Research Article Protective Effect of Decursin Extracted ...downloads.hindawi.com/journals/omcl/2016/5901098.pdf · anti-Nrf (/), anti-HO- (/), anti-Bax (/), and anti-Bcl- (:) polyclonal

Research ArticleProtective Effect of Decursin Extracted from Angelica gigas inMale Infertility via Nrf2HO-1 Signaling Pathway

Woong Jin Bae12 U Syn Ha2 Jin Bong Choi2 Kang Sup Kim2 Su Jin Kim2 Hyuk Jin Cho2

Sung Hoo Hong2 Ji Youl Lee2 Zhiping Wang3 Sung Yeoun Hwang4 and Sae Woong Kim12

1Catholic Integrative Medicine Research Institute College of Medicine The Catholic University of KoreaSeoul 137-701 Republic of Korea2Department of Urology College of Medicine The Catholic University of Korea Banpo-daero 222 Seocho-guSeoul 137-701 Republic of Korea3Department of Urology Second Hospital of Lanzhou University Lanzhou China4Korea Bio Medical Science Institute Seoul Republic of Korea

Correspondence should be addressed to Sae Woong Kim ksw1227catholicackr

Received 21 September 2015 Revised 17 November 2015 Accepted 6 December 2015

Academic Editor Sergio Di Meo

Copyright copy 2016 Woong Jin Bae 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

Higher testicular temperature results in altered spermatogenesis due to heat-related oxidative stress We examined the effects ofdecursin extracted from Angelica gigasNakai on antioxidant activity in vitro and in a cryptorchidism-induced infertility rat modelTM3 Leydig cell viability was measured based on oxidative stress according to treatment Either distilled water or AG 400mgkgof A gigas extract was administered orally for 4 weeks after unilateral cryptorchidism was induced After 1 2 and 4 weekssix rats from the control group and six rats from treatment group were sacrificed Testicular weight semen quality antioxidantactivities nuclear factor erythroid 2-related factor 2 (Nrf2) protein and mRNA expression of Nrf2-regulated genes were analyzedTreatment with A gigas extract (1) protected TM3 cells against oxidative stress in a dose-dependent manner (2) improved themean weight of the cryptorchid testis (3) maintained sperm counts motility and spermatogenic cell density (4) decreased levelsof 8-hydroxy-2-deoxyguanosine (8-OHdG) and increased levels of superoxide dismutase (SOD) (5) significantly increased Nrf2and heme oxygenase-1 (HO-1) and (6) significantly decreased apoptosis This study suggests that decursin extracted from A gigasis a supplemental agent that can reduce oxidative stress by Nrf2-mediated upregulation of HO-1 in rat experimentally inducedunilateral cryptorchidism and may improve cryptorchidism-induced infertility

1 Introduction

Infertility which can be explained as the failure of a couplein trying to conceive after one year of frequent unprotectedsexual intercourse is a serious clinical issue that affects 13ndash15 of couples worldwide [1] Male infertility is respon-sible for 60 of cases involving conceptive couples withpregnancy-related problems [2] Sperm is produced by thehighly complicated process of spermatogenesis and partialor complete interruption of spermatogenesis ultimately leadsto oligospermia or azoospermia

Observation study on male infertility with oligozoosper-mia or azoospermia in particular suggests that some patients

may have testicular heat exposure due to an intrinsic defect inscrotal thermoregulation varicocele or work hazard [3] Sev-eral studies report that testicular hyperthermia above normalranges causes impaired spermatogenesis due to heat-relatedoxidative stress on the seminiferous tubules [4 5] Moreovernuclear factor erythroid 2-related factor 2 (Nrf2) plays asignificant role in preventing the development of oxidativestress in spermatogenesis [6] Yu et al [7] also demonstrateda strong correlation between functional discrepancy in Nrf2promoter gene and abnormal spermatogenesis in humans

Decursin a major active ingredient from Angelica gigasNakai (Apiaceae) has been reported to inhibit the growth ofvarious cancer cells through cell cycle arrest and apoptosis

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 5901098 9 pageshttpdxdoiorg10115520165901098

2 Oxidative Medicine and Cellular Longevity

[8 9] In addition a protective effect of decursin has beensuggested against the neurotoxicity in animal cortical cells[10] Even further decursin plays a major role as free radicalscavenger and activated the upregulation of heme oxygenase-1 (HO-1) expression through stimulation of Nrf2 conferringprotection against oxidative damage in rat pheochromocy-toma (PC12) cells [11]

We examined the effects of decursin extracted fromAngelica gigas on antioxidant activity in vitro and in acryptorchidism-induced infertility rat model We hypothe-sized that decursin-induced HO-1 expression would protectagainst heat stress-induced degeneration of testicular germcells and apoptosis

2 Methods

21 Preparation of Angelica gigas Extract and Characteriza-tion of Decursin The extract of Angelica gigas used in ourstudy was produced using the followingmethod commercialAngelica gigas roots were extracted with 12000mL of 30ethanol for 3 hours at 90ndash100∘C The extracts were filteredtwice through a 50120583m and a 1 120583m filter and concentrated invacuo and then lyophilized Decursin in the Angelica gigasextract was analyzed and quantified by high performanceliquid chromatography (HPLC) using Waters 2695 Prepara-tion Module HPLC system (Waters Corporation MA USA)Several peaks were obtained in the HPLC chromatogramby diode array detection (DAD) at 230 nm The major peakwas identified as decursin by comparison with the standardcompound (Figure 1) As a result of this assay decursincontent was quantified as 376 plusmn 22mgg

