 8 pages 2010

[30] M Otranto A P do Nascimento and A Monte-Alto-CostaldquoEffects of supplementation with different edible oils on cuta-neous wound healingrdquoWound Repair and Regeneration vol 18no 6 pp 629ndash636 2010

[31] X Dolcet D Llobet J Pallares and X Matias-Guiu ldquoNF-kBin development and progression of human cancerrdquo VirchowsArchiv vol 446 no 5 pp 475ndash482 2005

[32] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[33] T Luedde and R F Schwabe ldquoNF-120581B in the livermdashlinkinginjury fibrosis and hepatocellular carcinomardquo Nature ReviewsGastroenterology amp Hepatology vol 8 no 2 pp 108ndash118 2011

[34] J A Schmid and A Birbach ldquoIkappaB kinase beta (IKKbetaIKK2IKBKB)mdasha keymolecule in signaling to the transcriptionfactor NF-kappaBrdquo Cytokine amp Growth Factor Reviews vol 19no 2 pp 157ndash165 2008

[35] S Yates and T E Rayner ldquoTranscription factor activation inresponse to cutaneous injury role of AP-1 in reepithelializa-tionrdquo Wound Repair and Regeneration vol 10 no 1 pp 5ndash152002

[36] A F Haas R R Isseroff R G Wheeland P A Rood and PJ Graves ldquoLow-energy helium-neon laser irradiation increasesthe motility of cultured human keratinocytesrdquo The Journal ofInvestigative Dermatology vol 94 no 6 pp 822ndash826 1990

[37] C-H Lin Y-M Hsiao C-C Ou et al ldquoGMI a Ganodermaimmunomodulatory protein down-regulates tumor necrosisfactor 120572-induced expression of matrix metalloproteinase 9 viaNF-120581Bpathway in human alveolar epithelial A549 cellsrdquo Journalof agricultural and food chemistry vol 58 no 22 pp 12014ndash12021 2010

[38] L Cai W Li G Wang L Guo Y Jiang and Y J KangldquoHyperglycemia-induced apoptosis in mouse myocardiummitochondrial cytochrome c-mediated caspase-3 activationpathwayrdquo Diabetes vol 51 no 6 pp 1938ndash1948 2002

[39] N K Rai K Tripathi D Sharma andV K Shukla ldquoApoptosis abasic physiologic process in wound healingrdquo The InternationalJournal of Lower Extremity Wounds vol 4 no 3 pp 138ndash1442005

[40] J Bian and Y Sun ldquoTranscriptional activation by p53 of thehuman type IV collagenase (gelatinase A or matrix metallopro-teinase 2) promoterrdquoMolecular and Cellular Biology vol 17 no11 pp 6330ndash6338 1997

[41] S Wang MMiura Y-K Jung et al ldquoIdentification and charac-terization of ich-3 a member of the interleukin-1120573 convertingenzyme (ICE)Ced-3 family and an upstream regulator of ICErdquoThe Journal of Biological Chemistry vol 271 no 34 pp 20580ndash20587 1996

[42] E S Alnemri D J Livingston D W Nicholson et al ldquoHumanICECED-3 protease nomenclaturerdquo Cell vol 87 no 2 article171 1996

[43] T D Hewitson T Bisucci and I A Darby ldquoHistochemicallocalization of apoptosis with in situ labeling of fragmentedDNArdquo Methods in Molecular Biology vol 326 pp 227ndash2342006

[44] D T Loo ldquoIn situ detection of apoptosis by the TUNEL assayan overview of techniquesrdquo in DNA Damage Detection In SituEx Vivo and In Vivo vol 682 ofMethods in Molecular Biologypp 3ndash13 Humana Press New Jersey NJ USA 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 10: Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014 › 916531.pdfResearch Article An Immunomodulatory Protein (Ling Zhi-8) from a

10 Evidence-Based Complementary and Alternative Medicine

TUNEL DAPI Merge

SSSS

+ L

Z-8

250 120583m 250 120583m 250 120583m

250 120583m 250 120583m 250 120583m

Figure 8 The TUNEL staining assay results from rats liver treated with SS group and SS + LZ-8 group after 7 days of healing

been operated with SS-needles secreted higher levels of NF-120581B expression and its nuclear translocation than that of CrNoperated tissues [13] Fibrosis is an important component ofadvanced chronic inflammatory liver disease Although thechanges in hepatic structures that occur in liver cirrhosis havebeen described in detail the pathogenesis of fibrosis in duringelectrosurgery process is largely unknown Our latest reportindicates that monopolar elecrtrosurgery-induced hepaticfibrosis after repeated liver injury and the mechanisms aremainly based on the upregulation of NF-120581B and caspase-3expression in the liver tissues [13]

