Research Article Epimedium Flavonoids Counteract the Side...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 938425 9 pageshttpdxdoiorg1011552013938425

Research ArticleEpimedium Flavonoids Counteract the Side Effects ofGlucocorticoids on Hypothalamic-Pituitary-Adrenal Axis

Jianhua Huang1 Jijun Li2 Songbai Zheng3 Junzhen Wu3 Wei Zhang3 Tao Sun3

Sheilesh Kumar Dewan3 Bill Kalionis4 Ziyin Shen1 Xiantao Tai5 and Shijin Xia6

1 Key Laboratory of Cellular and Molecular Biology Huashan Hospital Fudan University Shanghai 200040 China2Department of Integrative Medicine Shanghai Childrenrsquos Medical Center Shanghai Jiaotong University School of MedicineShanghai 200127 China

3Department of Geriatrics Huadong Hospital Fudan University Shanghai 200040 China4Department of Perinatal Medicine The Royal Womenrsquos Hospital University of Melbourne Parkville VIC 3052 Australia5 School of Acupuncture Massage and Rehabilitation Yunnan University of Traditional Chinese Medicine Kunming 650500 China6 Shanghai Institute of Geriatrics Huadong Hospital Fudan University Shanghai 200040 China

Correspondence should be addressed to Xiantao Tai taixiantao163com and Shijin Xia xiashijinhd163com

Received 19 April 2013 Revised 10 August 2013 Accepted 19 August 2013

Academic Editor Jenny M Wilkinson

Copyright copy 2013 Jianhua Huang et alThis 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

Our previous studies demonstrated that the epimediumherb when simultaneously usedwithGCs counteracted suppressive effectsof GCs on the HPA axis without adverse influence on the therapeutic action of GCs Here total flavones were extracted from theepimedium flavonoids (EFs) and then used to investigate whether EFs provide protective effects on the HPA axis We found thatGCs induced a significant decrease in body weight gain adrenal gland weight gain and plasma adrenocorticotropin (ACTH) andcorticosterone levels After treatment with EFs body weight gain adrenal gland weight gain and plasma corticosterone level weresignificantly restored whilst plasma ACTH level was partially elevated EFs were also shown to promote cell proliferation in theouter layer of adrenal cortex and to enhance themigration of newly divided cells toward the inner layer To elucidate the underlyingmechanisms themRNA expression of insulin-like growth factor II (IGF-II) wasmeasured and EFs significantly upregulated IGF-IIexpression Our results indicated that EFs counteract the suppression of the HPA axis induced by GCs This may involve both theACTH and IGF-II pathways and thereby promote regeneration of the adrenal cortex suggesting a potential clinical application ofEFs against the suppressive effects of GCs on the HPA axis

1 Introduction

Glucocorticoids (GCs) are widely used and have been suc-cessful for more than 50 years in the treatment of someinflammatory or autoimmune diseases including asthmanephrotic syndrome lupus and nephritis [1 2] Unfor-tunately the beneficial effects are often accompanied bymultiple undesirable adverse effects These include diabetesmellitus peptic ulcer Cushingrsquos syndrome and osteoporosisThe use of GCs also results in suppression of hypothalamic-pituitary-adrenal (HPA) axis which may persist after treat-ment withdrawal [3ndash5] Full restoration of HPA functionmayrequire several months or in some cases more than one year[6]

The adverse effects of HPA suppression are particularlysevere and correlate highly with the failure of GC withdrawaland reactivation of diseases HPA suppression is the directcause of the acute adrenal insufficiency syndrome which canbe precipitated by surgery or major stress and is associatedwith the life-threatening condition of hemodynamic collapse[7] Thus there is a need for the development of compoundswith a protective effect on the HPA axis which can be usedin combination with GCs

There are several reports suggesting a potential therapeu-tic benefit of some agents for preventing the HPA axis sup-pression induced by the long-term use of GCs Polypeptidepneumadyne (PNM) can elevate the content of corticosteronein adrenal tissue and increase cell volume and cell number

2 Evidence-Based Complementary and Alternative Medicine

in the adrenal possibly by stimulating the release of argi-nine vasopressin (AVP) and adrenocorticotropic hormone(ACTH) however PNM did not increase the serum level ofcorticosterone [7] Buspirone an antidepression drug and a5-HT receptor agonist was shown to enhance the synthesisand release of ACTH suggesting an antagonistic role againstthe suppression of the HPA axis by dexamethasone whichwas used in the treatment of depression [8] Huang reportedthat ovine corticotrophin releasing hormone vasopressinand exogenous ACTH can partially restore the suppressedHPA function in animals [9] However none of these drugshave been used in clinical settings specifically to treat thesuppression of the HPA axis

An agent that not only resists the suppression of HPA axisinduced by GCs but also has no adverse influence on thetherapeutic action of GCs is required Our previous studiesrevealed that a herbal compound prescribed in traditionalChinese medicine and already used in clinical treatmentmay be a suitable agent [10] Oral GCs were successfullyused to control asthma but due to their suppression of theHPA axis GCs dependence was frequently developed Thesuccessful withdrawal rate of GCs was very low at about12 [11 12] Subsequently inhalation of large doses of GCswas used to attenuate the adverse effects on the HPA axisand the successful withdrawal rate increased up to 27sim44[13 14] In order to further promote the success rate GCswereused in combination with a herbal compound prescriptionKidney-tonifying decoction Consequently we found that thesuccessful withdrawal rate was significantly increased to 70[10]

The encouraging results from clinical studies promptedus to isolate the possible effective components through whichthe Kidney-tonifying decoction exerts protective efficacy onHPA axis Among the numerous ingredients we identifiedepimedium flavones (EFs) extracted from epimedium herbcontained in the Kidney-tonifying decoction as a majorcomponentWepostulated that EFswere the effective compo-nents within the Kidney-tonifying decoction In the presentstudy we examined the protective role of EFs on theHPA axisin animals treated with large doses of GCs