22 Cell Viability Test In Vitro TM3 cells an imma-ture mouse Leydig cell line (Korean Cell Line BankSeoul Korea) were cultured in Dulbeccorsquos modified Eaglersquosmedium (DMEM)F-12 medium (GIBCO Life TechnologiesCo USA) supplemented with 10 heat-inactivated fetalbovine serum (FBS GIBCO) at 37∘C Cells (75000 cellsmLmediumwell) were plated in 24-well cluster dishes unlessotherwise specified They were seeded on 96-well plates in10 FBSDMEMF-12 and breeded for 24 hours They werepretreated with the Angelica gigas extract for two hours andtreated with 40 uM hydrogen peroxide (H

2O2) for two hours

to create oxidative cellular stress Afterwards alamarBlue(Invitrogen USA) was added to the cells and the intensityof the presented color was measured at 570 nm using ELISAReader (Molecular Devices USA) after incubating for 3hours Cell viability was calculated as previously described[12]

23 Animal Groups and Treatment Protocol This study wasinvestigated in strict accordance with the recommenda-tions in the Guide for the Care and Use of LaboratoryAnimals of the National Institutes of Health The protocolwas approved by the Institutional Animal Care and UseCommittee in School of Medicine The Catholic Univer-sity of Korea (CUMC-2012-0168-01) Thirty-six 8-week-oldmale Sprague-Dawley rats were treated under an approved

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200

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250

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202

92

187

287

23

428

366

74

099

435

5

569

36

068

707

07

291

861

7

107

40

118

62

216

16

227

82

247

74

(min)

(AU

)

(a)

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002

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400

600

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012

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140

016

00

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028

00

180

4 213

52

868

239

51

252

88

(min)

(AU

)

(b)

Figure 1 HPLC chromatogram of the extract of Angelica gigas (a)and the standard solution (b) The peak with the arrow indicatesdecursin in standard compounds A corresponding peak was seenin the extract HPLC chromatogram

protocol Unilateral cryptorchidism in rats was surgicallyinduced as previously described [13] Animals were anes-thetized and a midline abdominal incision was performedThe gubernaculum on the left side was cut to displace thetestis into the abdomen and the inguinal canal was closedThe contralateral testis was sham-operated to act as thepaired control the gubernaculum was cut and the inguinalcanal reconstructed Either distilled water (119899 = 18 controlgroups) or 400mgkg of Angelica gigas extract (119899 = 18treatment groups) was administered orally for 4 weeks afterunilateral cryptorchidism was inducedAngelica gigas extractwas dissolved in distilled water and administrated orally oncea day After 1 2 and 4 weeks six rats from the control groupand six rats from treatment group were sacrificed and bloodsamples were collected The testes and epididymides wereresected after anesthesia and measured

24 Evaluation of Cauda Epididymal Sperm Count and Motil-ity Samples of spermatozoa were collected from the caudalregion of epididymis by mincing it finely in normal salinecontaining 05 bovine serum albumin at 37∘C and then

Oxidative Medicine and Cellular Longevity 3

Table 1 Primers used for real-time PCR

Gene GenBank accession number Sequence (51015840 rarr 31015840) Length of DNA product (bp)

Beta-actin (ACTB) NM 007393 F CTGTCCCTGTATGCCTCTG 218R ATGTCACGCACGATTTCC

Nuclear factor erythroid 2-related factor 2 NM 010902 F CAGTGCTCCTATGCGTGAA 109R GCGGCTTGAATGTTTGTC

Heme oxygenase-1 NM 010442 F ACAGATGGCGTCACTTCG 128R TGAGGACCCACTGGAGGA

were filtered Sperm suspensions were analyzed as previouslydescribed [14] The sperm count represents the number ofsperms in 1mL of the medium Sperm motility is expressedas the percentage of sperm that showed any movement

25 Measurement of Spermatogenic Cell Density Testiculartissues procured were fixed embedded in paraffin stainedwith haematoxylin-eosin and inspected under a light micro-scope at times400 magnification Ten characteristic sites in sem-iniferous tubules were selected randomly and spermatogeniccell density wasmeasured It was calculated as the diameter ofgerminal cell layer divided by the width of the seminiferoustubule [15]

26 Measurement of Oxidative Stress Oxidative stress wasassessed by measuring the 8-hydroxy-2-deoxyguanosine (8-OHdG) content and superoxide dismutase (SOD) activityquantitatively Total DNA was extracted from the testis usingthe DNeasy Blood amp Tissue kit (Qiagen Valencia CAUSA) The level of 8-OHdG was measured with a DNAoxidation kit (Highly Sensitive 8-OHdG Check ELISA JapanInstitute for the Control of Aging Fukuroi Japan) Afterthe final color was developed with the addition of 331015840551015840-tetramethylbenzidine absorbance was measured at 450 nmTissue sample concentration was measured from a standardcurve and corrected for DNA concentration SOD activity(CuZnSOD and Mn SOD) in tissue was determined usingSOD Assay Kit-WST (Dojindo) and the decrease in the rateof superoxide-mediated reduction of nitroblue tetrazoliummonitored at 450 nm using a spectrophotometer

27 Western Blot Analysis Western blot was performedby the standard method Equal amounts of proteins werefractionated by SDS-PAGE gel electrophoresis and electro-transferred to Immun-Blot PVDF membrane (02 120583M poresize Bio-Rad) Membranes were blocked overnight at 4∘C inTris-buffered saline (TBS) 005 (vv) Tween-20 150mMNaCl and 5 (wv) bovine serum albumin (BSA SantaCruz Biotechnology Santa Cruz CA USA) followed by 2hours of incubation with primary antibody diluted in thesame buffer Immunoblot analysis was carried out usinganti-Nrf2 (1250) anti-HO-1 (11000) anti-Bax (11000)and anti-Bcl-2 (1 1000) polyclonal antibody (Abcam CoUK) After washing with TBS-T (TBS 01 Tween 20)the membrane was incubated with anti-rabbit IgG AP-linked secondary antibody and then washed with the same

buffer The immunoblotted membrane was developed withBCIPNBT color-developing solution The blots in the sam-ples were quantified by densitometry analysis using PDQuestsoftware (Version 70 Bio-Rad Hercules CA USA)