In this study we thus evaluated whether LZ-8 had anyeffect on nuclear translocation of NF-120581B during monopolarelectrosurgery and the results found for the first time thatLZ-8 inhibits nuclear translocation of NF-120581B in hepaticcells after 3 and 7 days of monopolar electrosurgery Yatesand Rayner [35] studied the effects of fatty acid supple-mentation on activation of NF-120581B and AP-1 after a skininjury since these transcription factors are highly associ-ated with wound healing They have also reported a novelfinding which indicates that wounding of normal humankeratinocytes in culture causes NF-120581B translocation Therelevance of NF-120581B activation to wound healing is sug-gested by the results that HeNe laser treatment a processshown to accelerate healing in a model of wounding [36]promoted the translocation of NF-120581B A previous studyalso proved that a ganoderma immunomodulatory proteincloned from Ganoderma microsporum holds anti-invasiveand anti-inflammatory activities in TNF-120572 induced humanalveolar epithelial A549 cells via inhibition of the I120581B120572 andNF-120581B pathway [37]

Apoptosis in wound healing is a promising issue Therehas been some evaluation of chronic diabetic wounds in

mice that suggests that there is increased cellular apoptosislikely due to hyperglycemia which produces reactive oxygenspecies that mediate mitochondrial release of cytochrome cand activate caspase-3 [37 38] Apoptosis is also involved inthe regulation of collagen synthesis and degradation withinwounds through regulation of fibroblast numbers and colla-genase activity [39] Recent work in another mouse modelsuggests that hypertrophic scars demonstrate decreased ratesof apoptosis with decreased expression of cleaved caspase-3Over expression of various caspase family members inducesapoptosis in cultured mammalian cells [40] Among thecysteine proteases caspase-3 is believed to be one of themost commonly involved in the execution of apoptosisin various cell types [41] The assay of TUNEL stainingcan identify the apoptotic cells via DNA fragmentation bylabeling the terminal ends of nucleic acids [42 43] Fromour latest study of TUNEL assay it can be clearly foundthat the apoptosis event was more significant in SS-needleoperated liver tissues [13] In this study TUNEL stainingprovides direct evidence of apoptosis in SS operated livertissues after 7 days Both the activation of caspase-3 and theinduction of apoptotic cell death could be suppressed by theaddition of antioxidants in the cultures [44] It has also beendemonstrated that LZ-8 is able to effectively promote theactivation and maturation of immature dendritic cells via thenuclear factor-kappaB and mitogen-activated protein kinasepathways [18] Moreover the present study observed theeffects of (i) inhibiting expression and nuclear translocationof NF-120581B and (ii) inhibiting expression of caspase-3 andreduction of apoptotic cell death by LZ-8 in liver tissuesafter electrosurgery which indicates that the wound healingproperty of LZ-8 may possibly occur through inhibition oftranscription factor NF-120581B and apoptotic factor caspase-3

Evidence-Based Complementary and Alternative Medicine 11

5 Conclusion

Under the same conditions LZ-8 treatment potentiates onreducing wound size and thermal injury An increasing fibro-sis and reducing apoptosis were found in LZ-8 treated livertissues during electrosurgery LZ-8 treatment significantlyinhibits NF-120581B and caspase-3 expressions and also reducesNF-120581B nuclear translocation and apoptotic cell death Theresults generated from this study suggest that LZ-8 has potenteffects on increasing wound healing action against the elec-trosurgical induced liver injury via the possible mechanismof inhibiting NF-120581B and caspase-3 expressions

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors would like to thank the Department of HealthExecutive Yuan Taiwan (Contract no MOHW103-TDU-N-211-133001) and TaipeiMedical University and TaipeiMedicalHospital (Contract no 100TMU-TMUH-05) for financiallysupporting this research Dr Hao-Jan Lin and Dr Yushan-Sophie Chang are considered the first coauthors of the paper

References

[1] J Liboon W Funkhouser and D J Terris ldquoA comparison ofmucosal incisions made by scalpel CO

2

laser electrocauteryand constant-voltage electrocauteryrdquo OtolaryngologymdashHeadand Neck Surgery vol 116 no 3 pp 379ndash385 1997

[2] J F Carew R F Ward A LaBruna P A Torzilli and W SSchley ldquoEffects of scalpel electrocautery and CO

