Research Article Warming Moxibustion Relieves Chronic ...LiQi, 1,2 Hui-RongLiu, 1 TaoYi, 3 Lu-YiWu,...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 920675 10 pageshttpdxdoiorg1011552013920675

Research ArticleWarming Moxibustion Relieves Chronic Visceral Hyperalgesia inRats Relations to Spinal Dynorphin and Orphanin-FQ System

Li Qi12 Hui-Rong Liu1 Tao Yi3 Lu-Yi Wu1 Xi-Ru Liu4 Chen Zhao5 Yin Shi5

Xiao-Peng Ma6 and Huan-Gan Wu1

1 Key Laboratory of Acupuncture-Moxibustion and Immunological EffectsShanghai University of Traditional Chinese Medicine Shanghai 200030 China

2 E-Institute of Shanghai Municipal Education Committee Shanghai University of Traditional Chinese MedicineShanghai 201203 China

3 Institute of Integrative Medicine Huashan Hospital Affiliated to Fudan University Shanghai 200040 China4 Shuguang Hospital Shanghai University of Traditional Chinese Medicine Shanghai 201203 China5 Yueyang Hospital of Integrated Traditional Chinese and Western MedicineShanghai University of Traditional Chinese Medicine Shanghai 200437 China

6 Shanghai Institute of Acupuncture-Moxibustion and Meridian Shanghai 200030 China

Correspondence should be addressed to Huan-Gan Wu wuhuangan2012163com

Received 1 December 2012 Revised 25 January 2013 Accepted 7 February 2013

Academic Editor Ke Ren

Copyright copy 2013 Li Qi et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

As a twin therapy of acupuncture in traditional Chinese medicine moxibustion has shown its effects in relieving abdominal pain inirritable bowel syndrome (IBS) patients and IBS rat models but its mechanisms are largely unknown In this paper we determinedthe role of spinal dynorphin and orphanin-FQ system in analgesic effect of warming moxibustion (WM) on chronic visceralhyperalgesia (CVH) in IBS-like rat model Here we show that (1) repeated WM at bilateral ST25 and ST37 acupoints markedlyattenuated the abdominal withdrawal reflex scores in CVH rats (2) intrathecal administration of 120581 receptor antagonist prior toWM significantly attenuated the WM analgesia and dynorphinA (1-17) enhanced the WM analgesia WM significantly reinforcedthe upregulation of spinal dynorphin mRNAprotein and 120581 receptor mRNA levels in CVH rats (3) intrathecal administration oforphanin-FQ receptor antagonist prior to WM significantly attenuated the WM analgesia and orphanin-FQ enhanced the WManalgesia WM reinforced the upregulation of spinal orphanin-FQ mRNAprotein and orphanin-FQ receptor mRNA levels inCVH rats These results suggest that moxibustion may relieve CVH at least in part by activating spinal dynorphin and orphanin-FQ system

1 Introduction

Irritable bowel syndrome (IBS) is a highly prevalent chronicgastrointestinal dysfunctional disease Chronic visceralhyperalgesia (CVH) has been identified as an importantaspect of IBS pathophysiology and abdominal pain is aprimary factor related to the quality of life in IBS patients[1 2] Despite multiple therapeutic approaches the treatmentof visceral pain remains a significant challenge [3]

Moxibustion an important component of traditionalChinese medicine is a therapeutic method in which burning

moxa produces thermal stimulation to the human bodyand it affects the function of the meridians and acupointsto prevent and treat the disease [4] Moxibustion has beenadopted as an analgesic method for thousands of years inChina and other AsiaN countries and is still frequently usedin the present clinical practice because of its advantages ofsafety effectiveness and no side effects [5ndash7]

Moxibustions include scarring moxibustion (the directburning of small moxa on the skin) warming moxibustion(also named suspended moxibustion the direct burning

2 Evidence-Based Complementary and Alternative Medicine

above the skin) and herb-partition moxibustion (indirectburning interposed by various materials) warming moxibus-tion (WM) is the most practical and convenient one from theperspective of clinical application

Our previous studies also revealed the analgesic effectsof moxibustion in reducing abdominal pain in IBS patients[8 9] and IBS rat models [10ndash15] We also reported that WMtreatment could alleviate CVH in IBS-like rats by suppressinghypothalamic corticotrophin-releasing-hormone level [11]which provides the evidence that the analgesic effect ofmoxibustion may be associated with the central nervoussystem (CNS)

It has been proved that the afferent signals of viscera couldbe converged and affected by the signals of superficial bodyin CNS This is the essential mechanism of stimulating acu-points for treating visceral disease [16]The spinal dorsal hornis the first integration center in CNS and then what role doesthe spinal cord play in the analgesic effect of moxibustionWhat are the kinds of chemicals that participate in the processof CVH and the analgesic effect of moxibustion in IBS-likerats

Many experimental models of pathological pain includ-ing inflammatory pain [17 18] neuropathic pain [19] andbone cancer pain [20] show a significant regional elevationof dynorphin and 120581 receptor in the spinal cord The earlyresearch has reported that dynorphin could act on 120581 recep-tor to impede nociceptive signals in the spinal cord andacupuncture could exert its analgesic action by activatingthe spinal dynorphin-120581 opioid system [21 22] A variety ofpain assays in rodents have shown the effect of intrathe-cally administered orphanin-FQ (OFQ) to be antinoci-ceptive [23 24] and it was also reported that acupunc-ture could exert an analgesic effect by activating spinalOFQ-OFQ receptor system in inflammatory pain model[25]

Both acupuncture and moxibustion are considered to bethe external therapy [26] and based on the mechanism ofacupuncture analgesia we presumed that the moxibustionanalgesia in CVH rats might be due to the induction ofdynorphin and orphanin-FQ system in the spinal cord Weperformed the pilot experiment and found that WM couldincrease the level of spinal dynorphin in CVH rats [13] whichholds our interests to further study the spinal dynorphin andorphanin-FQ system in moxibustion analgesia

Therefore using a well characterized rat model of IBS-like CVH the present study was intended to unfold theinvolvement of spinal dynorphin and orphanin-FQ system inmoxibustion analgesia

2 Materials and Methods

21 Animals Experiments were conducted strictly in accor-dance with the National Institute of Health Guide for theCare and Use of Laboratory Animals and the Guidelinesof the International Association for the Study of Pain [27]All efforts were made to minimize the number of animalsused and their suffering Rat model of CVH was inducedby mechanical colorectal distention (CRD) during postnatal

Table 1 AWR scoring criteria

Score 0 No behavioral response to CRD

Score 1 Immobile during the CRD and occasionally clicked thehead at the onset of the stimulus

Score 2 A mild contraction of the abdominal muscles but nolifting the abdomen off the platform

Score 3A strong contraction of the abdominal muscles andlifting the abdomen off the platform no lifting thepelvic structure off the platform

Score 4 Arching body and lifting the pelvic structure andscrotum

AWR abdominal withdrawal reflex CRD colorectal distention

development Neonatal male Sprague-Dawley rats (5-day-old) were supplied by the Experiment Animal Center Shang-hai Medical College Fudan University (permit no SCXK(Hu) 2009-0019) Prior to experimental manipulation ratswere allowed to acclimate for 3 days and were maintainedunder controlled conditions (20 plusmn 2∘C 60 plusmn 5 humidity)Every 6ndash8 neonatal rats were kept with a maternal rat in acage till 28 days old The neonatal rats were fed by rat milkand the maternal rats were fed with food pellets and water adlibitum

22 Induction of CVH and Behavioral Testing for CVHNeonatal rats were subjected to daily mechanical CRD atthe age of 8ndash21 days The procedure has been describedpreviously [23]Mainly balloon (constructed froma condomlength 20mm diameter 3mm)was inserted rectally into thedescending colonThe balloon was distended with 05mL airfor 1 minute and then deflated and withdrawnThe distentionwas repeated twice daily at a 30-minute intervalThe rats werereared until they reached adulthood (at least 6 week old) andbehavioral responses to visceral pain induced by acute CRDwere then examined