2 Experimental Section

21 Study Design Four-month-old male Sprague-Dawleyrats weighting 200 plusmn 15 g were randomly assigned into 3groups that is a normal control group a corticosterone only-treated group and a combined corticosterone and EF -treatedgroup Each group consisted of 16 rats All rats were housedin a temperature-controlled room (24∘C) with lights on from0600 hsim1800 h daily andwere allowed free access towater andrat pellet chow The corticosterone only-treated group wassubcutaneously injected with 01mL corticosterone dissolvedin olive pomace oil at a dose of 10mgkg body weight perday for 14 days This group received simultaneous oral saline(02mL) for 14 days The combined corticosterone and EF-treated group was injected with the same dose of corticos-terone as the corticosterone-treated group but received EForally at a daily dose of 60mgkg body weight In the control

group rats were subcutaneously injected with 01mL olivepomace oil and orally administrated with the same volumeof saline as above Fourteen days later the rats were killed bydecapitation The animal protocols were in accordance withAnimal Care and Use Committee of Fudan University

22 Preparation of EFs Epimedium is a major component inKidney-tonifying decoction which has been used to increasethe success rate of GCs withdrawal The Epimedium waspurchased from a traditional chinese medicine distributor(Shanghai U-sea Biotech Co Ltd Shanghai China) Theorigin of epimedium was in Liaoning province ChinaThe epimedium was verified by De-yun Kong a professorof Shanghai Institute of Pharmaceutical Industry ChinaA voucher specimen was deposited in the Herbarium ofShanghai Institute of Pharmaceutical Industry China Onlythe leaves and stems of Epimedium were used in our studyThe total flavonoids of epimediumwere extracted and used aspreviously [15] Epimedium was ground to a powder (about30 meshes) by a disintegrator and the powder (1000 g) wasextracted twicewith 10 L 75 ethanol for 2 h under refluxTheextracts were combined together and filtrated with cottonand the filtrate was concentrated under vacuum to give anaqueous fluid The aqueous fluid was subjected to a glasscolumn (5 cm times 80 cm) of macroporous resin (D101 400 g)and washed with water and 70 ethanol respectively Theeluent of 70 ethanol was concentrated in vacuum using arotary evaporator to dry the EFs powder (115 g puritygt70)The EFs were solvated with water to a 3 concentrationbefore orally administered to the rats [15] The content ofingredients of EFs was determined by high-performanceliquid chromatography (HPLC)

23 Plasma ACTH Assay After rats were sacrificed bloodsamples of 2mL per rat were immediately collected intoheparin-pretreated tubes and tubes were incubated for30min at room temperature followed by centrifugationat 1000 g for 15min The supernatant was obtained andaliquots of 015mL were prepared and stored at minus80∘C priorto assay The assay was performed using the DSL-10-5100ACTIVE ELISA kit (Diagnostic Systems Laboratories TXUSA) The detailed protocol was carried out according tothe manufacturerrsquos instructions In brief 100 120583L of variousconcentrations of ACTH standards positive control andsamples were used respectively followed by addition ofantibody-biotin conjugate streptavidin-enzyme conjugatechromogen solution and stopping solution in turn Finallyall samples were assayed at wavelength of 405 nm and awavelength of 530 nm was used as a reference

In order to determine the interassay coefficient of varia-tion (CV) three sampleswere assayed in the same experiment16 times then the CV was calculated In order to determineintrassay CV three samples were assayed in 8 separatedexperiments and the CV value was calculated

24 Plasma Corticosterone Assay The sample preparationwas the same as the ACTH assay For determining intra-or interassay CV the same method as that in ACTH assay

Evidence-Based Complementary and Alternative Medicine 3

00 50 100 150 200

0

25

50

75

100

(mAU

)

270nm 4nm (100)smth

(min)

(a)

00 50 100 150

0

25

50

75

(mAU

)

270nm 4nm (100)smth

(min)

(b)

Figure 1 Content of epimediumflavonoids (EFs) by high-performance liquid chromatography (HPLC) (a) HPLC profiles of EFs the contentof two components is more than 10 (b) HPLC profiles of standard samples of icariin The content of icariin is 437 of the preparation

was usedThe EIA kit for corticosterone assay was purchasedfromCayman Chemical Company (MI USA) In brief 50120583Lof corticosterone standards and samples were used Thedetection was consistent with the protocols provided by themanufacturer Finally all samples were assayed at wavelengthof 412 nm

25 Immunohistochemical Detection of 5-Bromo-21015840-deoxyuri-dine (BrdU) Incorporated Nuclei Twelve hours before ratswere sacrificed the rats were injected with 50mgBrdU kgbody weight 3 times at a time interval of 3 hours Theadrenal glands were excised fixed in formalin overnightand embedded in paraffin Slides containing 4120583m sectionswere prepared and subjected to 4 N HCL to denature theDNA The sections were incubated with a mouse anti-BrdUantibody (Sigma) and then incubated with a biotinylatedsecondary antibody and horseradish peroxidase-conjugatedstreptavidin (Boster Co Wuhan China) Colour detectionwas by addition of 005 331015840-diaminobenzidine (Sigma)and 001 hydrogen peroxide at room temperature Sectionswere then lightly counterstained with 01 hematoxylin Atleast three fields in the outer layer of adrenal gland werechosen for assessmentThe ratio of BrdU-positive cells versustotal cell numbers defined by hematoxylin-stained cells wasdetermined

To observe the migration of progenitor cells of adreno-cytes BrdU was injected before the beginning of the exper-iment 14 days later rats were sacrificed and the sampleswere prepared and treated as described above The migrationof BrdU-positive cells was observed by microscopy at amagnification of 400x