2

and KTPlasers on wound healing in rat tonguesrdquoThe Laryngoscope vol108 no 3 pp 373ndash380 1998

[3] J A Pearce Electrosurgery John Wiley amp Sons New York NYUSA 1986

[4] W L Maness F W Rober R E Clark E Cataldo andA W Haddad ldquoTissue damage from electrosurgical poweroutput variations in hamster tonguesrdquoThe Journal of ProstheticDentistry vol 42 no 4 pp 456ndash460 1979

[5] W L Maness F W Roeber R E Clark E Cataldo D Riisand A W Haddad ldquoHistologic evaluation of electrosurgerywith varying frequency andwaveformrdquoThe Journal of ProstheticDentistry vol 40 no 3 pp 304ndash308 1978

[6] J Friedman J Margolin and S Piliero ldquoA preliminary study ofthe histological effects of three different types of electrosurgicalcurrentsrdquoThe New York State Dental Journal vol 40 no 6 pp349ndash353 1974

[7] W H Noble K D McClatchey and G D Douglass ldquoAhistologic comparison of effects of electrosurgical resectionusing different electrodesrdquo The Journal of Prosthetic Dentistryvol 35 no 5 pp 575ndash579 1976

[8] K C Nixon K F Adkins and D W Keys ldquoHistologicalevaluation of effects produced in alveolar bone followinggingival incision with an electrosurgical scalpelrdquo Journal ofPeriodontology vol 46 no 1 pp 40ndash44 1975

[9] I Glickman and L R Imber ldquoComparison of gingival resec-tion with electrosurgery and periodontal knivesmdasha biometric

and histologic studyrdquo Journal of Periodontology vol 41 no 3pp 142ndash148 1970

[10] D H Coelho J Cavallaro and E A Rothschild ldquoGingivalrecession with electrosurgery for impression makingrdquo TheJournal of Prosthetic Dentistry vol 33 no 4 pp 422ndash426 1975

[11] P B Robertson B Luscher L S Spangberg and B M LevyldquoPulpal and periodontal effects of electrosurgery involvingcervical metallic restorationsrdquo Oral Surgery Oral Medicine andOral Pathology vol 46 no 5 pp 702ndash710 1978

[12] R DrsquoSouza ldquoPulpal and periapical immune response to electro-surgical contact of cervical metallic restorations in monkeysrdquoQuintessence International vol 17 no 12 pp 803ndash808 1986

[13] K-L Ou J-S Chu H Hosseinkhani J-F Chiou and C-HYu ldquoBiomedical nanostructured coating for minimally invasivesurgery devices applications characterization cell cytotoxicityevaluation and an animal study in ratrdquo Surgical Endoscopy 2014

[14] K Kino A Yamashita K Yamaoka et al ldquoIsolation and char-acterization of a new immunomodulatory protein Ling Zhi-8(LZ-8) from Ganoderma lucidiumrdquo The Journal of BiologicalChemistry vol 264 no 1 pp 472ndash478 1989

[15] AMurasugi S Tanaka N Komiyama et al ldquoMolecular cloningof a cDNAand a gene encoding an immunomodulatory proteinLing Zhi-8 from a fungusGanoderma lucidiumrdquoThe Journal ofBiological Chemistry vol 266 no 4 pp 2486ndash2493 1991

[16] M-H Tong P-J Chien H-H Chang M-J Tsai and F SheuldquoHigh processing tolerances of immunomodulatory proteins inEnoki and Reishi mushroomsrdquo Journal of Agricultural and FoodChemistry vol 56 no 9 pp 3160ndash3166 2008

[17] L Huang F Sun C Liang et al ldquoCrystal structure of LZ-8 fromthemedicinal fungusGanoderma lucidiumrdquo Proteins StructureFunction and Bioinformatics vol 75 no 2 pp 524ndash527 2009

[18] Y-L Lin Y-C Liang Y-S Tseng et al ldquoAn immunomodulatoryprotein Ling Zhi-8 induced activation and maturation ofhuman monocyte-derived dendritic cells by the NF-120581B andMAPK pathwaysrdquo Journal of Leukocyte Biology vol 86 no 4pp 877ndash889 2009

[19] R A D Drury and E A Wallington Carletonrsquos HistologicalTechnique 1981

[20] Y Du D-W Guan and R Zhao ldquoAn immunohistochemicalstudy on the expression of caspase-3 in the skin wound healingrdquoFa Yi Xue Za Zhi vol 20 no 1 pp 1ndash3 2004