28 Quantitative Real-Time PCR The frozen testes werehomogenized with TRIzol reagent (Invitrogen Carlsbad CAUS) to extract mRNA and cDNA synthesis was performedusing the SuperScript 3 First-Strand kit (Invitrogen CarlsbadCA US) according to the manufacturerrsquos information Gene-specific primers were determined based on the correspond-ing mRNA sequences with Primer Version 50 (Table 1) PCRamplification of cDNA was performed in a real-time PCRmachine step on plus (Applied Biosystems) with SYBRGreenPCR Master Mix (Invitrogen) as indicated 2 minutes at50∘C for dUTP activation and 10 minutes at 95∘C for initialdenaturation of cDNA followed by 40 cycles each consistingof 15 s of denaturation at 95∘C and 60 s at 60∘C for primerannealing and chain extension

29 Statistical Analysis Statistical analyses were performedusing SPSS 160 (SPSS Inc Chicago USA) The data wasexpressed as mean plusmn standard deviation Statistical signif-icance was analyzed by ANOVA test and 119901 lt 005 wasconsidered to be significant

3 Results

31 Protective Effect against Oxidative Stress in TM3 Cells Toevaluate whether Angelica gigas extract could protect TM3cells against H

2O2-induced damage alamarBlue assay was

performed IncubationwithH2O2significantly decreased cell

viability by 40 compared to untreated cells Increased cellviability was found in a dose-dependent manner (Figure 2)The viabilities were increased to 140 and 165 by treatmentwith Angelica gigas extract concentrations of 10 120583gmL and50 120583gmL respectively

32 Body and Testes Weights There was no significant differ-ence in contralateral testicular weights for four weeks Uponcryptorchidism induction in the first week there were alsono significant differences in left testicular weights betweenthe control group and the treatment group However themean weight of the left testes from the treatment groupwas significantly larger compared with the control group on


Table 2 Comparisons of parameters of the cryptorchid testicular health

Left testicular weight (g) Sperm count (times106g cauda) of motile spermatozoa Spermatogenic cell density

1st week Control 1526 plusmn 0225 3502 plusmn 109 207 plusmn 33 0343 plusmn 0016AG 1612 plusmn 0033 3606 plusmn 140 225 plusmn 52 0305 plusmn 0031

2nd week Control 1232 plusmn 0165 2402 plusmn 136 76 plusmn 23 0275 plusmn 0043AG 1515 plusmn 0367lowast 3008 plusmn 125lowast 145 plusmn 27lowast 0314 plusmn 0074lowast

4th week Control 0858 plusmn 0285 752 plusmn 182 57 plusmn 31 0154 plusmn 0028AG 1433 plusmn 0634lowast 3105 plusmn 147lowast 132 plusmn 82lowast 0269 plusmn 0052lowast

Data show the mean plusmn SD AG the extract of Angelica gigas treatmentlowast

119901 lt 005 as compared with the control group at the same period of time

0

20

40

60

80

100

120

140

160

180

200

Control

Cel

l via

bilit

y (

of c

ontro

l)

AG50120583gmL

AG10120583gmL

lowast

lowast

Figure 2 Protective effects of Angelica gigas extract against H2

O2

The cells were pretreated with the extract of Angelica gigas 2 hoursbefore H

2

O2

treatment lowast119901 lt 005 as compared with the controlgroup

the second and fourth weeks (119901 lt 005)Themean weights ofthe left testes are listed in Table 2

33 Sperm Counts and Motility Mean sperm counts andthe percentage of motile spermatozoa in the left epididymisare shown in Table 2 There were no significant differencesbetween the control and treatment groups in the first weekbut mean sperm counts and the percentage of motile sper-matozoa from the control groupwere significantly lower thanthose in the treatment group by the second and fourth weeks(119901 lt 005)

34 Spermatogenic Cell Density Upon cryptorchidism in-duction in the first week considerable spermatocytes linedup the germinal cell layer in the control and the treatmentgroups (Figure 3) On the other hand the seminiferous tubuleis shrunken and the germinal cell layers are decreased inthe control group after 2 weeks The germinal cell layerin the treatment group was thicker compared with that ofthe control group by the second and fourth weeks (Table 2

119901 lt 005)There was no significant difference in contralateraltesticular spermatogenic cell density

35 Measurement of Oxidative Stress Mean expression ofoxidative stress markers in the left testes is shown in Figure 4There were no significant differences in 8-OHdG and SODexpression between the two groups in the first week Atime-dependent increase in 8-OHdG and a decrease in SODwere examined in the control group but oxidative stress wasobserved to be significantly lower in the treatment group atthe same period of time (119901 lt 005)

36 Expression of Nrf2 and HO-1 We found that significantincreases of Nrf2 and HO-1 proteins were exhibited inthe treatment group compared with the control group bythe second and fourth weeks (Figure 5(a)) There were nosignificant differences inNrf2 andHO-1mRNA in the controlgroup across time points but mRNA transcript levels weresignificantly higher in the treatment group comparedwith thecontrol group by the second and fourth weeks (Figure 5(b))Moreover the progressive increase in Nrf2 levels was firmlyassociated with an increase in HO-1 expression

37 Apoptosis-Related Protein Expression The expression ofproapoptotic (Bax) and antiapoptotic (Bcl-2) proteins wasinvestigated Treatment with Angelica gigas extract signif-icantly declined the level of Bax protein with a collateralincrease in Bcl-2 protein compared with the control groupover the whole period (Figure 6(a))These changes decreasedthe BaxBcl-2 ratio which is considered to be an index toevaluate apoptosis (Figure 6(b))

4 Discussion

Themain findings of our study were as followsThe treatmentwith Angelica gigas extract (1) protected TM3 cells againstH2O2-induced oxidative stress in a dose-dependent manner