26 Measure of IGF-II mRNA Expression Using QuantitativePCR Adrenocytes isolated from normal 4-month-old ratswere cultured in RPMI 1640 and divided into a control groupand an EF-treated group In the latter 10minus7M EF was addedinto RPMI 1640 and coincubated with adrenocytes for 24hours Then cells were harvested and total RNA was isolated

with Trizol reagent (Invitrogen USA) Real-time two-stepRT-PCR was performed with a QuantiTect SYBR Green PCRkit (Qiagen Hilden Germany) on an ABI GeneAmp 7300Sequence detection system (Applied Biosystems USA) Theforward primer was 51015840-TTGGCCAGATAAGGAGATGG-31015840the reverse primer was 51015840-AGAGATGGCCCATAGGTGTG-31015840 The parameters for PCR were the following 50∘C 2min95∘C 15min 35 cycles of 94∘C 10 sec 55∘C 30 sec 72∘C30 sec followed by hold at 72∘C for 10min IGF-II mRNAwasisolated from rat liver and reversed into cDNA as standardAmplification specificity was confirmed by melting curveanalysis using incorporated StepOne software v 20

27 Statistical Analysis Data were presented as the mean plusmnSD Statistical analysis was by one-way ANOVA or unpaired119905-test (two-tailed) and significance was assumed at 119875 lt 005

3 Results

31 The Content of Epimedium Flavonoids (EFs) The mainingredient of EFs was determined by high-performanceliquid chromatography (HPLC) Results showed that thereare two ingredients which contents are more than 10(Figure 1(a)) Using the standard of icariin we found thecontent of icariin accounts for up to 437 (Figure 1) Thusour drugs are qualitatively controllable

32 Effects of EFs on Body Weight Gain The increase inbody weight for control group on the 7th day was 10225 plusmn387 g Corticosterone greatly delayed the growth of therats (66 plusmn 752 g) suggesting a potent growth retardationeffect of GCs The weight gain in the combined corticos-terone and EFs-treated rats was 98 plusmn 374 g on the 7thday and the value was not statistically different when com-pared with control rats (119875 gt 005) but significantly different(119875 lt 001) when compared with corticosterone only-treatedrats These data suggest a protective effect for body weight


120

100

80

60

40

20

0

Control Corticosterone EFs

lowastlowast

Body

wei

ght g

ain

on th

e 7th

day

(g)

(a)

160

140

120

100

80

60

40

20

0


lowastlowast

Body

wei

ght g

ain

on th

e 14t

h da

y (g

)(b)

Figure 2 Effects of EFs on body weight gain of rats (a)The body weight gain on 7th day for control corticosterone only and combined use ofcorticosterone and EFs was shown (b)The bodyweight gain on 14th day for control corticosterone alone and combined use of corticosteroneand EFs was shown lowastlowast119875 lt 001 versus control group

119875 lt 005 119875 lt 001 versus corticosterone-only group

of EFs in corticosterone-treated rats 7 days after treatment(Figure 2(a))

On the 14th day similar results were obtained(Figure 2(b)) body weight gains for control corticosteroneonly-treated and the combined corticosterone and EFs-treated group were 13425 plusmn 454 g 10335 plusmn 1085 g and119 plusmn 53 g respectively Although the gain in the combinedcorticosterone and EFs-treated group was lower than thatin control rats it was higher than that in corticosteroneonly-treated group (119875 lt 005 versus control group and co-rticosterone only-treated group) indicating a partiallyprotective effect of EFs on body weight gain after 14 days

33 Effects of EFs on Adrenal Gland Weight Treatment withcorticosterone significantly decreased organ weight of theadrenal gland the value for control and corticosterone only-treated group was 4963 plusmn 600mg and 2888 plusmn 945mgrespectively But in the combined corticosterone and EFs-treated group the adrenal weight was increased to 3497 plusmn484mg When compared with corticosterone only-treatedgroup the difference was significant (119875 lt 005)

34 Plasma ACTH and Corticosterone Level The ACTHlevel in the control group was 14315 plusmn 1576 pgmL butin corticosterone only-treated group the level was muchlower at 389 plusmn 957 pgmL After treatment with combinedcorticosterone and EFs there was a trend to an increase butthe difference was not significant when compared with thecorticosterone only-treated group (Figure 3(a))

The plasma corticosterone level in the control groupwas 9603 plusmn 3095 ngmL In corticosterone only-treated

rats the plasma corticosterone level was very low (3072 plusmn2173 ngmL) but after combined corticosterone and EFstreatment the level (5379 plusmn 2563 ngmL) of plasma corti-costerone level was significantly elevated compared with thecorticosterone only-treated group (119875 lt 005) although thelevels were still lower than that of control group (Figure 3(b))

35 Progenitor Cell Proliferation in the Outer Layer of AdrenalCortex BrdU is an analog of uridine and is incorporated intonuclei of cells in which DNA synthesis is occurring thus byusing a BrdUmonoclonal antibody and immunohistochemi-cal techniques proliferating cells can be visualized As shownin Figures 4(a) 4(b) and 4(c) BrdU-positive cells were con-centrated in the outer layer of adrenal cortex in each groupAfter treatment with corticosterone the percentage of BrdU-stained cells was decreased significantly (1571 plusmn 758)But in the combined corticosterone and EFs-treated ratsthe percentage of BrdU-positive cells (4852 plusmn 1059) wassignificantly increased (119875 lt 001) when compared with thecorticosterone only-treated group and actually exceeded thatin control group (3515plusmn 1391) (Figures 4(a) 4(b) 4(c) and4(d)) These results suggest that EFs enhance proliferation ofcells located in the outer layer of adrenal cortex

36 Immigration of BrdU-Positive Cells toward the InnerRegion of Adrenal Cortex To further investigate whether EFsaffect the immigration of proliferating cells toward functionalzones of adrenal cortex in the inner region to replenishthe lost cells after corticosterone treatment we injected ratswith BrdU before the experiment and then the rats weresubjected to treatment with corticosterone only or combined


200

160

120

80

40

0


lowastlowast

Plas

ma A

CTH

leve

l (pg

mL)

(a)

160

120

80

40

0


lowastlowast

Plas

ma c

ortic

oste

rone

leve

l (ng

mL)

(b)

Figure 3 Effects of EFs on plasma ACTH and corticosterone level of rats (a) Plasma ACTH level was measured by ELISA in controlcorticosterone and combined use of corticosterone and EFs (b) Plasma corticosterone level was measured by ELISA after treatment withsaline (control) corticosterone and combined use of corticosterone and EFs lowast119875 lt 005 lowastlowast119875 lt 001 versus control group