Abdominal withdrawal reflex (AWR) scoring systemwas adopted for behavioral assessment in the evaluationof visceral pain (Table 1) using a procedure as previouslydescribed [28] Briefly after anesthesia with ether a balloon(3 cm in length made using one finger of a latex glove)was inserted into the descending colon The rats were thenhoused in a small Lucite cubicles (20 cm times 8 cm times 8 cm)on a platform and allowed to wake up and adapt for 20minutes CRD was produced by rapidly inflating the balloonat different levels of CRD pressure (266 532 798 and1064 kPa) for 20 seconds respectively Each score was testedthree times and therewas a 5-minute interval between the twotests to allow the rats to adapt AWR scores were observedby two observers who were blinded to the experimentalconditions

23WarmingMoxibustion Treatment ThedetailedWMpro-cedure has been described previously [13] In brief warmingmoxibustion was given to bilateral TianShu (ST25 10mmlateral to the navel) and ShangJuXu (ST37 5mm lateral tothe anterior tubercle of the tibia and 20mm below the knee

Evidence-Based Complementary and Alternative Medicine 3

joint) igniting refined moxa stick (Nanyang Hanyi MoxaCo Ltd Nanyang China) (05 cm in diameter made ofrefined mugwort floss) (2 cm high from the acupoints) for10 minutes once daily 7 consecutive days Rats were notanesthetized before WM treatment and they were held ina supine position on one gloved hand Rats of both normalgroup and model group were also held on one glovedhand in supine position as controls but not given the WMtreatment

ST25 and ST37 were the two key acupoints chosen inthis study based on our clinical treatment of patients withIBS since the 1980s [29 30] In the theory of TCM ST25is the mu point of the large intestine and ST37 is the xiahepoint of the large intestine and they are also used fortreatment of abdominal pain diarrhea and constipationThe two pairs of acupoints (ST25 and ST37) were simul-taneously treated with WM Each treatment consisted of20 minutes of moxibustion (10 minutes for each pair ofacupoints)

24 Intrathecal Administration To investigate the influenceof the spinal dynorphin-120581 system in CRD-induced CVHit 30 nmoL10120583L dynorphinA (1-17) (Sigma USA) it100 nmoL10120583L 120581 antagonist nor-binaltorphimine (nor-BNISigma USA) 5-minute prior to dynorphinA (1-17) and it100 nmoL10120583L nor-BNI were performed to the CVH rats atthe age of 43 days respectively Each injection was followedby 10 120583L normal saline (NS) flush Control experimentswere performed by intrathecally injecting CVH rats with NSalone

To observe the influence of the spinal dynorphin-120581 systeminmoxibustion analgesia 30 nmoL10 120583L dynorphinA (1-17)and 100 nmoL10120583Lnor-BNIwere respectively administeredintrathecally to the CVH rats 15-minute before moxibustionat the age of 43ndash49 days

To investigate the influence of the spinal OFQ-OFQreceptor system in CRD-induced CVH it 5120583g10 120583L OFQ(Sigma USA) it 5120583g10 120583L OFQ receptor antagonist[Nphe1]nociceptin(1-13)NH

2(SigmaUSA) 5-minute prior to

OFQ and it 5 120583g10 120583L [Nphe1]nociceptin(1-13)NH2were

performed to the CVH rats at the age of 43 days respectivelyEach injection was followed by 10 120583L normal saline (NS)flush Control experiments were performed by intrathecallyinjecting CVH rats with NS alone

To observe the influence of the spinal OFQ-OFQ recep-tor system in moxibustion analgesia 5 120583g10 120583L OFQ and5 120583g10 120583L [Nphe1]nociceptin(1-13)NH

2were respectively

administered intrathecally to the CVH rats 15-minute beforemoxibustion at the age of 43ndash49 days

The procedures of intrathecal injection were as followsthe rat was put into a custom-made organic-glass box with02mL ether injected for anesthesiaThe lower body of the ratwas then pulled out from the box with the head kept in thebox for sustaining anesthesia A microsyringe of 25 120583L wasinserted through the gap between the L4 and L5 vertebraeand extended to the subarachnoid space of the lumbarenlargement Retain the needle for 10 seconds after injection

Disinfect the local area after withdrawing the syringe Therat usually come back to conscious within 3 minutes afterstaying free from ether which will not disturb the followingassessment of pain behavior Then CVH was assessed within30ndash90 minutes after the intervention

25 In Situ Hybridization In situ hybridization (ISH) wasutilized to identify the mRNA levels of preprodynorphin(PPD) 120581 receptor (KOR) OFQ and OFQ receptor Afterthe behavioral measurement the rats in each group weredeeply anaesthetized with sodium pentobarbital (100mgkgip) and perfused via the aorta with 200mL NS followedby 200mL 4 paraformaldehyde in 01M phosphate buffer(PB pH 74) The spinal cord from the L6-S1 segments wasremoved and postfixed in the fixative solution Each samplewas embedded in paraffin serially sliced into 5 120583m slicesSlices were passed through a dehydrating ethanol gradientand incubated with probe in hybridization solution at asaturated concentration under glass coverslips at 47∘C for18 h Coverslips were removed in 4times standard sodium citrateand nonspecifically bound probe was removed by treatmentwith RNase (Sigma USA) for 30 minutes Sections were runthrough stringency washes of 1times SSC and 05times SSC at 37∘Cand 01times SSC at 42∘C The antisense probes were providedby ShangHai DaWeiKe Biotechnology Co Ltd The PPDprobes are as follows 51015840-AGAGCCTGTCGCAGATGA-GGCCGATGAGGTGGAGCAGAAG-31015840 The KOR probesare as follows 51015840-GGTGGGCTTAGTGGGCAATTCCCT-GGTCATGTTTGTCATC-31015840TheOFQprobes are as follows51015840-ATTCGACGACTTCGTGGTGTCCGTCTGCTTTCA-GGCTCCC-31015840 The OFQ receptor probes are as follows51015840-GCGGCGTCAGTTCAAGGTGGTGACTCGGTCCCA-GGAG-31015840

26 Immunohistochemistry The expression of dynorphinand of OFQ in the spinal dorsal horn was detected byimmunohistochemistry Tissue sections were incubated inprimary polyclonal antidynorphin antibody (diluted 1 500Abcam USA) for 36 hours at 4∘C Then the bound pri-mary antibody was localized by a biotinylated secondaryantirabbit IgG (Boster CN) incubated for 1 hour in normalgoat serum in PBS and subsequently with the avidin biotincomplex (ABC kits) at room temperature for 1 hour Visu-alization of the antigen-antibody complex was performedusing DAB kits (Boster CN) The same immunohistochem-ical procedure was followed to determine OFQ (primaryantibody rabbit polyclonal to OFQ 1 500 Abcam USAsecondary antibody antirabbit IgG 1 400) All sections werethen cover-slipped with distyrene-plasticizer-xylene (SigmaUSA)

Three sections randomly selected from each animal werecaptured on theOlympusmicroscope equippedwith a Canoncamera The area and opacity density (OD) of immunoreac-tion in the spinal cord were measured analyzed by MoticMed 60 (Motic China Group China) and averaged forsections of each rat and then averaged for the group Yellow-brown staining granulation was observed with a purple-bluebackground


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Figure 1 The antiCVH effects of repeated WM in IBS-like rats WM treatments were performed beginning on the 43rd day and continuingto the 49th day (10minutes once daily) Data are presented as the mean plusmn SEM (119899 = 8 per group) lowastlowast119875 lt 001 lowast119875 lt 005 versus normalgroup 119875 lt 001 versus model group WM warming moxibustion AWR abdominal withdrawal reflex