[21] K E Grund T Straub and G Farin ldquoNew haemostatictechniques argon plasma coagulationrdquo Baillierersquos Best Practiceand Research in Clinical Gastroenterology vol 13 no 1 pp 67ndash84 1999

[22] D Hellwig R Haag V Bartel et al ldquoApplication of newelectrosurgical devices and probes in endoscopic neurosurgeryrdquoNeurological Research vol 21 no 1 pp 67ndash72 1999

[23] B Chehrazi and W F Collins Jr ldquoA comparison of effects ofbipolar and monopolar electrocoagulation in brainrdquo Journal ofNeurosurgery vol 54 no 2 pp 197ndash203 1981

[24] J Kerl and J Staubesand ldquoAn ultrastructural report on thechoice between monopolar and bipolar electrocoagulationrdquoActa Neurochirurgica vol 92 no 1ndash4 pp 123ndash131 1988

[25] Y Temel V Visser-Vandewalle M van derWolf et al ldquoMonop-olar versus bipolar high frequency stimulation in the rat subtha-lamic nucleus differences in histological damagerdquoNeuroscienceLetters vol 367 no 1 pp 92ndash96 2004

[26] L I Malis ldquoNew trends in microsurgery and applied technol-ogyrdquo in Advanced Technology in Neurosurgery F Pluchino andG Broggi Eds pp 1ndash16 Springer Berlin Germany 1988

12 Evidence-Based Complementary and Alternative Medicine

[27] C R B Cardoso M A Souza E A V Ferro S Favoreto Jrand J D O Pena ldquoInfluence of topical administration of n-3and n-6 essential and n-9 nonessential fatty acids on the healingof cutaneous woundsrdquoWound Repair and Regeneration vol 12no 2 pp 235ndash243 2004

[28] L M Pereira E Hatanaka E F Martins et al ldquoEffect of oleicand linoleic acids on the inflammatory phase of wound healingin ratsrdquo Cell Biochemistry and Function vol 26 no 2 pp 197ndash204 2008

[29] N-Y ParkGValacchi andY Lim ldquoEffect of dietary conjugatedlinoleic acid supplementation on early inflammatory responsesduring cutaneous wound healingrdquo Mediators of Inflammationvol 2010 Article ID 342328 8 pages 2010

[30] M Otranto A P do Nascimento and A Monte-Alto-CostaldquoEffects of supplementation with different edible oils on cuta-neous wound healingrdquoWound Repair and Regeneration vol 18no 6 pp 629ndash636 2010

[31] X Dolcet D Llobet J Pallares and X Matias-Guiu ldquoNF-kBin development and progression of human cancerrdquo VirchowsArchiv vol 446 no 5 pp 475ndash482 2005

[32] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[33] T Luedde and R F Schwabe ldquoNF-120581B in the livermdashlinkinginjury fibrosis and hepatocellular carcinomardquo Nature ReviewsGastroenterology amp Hepatology vol 8 no 2 pp 108ndash118 2011

[34] J A Schmid and A Birbach ldquoIkappaB kinase beta (IKKbetaIKK2IKBKB)mdasha keymolecule in signaling to the transcriptionfactor NF-kappaBrdquo Cytokine amp Growth Factor Reviews vol 19no 2 pp 157ndash165 2008

[35] S Yates and T E Rayner ldquoTranscription factor activation inresponse to cutaneous injury role of AP-1 in reepithelializa-tionrdquo Wound Repair and Regeneration vol 10 no 1 pp 5ndash152002

[36] A F Haas R R Isseroff R G Wheeland P A Rood and PJ Graves ldquoLow-energy helium-neon laser irradiation increasesthe motility of cultured human keratinocytesrdquo The Journal ofInvestigative Dermatology vol 94 no 6 pp 822ndash826 1990

[37] C-H Lin Y-M Hsiao C-C Ou et al ldquoGMI a Ganodermaimmunomodulatory protein down-regulates tumor necrosisfactor 120572-induced expression of matrix metalloproteinase 9 viaNF-120581Bpathway in human alveolar epithelial A549 cellsrdquo Journalof agricultural and food chemistry vol 58 no 22 pp 12014ndash12021 2010

[38] L Cai W Li G Wang L Guo Y Jiang and Y J KangldquoHyperglycemia-induced apoptosis in mouse myocardiummitochondrial cytochrome c-mediated caspase-3 activationpathwayrdquo Diabetes vol 51 no 6 pp 1938ndash1948 2002

[39] N K Rai K Tripathi D Sharma andV K Shukla ldquoApoptosis abasic physiologic process in wound healingrdquo The InternationalJournal of Lower Extremity Wounds vol 4 no 3 pp 138ndash1442005