(2) improved the mean weight of the cryptorchid testis (3)maintained sperm counts motility and spermatogenic celldensity (4) decreased levels of 8-OHdG and increased levelsof SOD demonstrating its antioxidant effect (5) significantlyincreased Nrf2 and HO-1 and (6) significantly decreasedapoptosis

We evaluated the protective effect of Angelica gigasextract against H

2O2-induced oxidative stress in TM3 cells


1st weekAG

Con

trol

2nd week 4th week

Figure 3 Time-dependent histopathological findings of left testis (haematoxylin and eosin stain) Scale bars shown in each figure represent100120583m AG the extract of Angelica gigas treatment

0

10

20

30

40

50

60

Control AG Control AG Control AG1st week 2nd week 4th week

SOD

activ

ity (

) lowastlowast

(a)

0

1

2

3

4

5


8-O

HdG

(ng

mL)

lowastlowast

(b)

Figure 4 Comparison of the expression levels of SOD (a) and 8-OHdG (b) AG the extract ofAngelica gigas treatment lowast119901 lt 005 as comparedwith the control group at the same period of time

In addition we demonstrated that the mean weight of thecryptorchid testis in the control group was significantlydecreased compared with that of the treatment group bythe second and fourth weeks Most mammal testes are moresensitive to heat than other organs and scrotal temperatureis lower than core body temperature [16 17] Elevated intrat-esticular temperature induces oxidative stress resulting inapoptosis and impairment of spermatogenesis [18 19] It iscorrelated with a decrease in cellular antioxidant defensesor an increase in the production of reactive oxygen species(ROS) [20] Several studies demonstrated that increased ROSimpaired the physiological processes such as capacitationhyperactivation acrosome reactions and signaling processesto provide appropriate fertilization [21] Thus antioxidantsupplement may help improve the imbalance of an excessiveROS and restore sperm parameters

Oral supplements and herbal medicines have been pro-posed to recover male factor infertility [22] These sup-plements were reported to improve sperm quality andtheir antioxidants are thought to decrease ROS in oligoas-thenospermia patients [23ndash26] We found that treatmentwith Angelica gigas extract decreases oxidative stress incryptorchidism-induced rats and posit that the curativeeffects may contribute to suppression of ROS productionIn addition we examined the detailed mechanism of theantioxidant activity in Angelica gigas extract

HO-1 is a stress-responsive protein shortly activated byvariable noxious stimuli as well as oxidative stress HO-1 hasbeen known as the cytoprotective effect resistant to oxidativestress [27] The Nrf2 antioxidant system also has been rec-ognized as an important therapeutic target against oxidativestress producing expression of cytoprotective enzymes and


HO-1

Nrf2

120573-actin

1st week

AG

Con

trol

AG

Con

trol

AG

Con

trol

2nd week 4th week

0

004

008

012

016


Relat

ive N

rf2 ex

pres

sion


0

004

008

012

016

Relat

ive H

O-1

expr

essio

n

lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25


Relat

ive N

rf2 m

RNA

expr

essio

n

0

04

08

12

16

2

24


Relat

ive H

O-1

mRN

A ex

pres

sion

lowastlowast

lowast

lowast

(b)

Figure 5 Effect of Angelica gigas extract on protein expression (a) and mRNA expression (b) of Nrf2-regulated genes AG the extract ofAngelica gigas treatment HO-1 heme oxygenase-1 Nrf2 nuclear factor erythroid 2-related factor 2 lowast119901 lt 005 as compared with the controlgroup at the same period of time

related proteins [28] Previous studies have reported thatoxdative stress resulted in lower epididymal sperm motilityin Nrf2 knockout mice [6] Nrf2 is released and transmits thestress signal to the nucleus for activation of a specific set ofgenes encoding phase II antioxidant enzymes as well as stressresponsive proteins such as HO-1 [29]

Li et al [30] studied about the oxidative damage on malereproductive organ The whole body heat stress upregulatedNrf2 expression for about aweek but the significant increasedoxidative stress was identified thereafter Several supplements(eg sulforaphane curcumin and caffeic acid phenethylester) activate the Nrf2 antioxidant response element (ARE)


Bax

Bcl-2

120573-actin

1st week

AG

Con

trol

AG

Con

trol

AG

Con

trol

2nd week 4th week(a)

0

05

1

15

2

25

3

Con

trol

AG

Con

trol

AG

Con

trol

AG

1st week 2nd week 4th week

Relat

ive B

ax ex

pres

sion

Relat

ive B

cl-2

expr

essio

n

0

004

008

012

016

Con

trol

AG

Con

trol

AG

Con

trol

AG


0

004

008

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016

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trol

AG

Con

trol

AG

Con

trol

AG


Relat

ive B

axB

cl-2

expr

essio

n

lowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

(b)

Figure 6 Comparison of the expression levels of Bax and Bcl-2 expression in the testicular tissue (a) Western blot analysis of Bax and Bcl-2(b) Densitometric analysis of Bax Bcl-2 and BaxBcl-2 ratio relative to beta-actin AG the extract of Angelica gigas treatment lowast119901 lt 005 ascompared with the control group at the same period of time

system [31 32] Li et al [11] reported that decursin treat-ment leads to the upregulation of Nrf2HO-1 expressionin PC12 cells Decursin may have the possibilities to pre-vent chemotherapy-induced cytotoxicity via the activation

of antioxidant enzyme We found that decursin extractedfrom Angelica gigas increased Nrf2 and HO-1 in the cryp-torchid testis Moreover a prevention of decrease in SODwas observed in the treatment group Therefore our results


suggest that antioxidant defense is reinforced by decursin viaactivation of Nrf2 and upregulation of antioxidant enzymeactivity