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Figure 2 (a) AWR was used for behavioral assessment to evaluation effects of it dynorphinA (1-17) orand 120581 receptor antagonist on CVH atdifferent levels of CRD stimuli (266 532 798 and 1064 kPa) Data are presented as the mean plusmn SEM (119899 = 6 per group) lowastlowast119875 lt 001 versusnormal group 119875 lt 001 119875 lt 005 versus model group 998771998771119875 lt 001 998771119875 lt 005 versus DYN group DYN dynorphinA (1-17) nor-BNI 120581antagonist nor-binaltorphimine (b) AWRwas used for behavioral assessment to evaluation effects of it dynorphinA (1-17) orand 120581 receptorantagonist on moxibustion analgesia at different levels of CRD stimuli (266 532 798 and 1064 kPa) Data are presented as the mean plusmnSEM (119899 = 6 per group) lowastlowast119875 lt 001 versus normal group 119875 lt 001 versus model group 998771998771119875 lt 001 998771119875 lt 005 versus WM group DYNdynorphinA (1-17) nor-BNI 120581 antagonist nor-binaltorphimine WM warming moxibustion

27 Enzyme-Linked Immunosorbent Assay Concentrationsof spinal dynorphin and OFQ were detected by enzyme-linked immunosorbent assay (ELISA) The L6-S1 spinalcord was removed and stored at minus80∘C until sonicationTotal protein was dissociated mechanically from tissueusing an ultrasonic cell disruptor and then centrifuged at

3000timesg for 15 minutes Supernatant was removed andstored at minus20∘C until analysis Dynorphin and OFQ werequantified using ELISA kits (RayBiotech USA) accordingto the manufacturerrsquos protocol Measurement was com-pleted using an ELISA with an absorbency maximum at450 nm


28 Statistical Analysis All data were analyzed using SPSS110 statistical software (SPSS Inc USA) and expressed asmean plusmn SEM One-way ANOVA-LSD test was used if thevariance was homogenous Dunnettrsquos T3 test was used ifthe variance was heterogenous 119875 lt 005 was consideredstatistically significant

3 Results

31 Experiment 1 Analgesic Effects of Moxibustion on CVH inIBS-Like Rat Model As shown in Figure 1 at different levelsof CRD stimuli (266 532 798 and 1064KPa) the AWRscores were remarkably higher in model group than that innormal group (119875 lt 001) and lower in WM group than thatin model group (119875 lt 001) indicating a therapeutic antiCVHeffect of WM treatment in IBS-like rats induced by CRD

32 Experiment 2 Moxibustion Exert Analgesic Effect byActivating Spinal Dynorphin-120581 System First we investi-gated whether the spinal dynorphin-120581 system could produceinhibitory effect on CVH by a behavioral test As shownin Figure 2(a) at different levels of CRD stimuli (266 532798 and 1064 kPa) the AWR scores in model group wereobviously higher than in normal group (119875 lt 001) and inDYN group (119875 lt 001 119875 lt 005) and higher in DYN+nor-BNI group than that in DYN group (119875 lt 001 119875 lt 005)and there was no significant difference among DYN+nor-BNI model and NS group These data indicated that itdynorphin alleviated the CRD-induced CVH by binding tothe 120581 receptor

Next we investigated whether the spinal dynorphin-120581system was associated with the moxibustion analgesia Asshown in Figure 2(b) at different levels of CRD stimuli anobvious increase of AWR scores was shown in nor-BNI+WMgroup compared to WM group (119875 lt 001) and at stimuliof 532 and 798 kPa the AWR scores in DYN+WM groupwere lower than that in WM group (119875 lt 005) It suggestedthat it 120581 antagonist could attenuate the analgesic effect ofmoxibustion and it dynorphin may enhance it

We further tested the expression of dynorphin and 120581receptor in L6-S1 spinal dorsal horn by ISH immunohis-tochemistry and ELISA The expression of PPD mRNA inmodel group was higher than that in normal group (119875 lt001) and WM treatment further produced a marked eleva-tion of PPD mRNA (119875 lt 001 119875 lt 005) (Figure 3) At theprotein level the expression of dynorphin-immunoreactionwas higher in model group than that in normal group(119875 lt 005) and dense immunoreactivity for dynorphinwas shown after WM treatment (119875 lt 005) Similarly theresults of the ELISA showed that the dynorphin increasedremarkably during CVH and was further enhanced by WMtreatment (119875 lt 005) (Figure 4) ISH showed that theexpression of spinal KOR mRNA increased during CVHand was further remarkably enhanced by WM treatment(119875 lt 001 119875 lt 005) (Figure 5) These data indicatedthat WM treatment induced the expression of dynorphinand enhanced the transcription of KOR mRNA in CVHrats

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119875 lt 001 versus normal group 119875 lt 001 119875 lt 005 versusmodelgroup WM warming moxibustion

33 Experiment 3 Moxibustion Exert Analgesic Effect byActivating Spinal OFQ-OFQ Receptor System As shown inFigure 6(a) at different levels of CRD stimuli (266 532 798and 1064 kPa) the AWR scores in model group were higherthan that in OFQ group (119875 lt 001 119875 lt 005) The AWRscores were higher in OFQ+Nphe1 group than that in OFQgroup (119875 lt 001 119875 lt 005) These data indicated that itOFQ alleviated the CRD-induced CVH by binding to theOFQ receptor

As shown in Figure 6(b) at different levels of CRDstimuli an obvious increase of AWR scores was shown inNphe1+WM group compared to WM group (119875 lt 001 119875 lt005) and the AWR scores in OFQ+WM group was lowerthan that in WM group (119875 lt 005) It suggested that it[Nphe1]NC(1-13)NH

2could attenuate the analgesic effect of

moxibustion on CVH and it OFQ could enhance itThe expression of OFQ and of its receptor in L6-S1 spinal

dorsal horn was also tested by ISH immunohistochemistryand ELISA As shown in Figure 7 ISH examination revealedthat the expression ofOFQmRNA inmodel groupwas higherthan that in normal group (119875 lt 001) and WM treatmentfurther produced a marked elevation of OFQ mRNA (119875 lt001) As shown in Figure 8 the level of OFQ protein washigher in model group than that in normal group (119875 lt 005)


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and significantly increased after WM treatment (119875 lt 005)ISH showed that the expression of spinal OFQ receptormRNA increased during CVH and was further remarkablyenhanced byWM treatment (119875 lt 001) (Figure 9)These dataindicated thatWM treatment induced the expression of OFQand enhanced the transcription of OFQ receptor mRNA inCVH rats

4 Discussion

Moxibustion has been adopted as an analgesic method forthousands of years in China and it originated in the applica-tion of fire and then continuously developed in practice [31]The primitive men living in damp-cold caves were sufferingfrom pain in joints and the symptoms could be relievedby basting in sunshine or fire pressed by heated sand orsoil abdomen could be relieved from pain applied fire to acertain part of the skin toothache and stomachache couldbe attenuated All of these archaeological investigations werethe embryonic form of moxibustion therapy and also the

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beginning of moxibustion analgesia [32] It has been shownthat moxibustion could effectively treat human primarydysmenorrheal [33] knee osteoarthritis [34] rheumatoidarthritis [35] and cancer pain [36]However themechanismsof moxibustion analgesia are not elucidated

In the present study the behavioral results showed that 7times ofWMat ST25 and ST37 significantly attenuated CRD-induced CVH in IBS-like rats which is consistent with ourprevious studies [10ndash14] It should be mentioned that WMtreatment in the present and previous animal studies onlypartially inhibited CVHThismay be due to the fact thatWMtreatment in animal studies only involves stimulation of twoacupoints which also supports the clinical usage of multipleacupoints

Here the behavioral results demonstrated that intrathe-cal administration of dynorphinA (1-17) in analgesic doserelieved CVH and 120581 receptor antagonist reversed the anti-hyperalgesic effect indicating that dynorphinA (1-17) acts on120581 receptors to impede nociceptive signals at the spinal level inthis rat modelThe expression of dynorphin in the superficialdorsal horn was found to be increased in both mRNA andprotein levels in model rats and the data also indicatedthat KOR mRNA transcription was induced in CHV Thissuggests that spinal dynorphin-120581 system could be an initiatedexposure to nociceptive stimuli in CVH rats It was reportedthat the increases of endogenous analgesic substances during