[40] J Bian and Y Sun ldquoTranscriptional activation by p53 of thehuman type IV collagenase (gelatinase A or matrix metallopro-teinase 2) promoterrdquoMolecular and Cellular Biology vol 17 no11 pp 6330ndash6338 1997

[41] S Wang MMiura Y-K Jung et al ldquoIdentification and charac-terization of ich-3 a member of the interleukin-1120573 convertingenzyme (ICE)Ced-3 family and an upstream regulator of ICErdquoThe Journal of Biological Chemistry vol 271 no 34 pp 20580ndash20587 1996

[42] E S Alnemri D J Livingston D W Nicholson et al ldquoHumanICECED-3 protease nomenclaturerdquo Cell vol 87 no 2 article171 1996

[43] T D Hewitson T Bisucci and I A Darby ldquoHistochemicallocalization of apoptosis with in situ labeling of fragmentedDNArdquo Methods in Molecular Biology vol 326 pp 227ndash2342006

[44] D T Loo ldquoIn situ detection of apoptosis by the TUNEL assayan overview of techniquesrdquo in DNA Damage Detection In SituEx Vivo and In Vivo vol 682 ofMethods in Molecular Biologypp 3ndash13 Humana Press New Jersey NJ USA 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 11: Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014 › 916531.pdfResearch Article An Immunomodulatory Protein (Ling Zhi-8) from a

Evidence-Based Complementary and Alternative Medicine 11

5 Conclusion

Under the same conditions LZ-8 treatment potentiates onreducing wound size and thermal injury An increasing fibro-sis and reducing apoptosis were found in LZ-8 treated livertissues during electrosurgery LZ-8 treatment significantlyinhibits NF-120581B and caspase-3 expressions and also reducesNF-120581B nuclear translocation and apoptotic cell death Theresults generated from this study suggest that LZ-8 has potenteffects on increasing wound healing action against the elec-trosurgical induced liver injury via the possible mechanismof inhibiting NF-120581B and caspase-3 expressions

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors would like to thank the Department of HealthExecutive Yuan Taiwan (Contract no MOHW103-TDU-N-211-133001) and TaipeiMedical University and TaipeiMedicalHospital (Contract no 100TMU-TMUH-05) for financiallysupporting this research Dr Hao-Jan Lin and Dr Yushan-Sophie Chang are considered the first coauthors of the paper

References

[1] J Liboon W Funkhouser and D J Terris ldquoA comparison ofmucosal incisions made by scalpel CO

2

laser electrocauteryand constant-voltage electrocauteryrdquo OtolaryngologymdashHeadand Neck Surgery vol 116 no 3 pp 379ndash385 1997

[2] J F Carew R F Ward A LaBruna P A Torzilli and W SSchley ldquoEffects of scalpel electrocautery and CO

2

and KTPlasers on wound healing in rat tonguesrdquoThe Laryngoscope vol108 no 3 pp 373ndash380 1998

[3] J A Pearce Electrosurgery John Wiley amp Sons New York NYUSA 1986

[4] W L Maness F W Rober R E Clark E Cataldo andA W Haddad ldquoTissue damage from electrosurgical poweroutput variations in hamster tonguesrdquoThe Journal of ProstheticDentistry vol 42 no 4 pp 456ndash460 1979

[5] W L Maness F W Roeber R E Clark E Cataldo D Riisand A W Haddad ldquoHistologic evaluation of electrosurgerywith varying frequency andwaveformrdquoThe Journal of ProstheticDentistry vol 40 no 3 pp 304ndash308 1978

[6] J Friedman J Margolin and S Piliero ldquoA preliminary study ofthe histological effects of three different types of electrosurgicalcurrentsrdquoThe New York State Dental Journal vol 40 no 6 pp349ndash353 1974

[7] W H Noble K D McClatchey and G D Douglass ldquoAhistologic comparison of effects of electrosurgical resectionusing different electrodesrdquo The Journal of Prosthetic Dentistryvol 35 no 5 pp 575ndash579 1976

[8] K C Nixon K F Adkins and D W Keys ldquoHistologicalevaluation of effects produced in alveolar bone followinggingival incision with an electrosurgical scalpelrdquo Journal ofPeriodontology vol 46 no 1 pp 40ndash44 1975

[9] I Glickman and L R Imber ldquoComparison of gingival resec-tion with electrosurgery and periodontal knivesmdasha biometric

and histologic studyrdquo Journal of Periodontology vol 41 no 3pp 142ndash148 1970