The Bcl-2 family and related proteins are key regulatorsof apoptosis [33] They can be classified into two groupsBcl-2 an antiapoptotic protein and Bax a proapoptoticprotein [34] Knudson et al [35] demonstrated that the Bcl-2 family mainly plays a role during spermatogenesis in Bax-deficient mice Several studies have reported the importanceof the Bcl-2 family in heat-induced oxidative stress on malereproductive function [36 37] They suggested the possibleinvolvement of Bcl-2 and Bax in the germ cell apoptosis Wealso investigated the germ cell apoptosis induced by cryp-torchidism and the antiapoptotic effect of decursin extractedfrom Angelica gigas

We examined the role of decursin extracted from Angel-ica gigas as a supplemental agent to prevent heat-inducedoxidative stress in cryptorchidism Our limitation is that themore accurate infertility model due to oxidative stress isneeded More defense mechanisms such as Nrf2 and relatedantioxidative enzyme are required if oxidative stress causesimpaired semen parameters DNA damage and apoptosisAntioxidant supplementation as well as lifestyle change likeno smoking or balanced diet can be helpful for the naturalbalance between ROS and antioxidant Therefore our studymay support appropriate evidence for the use of a newcomplementary and alternative medicine for treating maleinfertility In addition future work should investigate adetailed mechanism of decursin with or without Nrf2 relatedenzyme activity

The present study suggests that decursin extracted fromAngelica gigas is a supplemental agent that can reduceoxidative stress by Nrf2-mediated upregulation of HO-1 andmay improve cryptorchidism-induced infertility

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by a grant of the Korea HealthTechnology RampD Project through the Korea Health IndustryDevelopment Institute (KHIDI) funded by the Ministry ofHealth ampWelfare Republic of Korea (Grant no HI15C0099)This study was supported by Research Fund of Seoul StMaryrsquos Hospital The Catholic University of Korea

References

[1] J P Jarow I D Sharlip AM Belker et al ldquoBest practice policiesfor male infertilityrdquo The Journal of Urology vol 167 no 5 pp2138ndash2144 2002

[2] PThonneau S Marchand A Tallec et al ldquoIncidence and maincauses of infertility in a resident population (1850000) of threeFrench regions (1988-1989)rdquoHuman Reproduction vol 6 no 6pp 811ndash816 1991

[3] R Dada N P Gupta and K Kucheria ldquoSpermatogenic arrest inmen with testicular hyperthermiardquo Teratog Carcinog Mutagensupplement 1 pp 235ndash243 2003

[4] A Kumagai H Kodama J Kumagai et al ldquoXanthine oxidaseinhibitors suppress testicular germ cell apoptosis induced byexperimental cryptorchidismrdquoMolecularHumanReproductionvol 8 no 2 pp 118ndash123 2002

[5] R M Vigueras-Villasenor I Ojeda O Gutierrez-Perez et alldquoProtective effect of 120572-tocopherol on damage to rat testes byexperimental cryptorchidismrdquo International Journal of Exper-imental Pathology vol 92 no 2 pp 131ndash139 2011

[6] B N Nakamura G Lawson J Y Chan et al ldquoKnockout of thetranscription factor NRF2 disrupts spermatogenesis in an age-dependent mannerrdquo Free Radical Biology and Medicine vol 49no 9 pp 1368ndash1379 2010

[7] B Yu H Lin L Yang et al ldquoGenetic variation in the Nrf2 pro-moter associates with defective spermatogenesis in humansrdquoJournal of Molecular Medicine vol 90 no 11 pp 1333ndash13422012

[8] J Guo C Jiang ZWang et al ldquoA novel class of pyranocoumarinanti-androgen receptor signaling compoundsrdquoMolecular Can-cer Therapeutics vol 6 no 3 pp 907ndash917 2007

[9] H J Lee H J Lee E O Lee et al ldquoIn vivo anti-cancer activity ofKoreanAngelica gigas and its major pyranocoumarin decursinrdquoTheAmerican Journal of ChineseMedicine vol 37 no 1 pp 127ndash142 2009

[10] S Y Kang and Y C Kim ldquoDecursinol and decursin protectprimary cultured rat cortical cells from glutamate-inducedneurotoxicityrdquo Journal of Pharmacy and Pharmacology vol 59no 6 pp 863ndash870 2007

[11] L Li J-K Du L-Y Zou T Wu Y-W Lee and Y-H KimldquoDecursin isolated from angelica gigas Nakai rescues PC12 cellsfrom amyloid 120573-protein-induced neurotoxicity through Nrf2-mediated upregulation of heme oxygenase-1 potential rolesof MAPKrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 467245 14 pages 2013

[12] H J Lee J-H Bach H-S Chae et al ldquoMitogen-activatedprotein kinaseextracellular signal-regulated kinase attenuates3-hydroxykynurenine-induced neuronal cell deathrdquo Journal ofNeurochemistry vol 88 no 3 pp 647ndash656 2004

[13] W J Bae U S Ha K S Kim et al ldquoEffects of KH-204 on theexpression of heat shock protein 70 and germ cell apoptosisin infertility rat modelsrdquo BMC Complementary and AlternativeMedicine vol 14 article 367 2014

[14] K W Ko J Y Chae S W Kim et al ldquoThe effect of the partialobstruction site of the renal vein on testis and kidney in rats isthe traditional animal model suitable for varicocele researchrdquoKorean Journal of Urology vol 51 no 8 pp 565ndash571 2010

[15] G Turk M Sonmez M Aydin et al ldquoEffects of pomegranatejuice consumption on spermquality spermatogenic cell densityantioxidant activity and testosterone level in male ratsrdquo ClinicalNutrition vol 27 no 2 pp 289ndash296 2008

[16] B P Setchell ldquoThe Parkes lecture Heat and the testisrdquo Journalof Reproduction and Fertility vol 114 no 2 pp 179ndash194 1998