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phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

998771998771998771998771

998771

998771

998771

998771

(b)

Figure 6 (a) AWR was used for behavioral assessment to evaluation the effects of it orphanin FQ orand OFQ receptor antagonist[Nphe1]nociception(1-13)NH

2

on CVH at different levels of CRD stimuli (266 532 798 and 1064 kPa) Data are presented as the meanplusmn SEM (119899 = 6 per group) lowastlowast119875 lt 001 versus normal group 119875 lt 001 119875 lt 005 versus model group 998771998771119875 lt 001 998771119875 lt 005 versus OFQgroup OFQ orphanin FQ Nphe1 OFQ receptor antagonist [Nphe1]nociceptin (1-13)NH

2

(b) AWR was used for behavioral assessment toevaluate the effects of it orphanin FQ orand [Nphe1]nociception(1-13)NH

2

onmoxibustion analgesia at different levels of CRD stimuli (266532 798 and 1064 kPa) Data are presented as the mean plusmn SEM (119899 = 6 per group) lowastlowast119875 lt 001 versus normal group 119875 lt 001 versus modelgroup 998771998771119875 lt 001 998771119875 lt 005 versus WM group OFQ orphanin FQ Nphe1 OFQ receptor antagonist [Nphe1]nociception(1-13)NH

2

WMwarming moxibustion

pain are important to maintain homeostasis [37] Thusalthough dynorphin-120581 system is known to be an antihyperal-gesic agent it may be upregulated following exposure to thenociceptive stimulusThis result is in accordance with studiesthat demonstrated that the expression of antihyperalgesicagents such as neurotrophin-3 cannabinoid receptor-2 andinterleukin-10 is significantly increased in rats with pain [37]

However our pilot study employed ELISA to determinespinal dynorphin and found a decreased dynorphin conflictto the present result in CVH rats [13]Then we again adoptedELISA to determine the concentration of the dynorphinThe data showed that dynorphin concentration in L6-S1spinal cord was largely increased in model group than thatin normal group which was in favor of the morphologicalresults The decreased dynorphin level in the pilot studymight be due to a comparative shortage of dynorphinproteins produced by the imbalance between release andsynthesis

We also found that WMrsquos analgesic effects in CVH ratswere enhanced by it dynorphinA (1-17) and partly attenuatedfollowing 120581 antagonist intervention indicating the involve-ment of spinal that dynorphin-120581 system in moxibustionanalgesia Also the data showed dynorphin mRNAproteinand KOR mRNA were largely increased following 7 times ofWM treatment indicating that WM relieved visceral pain byactivating spinal dynorphin-120581 system

Notably regarding the behavioral results in Figure 2(b)the AWR score was increased under the it 120581 antagonistintervention but without statistical significance at 532 KPastimuli and the AWR score was reduced under dynorphinA(1-17) intervention but without statistical significance at1064KPa stimuli In our opinion this change may be due tothe dose of dynorphinA (1-17) and 120581 antagonist

In agreement with our findings numerous studiesdemonstrate that dynorphin-120581 system acts in an antinocicep-tive manner in the spinal cord [38ndash40] However conflicting


Normal

(a)

(b)

(c)

Model WM

Normal Model WM

OFQ

mRN

A (a

rea)

OFQ

mRN

A (O

D)

15

10

5

0

20000

15000

10000

5000

0

lowastlowast

lowastlowast

Figure 7 Effects of WM on the expression of OFQ mRNA in L6-S1 spinal cord (a) Spinal dorsal horn from normal group (times100)(b) Spinal dorsal horn from model group (times100) (c) Spinal dorsalhorn fromWMgroup (times100) Data are presented as themeanplusmn SEM(119899 = 6 per group) lowastlowast119875 lt 001 versus normal group

119875 lt 001

versus model group WM warming moxibustion

reports showed that 120581 receptor cannot modulate visceralnociceptive transmission at the spinal level [41 42] Causes ofthese conflicting reports are still unclear Different rat modelsof visceral pain and the use of kappa receptor agonist may beinvolved so further investigation would be necessary

The outcome of research on dynorphin-120581 system inspiredour study on OFQ-OFQ receptor system and we presumedthat the effect of moxibustion analgesia might be due to theinduction of OFQ-OFQ receptor system in the spinal cordThe present study provides the convincing evidence for ourpresumption and we found that WMrsquos analgesic effects inCVH rats were enhanced by intrathecal administration ofOFQ and partly attenuated following OFQ receptor antago-nist indicating the involvement of spinal OFQ-OFQ receptorsystem in moxibustion analgesia Also the data showedthat OFQ and OFQ receptor mRNA were largely increasedfollowing 7 times ofWM treatment indicating thatWMmayrelieve visceral pain by activating spinal OFQ-OFQ receptorsystem

Taken together and based on the mechanism of acupunc-ture analgesia we infer further that moxibustion at ST 37and ST 25 could stimulate the special receptors in acupointsand produce heat signals projected to CNS in the processof transmission the heat signals may partly block the inputof pain sensation in the spinal dorsal horn by activatingthe segmental inhibition (including both presynaptic andpostsynaptic inhibition) or initiating the descending inhi-bition system or both The integrated action of segmental

Normal(a)

(b)

(c)

Model WM

OFQ

pro

tein

(are

a)

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

OFQ

pro

tein

(OD

)

lowastlowast

15

10

5

0

Normal Model WM

OFQ

pro

tein

(pg

mL)

lowastlowast

40

30

20

10

0

Figure 8 Effects of WM on the expression of orphanin FQ in L6-S1spinal cord tested by immunohistochemistry and ELISA (a) Spinaldorsal horn from normal group (times100) (b) Spinal dorsal horn frommodel group (times100) (c) Spinal dorsal horn fromWM group (times100)Data are presented as themeanplusmn SEM (119899 = 6 per group) lowastlowast119875 lt 001versus normal group 119875 lt 001 versusmodel groupWMwarmingmoxibustion

inhibition and descending inhibition may activate the spinaldynorphin and OFQ system modulate the release of excita-tory neurotransmitter in primary afferent fibers and suppressthe response of spinal neurons to the noxious stimuli Thepathway of the thermal stimulation signal is similar to thatof algesic signal [43] which provides a morphological basisfor the interaction of the two signalsThe neural mechanismsof moxibustion analgesia should be complicated and it is pos-sible that moxibustion may modulate the activities of variouspeptides or neurotransmitters to inhibit pain However moreconcrete evidence is required from future investigations

5 Conclusions

Our results demonstrate that WM treatment may attenuateCRD-induced chronic visceral hyperalgesia in IBS-like ratsand that this effect may be due at least in part to WMinduction of spinal dynorphin and orphanin-FQ system

Authorsrsquo Contribution

L Qi and H-R Liu contributed equally to this work


OFQ

rece

ptor

mRN

A (a

rea)

OFQ

rece

ptor

mRN

A (O

D)

20

15

10

5

0

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

Normal Model WM

(a)

(b)

(c)

Figure 9 Effects of WM on the expression of OFQ receptor mRNAin L6-S1 spinal cord (a) Spinal dorsal horn from normal group(times100) (b) Spinal dorsal horn from model group (times100) (c) Spinaldorsal horn fromWMgroup (times100) Data are presented as themeanplusmn SEM (119899 = 6 per group) lowastlowast119875 lt 001 versus normal group119875 lt 001 versus model group WM warming moxibustion

Acknowledgments

Many thanks go to Dr Yong Tang of Chengdu University ofTraditional Chinese Medicine for his invaluable suggestionsand advice for revision of this paper Many thanks go toDr Xiu li of Shanghai Medical College of Fudan Universityfor providing information on intrathecal administrationThiswork was supported by the National Basic Research Programof China (2009CB522905) and National Natural ScienceFoundation (30973783)