[10] D H Coelho J Cavallaro and E A Rothschild ldquoGingivalrecession with electrosurgery for impression makingrdquo TheJournal of Prosthetic Dentistry vol 33 no 4 pp 422ndash426 1975

[11] P B Robertson B Luscher L S Spangberg and B M LevyldquoPulpal and periodontal effects of electrosurgery involvingcervical metallic restorationsrdquo Oral Surgery Oral Medicine andOral Pathology vol 46 no 5 pp 702ndash710 1978

[12] R DrsquoSouza ldquoPulpal and periapical immune response to electro-surgical contact of cervical metallic restorations in monkeysrdquoQuintessence International vol 17 no 12 pp 803ndash808 1986

[13] K-L Ou J-S Chu H Hosseinkhani J-F Chiou and C-HYu ldquoBiomedical nanostructured coating for minimally invasivesurgery devices applications characterization cell cytotoxicityevaluation and an animal study in ratrdquo Surgical Endoscopy 2014

[14] K Kino A Yamashita K Yamaoka et al ldquoIsolation and char-acterization of a new immunomodulatory protein Ling Zhi-8(LZ-8) from Ganoderma lucidiumrdquo The Journal of BiologicalChemistry vol 264 no 1 pp 472ndash478 1989

[15] AMurasugi S Tanaka N Komiyama et al ldquoMolecular cloningof a cDNAand a gene encoding an immunomodulatory proteinLing Zhi-8 from a fungusGanoderma lucidiumrdquoThe Journal ofBiological Chemistry vol 266 no 4 pp 2486ndash2493 1991

[16] M-H Tong P-J Chien H-H Chang M-J Tsai and F SheuldquoHigh processing tolerances of immunomodulatory proteins inEnoki and Reishi mushroomsrdquo Journal of Agricultural and FoodChemistry vol 56 no 9 pp 3160ndash3166 2008

[17] L Huang F Sun C Liang et al ldquoCrystal structure of LZ-8 fromthemedicinal fungusGanoderma lucidiumrdquo Proteins StructureFunction and Bioinformatics vol 75 no 2 pp 524ndash527 2009

[18] Y-L Lin Y-C Liang Y-S Tseng et al ldquoAn immunomodulatoryprotein Ling Zhi-8 induced activation and maturation ofhuman monocyte-derived dendritic cells by the NF-120581B andMAPK pathwaysrdquo Journal of Leukocyte Biology vol 86 no 4pp 877ndash889 2009

[19] R A D Drury and E A Wallington Carletonrsquos HistologicalTechnique 1981

[20] Y Du D-W Guan and R Zhao ldquoAn immunohistochemicalstudy on the expression of caspase-3 in the skin wound healingrdquoFa Yi Xue Za Zhi vol 20 no 1 pp 1ndash3 2004

[21] K E Grund T Straub and G Farin ldquoNew haemostatictechniques argon plasma coagulationrdquo Baillierersquos Best Practiceand Research in Clinical Gastroenterology vol 13 no 1 pp 67ndash84 1999

[22] D Hellwig R Haag V Bartel et al ldquoApplication of newelectrosurgical devices and probes in endoscopic neurosurgeryrdquoNeurological Research vol 21 no 1 pp 67ndash72 1999

[23] B Chehrazi and W F Collins Jr ldquoA comparison of effects ofbipolar and monopolar electrocoagulation in brainrdquo Journal ofNeurosurgery vol 54 no 2 pp 197ndash203 1981

[24] J Kerl and J Staubesand ldquoAn ultrastructural report on thechoice between monopolar and bipolar electrocoagulationrdquoActa Neurochirurgica vol 92 no 1ndash4 pp 123ndash131 1988

[25] Y Temel V Visser-Vandewalle M van derWolf et al ldquoMonop-olar versus bipolar high frequency stimulation in the rat subtha-lamic nucleus differences in histological damagerdquoNeuroscienceLetters vol 367 no 1 pp 92ndash96 2004

[26] L I Malis ldquoNew trends in microsurgery and applied technol-ogyrdquo in Advanced Technology in Neurosurgery F Pluchino andG Broggi Eds pp 1ndash16 Springer Berlin Germany 1988

12 Evidence-Based Complementary and Alternative Medicine

[27] C R B Cardoso M A Souza E A V Ferro S Favoreto Jrand J D O Pena ldquoInfluence of topical administration of n-3and n-6 essential and n-9 nonessential fatty acids on the healingof cutaneous woundsrdquoWound Repair and Regeneration vol 12no 2 pp 235ndash243 2004