[17] P J Hansen ldquoEffects of heat stress on mammalian reproduc-tionrdquoPhilosophical Transactions of the Royal Society B BiologicalSciences vol 364 no 1534 pp 3341ndash3350 2009

[18] C Paul S Teng and P T K Saunders ldquoA single mild transientscrotal heat stress causes hypoxia and oxidative stress in mousetestes which induces germ cell deathrdquo Biology of Reproductionvol 80 no 5 pp 913ndash919 2009


[19] K Shiraishi H Takihara and H Matsuyama ldquoElevated scrotaltemperature but not varicocele grade reflects testicular oxida-tive stress-mediated apoptosisrdquo World Journal of Urology vol28 no 3 pp 359ndash364 2010

[20] R J Aitken and S D Roman ldquoAntioxidant systems andoxidative stress in the testesrdquo Oxidative Medicine and CellularLongevity vol 1 no 1 pp 15ndash24 2008

[21] A Agarwal G Virk C Ong and S S du Plessis ldquoEffect ofoxidative stress on male reproductionrdquo The World Journal ofMenrsquos Health vol 32 no 1 pp 1ndash17 2014

[22] N A Clark M Will M B Moravek and S Fisseha ldquoA system-atic review of the evidence for complementary and alternativemedicine in infertilityrdquo International Journal of Gynecology andObstetrics vol 122 no 3 pp 202ndash206 2013

[23] A Agarwal and L H Sekhon ldquoThe role of antioxidant therapyin the treatment of male infertilityrdquoHuman Fertility vol 13 no4 pp 217ndash225 2010

[24] E Greco M Iacobelli L Rienzi F Ubaldi S Ferrero and JTesarik ldquoReduction of the incidence of sperm DNA fragmen-tation by oral antioxidant treatmentrdquo Journal of Andrology vol26 no 3 pp 349ndash353 2005

[25] A E Omu M K Al-Azemi E O Kehinde J T Anim MA Oriowo and T C Mathew ldquoIndications of the mechanismsinvolved in improved sperm parameters by zinc therapyrdquoMedical Principles and Practice vol 17 no 2 pp 108ndash116 2008

[26] M R Safarinejad ldquoEfficacy of coenzyme Q10 on semen param-eters sperm function and reproductive hormones in infertilemenrdquo Journal of Urology vol 182 no 1 pp 237ndash248 2009

[27] L E Otterbein and A M K Choi ldquoHeme oxygenase colors ofdefense against cellular stressrdquoAmerican Journal of PhysiologymdashLung Cellular and Molecular Physiology vol 279 no 6 ppL1029ndashL1037 2000

[28] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[29] K Itoh N Wakabayashi Y Katoh T Ishii T OrsquoConnorand M Yamamoto ldquoKeap1 regulates both cytoplasmic-nuclearshuttling and degradation of Nrf2 in response to electrophilesrdquoGenes to Cells vol 8 no 4 pp 379ndash391 2003

[30] Y Li Y Huang Y Piao et al ldquoProtective effects of nuclear factorerythroid 2-related factor 2 on whole body heat stress-inducedoxidative damage in the mouse testisrdquo Reproductive Biology andEndocrinology vol 11 article 23 2013

[31] C Chen and A-N T Kong ldquoDietary chemopreventive com-pounds and AREEpRE signalingrdquo Free Radical Biology andMedicine vol 36 no 12 pp 1505ndash1516 2004

[32] Y Morimitsu Y Nakagawa K Hayashi et al ldquoA sulforaphaneanalogue that potently activates the Nrf2-dependent detoxifica-tion pathwayrdquo The Journal of Biological Chemistry vol 277 no5 pp 3456ndash3463 2002

[33] J M Adams and S Cory ldquoThe Bcl-2 protein family arbiters ofcell survivalrdquo Science vol 281 no 5381 pp 1322ndash1326 1998

[34] YAkao YOtsuki S Kataoka Y Ito andY Tsujimoto ldquoMultiplesubcellular localization of bcl-2 detection in nuclear outermembrane endoplasmic reticulum membrane and mitochon-drial membranesrdquo Cancer Research vol 54 no 9 pp 2468ndash2471 1994

[35] C M Knudson K S K Tung W G Tourtellotte G A JBrown and S J Korsmeyer ldquoBax-deficient mice with lymphoidhyperplasia and male germ cell deathrdquo Science vol 270 no5233 pp 96ndash99 1995

[36] C M Yamamoto A P Sinha Hikim P N Huynh et alldquoRedistribution of Bax is an early step in an apoptotic pathwayleading to germ cell death in rats triggered by mild testicularhyperthermiardquo Biology of Reproduction vol 63 no 6 pp 1683ndash1690 2000

[37] Z-H Zhang X Jin X-S Zhang et al ldquoBcl-2 and Bax areinvolved in experimental cryptorchidism-induced testiculargerm cell apoptosis in rhesus monkeyrdquo Contraception vol 68no 4 pp 297ndash301 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


[8 9] In addition a protective effect of decursin has beensuggested against the neurotoxicity in animal cortical cells[10] Even further decursin plays a major role as free radicalscavenger and activated the upregulation of heme oxygenase-1 (HO-1) expression through stimulation of Nrf2 conferringprotection against oxidative damage in rat pheochromocy-toma (PC12) cells [11]

We examined the effects of decursin extracted fromAngelica gigas on antioxidant activity in vitro and in acryptorchidism-induced infertility rat model We hypothe-sized that decursin-induced HO-1 expression would protectagainst heat stress-induced degeneration of testicular germcells and apoptosis