119875 lt 005 versuscorticosterone-only group

corticosterone and EFs After 14 days immunohistochemicaldetection of BrdU was used to determine the migration ofBrdU-stained cellsWe found thatmost BrdU-positive cells incontrol and corticosterone only-treated rats still resided in theouter layer of adrenal cortex while the percentage of BrdU-positive cells residing in the fasciculata in control or corticos-terone only-treated groupwas (88 plusmn 35) and (200 plusmn 49)respectively In the combined corticosterone and EFs-treatedgroup themajority of BrdU-positive cells (792 plusmn 116) hadmigrated into the inner region corresponding to fasciculatazone of adrenal cortex as shown in Figures 5(a) 5(b) 5(c)and 5(d) with 119875 lt 001 when compared with control orcorticosterone only-treated group

37 IGF-II mRNA Expression after Induction by EFs Quanti-tative real-time analysis showed that EFs significantly upreg-ulated the gene expression of IGF-II In adrenocytes withoutaddition of EFs IGF-II mRNA was almost not detectablewith only (019 plusmn 024) times 103 copies per 1 120583g initial total RNABut after induction by EFs the values were increased to (535plusmn 352) times 103 copies per 1 120583g initial total RNA respectivelyand thus increased more than 30-fold than that in normaladrenocytes The experiment was repeated four times inde-pendently Results exhibited statistical significancewhenEFs-treated adrenocyteswere comparedwith adrenocyteswithoutinduction by EFs (119875 lt 005)

4 Discussion

GCs suppress the HPA axis at various sites of the axisincluding the secretion of corticotropin (CRH) and ACTH

The suprahypothalamic brain regions such as the amygdalaand the hippocampus are also involved in regulating the HPAaxis [16ndash18] In addition long-term use of supraphysiologicaldoses of GCs leads to atrophy of adrenal gland due toa decrease of cell number cell volume and reduced cellproliferation in the adrenal cortex [19ndash21] In the presentstudy we demonstrated that the plasma corticosterone levelsdecreased when rats were treated by corticosterone aloneBut after treatment with a combination of corticosteroneand EFs the levels of ACTH and plasma corticosteronewere increased Moreover we found that EFs significantlycounteracted the involution of the adrenal gland induced bycorticosterone treatment The results above suggest that EFsprotect the HPA axis function from the effects of GCs In thepresent study the plasma ACTH level only showed a trend ofan increase after combined corticosterone and EFs treatmentwithout statistical significance Dupouy et al reported thatheparin could slightly degrade the plasma ACTH level [22]Whether the heparin interfered with the measurement ofACTH in our study remained unknown But this resultmightalso suggest another possibility that there is an alternatemechanism(s) by which EFs exert their effects on maximallyincreased corticosterone levels for instance by acting onadrenocortex directly

To further investigate the cellular and molecular mecha-nisms by which EFs may counteract the atrophy induced byGCs we performed cytogenesis and gene expression analysisin adrenal cortex Results showed that EFs promoted theproliferation and migration inward of stem cells of adrenalcortex suggesting a promoted regeneration of adrenal cortexby EFs The adrenal cortex of mammals is composed ofthree morphologically and functionally distinct zones from


Cap

G

F

(a)

Cap G F

(b)

Cap

G

F

(c)

70

60

50

40

30

20

10

0


lowastlowast

BrdU

-pos

itive

cells

()

(d)

Figure 4 Localization and the number of BrdU-incorporated nuclei in the adrenal gland ((a) (b) (c)) Sections of the adrenal gland fromrats of the control group corticosterone only-treated group and combined corticosterone and EFs-treated group respectively were stainedwith anti-BrdU antibody and visualized withmicroscope (magnificationtimes400) (d)The percentage of BrdU-positive against total cells in zonaglomerulosawas calculated Cells with brownnuclei are considered as BrdU-positive cells CapG andFdenote the capsule zona glomerulosaand zona fasciculata respectively All sections were counterstained with hematoxylin lowastlowast119875 lt 001 versus control group


the outer side to the inner side the zona glomerulosa thezona fasciculate and the zona reticularis [23 24] The cellmigration theory was proposed by Gottschau where adreno-cytes derive from the outer part of adrenal cortex migratecentripetally and differentiate into each functional zone[25] Researchers confirmed that the precise compartmentof the adrenocyte progenitors is located between the zonaglomerulosa and zona fasciculata and the newly divided cellsmigrate inwards [26] This zone was identified as a stem cellzone for adrenal cortex [27] which builds a new field ofadrenal cortex regeneration [28 29]The regeneration is char-acterised by cell proliferation cell migration and differentia-tion [30] Therefore we determined whether the cytogenesisof adrenal cortex can be enhanced by EFs ConsequentlyBrdU-positive cells were confined to the subcapsular part

of adrenal gland consistent with previous reports [25] Wecalculated the percentage of BrdU-positive cells to total cellsto indicate the proliferation capability Results showed thatthe percentage in the corticosterone-treated group decreasedBut surprisingly in rats simultaneously treated with GCsand EFs there was an increased percentage of BrdU-positivecells which exceeded that in normal rats suggesting a strongproliferation-promoting property of EFs We also analyzedthe migration of BrdU-positive cells In general cells formedin the first compartment of zona glomerulosa traverse thesecond and into the innermost zona reticularis Half of thecells are eliminated during the migration and the rest diedin the reticularis by apoptosis [31] A cell which does notdie during migration will take about 104 days to reach thereticularis [25] Here we found that most BrdU-positive


Cap

G

F

(a)

CapG

F

(b)

CapG

F

(c)

100

80

60

20

0

40


GlomerulosaFasciculata

BrdU

-pos

itive

cells

()

(d)