[28] L M Pereira E Hatanaka E F Martins et al ldquoEffect of oleicand linoleic acids on the inflammatory phase of wound healingin ratsrdquo Cell Biochemistry and Function vol 26 no 2 pp 197ndash204 2008

[29] N-Y ParkGValacchi andY Lim ldquoEffect of dietary conjugatedlinoleic acid supplementation on early inflammatory responsesduring cutaneous wound healingrdquo Mediators of Inflammationvol 2010 Article ID 342328 8 pages 2010

[30] M Otranto A P do Nascimento and A Monte-Alto-CostaldquoEffects of supplementation with different edible oils on cuta-neous wound healingrdquoWound Repair and Regeneration vol 18no 6 pp 629ndash636 2010

[31] X Dolcet D Llobet J Pallares and X Matias-Guiu ldquoNF-kBin development and progression of human cancerrdquo VirchowsArchiv vol 446 no 5 pp 475ndash482 2005

[32] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[33] T Luedde and R F Schwabe ldquoNF-120581B in the livermdashlinkinginjury fibrosis and hepatocellular carcinomardquo Nature ReviewsGastroenterology amp Hepatology vol 8 no 2 pp 108ndash118 2011

[34] J A Schmid and A Birbach ldquoIkappaB kinase beta (IKKbetaIKK2IKBKB)mdasha keymolecule in signaling to the transcriptionfactor NF-kappaBrdquo Cytokine amp Growth Factor Reviews vol 19no 2 pp 157ndash165 2008

[35] S Yates and T E Rayner ldquoTranscription factor activation inresponse to cutaneous injury role of AP-1 in reepithelializa-tionrdquo Wound Repair and Regeneration vol 10 no 1 pp 5ndash152002

[36] A F Haas R R Isseroff R G Wheeland P A Rood and PJ Graves ldquoLow-energy helium-neon laser irradiation increasesthe motility of cultured human keratinocytesrdquo The Journal ofInvestigative Dermatology vol 94 no 6 pp 822ndash826 1990

[37] C-H Lin Y-M Hsiao C-C Ou et al ldquoGMI a Ganodermaimmunomodulatory protein down-regulates tumor necrosisfactor 120572-induced expression of matrix metalloproteinase 9 viaNF-120581Bpathway in human alveolar epithelial A549 cellsrdquo Journalof agricultural and food chemistry vol 58 no 22 pp 12014ndash12021 2010

[38] L Cai W Li G Wang L Guo Y Jiang and Y J KangldquoHyperglycemia-induced apoptosis in mouse myocardiummitochondrial cytochrome c-mediated caspase-3 activationpathwayrdquo Diabetes vol 51 no 6 pp 1938ndash1948 2002

[39] N K Rai K Tripathi D Sharma andV K Shukla ldquoApoptosis abasic physiologic process in wound healingrdquo The InternationalJournal of Lower Extremity Wounds vol 4 no 3 pp 138ndash1442005

[40] J Bian and Y Sun ldquoTranscriptional activation by p53 of thehuman type IV collagenase (gelatinase A or matrix metallopro-teinase 2) promoterrdquoMolecular and Cellular Biology vol 17 no11 pp 6330ndash6338 1997

[41] S Wang MMiura Y-K Jung et al ldquoIdentification and charac-terization of ich-3 a member of the interleukin-1120573 convertingenzyme (ICE)Ced-3 family and an upstream regulator of ICErdquoThe Journal of Biological Chemistry vol 271 no 34 pp 20580ndash20587 1996

[42] E S Alnemri D J Livingston D W Nicholson et al ldquoHumanICECED-3 protease nomenclaturerdquo Cell vol 87 no 2 article171 1996

[43] T D Hewitson T Bisucci and I A Darby ldquoHistochemicallocalization of apoptosis with in situ labeling of fragmentedDNArdquo Methods in Molecular Biology vol 326 pp 227ndash2342006

[44] D T Loo ldquoIn situ detection of apoptosis by the TUNEL assayan overview of techniquesrdquo in DNA Damage Detection In SituEx Vivo and In Vivo vol 682 ofMethods in Molecular Biologypp 3ndash13 Humana Press New Jersey NJ USA 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 12: Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014 › 916531.pdfResearch Article An Immunomodulatory Protein (Ling Zhi-8) from a

12 Evidence-Based Complementary and Alternative Medicine

[27] C R B Cardoso M A Souza E A V Ferro S Favoreto Jrand J D O Pena ldquoInfluence of topical administration of n-3and n-6 essential and n-9 nonessential fatty acids on the healingof cutaneous woundsrdquoWound Repair and Regeneration vol 12no 2 pp 235ndash243 2004