References

[1] F Azpiroz M Bouin M Camilleri et al ldquoMechanisms ofhypersensitivity in IBS and functional disordersrdquo Neurogas-troenterology and Motility vol 19 no 1 supplement pp 62ndash882007

[2] K Wolitzky-Taylor M G Craske J S Labus E A Mayer andB D Naliboff ldquoVisceral sensitivity as a mediator of outcome inthe treatment of irritable bowel syndromerdquo Behaviour ResearchandTherapy vol 50 no 10 pp 647ndash650 2012

[3] S Bradesi J Herman and E A Mayer ldquoVisceral analgesicsdrugs with a great potential in functional disordersrdquo CurrentOpinion in Pharmacology vol 8 no 6 pp 697ndash703 2008

[4] X Wang S Zhou W Yao et al ldquoEffects of moxibustionstimulation on the intensity of infrared radiation of tianshu(ST25) acupoints in rats with ulcerative colitisrdquo Evidence-BasedComplementary and Alternative Medicine vol 2012 Article ID704584 13 pages 2012

[5] X M Shi ldquoIndications and prospects of acupuncture-moxibustionrdquo Zhongguo Zhen Jiu vol 31 no 11 pp 961ndash9642011

[6] C H HanM S Shin S H Shin KW Kang B K Kang and SH Park ldquoAn in-depth interview for use of moxibustion therapyin Koreardquo Journal of Meridian and Acupoint vol 25 pp 85ndash972008

[7] O Kaoru and K Kenj ldquoAnalgesic action of acupuncture andmoxibustion a reviewof unique approaches in JapanrdquoEvidence-Based Complementary andAlternativeMedicine vol 6 no 1 pp7ndash11 2009

[8] H G Wu C Zhao and Z Shi ldquoClinical study on spleen-stomach-reinforcing moxibustion treatment of diarrhea-typeirritable bowel syndromerdquo World Journal of Acupuncture-Moxibustion vol 12 pp 10ndash15 2002

[9] H R Liu X G Hua and Y Yang ldquoClinical study on theexpression of 5-HT in colon mucosa and herb-partitionedmoxibustion in diarrhea-predominant IBSrdquo Liao Ning Zhong YiYao Za Zhi vol 33 pp 984ndash985 2006

[10] L Y Wu C H Bao L B Ge et al ldquoMild moxibustion atTianshu (ST 25) decreases expression of prokineticin-1 andprokineticin receptor-1 in colon tissue of rats with chronicvisceral hyperalgesiardquo Neural Regeneration Research vol 6 pp2600ndash2604 2011

[11] E H Zhou X M Wang G H Ding et al ldquoSuspended mox-ibustion relieves chronic visceral hyperalgesia and decreaseshypothalamic corticotropin-releasing hormone levelsrdquo WorldJournal of Gastroenterology vol 17 no 5 pp 662ndash665 2011

[12] T Yi L Qi H G Wu X P Ma H R Liu and X MWang ldquoAnalgesic action of suspended moxibustion in rats withchronic visceral hyperalgesia correlates with enkephalins in thespinal cordrdquo Neural Regeneration Research vol 3 pp 219ndash2222012

[13] H R Liu L Qi L Y Wu et al ldquoEffects of moxibustionon dynorphin and endomorphin in rats with chronic visceralhyperalgesiardquoWorld Journal of Gastroenterology vol 16 no 32pp 4079ndash4083 2010

[14] E H Zhou H R Liu H GWu et al ldquoSuspended moxibustionrelieves chronic visceral hyperalgesia via serotonin pathway inthe colonrdquoNeuroscience Letters vol 451 no 2 pp 144ndash147 2009

[15] E H Zhou H R Liu H G Wu et al ldquoHerb-partitionmoxibustion relieves chronic visceral hyperalgesia and 5-HTconcentration in colon mucosa of ratsrdquo Neurological Researchvol 31 no 7 pp 734ndash737 2009

[16] P M Lang J Stoer G M Schober J F Audette and D IrnichldquoBilateral acupuncture analgesia observed by quantitative sen-sory testing in healthy volunteersrdquo Anesthesia and Analgesiavol 110 no 5 pp 1448ndash1456 2010

[17] MC LuoQChenMHOssipovD R Rankin F Porreca andJ Lai ldquoSpinal dynorphin and bradykinin receptors maintaininflammatory hyperalgesiardquo Journal of Pain vol 9 no 12 pp1096ndash1105 2008

[18] R X Zhang B Liu L Lao J T Qiao and M A Ruda ldquoSpinalpreprodynorphin mRNA expression in neonatal rats followingperipheral inflammationrdquo Brain Research vol 1038 no 2 pp238ndash242 2005

[19] T P Malan M H Ossipov L R Gardell et al ldquoExtraterritorialneuropathic pain correlates with multisegmental elevation ofspinal dynorphin in nerve-injured ratsrdquo Pain vol 86 no 1-2pp 185ndash194 2000

[20] M J Schwei P Honore S D Rogers et al ldquoNeurochemical andcellular reorganization of the spinal cord in a murine model of


bone cancer painrdquo Journal of Neuroscience vol 19 no 24 pp10886ndash10897 1999

[21] J S Han and G X Xie ldquoDynorphin important mediator forelectroacupuncture analgesia in the spinal cord of the rabbitrdquoPain vol 18 no 4 pp 367ndash376 1984

[22] J G Lin and W L Chen ldquoAcupuncture analgesia a review ofits mechanisms of actionsrdquo The American Journal of ChineseMedicine vol 36 no 4 pp 635ndash645 2008

[23] K Erb J T Liebel I Tegeder H U Zeilhofer K Bruneand G Geisslinger ldquoSpinally delivered nociceptinorphaninFQ reduces flinching behaviour in the rat formalin testrdquoNeuroReport vol 8 no 8 pp 1967ndash1970 1997

[24] I Obara R Przewlocki and B Przewlocka ldquoSpinal and localperipheral antiallodynic activity of Ro64-6198 in neuropathicpain in the ratrdquo Pain vol 116 no 1-2 pp 17ndash25 2005

[25] X Fu Y Q Wang J Wang J Yu and G C Wu ldquoChanges inexpression of nociceptinorphanin FQ and its receptor in spinaldorsal horn during electroacupuncture treatment for peripheralinflammatory pain in ratsrdquo Peptides vol 28 no 6 pp 1220ndash1228 2007

[26] C S Jia J Xu X F Li and J L Wang ldquoSpecificity ofacupuncture-moxibustion effect and its research valuerdquo Zhong-guo Zhen Jiu vol 31 no 7 pp 577ndash579 2011

[27] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983

[28] E D Al-ChaerM Kawasaki and P J Pasricha ldquoA newmodel ofchronic visceral hypersensitivity in adult rats induced by colonirritation during postnatal developmentrdquo Gastroenterology vol119 no 5 pp 1276ndash1285 2000

[29] H G Wu J H Wang H P Chen and H Z Shi ldquoExplorationof efficacy and immunological mechanism for IBS treated byherb-partition moxibustionrdquo Zhongguo Zhen Ji vol 16 pp 43ndash45 1996

[30] J H Wang H G Wu and H P Chen ldquoClinical research of 28cases of patients with irritable bowel syndrome treated by herb-partition moxibustionrdquo Shanghai Journal of Acu-Mox vol 14pp 6ndash7 1995

[31] R Zhang ldquoHistory and current state of moxibustionrdquo Journal ofChinese Integrative Medicine vol 2 no 6 pp 466ndash473 2004

[32] D Harper Early Chinese Medical Literature The MawangduiMedical Manuscripts Kegan Paul International London UK1998

[33] Y Zhu R L Chen J I Le and F R Miao ldquoEfficacy obser-vation of primary dysmenorrhea treated with isolated-herbalmoxibustion on Shenque (CV 8)rdquo Chinese Acupuncture ampMoxibustion vol 30 no 6 pp 453ndash455 2010