Figure 5Migration of BrdU-positive cells toward the inner layer of adrenal gland Rats were injected with BrdU 14 days later rats were killedand adrenal glands were excised and the BrdU incorporation assay was carried out ((a) (b) (c)) Sections of the adrenal gland from rats ofthe control group corticosterone only-treated group and combined corticosterone and EFs-treated group respectively were stained withanti-BrdU antibody and visualized with microscope (magnification times400) (d)The percentage of BrdU-positive cells in zona glomerulosa orzona fasciculata against total BrdU-positive cells was calculated respectively Cells with brown nuclei are considered as BrdU-positive cellsCap G and F denote the capsule and zona glomerulosa zona fasciculata respectively All sections were counterstained with hematoxylin

cells either in control rats or GCs only-treated rats remainedin the zona glomerulosa perhaps due to too short timeinterval than 104 days to observe the migration In contrastin the rats treated with GCs and EFs most of BrdU-stainingcells migrated into zona fasciculata suggesting increasedmigration

Numerous studies established that ACTH is the majorregulator of the adrenal cortex Upon the use of GCsACTH can counteract the atrophy of adrenal gland throughenhanced proliferation and migration of adrenocytesbetween zona glomerulosa and zona fasciculata [32 33]

Recent studies have provided a new concept that theadrenocortex is also regulated by autocrine andor paracrinefactors including IGFs [34 35] fibroblast growth factors(FGFs) [36] endothelin [37] cholecystokinin [38] adren-omedullin [39] and cytokines [40] Among these IGF II isthe most important and is involved in cell division steroidsynthesis and cell differentiation of adrenocytes [41] Thuswe measured the IGF II gene expression using isolatedadrenocytes coincubated with EFs for a specified timeResults showed that EFs significantly upregulated IGF IIgene expression In our study the increase of ACTH by EFs


treatment was not statistically significant This unexpectedresult may be due to the interference of heparin Thus ourdata do not preclude a role for ACTH in mediating effectsof EFs We believe that our data are consistent with EFsexerting their effect on adrenocytes through both ACTH andthe autocrine andor paracrine factor IGF II

Another striking result was that EFs markedly counter-acted the decrease of body weight gain induced by corticos-terone which is the consequence of the nonspecific cataboliceffect of the steroidThere are reports which provide evidencethat thyroid hormone or growth hormone administrationcould prevent the decrease in body and organ weight gains[42] In the present study we showed that EFs upregulatedIGF II which is a component of growth hormone-insulin-like growth factor (GHIGF) axis Increased bodyweight gainas a result of EFs treatment may be due to elevated GHIGFaxis activity But the precise molecular mechanism needs tobe further investigated

According to our experimental design the EFs treatmenton normal rats was omittedThus there was a possibility thateffects of EFs were achieved through altering the pharma-cokinetics of the oil or corticosterone However consideringour accumulated data we believe that these effects wereinduced by EFs For example IGF II mRNA was significantlyincreased by more than 30 fold which can be attributedto specific pharmacological effects of EFs In summary EFsextract from a herbal origin proved to be effective againstthe suppressive effects of GCs EFs could be considered forclinical use to selectively counteract the side effects of GCson the HPA axis and thus provide an effective and safealternative treatment

Authorsrsquo Contribution

Jianhua Huang and Jijun Li contributed equally to this workand should be considered the first authors

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (no 81270115 no 31171129 and no30901888) Shanghai Municipal Health Bureau (2004J014A)and the National Basic Research Program of China (no2010CB540302)

References

[1] R A Hughes and M M Mehndiratta ldquoCorticosteroids forchronic inflammatory demyelinating polyradiculoneuropathyrdquoCochrane Database of Systematic Reviews no 8 Article IDCD002062 2002

[2] K Suzuki ldquoCurrent therapeutic strategy for multiple myelomardquoJapanese Journal of Clinical Oncology vol 43 no 2 pp 116ndash1242013

[3] B M Vayssiere S Dupont A Choquart et al ldquoSynthetic glu-cocorticoids that dissociate transactivation and AP-1 transre-pression exhibit antiinflammatory activity in vivordquo MolecularEndocrinology vol 11 no 9 pp 1245ndash1255 1997

[4] M Resche-Rigon and H Gronemeyer ldquoTherapeutic potentialof selective modulators of nuclear receptor actionrdquo CurrentOpinion in Chemical Biology vol 2 no 4 pp 501ndash507 1998

[5] R L Hopkins and M C Leinung ldquoExogenous Cushingrsquos syn-drome and glucocorticoid withdrawalrdquo Endocrinology andMetabolism Clinics of North America vol 34 no 2 pp 371ndash3842005

[6] T Livanou D Ferriman and V H James ldquoRecovery of hypo-thalamo-pituitary-adrenal function after corticosteroid ther-apyrdquoThe Lancet vol 2 no 7521 pp 856ndash859 1967

[7] B Richter G Neises and C Clar ldquoGlucocorticoid withdrawalschemes in chronic medical disorders A systematic reviewrdquoEndocrinology andMetabolism Clinics of North America vol 31no 3 pp 751ndash778 2002

[8] M Maes A van Gastel H Y Meltzer P Cosyns P Blockx andR Desnyder ldquoAcute administration of buspirone increases theescape of hypothalamic-pituitary-adrenal-axis hormones fromsuppression by dexamethasone in depressionrdquo Psychoneuroen-docrinology vol 21 no 1 pp 67ndash81 1996

[9] T S Huang ldquoCorticotropin secretagogues facilitate recoveryof the hypothalamus-pituitary-adrenal axis suppressed by pro-longed treatment with dexamethasonerdquoMetabolism vol 43 no5 pp 544ndash548 1994

[10] J C Dong Z Y Shi and Z Y Shen ldquoInvestigation of treatingsteroids dependent asthmatic patients with kidney-tonifyingherbs and high dose beclomethasone dipropionate aerosolrdquoZhongguo Zhong Xi Yi Jie He Za Zhi vol 14 no 8 pp 458ndash4611994

[11] R S Pearson J H Baylis and H C Smellie ldquoTreatment ofchronic asthma with prednisolone and the newer steroidsrdquoBritish Medical Journal vol 1 no 5222 pp 315ndash319 1961