2 Methods

21 Preparation of Angelica gigas Extract and Characteriza-tion of Decursin The extract of Angelica gigas used in ourstudy was produced using the followingmethod commercialAngelica gigas roots were extracted with 12000mL of 30ethanol for 3 hours at 90ndash100∘C The extracts were filteredtwice through a 50120583m and a 1 120583m filter and concentrated invacuo and then lyophilized Decursin in the Angelica gigasextract was analyzed and quantified by high performanceliquid chromatography (HPLC) using Waters 2695 Prepara-tion Module HPLC system (Waters Corporation MA USA)Several peaks were obtained in the HPLC chromatogramby diode array detection (DAD) at 230 nm The major peakwas identified as decursin by comparison with the standardcompound (Figure 1) As a result of this assay decursincontent was quantified as 376 plusmn 22mgg

22 Cell Viability Test In Vitro TM3 cells an imma-ture mouse Leydig cell line (Korean Cell Line BankSeoul Korea) were cultured in Dulbeccorsquos modified Eaglersquosmedium (DMEM)F-12 medium (GIBCO Life TechnologiesCo USA) supplemented with 10 heat-inactivated fetalbovine serum (FBS GIBCO) at 37∘C Cells (75000 cellsmLmediumwell) were plated in 24-well cluster dishes unlessotherwise specified They were seeded on 96-well plates in10 FBSDMEMF-12 and breeded for 24 hours They werepretreated with the Angelica gigas extract for two hours andtreated with 40 uM hydrogen peroxide (H

2O2) for two hours

to create oxidative cellular stress Afterwards alamarBlue(Invitrogen USA) was added to the cells and the intensityof the presented color was measured at 570 nm using ELISAReader (Molecular Devices USA) after incubating for 3hours Cell viability was calculated as previously described[12]

23 Animal Groups and Treatment Protocol This study wasinvestigated in strict accordance with the recommenda-tions in the Guide for the Care and Use of LaboratoryAnimals of the National Institutes of Health The protocolwas approved by the Institutional Animal Care and UseCommittee in School of Medicine The Catholic Univer-sity of Korea (CUMC-2012-0168-01) Thirty-six 8-week-oldmale Sprague-Dawley rats were treated under an approved

000

005

010

015

020

025

030

035

000

500

100

0

150

0

200

0

250

0

202

92

187

287

23

428

366

74

099

435

5

569

36

068

707

07

291

861

7

107

40

118

62

216

16

227

82

247

74

(min)

(AU

)

(a)

000

002

004

006

008

010

200

400

600

800

100

012

00

140

016

00

180

020

00

220

024

00

260

028

00

180

4 213

52

868

239

51

252

88

(min)

(AU

)

(b)

Figure 1 HPLC chromatogram of the extract of Angelica gigas (a)and the standard solution (b) The peak with the arrow indicatesdecursin in standard compounds A corresponding peak was seenin the extract HPLC chromatogram

protocol Unilateral cryptorchidism in rats was surgicallyinduced as previously described [13] Animals were anes-thetized and a midline abdominal incision was performedThe gubernaculum on the left side was cut to displace thetestis into the abdomen and the inguinal canal was closedThe contralateral testis was sham-operated to act as thepaired control the gubernaculum was cut and the inguinalcanal reconstructed Either distilled water (119899 = 18 controlgroups) or 400mgkg of Angelica gigas extract (119899 = 18treatment groups) was administered orally for 4 weeks afterunilateral cryptorchidism was inducedAngelica gigas extractwas dissolved in distilled water and administrated orally oncea day After 1 2 and 4 weeks six rats from the control groupand six rats from treatment group were sacrificed and bloodsamples were collected The testes and epididymides wereresected after anesthesia and measured

24 Evaluation of Cauda Epididymal Sperm Count and Motil-ity Samples of spermatozoa were collected from the caudalregion of epididymis by mincing it finely in normal salinecontaining 05 bovine serum albumin at 37∘C and then














3 Results














0

20

40

60

80

100

120

140

160

180

200

Control

Cel

l via

bilit

y (

of c

ontro

l)

AG50120583gmL

AG10120583gmL

lowast

lowast


O2


2

O2









4 Discussion






1st weekAG

Con

trol

2nd week 4th week


0

10

20

30

40

50

60


SOD

activ

ity (

) lowastlowast

(a)

0

1

2

3

4

5


8-O

HdG

(ng

mL)

lowastlowast

(b)






HO-1

Nrf2

120573-actin

1st week

AG

Con

trol

AG

Con

trol

AG

Con

trol

2nd week 4th week

0

004

008

012

016


Relat

ive N

rf2 ex

pres

sion


0

004

008

012

016

Relat

ive H

O-1

expr

essio

n

lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25


Relat

ive N

rf2 m

RNA

expr

essio

n

0

04

08

12

16

2

24


Relat

ive H

O-1

mRN

A ex

pres

sion

lowastlowast

lowast

lowast

(b)





Bax

Bcl-2

120573-actin

1st week

AG

Con

trol

AG

Con

trol

AG

Con

trol


0

05

1

15

2

25

3

Con

trol

AG

Con

trol

AG

Con

trol

AG


Relat

ive B

ax ex

pres

sion

Relat

ive B

cl-2

expr

essio

n

0

004

008

012

016

Con

trol

AG

Con

trol

AG

Con

trol

AG


0

004

008

012

016

Con

trol

AG

Con

trol

AG

Con

trol

AG


Relat

ive B

axB

cl-2

expr

essio

n

lowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

(b)











Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























3 Results














0

20

40

60

80

100

120

140

160

180

200

Control

Cel

l via

bilit

y (

of c

ontro

l)

AG50120583gmL

AG10120583gmL

lowast

lowast


O2


2

O2









4 Discussion






1st weekAG

Con

trol

2nd week 4th week


0

10

20

30

40

50

60


SOD

activ

ity (

) lowastlowast

(a)

0

1

2

3

4

5


8-O

HdG

(ng

mL)

lowastlowast

(b)