[34] R Chen M Chen M Kang et al ldquoThe design and protocolof heat-sensitive moxibustion for knee osteoarthritis a mul-ticenter randomized controlled trial on the rules of selectingmoxibustion locationrdquo BMC Complementary and AlternativeMedicine vol 25 no 10 article 32 2010

[35] C Li Z Jiang and Y Li ldquoTherapeutic effect of needle warmingthrough moxibustion at twelve shu points on rheumatoidarthritisrdquo Journal of Traditional Chinese Medicine vol 19 no1 pp 22ndash26 1999

[36] Z J Chen Y P Guo and Z C Wu ldquoAdvances of clinical studyon acupuncture and moxibustion for treatment of cancer painrdquoZhongguo Zhen Jiu vol 28 no 5 pp 392ndash394 2008

[37] W L Mi Q L Mao-Ying X W Wang et al ldquoInvolvementof spinal neurotrophin-3 in electroacupuncture analgesia and

inhibition of spinal glial activation in rat model of monoarthri-tisrdquoThe Journal of Pain vol 12 no 9 pp 974ndash984 2011

[38] R Przewłocki L Stala M Greczek G T Shearman BPrzewłocka and A Herz ldquoAnalgesic effects of mu- delta- andkappa-opiate agonists and in particular dynorphin at the spinallevelrdquo Life Sciences vol 33 supplement 1 pp 649ndash652 1983

[39] T Pelissier C Paeile R Soto-Moyano H Saavedra and AHernandez ldquoAnalgesia produced by intrathecal administrationof the 120581 opioid agonist U-50488H on formalin-evoked cuta-neous pain in the ratrdquo European Journal of Pharmacology vol190 no 3 pp 287ndash293 1990

[40] H Nagasaka H Awad and T L Yaksh ldquoPeripheral andspinal actions of opioids in the blockade of the autonomicresponse evoked by compression of the inflamed knee jointrdquoAnesthesiology vol 85 no 4 pp 808ndash816 1996

[41] R M Danzebrink S A Green and G F Gebhart ldquoSpinal muand delta but not kappa opioid-receptor agonists attenuateresponses to noxious colorectal distension in the ratrdquo Pain vol63 no 1 pp 39ndash47 1995

[42] K Tan-No A Esashi O Nakagawasai et al ldquoIntrathecallyadministered big dynorphin a prodynorphin-derived pep-tide produces nociceptive behavior through an N-methyl-D-aspartate receptor mechanismrdquo Brain Research vol 952 no 1pp 7ndash14 2002

[43] M F Bear B W Connors and M A Paradiso NeuroscienceExploring the Brain 2nd edition 2004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


above the skin) and herb-partition moxibustion (indirectburning interposed by various materials) warming moxibus-tion (WM) is the most practical and convenient one from theperspective of clinical application

Our previous studies also revealed the analgesic effectsof moxibustion in reducing abdominal pain in IBS patients[8 9] and IBS rat models [10ndash15] We also reported that WMtreatment could alleviate CVH in IBS-like rats by suppressinghypothalamic corticotrophin-releasing-hormone level [11]which provides the evidence that the analgesic effect ofmoxibustion may be associated with the central nervoussystem (CNS)

It has been proved that the afferent signals of viscera couldbe converged and affected by the signals of superficial bodyin CNS This is the essential mechanism of stimulating acu-points for treating visceral disease [16]The spinal dorsal hornis the first integration center in CNS and then what role doesthe spinal cord play in the analgesic effect of moxibustionWhat are the kinds of chemicals that participate in the processof CVH and the analgesic effect of moxibustion in IBS-likerats

Many experimental models of pathological pain includ-ing inflammatory pain [17 18] neuropathic pain [19] andbone cancer pain [20] show a significant regional elevationof dynorphin and 120581 receptor in the spinal cord The earlyresearch has reported that dynorphin could act on 120581 recep-tor to impede nociceptive signals in the spinal cord andacupuncture could exert its analgesic action by activatingthe spinal dynorphin-120581 opioid system [21 22] A variety ofpain assays in rodents have shown the effect of intrathe-cally administered orphanin-FQ (OFQ) to be antinoci-ceptive [23 24] and it was also reported that acupunc-ture could exert an analgesic effect by activating spinalOFQ-OFQ receptor system in inflammatory pain model[25]

Both acupuncture and moxibustion are considered to bethe external therapy [26] and based on the mechanism ofacupuncture analgesia we presumed that the moxibustionanalgesia in CVH rats might be due to the induction ofdynorphin and orphanin-FQ system in the spinal cord Weperformed the pilot experiment and found that WM couldincrease the level of spinal dynorphin in CVH rats [13] whichholds our interests to further study the spinal dynorphin andorphanin-FQ system in moxibustion analgesia

Therefore using a well characterized rat model of IBS-like CVH the present study was intended to unfold theinvolvement of spinal dynorphin and orphanin-FQ system inmoxibustion analgesia

2 Materials and Methods

21 Animals Experiments were conducted strictly in accor-dance with the National Institute of Health Guide for theCare and Use of Laboratory Animals and the Guidelinesof the International Association for the Study of Pain [27]All efforts were made to minimize the number of animalsused and their suffering Rat model of CVH was inducedby mechanical colorectal distention (CRD) during postnatal

Table 1 AWR scoring criteria

Score 0 No behavioral response to CRD

Score 1 Immobile during the CRD and occasionally clicked thehead at the onset of the stimulus

Score 2 A mild contraction of the abdominal muscles but nolifting the abdomen off the platform

Score 3A strong contraction of the abdominal muscles andlifting the abdomen off the platform no lifting thepelvic structure off the platform

Score 4 Arching body and lifting the pelvic structure andscrotum

AWR abdominal withdrawal reflex CRD colorectal distention

development Neonatal male Sprague-Dawley rats (5-day-old) were supplied by the Experiment Animal Center Shang-hai Medical College Fudan University (permit no SCXK(Hu) 2009-0019) Prior to experimental manipulation ratswere allowed to acclimate for 3 days and were maintainedunder controlled conditions (20 plusmn 2∘C 60 plusmn 5 humidity)Every 6ndash8 neonatal rats were kept with a maternal rat in acage till 28 days old The neonatal rats were fed by rat milkand the maternal rats were fed with food pellets and water adlibitum

22 Induction of CVH and Behavioral Testing for CVHNeonatal rats were subjected to daily mechanical CRD atthe age of 8ndash21 days The procedure has been describedpreviously [23]Mainly balloon (constructed froma condomlength 20mm diameter 3mm)was inserted rectally into thedescending colonThe balloon was distended with 05mL airfor 1 minute and then deflated and withdrawnThe distentionwas repeated twice daily at a 30-minute intervalThe rats werereared until they reached adulthood (at least 6 week old) andbehavioral responses to visceral pain induced by acute CRDwere then examined

Abdominal withdrawal reflex (AWR) scoring systemwas adopted for behavioral assessment in the evaluationof visceral pain (Table 1) using a procedure as previouslydescribed [28] Briefly after anesthesia with ether a balloon(3 cm in length made using one finger of a latex glove)was inserted into the descending colon The rats were thenhoused in a small Lucite cubicles (20 cm times 8 cm times 8 cm)on a platform and allowed to wake up and adapt for 20minutes CRD was produced by rapidly inflating the balloonat different levels of CRD pressure (266 532 798 and1064 kPa) for 20 seconds respectively Each score was testedthree times and therewas a 5-minute interval between the twotests to allow the rats to adapt AWR scores were observedby two observers who were blinded to the experimentalconditions

23WarmingMoxibustion Treatment ThedetailedWMpro-cedure has been described previously [13] In brief warmingmoxibustion was given to bilateral TianShu (ST25 10mmlateral to the navel) and ShangJuXu (ST37 5mm lateral tothe anterior tubercle of the tibia and 20mm below the knee











2were respectively








2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

266 kPa 532 kPa 798 kPa 1064 kPa


lowastlowast

lowastlowast

lowast

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1



266 kPa 532 kPa 798 kPa 1064 kPa2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