[12] P H Henneman D M Wang J W Irwin and W S BurrageldquoSyndrome following abrupt cessation of prolonged cortisonetherapyrdquo Journal of the American Medical Association vol 158no 5 pp 384ndash386 1955

[13] C R Jenkins and A J Woolcock ldquoEffect of prednisoneand beclomethasone dipropionate on airway responsiveness inasthma a comparative studyrdquoThorax vol 43 no 5 pp 378ndash3841988

[14] S J Cameron E J Cooper and G K Crompton ldquoSubstitutionof beclomethasone aerosol for oral prednisolone in the treat-ment of chronic asthmardquo British Medical Journal vol 4 no5886 pp 205ndash207 1973

[15] B Wu S Yan Z Lin et al ldquoMetabonomic study on ageingNMR-based investigation into rat urinary metabolites and theeffect of the total flavone of epimediumrdquoMolecular BioSystemsvol 4 no 8 pp 855ndash861 2008

[16] K M Knigge ldquoAdrenocortical response to stress in rats withlesions in hippocampus and amygdalardquo Proceedings of theSociety for Experimental Biology and Medicine vol 108 no 1pp 18ndash21 1961

[17] J P Herman J M Mcklveen M B Solomon E Carvalho-Netto and B Myers ldquoNeural regulation of the stress responseglucocorticoid feedback mechanismsrdquo Brazilian Journal ofMedical andBiological Research vol 45 no 4 pp 292ndash298 2012

[18] S Feldman and N Conforti ldquoParticipation of the dorsalhippocampus in the glucocorticoid feedback effect on adreno-cortical activityrdquo Neuroendocrinology vol 30 no 1 pp 52ndash551980

[19] MQuinn YUenoH Y Pae et al ldquoSuppression of theHPA axisduring extrahepatic biliary obstruction induces cholangiocyte


proliferation in the ratrdquo American Journal of Physiology Gas-trointestinal and Liver Physiology vol 302 no 1 pp G182ndashG1932012

[20] G G Nussdorfer ldquoCytophysiology of the adrenal cortexrdquo Inter-national Review of Cytology vol 98 pp 1ndash405 1986

[21] B Lesniewska KWNowak and L KMalendowicz ldquoDexame-thasone-induced adrenal cortex atrophy and recovery of thegland from partial steroid-induced atrophyrdquo Experimental andClinical Endocrinology vol 100 no 3 pp 133ndash139 1992

[22] J P Dupouy A Chatelain and M Godaut ldquoInfluences ofheparin onACTHdistribution and immunoreactivity in plasmaof the rat in vivo and in vitro studiesrdquo Journal de Physiologie vol76 no 6 pp 631ndash635 1980

[23] G P Vinson ldquoAdrenocortical zonation and ACTHrdquoMicroscopyResearch and Technique vol 61 no 3 pp 227ndash239 2003

[24] J F Tait and S A Tait ldquoRecent perspectives on the history ofthe adrenal cortexThe Sir Henry Dale lecture for 1979rdquo Journalof Endocrinology vol 83 no 1 pp 3Pndash24P 1979

[25] G Zajicek I Ariel and N Arber ldquoThe streaming adrenalcortex direct evidence of centripetal migration of adrenocytesby estimation of cell turnover raterdquo Journal of Endocrinologyvol 111 no 3 pp 477ndash482 1986

[26] F Mitani H Suzuki J-I Hata T Ogishima H Shimadaand Y Ishimura ldquoA novel cell layer without corticosteroid-synthesizing enzymes in rat adrenal cortex histochemicaldetection and possible physiological rolerdquo Endocrinology vol135 no 1 pp 431ndash438 1994

[27] F Mitani K Mukai H Miyamoto M Suematsu and YIshimura ldquoThe undifferentiated cell zone is a stem cell zone inadult rat adrenal cortexrdquoBiochimica et BiophysicaActa vol 1619no 3 pp 317ndash324 2003

[28] W B Ennen B K Levay-Young and W C Engeland ldquoZone-specific cell proliferation during adrenocortical regenerationafter enucleation in ratsrdquo American Journal of PhysiologyEndocrinology and Metabolism vol 289 no 5 pp E883ndashE8912005

[29] M Thomas S R Northrup and P J Hornsby ldquoAdrenocorticaltissue formed by transplantation of normal clones of bovineadrenocortical cells in scidmice replaces the essential functionsof the animalsrsquo adrenal glandsrdquoNatureMedicine vol 3 no 9 pp978ndash983 1997

[30] T M Taki and P A Nickerson ldquoDifferentiation and pro-liferation of adrenocortical cells during the early stages ofregenerationrdquo Laboratory Investigation vol 53 no 1 pp 91ndash1001985

[31] A H Wyllie J F R Kerr I A M Macaskill and A R CurrieldquoAdrenocortical cell deletion the role of ACTHrdquo Journal ofPathology vol 111 no 2 pp 85ndash94 1973

[32] D Pignatelli J Ferreira P Vendeira M C Magalhaes and G PVinson ldquoProliferation of capsular stem cells induced by ACTHin the rat adrenal cortexrdquo Endocrine Research vol 28 no 4 pp683ndash691 2002

[33] A S Belloni G Mazzocchi V Meneghelli and G GNussdorfer ldquoCytogenesis in the rat adrenal cortex evi-dence for an ACTH-induced centripetal cell migration fromthe zona glomerulosardquo Archives drsquoAnatomie drsquoHistologie etdrsquoEmbryologie vol 61 pp 195ndash205 1978

[34] E JWhitworthOKosti D Renshaw and J PHinson ldquoAdrenalneuropeptides regulation and interaction with ACTH andother adrenal regulatorsrdquo Microscopy Research and Techniquevol 61 no 3 pp 259ndash267 2003

[35] M T Pham-Huu-Trung J M Villette A Bogyo J M DuclosJ Fiet and M Binoux ldquoEffects of insulin-like growth factor I(IGF-I) on enzymatic activity in human adrenocortical cellsInteractions with ACTHrdquo Journal of Steroid Biochemistry andMolecular Biology vol 39 no 6 pp 903ndash909 1991

[36] C Palmieri D Roberts-Clark A Assadi-Sabet et al ldquoFibrolastgrowth factor 7 secreted by breast fibroblasts is an interleukin-1120573-induced paracrine growth factor for human breast cellsrdquoJournal of Endocrinology vol 177 no 1 pp 65ndash81 2003

[37] G P Rossi P G Andreis S Colonna et al ldquoEndothelin-1[1-31]a novel autocrine-paracrine regulator of human adrenal cortexsecretion and growthrdquo Journal of Clinical Endocrinology andMetabolism vol 87 no 1 pp 322ndash328 2002

[38] G G Nussdorfer R Spinazzi and G Mazzocchi ldquoCholecys-tokinin and adrenal-cortex secretionrdquo Vitamins and Hormonesvol 71 pp 433ndash453 2005

[39] A Ziolkowska K Budzynska M Trejter C Tortorella A SBelloni and L KMalendowicz ldquoEffects of adrenomedullin andits fragment 22-52 on basal and ACTH-stimulated secretionof cultured rat adrenocortical cellsrdquo International Journal ofMolecular Medicine vol 11 no 5 pp 613ndash615 2003

[40] S R Bornstein H Rutkowski and I Vrezas ldquoCytokines andsteroidogenesisrdquoMolecular and Cellular Endocrinology vol 215no 1-2 pp 135ndash141 2004

[41] M M Weber P Simmler C Fottner and D EngelhardtldquoInsulin-like growth factor II (IGF-II) is more potent thanIGF-I in stimulating cortisol secretion from cultured bovineadrenocortical cells interaction with the IGF-I receptor andIGF-binding proteinsrdquo Endocrinology vol 136 no 9 pp 3714ndash3720 1995

[42] R P A Rooman G Kuijpers R Gresnigt R Bloemen J GKoster and S C van Buul-Offers ldquoDexamethasone differen-tially inhibits thyroxine- or growth hormone-induced body andorgan growth of snell dwarf micerdquo Endocrinology vol 144 no6 pp 2553ndash2558 2003

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


in the adrenal possibly by stimulating the release of argi-nine vasopressin (AVP) and adrenocorticotropic hormone(ACTH) however PNM did not increase the serum level ofcorticosterone [7] Buspirone an antidepression drug and a5-HT receptor agonist was shown to enhance the synthesisand release of ACTH suggesting an antagonistic role againstthe suppression of the HPA axis by dexamethasone whichwas used in the treatment of depression [8] Huang reportedthat ovine corticotrophin releasing hormone vasopressinand exogenous ACTH can partially restore the suppressedHPA function in animals [9] However none of these drugshave been used in clinical settings specifically to treat thesuppression of the HPA axis

An agent that not only resists the suppression of HPA axisinduced by GCs but also has no adverse influence on thetherapeutic action of GCs is required Our previous studiesrevealed that a herbal compound prescribed in traditionalChinese medicine and already used in clinical treatmentmay be a suitable agent [10] Oral GCs were successfullyused to control asthma but due to their suppression of theHPA axis GCs dependence was frequently developed Thesuccessful withdrawal rate of GCs was very low at about12 [11 12] Subsequently inhalation of large doses of GCswas used to attenuate the adverse effects on the HPA axisand the successful withdrawal rate increased up to 27sim44[13 14] In order to further promote the success rate GCswereused in combination with a herbal compound prescriptionKidney-tonifying decoction Consequently we found that thesuccessful withdrawal rate was significantly increased to 70[10]

The encouraging results from clinical studies promptedus to isolate the possible effective components through whichthe Kidney-tonifying decoction exerts protective efficacy onHPA axis Among the numerous ingredients we identifiedepimedium flavones (EFs) extracted from epimedium herbcontained in the Kidney-tonifying decoction as a majorcomponentWepostulated that EFswere the effective compo-nents within the Kidney-tonifying decoction In the presentstudy we examined the protective role of EFs on theHPA axisin animals treated with large doses of GCs

2 Experimental Section

21 Study Design Four-month-old male Sprague-Dawleyrats weighting 200 plusmn 15 g were randomly assigned into 3groups that is a normal control group a corticosterone only-treated group and a combined corticosterone and EF -treatedgroup Each group consisted of 16 rats All rats were housedin a temperature-controlled room (24∘C) with lights on from0600 hsim1800 h daily andwere allowed free access towater andrat pellet chow The corticosterone only-treated group wassubcutaneously injected with 01mL corticosterone dissolvedin olive pomace oil at a dose of 10mgkg body weight perday for 14 days This group received simultaneous oral saline(02mL) for 14 days The combined corticosterone and EF-treated group was injected with the same dose of corticos-terone as the corticosterone-treated group but received EForally at a daily dose of 60mgkg body weight In the control

group rats were subcutaneously injected with 01mL olivepomace oil and orally administrated with the same volumeof saline as above Fourteen days later the rats were killed bydecapitation The animal protocols were in accordance withAnimal Care and Use Committee of Fudan University

22 Preparation of EFs Epimedium is a major component inKidney-tonifying decoction which has been used to increasethe success rate of GCs withdrawal The Epimedium waspurchased from a traditional chinese medicine distributor(Shanghai U-sea Biotech Co Ltd Shanghai China) Theorigin of epimedium was in Liaoning province ChinaThe epimedium was verified by De-yun Kong a professorof Shanghai Institute of Pharmaceutical Industry ChinaA voucher specimen was deposited in the Herbarium ofShanghai Institute of Pharmaceutical Industry China Onlythe leaves and stems of Epimedium were used in our studyThe total flavonoids of epimediumwere extracted and used aspreviously [15] Epimedium was ground to a powder (about30 meshes) by a disintegrator and the powder (1000 g) wasextracted twicewith 10 L 75 ethanol for 2 h under refluxTheextracts were combined together and filtrated with cottonand the filtrate was concentrated under vacuum to give anaqueous fluid The aqueous fluid was subjected to a glasscolumn (5 cm times 80 cm) of macroporous resin (D101 400 g)and washed with water and 70 ethanol respectively Theeluent of 70 ethanol was concentrated in vacuum using arotary evaporator to dry the EFs powder (115 g puritygt70)The EFs were solvated with water to a 3 concentrationbefore orally administered to the rats [15] The content ofingredients of EFs was determined by high-performanceliquid chromatography (HPLC)

23 Plasma ACTH Assay After rats were sacrificed bloodsamples of 2mL per rat were immediately collected intoheparin-pretreated tubes and tubes were incubated for30min at room temperature followed by centrifugationat 1000 g for 15min The supernatant was obtained andaliquots of 015mL were prepared and stored at minus80∘C priorto assay The assay was performed using the DSL-10-5100ACTIVE ELISA kit (Diagnostic Systems Laboratories TXUSA) The detailed protocol was carried out according tothe manufacturerrsquos instructions In brief 100 120583L of variousconcentrations of ACTH standards positive control andsamples were used respectively followed by addition ofantibody-biotin conjugate streptavidin-enzyme conjugatechromogen solution and stopping solution in turn Finallyall samples were assayed at wavelength of 405 nm and awavelength of 530 nm was used as a reference

In order to determine the interassay coefficient of varia-tion (CV) three sampleswere assayed in the same experiment16 times then the CV was calculated In order to determineintrassay CV three samples were assayed in 8 separatedexperiments and the CV value was calculated

24 Plasma Corticosterone Assay The sample preparationwas the same as the ACTH assay For determining intra-or interassay CV the same method as that in ACTH assay


00 50 100 150 200

0

25

50

75

100

(mAU

)

270nm 4nm (100)smth

(min)

(a)

00 50 100 150

0

25

50

75

(mAU

)

270nm 4nm (100)smth

(min)

(b)








3 Results




120

100

80

60

40

20

0


lowastlowast

Body

wei

ght g

ain

on th

e 7th

day

(g)

(a)

160

140

120

100

80

60

40

20

0


lowastlowast

Body

wei

ght g

ain

on th

e 14t

h da

y (g

)(b)












200

160

120

80

40

0


lowastlowast

Plas

ma A

CTH

leve

l (pg

mL)

(a)

160

120

80

40

0


lowastlowast

Plas

ma c

ortic

oste

rone

leve

l (ng

mL)

(b)





4 Discussion





Cap

G

F

(a)

Cap G F

(b)

Cap

G

F

(c)

70

60

50

40

30

20

10

0


lowastlowast

BrdU

-pos

itive

cells

()

(d)






Cap

G

F

(a)

CapG

F

(b)

CapG

F

(c)

100

80

60

20

0

40



BrdU

-pos

itive

cells

()

(d)











Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00 50 100 150 200

0

25

50

75

100

(mAU

)

270nm 4nm (100)smth

(min)

(a)

00 50 100 150

0

25

50

75

(mAU

)

270nm 4nm (100)smth

(min)

(b)








3 Results




120

100

80

60

40

20

0


lowastlowast

Body

wei

ght g

ain

on th

e 7th

day

(g)

(a)

160

140

120

100

80

60

40

20

0


lowastlowast

Body

wei

ght g

ain

on th

e 14t

h da

y (g

)(b)












200

160

120

80

40

0


lowastlowast

Plas

ma A

CTH

leve

l (pg

mL)

(a)

160

120

80

40

0


lowastlowast

Plas

ma c

ortic

oste

rone

leve

l (ng

mL)

(b)





4 Discussion





Cap

G

F

(a)

Cap G F

(b)

Cap

G

F

(c)

70

60

50

40

30

20

10

0


lowastlowast

BrdU

-pos

itive

cells

()

(d)






Cap

G

F

(a)

CapG

F

(b)

CapG

F

(c)

100

80

60

20

0

40



BrdU

-pos

itive

cells

()

(d)











Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















120

100

80

60

40

20

0


lowastlowast

Body

wei

ght g

ain

on th

e 7th

day

(g)

(a)

160

140

120

100

80

60

40

20

0


lowastlowast

Body

wei

ght g

ain

on th

e 14t

h da

y (g

)(b)












200

160

120

80

40

0


lowastlowast

Plas

ma A

CTH

leve

l (pg

mL)

(a)

160

120

80

40

0


lowastlowast

Plas

ma c

ortic

oste

rone

leve

l (ng

mL)

(b)





4 Discussion





Cap

G

F

(a)

Cap G F

(b)

Cap

G

F

(c)

70

60

50

40

30

20

10

0


lowastlowast

BrdU

-pos

itive

cells

()

(d)






Cap

G

F

(a)

CapG

F

(b)

CapG

F

(c)

100

80

60

20

0

40



BrdU

-pos

itive

cells

()

(d)











Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















200

160

120

80

40

0


lowastlowast

Plas

ma A

CTH

leve

l (pg

mL)

(a)

160

120

80

40

0


lowastlowast

Plas

ma c

ortic

oste

rone

leve

l (ng

mL)

(b)





4 Discussion





Cap

G

F

(a)

Cap G F

(b)

Cap

G

F

(c)

70

60

50

40

30

20

10

0


lowastlowast

BrdU

-pos

itive

cells

()

(d)






Cap

G

F

(a)

CapG

F

(b)

CapG

F

(c)

100

80

60

20

0

40



BrdU

-pos

itive

cells

()

(d)











Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cap

G

F

(a)

Cap G F

(b)

Cap

G

F

(c)

70

60

50

40

30

20

10

0


lowastlowast

BrdU

-pos

itive

cells

()

(d)






Cap

G

F

(a)

CapG

F

(b)

CapG

F

(c)

100

80

60

20

0

40



BrdU

-pos

itive

cells

()

(d)











Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cap

G

F

(a)

CapG

F

(b)

CapG

F

(c)

100

80

60

20

0

40



BrdU

-pos

itive

cells

()

(d)











Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















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 Epimedium Flavonoids Counteract the Side...

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Transcript of Research Article Epimedium Flavonoids Counteract the Side...