HO-1

Nrf2

120573-actin

1st week

AG

Con

trol

AG

Con

trol

AG

Con

trol

2nd week 4th week

0

004

008

012

016


Relat

ive N

rf2 ex

pres

sion


0

004

008

012

016

Relat

ive H

O-1

expr

essio

n

lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25


Relat

ive N

rf2 m

RNA

expr

essio

n

0

04

08

12

16

2

24


Relat

ive H

O-1

mRN

A ex

pres

sion

lowastlowast

lowast

lowast

(b)





Bax

Bcl-2

120573-actin

1st week

AG

Con

trol

AG

Con

trol

AG

Con

trol


0

05

1

15

2

25

3

Con

trol

AG

Con

trol

AG

Con

trol

AG


Relat

ive B

ax ex

pres

sion

Relat

ive B

cl-2

expr

essio

n

0

004

008

012

016

Con

trol

AG

Con

trol

AG

Con

trol

AG


0

004

008

012

016

Con

trol

AG

Con

trol

AG

Con

trol

AG


Relat

ive B

axB

cl-2

expr

essio

n

lowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

(b)











Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























0

20

40

60

80

100

120

140

160

180

200

Control

Cel

l via

bilit

y (

of c

ontro

l)

AG50120583gmL

AG10120583gmL

lowast

lowast


O2


2

O2









4 Discussion






1st weekAG

Con

trol

2nd week 4th week


0

10

20

30

40

50

60


SOD

activ

ity (

) lowastlowast

(a)

0

1

2

3

4

5


8-O

HdG

(ng

mL)

lowastlowast

(b)






HO-1

Nrf2

120573-actin

1st week

AG

Con

trol

AG

Con

trol

AG

Con

trol

2nd week 4th week

0

004

008

012

016


Relat

ive N

rf2 ex

pres

sion


0

004

008

012

016

Relat

ive H

O-1

expr

essio

n

lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25


Relat

ive N

rf2 m

RNA

expr

essio

n

0

04

08

12

16

2

24


Relat

ive H

O-1

mRN

A ex

pres

sion

lowastlowast

lowast

lowast

(b)





Bax

Bcl-2

120573-actin

1st week

AG

Con

trol

AG

Con

trol

AG

Con

trol


0

05

1

15

2

25

3

Con

trol

AG

Con

trol

AG

Con

trol

AG


Relat

ive B

ax ex

pres

sion

Relat

ive B

cl-2

expr

essio

n

0

004

008

012

016

Con

trol

AG

Con

trol

AG

Con

trol

AG


0

004

008

012

016

Con

trol

AG

Con

trol

AG

Con

trol

AG


Relat

ive B

axB

cl-2

expr

essio

n

lowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

(b)











Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1st weekAG

Con

trol

2nd week 4th week


0

10

20

30

40

50

60


SOD

activ

ity (

) lowastlowast

(a)

0

1

2

3

4

5


8-O

HdG

(ng

mL)

lowastlowast

(b)






HO-1

Nrf2

120573-actin

1st week

AG

Con

trol

AG

Con

trol

AG

Con

trol

2nd week 4th week

0

004

008

012

016


Relat

ive N

rf2 ex

pres

sion


0

004

008

012

016

Relat

ive H

O-1

expr

essio

n

lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25


Relat

ive N

rf2 m

RNA

expr

essio

n

0

04

08

12

16

2

24


Relat

ive H

O-1

mRN

A ex

pres

sion

lowastlowast

lowast

lowast

(b)





Bax

Bcl-2

120573-actin

1st week

AG

Con

trol

AG

Con

trol

AG

Con

trol


0

05

1

15

2

25

3

Con

trol

AG

Con

trol

AG

Con

trol

AG


Relat

ive B

ax ex

pres

sion

Relat

ive B

cl-2

expr

essio

n

0

004

008

012

016

Con

trol

AG

Con

trol

AG

Con

trol

AG


0

004

008

012

016

Con

trol

AG

Con

trol

AG

Con

trol

AG


Relat

ive B

axB

cl-2

expr

essio

n

lowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

(b)











Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HO-1

Nrf2

120573-actin

1st week

AG

Con

trol

AG

Con

trol

AG

Con

trol

2nd week 4th week

0

004

008

012

016


Relat

ive N

rf2 ex

pres

sion


0

004

008

012

016

Relat

ive H

O-1

expr

essio

n

lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25


Relat

ive N

rf2 m

RNA

expr

essio

n

0

04

08

12

16

2

24


Relat

ive H

O-1

mRN

A ex

pres

sion

lowastlowast

lowast

lowast

(b)





Bax

Bcl-2

120573-actin

1st week

AG

Con

trol

AG

Con

trol

AG

Con

trol


0

05

1

15

2

25

3

Con

trol

AG

Con

trol

AG

Con

trol

AG


Relat

ive B

ax ex

pres

sion

Relat

ive B

cl-2

expr

essio

n

0

004

008

012

016

Con

trol

AG

Con

trol

AG

Con

trol

AG


0

004

008

012

016

Con

trol

AG

Con

trol

AG

Con

trol

AG


Relat

ive B

axB

cl-2

expr

essio

n

lowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

(b)











Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Bax

Bcl-2

120573-actin

1st week

AG

Con

trol

AG

Con

trol

AG

Con

trol


0

05

1

15

2

25

3

Con

trol

AG

Con

trol

AG

Con

trol

AG


Relat

ive B

ax ex

pres

sion

Relat

ive B

cl-2

expr

essio

n

0

004

008

012

016

Con

trol

AG

Con

trol

AG

Con

trol

AG


0

004

008

012

016

Con

trol

AG

Con

trol

AG

Con

trol

AG


Relat

ive B

axB

cl-2

expr

essio

n

lowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

(b)











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





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Protective Effect of Decursin Extracted ...downloads.hindawi.com/journals/omcl/2016/5901098.pdf · anti-Nrf (/), anti-HO- (/), anti-Bax (/), and anti-Bcl- (:) polyclonal