DYN

Nor

-BN

I

NS

DYN

+ n

or-B

NI

Nor

mal

Mod

el

DYN

Nor

-BN

I

NS

DYN

+ n

or-B

NI

Nor

mal

Mod

el

DYN

Nor

-BN

I

NS

DYN

+ n

or-B

NI

Nor

mal

Mod

el

DYN

Nor

-BN

I

NS

DYN

+ n

or-B

NI



998771998771

998771998771

998771998771998771

(a)

266 kPa 532 kPa 798 kPa 1064 kPa2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

WM

WM

+ D

YN

WM

+ n

or-B

NI

Nor

mal

Mod

el

WM

WM

+ D

YN

WM

+ n

or-B

NI

Nor

mal

Mod

el

WM

WM

+ D

YN

WM

+ n

or-B

NI

Nor

mal

Mod

el

WM

WM

+ D

YN

WM

+ n

or-B

NI

lowastlowast

lowastlowast

lowastlowast

lowastlowast

998771998771

998771

998771998771

998771

998771998771

(b)






3 Results





lowastlowast

PPD

mRN

A (a

rea)

(a)

(b)

(c)

(d)30000

20000

10000

0Normal Model WM

lowastlowast

PPD

mRN

A (O

D)

Normal Model WM

20

15

10

5

0









lowast

lowast

lowast

Normal Model WM

Normal Model WM

Normal(a)

(b)

(c)

Model WM20

15

10

5

0

6000

8000

4000

2000

0

300

200

100

0

Dyn

pro

tein

(pg

mL)

Dyn

pro

tein

(OD

)D

yn p

rote

in (a

rea)



4 Discussion


lowast

lowast

Normal

Normal

(a)

(b)

(c)Model

Model

WM

WM

20

15

10

5

0

KOR

mRN

A (O

D)

KOR

mRN

A (a

rea)

25000

20000

15000

10000

5000

0






2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

lowastlowast


lowastlowast

266 kPa 532 kPa 798 kPa 1064 kPa

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

998771998771998771 998771998771

998771

(a)

2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

lowastlowast

lowastlowast


266 kPa 532 kPa 798 kPa 1064 kPa

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

998771998771998771998771

998771

998771

998771

998771

(b)


2


2


2


2








Normal

(a)

(b)

(c)

Model WM

Normal Model WM

OFQ

mRN

A (a

rea)

OFQ

mRN

A (O

D)

15

10

5

0

20000

15000

10000

5000

0

lowastlowast

lowastlowast


119875 lt 001





Normal(a)

(b)

(c)

Model WM

OFQ

pro

tein

(are

a)

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

OFQ

pro

tein

(OD

)

lowastlowast

15

10

5

0

Normal Model WM

OFQ

pro

tein

(pg

mL)

lowastlowast

40

30

20

10

0



5 Conclusions





OFQ

rece

ptor

mRN

A (a

rea)

OFQ

rece

ptor

mRN

A (O

D)

20

15

10

5

0

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

Normal Model WM

(a)

(b)

(c)


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























2were respectively








2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

266 kPa 532 kPa 798 kPa 1064 kPa


lowastlowast

lowastlowast

lowast

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1



266 kPa 532 kPa 798 kPa 1064 kPa2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

DYN

Nor

-BN

I

NS

DYN

+ n

or-B

NI

Nor

mal

Mod

el

DYN

Nor

-BN

I

NS

DYN

+ n

or-B

NI

Nor

mal

Mod

el

DYN

Nor

-BN

I

NS

DYN

+ n

or-B

NI

Nor

mal

Mod

el

DYN

Nor

-BN

I

NS

DYN

+ n

or-B

NI



998771998771

998771998771

998771998771998771

(a)

266 kPa 532 kPa 798 kPa 1064 kPa2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

WM

WM

+ D

YN

WM

+ n

or-B

NI

Nor

mal

Mod

el

WM

WM

+ D

YN

WM

+ n

or-B

NI

Nor

mal

Mod

el

WM

WM

+ D

YN

WM

+ n

or-B

NI

Nor

mal

Mod

el

WM

WM

+ D

YN

WM

+ n

or-B

NI

lowastlowast

lowastlowast

lowastlowast

lowastlowast

998771998771

998771

998771998771

998771

998771998771

(b)






3 Results





lowastlowast

PPD

mRN

A (a

rea)

(a)

(b)

(c)

(d)30000

20000

10000

0Normal Model WM

lowastlowast

PPD

mRN

A (O

D)

Normal Model WM

20

15

10

5

0









lowast

lowast

lowast

Normal Model WM

Normal Model WM

Normal(a)

(b)

(c)

Model WM20

15

10

5

0

6000

8000

4000

2000

0

300

200

100

0

Dyn

pro

tein

(pg

mL)

Dyn

pro

tein

(OD

)D

yn p

rote

in (a

rea)



4 Discussion


lowast

lowast

Normal

Normal

(a)

(b)

(c)Model

Model

WM

WM

20

15

10

5

0

KOR

mRN

A (O

D)

KOR

mRN

A (a

rea)

25000

20000

15000

10000

5000

0






2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

lowastlowast


lowastlowast

266 kPa 532 kPa 798 kPa 1064 kPa

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

998771998771998771 998771998771

998771

(a)

2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

lowastlowast

lowastlowast


266 kPa 532 kPa 798 kPa 1064 kPa

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

998771998771998771998771

998771

998771

998771

998771

(b)


2


2


2


2








Normal

(a)

(b)

(c)

Model WM

Normal Model WM

OFQ

mRN

A (a

rea)

OFQ

mRN

A (O

D)

15

10

5

0

20000

15000

10000

5000

0

lowastlowast

lowastlowast


119875 lt 001





Normal(a)

(b)

(c)

Model WM

OFQ

pro

tein

(are

a)

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

OFQ

pro

tein

(OD

)

lowastlowast

15

10

5

0

Normal Model WM

OFQ

pro

tein

(pg

mL)

lowastlowast

40

30

20

10

0



5 Conclusions





OFQ

rece

ptor

mRN

A (a

rea)

OFQ

rece

ptor

mRN

A (O

D)

20

15

10

5

0

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

Normal Model WM

(a)

(b)

(c)


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

266 kPa 532 kPa 798 kPa 1064 kPa


lowastlowast

lowastlowast

lowast

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1



266 kPa 532 kPa 798 kPa 1064 kPa2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

DYN

Nor

-BN

I

NS

DYN

+ n

or-B

NI

Nor

mal

Mod

el

DYN

Nor

-BN

I

NS

DYN

+ n

or-B

NI

Nor

mal

Mod

el

DYN

Nor

-BN

I

NS

DYN

+ n

or-B

NI

Nor

mal

Mod

el

DYN

Nor

-BN

I

NS

DYN

+ n

or-B

NI



998771998771

998771998771

998771998771998771

(a)

266 kPa 532 kPa 798 kPa 1064 kPa2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

WM

WM

+ D

YN

WM

+ n

or-B

NI

Nor

mal

Mod

el

WM

WM

+ D

YN

WM

+ n

or-B

NI

Nor

mal

Mod

el

WM

WM

+ D

YN

WM

+ n

or-B

NI

Nor

mal

Mod

el

WM

WM

+ D

YN

WM

+ n

or-B

NI

lowastlowast

lowastlowast

lowastlowast

lowastlowast

998771998771

998771

998771998771

998771

998771998771

(b)






3 Results





lowastlowast

PPD

mRN

A (a

rea)

(a)

(b)

(c)

(d)30000

20000

10000

0Normal Model WM

lowastlowast

PPD

mRN

A (O

D)

Normal Model WM

20

15

10

5

0









lowast

lowast

lowast

Normal Model WM

Normal Model WM

Normal(a)

(b)

(c)

Model WM20

15

10

5

0

6000

8000

4000

2000

0

300

200

100

0

Dyn

pro

tein

(pg

mL)

Dyn

pro

tein

(OD

)D

yn p

rote

in (a

rea)



4 Discussion


lowast

lowast

Normal

Normal

(a)

(b)

(c)Model

Model

WM

WM

20

15

10

5

0

KOR

mRN

A (O

D)

KOR

mRN

A (a

rea)

25000

20000

15000

10000

5000

0






2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

lowastlowast


lowastlowast

266 kPa 532 kPa 798 kPa 1064 kPa

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

998771998771998771 998771998771

998771

(a)

2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

lowastlowast

lowastlowast


266 kPa 532 kPa 798 kPa 1064 kPa

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

998771998771998771998771

998771

998771

998771

998771

(b)


2


2


2


2








Normal

(a)

(b)

(c)

Model WM

Normal Model WM

OFQ

mRN

A (a

rea)

OFQ

mRN

A (O

D)

15

10

5

0

20000

15000

10000

5000

0

lowastlowast

lowastlowast


119875 lt 001





Normal(a)

(b)

(c)

Model WM

OFQ

pro

tein

(are

a)

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

OFQ

pro

tein

(OD

)

lowastlowast

15

10

5

0

Normal Model WM

OFQ

pro

tein

(pg

mL)

lowastlowast

40

30

20

10

0



5 Conclusions





OFQ

rece

ptor

mRN

A (a

rea)

OFQ

rece

ptor

mRN

A (O

D)

20

15

10

5

0

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

Normal Model WM

(a)

(b)

(c)


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3 Results





lowastlowast

PPD

mRN

A (a

rea)

(a)

(b)

(c)

(d)30000

20000

10000

0Normal Model WM

lowastlowast

PPD

mRN

A (O

D)

Normal Model WM

20

15

10

5

0









lowast

lowast

lowast

Normal Model WM

Normal Model WM

Normal(a)

(b)

(c)

Model WM20

15

10

5

0

6000

8000

4000

2000

0

300

200

100

0

Dyn

pro

tein

(pg

mL)

Dyn

pro

tein

(OD

)D

yn p

rote

in (a

rea)



4 Discussion


lowast

lowast

Normal

Normal

(a)

(b)

(c)Model

Model

WM

WM

20

15

10

5

0

KOR

mRN

A (O

D)

KOR

mRN

A (a

rea)

25000

20000

15000

10000

5000

0






2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

lowastlowast


lowastlowast

266 kPa 532 kPa 798 kPa 1064 kPa

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

998771998771998771 998771998771

998771

(a)

2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

lowastlowast

lowastlowast


266 kPa 532 kPa 798 kPa 1064 kPa

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

998771998771998771998771

998771

998771

998771

998771

(b)


2


2


2


2








Normal

(a)

(b)

(c)

Model WM

Normal Model WM

OFQ

mRN

A (a

rea)

OFQ

mRN

A (O

D)

15

10

5

0

20000

15000

10000

5000

0

lowastlowast

lowastlowast


119875 lt 001





Normal(a)

(b)

(c)

Model WM

OFQ

pro

tein

(are

a)

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

OFQ

pro

tein

(OD

)

lowastlowast

15

10

5

0

Normal Model WM

OFQ

pro

tein

(pg

mL)

lowastlowast

40

30

20

10

0



5 Conclusions





OFQ

rece

ptor

mRN

A (a

rea)

OFQ

rece

ptor

mRN

A (O

D)

20

15

10

5

0

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

Normal Model WM

(a)

(b)

(c)


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

lowast

lowast

Normal Model WM

Normal Model WM

Normal(a)

(b)

(c)

Model WM20

15

10

5

0

6000

8000

4000

2000

0

300

200

100

0

Dyn

pro

tein

(pg

mL)

Dyn

pro

tein

(OD

)D

yn p

rote

in (a

rea)



4 Discussion


lowast

lowast

Normal

Normal

(a)

(b)

(c)Model

Model

WM

WM

20

15

10

5

0

KOR

mRN

A (O

D)

KOR

mRN

A (a

rea)

25000

20000

15000

10000

5000

0






2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

lowastlowast


lowastlowast

266 kPa 532 kPa 798 kPa 1064 kPa

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

998771998771998771 998771998771

998771

(a)

2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

lowastlowast

lowastlowast


266 kPa 532 kPa 798 kPa 1064 kPa

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

998771998771998771998771

998771

998771

998771

998771

(b)


2


2


2


2








Normal

(a)

(b)

(c)

Model WM

Normal Model WM

OFQ

mRN

A (a

rea)

OFQ

mRN

A (O

D)

15

10

5

0

20000

15000

10000

5000

0

lowastlowast

lowastlowast


119875 lt 001





Normal(a)

(b)

(c)

Model WM

OFQ

pro

tein

(are

a)

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

OFQ

pro

tein

(OD

)

lowastlowast

15

10

5

0

Normal Model WM

OFQ

pro

tein

(pg

mL)

lowastlowast

40

30

20

10

0



5 Conclusions





OFQ

rece

ptor

mRN

A (a

rea)

OFQ

rece

ptor

mRN

A (O

D)

20

15

10

5

0

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

Normal Model WM

(a)

(b)

(c)


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

lowastlowast


lowastlowast

266 kPa 532 kPa 798 kPa 1064 kPa

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

Nor

mal

Mod

el

OFQ

OFQ

+N

phe1

Nph

e1 NS

998771998771998771 998771998771

998771

(a)

2

15

1

05

0

Scor

es o

f AW

R

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

4

3

2

1

0

Scor

es o

f AW

R

5

4

3

2

1

0

Nor

mal

Mod

el

lowastlowast

lowastlowast


266 kPa 532 kPa 798 kPa 1064 kPa

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

Nor

mal

Mod

el

WM

WM

+N

phe1

WM

+O

FQ

998771998771998771998771

998771

998771

998771

998771

(b)


2


2


2


2








Normal

(a)

(b)

(c)

Model WM

Normal Model WM

OFQ

mRN

A (a

rea)

OFQ

mRN

A (O

D)

15

10

5

0

20000

15000

10000

5000

0

lowastlowast

lowastlowast


119875 lt 001





Normal(a)

(b)

(c)

Model WM

OFQ

pro

tein

(are

a)

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

OFQ

pro

tein

(OD

)

lowastlowast

15

10

5

0

Normal Model WM

OFQ

pro

tein

(pg

mL)

lowastlowast

40

30

20

10

0



5 Conclusions





OFQ

rece

ptor

mRN

A (a

rea)

OFQ

rece

ptor

mRN

A (O

D)

20

15

10

5

0

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

Normal Model WM

(a)

(b)

(c)


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Normal

(a)

(b)

(c)

Model WM

Normal Model WM

OFQ

mRN

A (a

rea)

OFQ

mRN

A (O

D)

15

10

5

0

20000

15000

10000

5000

0

lowastlowast

lowastlowast


119875 lt 001





Normal(a)

(b)

(c)

Model WM

OFQ

pro

tein

(are

a)

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

OFQ

pro

tein

(OD

)

lowastlowast

15

10

5

0

Normal Model WM

OFQ

pro

tein

(pg

mL)

lowastlowast

40

30

20

10

0



5 Conclusions





OFQ

rece

ptor

mRN

A (a

rea)

OFQ

rece

ptor

mRN

A (O

D)

20

15

10

5

0

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

Normal Model WM

(a)

(b)

(c)


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OFQ

rece

ptor

mRN

A (a

rea)

OFQ

rece

ptor

mRN

A (O

D)

20

15

10

5

0

25000

20000

15000

10000

5000

0

lowastlowast

Normal Model WM

Normal Model WM

(a)

(b)

(c)


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Warming Moxibustion Relieves Chronic ...LiQi, 1,2 Hui-RongLiu, 1 TaoYi, 3 Lu-YiWu,...

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Transcript of Research Article Warming Moxibustion Relieves Chronic ...LiQi, 1,2 Hui-RongLiu, 1 TaoYi, 3 Lu-YiWu,...