[28] L M Pereira E Hatanaka E F Martins et al ldquoEffect of oleicand linoleic acids on the inflammatory phase of wound healingin ratsrdquo Cell Biochemistry and Function vol 26 no 2 pp 197ndash204 2008

[29] N-Y ParkGValacchi andY Lim ldquoEffect of dietary conjugatedlinoleic acid supplementation on early inflammatory responsesduring cutaneous wound healingrdquo Mediators of Inflammationvol 2010 Article ID 342328 8 pages 2010

[30] M Otranto A P do Nascimento and A Monte-Alto-CostaldquoEffects of supplementation with different edible oils on cuta-neous wound healingrdquoWound Repair and Regeneration vol 18no 6 pp 629ndash636 2010

[31] X Dolcet D Llobet J Pallares and X Matias-Guiu ldquoNF-kBin development and progression of human cancerrdquo VirchowsArchiv vol 446 no 5 pp 475ndash482 2005

[32] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[33] T Luedde and R F Schwabe ldquoNF-120581B in the livermdashlinkinginjury fibrosis and hepatocellular carcinomardquo Nature ReviewsGastroenterology amp Hepatology vol 8 no 2 pp 108ndash118 2011

[34] J A Schmid and A Birbach ldquoIkappaB kinase beta (IKKbetaIKK2IKBKB)mdasha keymolecule in signaling to the transcriptionfactor NF-kappaBrdquo Cytokine amp Growth Factor Reviews vol 19no 2 pp 157ndash165 2008

[35] S Yates and T E Rayner ldquoTranscription factor activation inresponse to cutaneous injury role of AP-1 in reepithelializa-tionrdquo Wound Repair and Regeneration vol 10 no 1 pp 5ndash152002

[36] A F Haas R R Isseroff R G Wheeland P A Rood and PJ Graves ldquoLow-energy helium-neon laser irradiation increasesthe motility of cultured human keratinocytesrdquo The Journal ofInvestigative Dermatology vol 94 no 6 pp 822ndash826 1990

[37] C-H Lin Y-M Hsiao C-C Ou et al ldquoGMI a Ganodermaimmunomodulatory protein down-regulates tumor necrosisfactor 120572-induced expression of matrix metalloproteinase 9 viaNF-120581Bpathway in human alveolar epithelial A549 cellsrdquo Journalof agricultural and food chemistry vol 58 no 22 pp 12014ndash12021 2010

[38] L Cai W Li G Wang L Guo Y Jiang and Y J KangldquoHyperglycemia-induced apoptosis in mouse myocardiummitochondrial cytochrome c-mediated caspase-3 activationpathwayrdquo Diabetes vol 51 no 6 pp 1938ndash1948 2002

[39] N K Rai K Tripathi D Sharma andV K Shukla ldquoApoptosis abasic physiologic process in wound healingrdquo The InternationalJournal of Lower Extremity Wounds vol 4 no 3 pp 138ndash1442005

[40] J Bian and Y Sun ldquoTranscriptional activation by p53 of thehuman type IV collagenase (gelatinase A or matrix metallopro-teinase 2) promoterrdquoMolecular and Cellular Biology vol 17 no11 pp 6330ndash6338 1997

[41] S Wang MMiura Y-K Jung et al ldquoIdentification and charac-terization of ich-3 a member of the interleukin-1120573 convertingenzyme (ICE)Ced-3 family and an upstream regulator of ICErdquoThe Journal of Biological Chemistry vol 271 no 34 pp 20580ndash20587 1996

[42] E S Alnemri D J Livingston D W Nicholson et al ldquoHumanICECED-3 protease nomenclaturerdquo Cell vol 87 no 2 article171 1996

[43] T D Hewitson T Bisucci and I A Darby ldquoHistochemicallocalization of apoptosis with in situ labeling of fragmentedDNArdquo Methods in Molecular Biology vol 326 pp 227ndash2342006

[44] D T Loo ldquoIn situ detection of apoptosis by the TUNEL assayan overview of techniquesrdquo in DNA Damage Detection In SituEx Vivo and In Vivo vol 682 ofMethods in Molecular Biologypp 3ndash13 Humana Press New Jersey NJ USA 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 13: Research Article An Immunomodulatory Protein …downloads.hindawi.com › journals › ecam › 2014 › 916531.pdfResearch Article An Immunomodulatory Protein (Ling Zhi-8) from a

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom