Himani Paper 1-Andrologia

ORIGINAL ARTICLE

Cytoprotective effects of fruit pulp of Eugenia jambolanaon H2O2-induced oxidative stress and apoptosis in ratLeydig cells in vitroH. Anand1,2, M. M. Misro1, S. B. Sharma3 & S. Prakash2

1 Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, New Delhi, India;

2 Department of Zoology, Dayalbagh Educational Institute, Dayalbagh, India;

3 Department of Biochemistry, University College of Medical Sciences, University of Delhi, India

Keywords

ApoptosisEugenia jambolanaH2O2

Leydig cellsoxidative stress

Correspondence

M. M. Misro, Department of Reproductive

Biomedicine, National Institute of Health and

Family Welfare, Baba Gangnath Marg,

Munirka New Delhi-110067, India.

Tel.: +91 11 26165959;

Fax: +91 11 26101623;

E-mail: [email protected]

Accepted: May 02, 2012

doi: 10.1111/j.1439-0272.2012.01323.x

Summary

This study was undertaken to investigate the cytoprotective effect of the fruit

pulp of Eugenia jambolana (50250 lg ml1) against the damage induced byH2O2 (100 lM) exposure to Leydig cells in vitro. Cell survival with extract wasfound comparable to similar effects by N-acetyl-L-cysteine. H2O2-induced rise

in thiobarbituric acid reactive substance formation and decline in the activity

and expression of antioxidant enzymes like superoxide dismutase, catalase and

glutathione-s-transferase were effectively checked. Cellular glutathione and total

antioxidant capacity demonstrated significant improvement. The increase in

expression of inducible nitric oxide (NO) synthase leading to NO production

was successfully countered. Co-treatment of the extract helped in the down-

regulation of caspase-3 and poly-ADP-ribose polymerase resulting in a signifi-

cant reduction in Leydig cell apoptosis induced by H2O2. Upstream marker

proteins of extrinsic (caspase-8, Fas, FasL) and intrinsic (caspase-9) pathway of

metazoan apoptosis were identically down-regulated. The Bcl-2 family of pro-

teins, though, remained unaffected. The extract also positively modulated the

other marker proteins like c-Jun NH2-terminal kinase, p38, Akt, nuclear factor-

jB, c-Fos, cellular FLICE-inhibitory protein, cyclooxygenase-2 and p53. Takentogether, the above-mentioned findings establish the anti-oxidative and anti-

apoptotic potency of the extract that ameliorates the H2O2-induced adverse

effects on rat Leydig cells in vitro.

Introduction

H2O2 as an oxidant has been recognised as a damaging

entity and a signalling agent mediating various pathogenic

processes in different cells and tissues (Stone & Yang,

2006). As reactive oxygen species (ROS), it is generated

from nearly all sources of oxidative cycle and has the

ability to diffuse in and out of cells (Barbouti et al.,

2002). Leydig cell steroidogenesis is oxygen dependent,

and oxidative stress due to ROS accumulation is shown

to disrupt mitochondria and steroid synthesis in MA-10

cells (Diemer et al., 2000). Besides its indigenous produc-

tion during steroid synthesis, Leydig cells particularly are

exposed to the risk of more external H2O2 as a secretory

product from the interstitial macrophages with whom

they share a close structural proximity in-vivo. Being an

ROS, H2O2 was reported to affect Leydig cell function

in vitro, even at physiological concentrations, inducing

oxidative stress and apoptosis (Gautam et al., 2006). ROS

production and accumulation leading to oxidative stress

in Leydig cells may arise due to altered hormonal or

other conditions leading to a decline in cellular function.

Even with persistent stimulation of Leydig cells with

human chorionic gonadotropin (hCG), the decline in

Leydig cell steroidogenesis was as a result of increased

oxidative stress and apoptosis in the target cells (Aggarwal

et al., 2009). Thus, appropriate interventions, which at

present are not many, must be developed so as to provide

the necessary protection to Leydig cells under such

adverse conditions.

In recent years, agents with anti-oxidative and anti-

apoptotic properties are utilised to counteract H2O2-

induced DNA damage leading to improved cell survival

through modulation of gene expressions. A synthetic

2012 Blackwell Verlag GmbH 1Andrologia 2012, XX, 113

agent, N-acetyl-L-cysteine (NAC), has emerged as an

effective antioxidant and has been reported to success-

fully mitigate the adverse effects of H2O2 on germ cell

survival by regulation of intrinsic and c-Jun NH2-termi-

nal kinase (JNK) pathway of apoptosis (Maheshwari

et al., 2009). In addition, it has been shown to amelio-

rate the adverse effects of oxidative stress and apoptosis

in Leydig cells following persistent stimulation with

hCG again mainly through the modulation of extrinsic

and JNK pathways of apoptosis (Aggarwal et al., 2010).

Use of natural products is always considered more

beneficial than chemo-therapeutic or chemo-preventive

agents, and therefore, there is an increasing interest for

the development of phytochemical anti-oxidants as

health benefactors (Krishnaiah et al., 2007). Eugenia

jambolana (Myrtaceae, common name: Black plum/Black

berry in English and Jamun/Jambul in Hindi) seeds have

been reported to possess anti-inflammatory, anti-bacte-

rial, anti-HIV, anti-diarrhoeal (Chaturvedi et al., 2007)

and strong antioxidant properties (Vasi & Austin, 2009).

Hypoglycaemic and hypolipidaemic activity of the aque-

ous and ethanolic extracts of seeds, at different dose lev-

els, have been tried in animals (Sharma et al., 2006,

2010). The seeds have also shown to have protective

effects on testis of streptozotocin-induced diabetic rats

(Mallick et al., 2007, 2008). The active ingredient from

the fruit pulp of Eugenia jambolana, which reportedly

improved the renal dysfunction in streptozotocin-

induced diabetic rats, has been chemically characterised

as a-hydroxy succinamic acid by HPLC (Tanwar et al.,2010) and patented (Sharma et al., 2009). At present,

there is no other product emerged so far, which has

established antioxidative effects comparable with NAC.

The active ingredient is not available in plenty either

commercially or otherwise; therefore, this study was

planned with the purified aqueous extract of Eugenia

jambolana to explore its cytoprotective potential against

H2O2-induced damage of Leydig cells in vitro. The asso-

ciated molecular mechanisms were also investigated.

Materials and methods

Animals

Adult male albino rats of Holtzman strain, 6090 daysweighing about 200220 g, maintained under controlledtemperature (25 2 C) and constant photoperiodic con-ditions (12h light/12h dark), were used. The animals were

allowed food and water ad libitum. The animals were sac-

rificed under strict compliance of the Institutional Guide-

lines for Animal Care issued by the Committee for the

Purpose of Control and Supervision on Experiments on

Animals, India.

Leydig cell isolation, viability and treatment

The Leydig cells were isolated as previously described

(Anakwe & Moger, 1986). The cells were incubated with

H2O2 (10250 lM) for different time periods (16 h) at37 C. The dose of H2O2 (100 lM) showing a cell viabil-ity of >75% following 4 h incubation was selected for fur-ther analysis. The aqueous extract from the fruit pulp of

Eugenia jambolana has been prepared as described (Tan-

war et al., 2010). Briefly, the fruit pulp in dH2O was grin-

ded, filtered, centrifuged and lyophilised in cold and

stored at 20 C until used. The cytoprotective effect ofthe extract was then examined at different concentrations

(50250 lg ml1) following co-incubation of cells withH2O2 (100 lM). The effective dose (100 lg ml

1) wasselected accordingly, which was later utilised in subse-

quent experimentation. The beneficial effect of Eugenia

jambola extract (EJE) on Leydig cell viability through try-

pan blue dye exclusion test (Gautam et al., 2006) was also

assessed in comparison with NAC, the known and recog-

nised antioxidant.

Lipid peroxidation, antioxidant enzyme activity and total

antioxidant capacity (TAC)

Leydig cells untreated and treated with only H2O2 or EJE

or with both were briefly sonicated for 30 s. One part of

the lysate was assayed for lipid peroxidation as described

(Ohkawa et al., 1979). The other part was centrifuged at

10 000 g for 5 min and activities of antioxidant enzymes,

superoxide dismutase (SOD; Das et al., 2000), catalase

(Aebi, 1984) and glutathione-s-transferase (GST; Habig

et al., 1974) were assayed using the supernatant. The pro-

tein was assayed using the Bradford method (Bradford,

1976).

Assessment of TAC of Leydig cells, treated or

untreated, was carried out using the commercial kit as

per the manufacturers (Cayman Chemical Company,

Ann Arbor, MI, USA) instructions. The assay exploits the

combined ability of various antioxidants (vitamin, pro-

tein, lipids, glutathione, uric acid, etc.) present in the cell

lysate to inhibit the oxidation of 2, 2-Azino-di-(3-ethyl-

benzthiazoline sulphonate) (ABTS) by metmyoglobin.

Absorbance at 750 nm was representative of the amount

of the oxidised ABTS produced, and TAC (mM) was cal-

culated using a Trolox standard curve.

Intracellular glutathione levels

Total intracellular cellular glutathione (GSH) levels were

measured using the commercial ApoGSH Glutathione

Detection Kit (Biovision, Mountain view, CA, USA) as per

the manufacturers instructions. Briefly, approximately

2 2012 Blackwell Verlag GmbHAndrologia 2012, XX, 113

Effects of Eugenia jambolana on rat Leydig cells H. Anand et al.

5 9 106 cells were lysed using 200 ll of lysis buffer (pro-vided in the kit) for 10 min at 4 C, centrifuged at12 000 g and the supernatant filtered through 10 kDa fil-

ters (R&D System, Inc., Minneapolis, MN, USA). 40 ll ofthe filtrate diluted with lysis buffer (final 100 ll) and GSHstandards (100 ll) were taken in a fluorometric plate towhich 2 ll of monochlorobimane dye was added, shakenwell and incubated for 30 min at 37 C. Fluorescence wasmeasured using microtitre plate reader (BioTek, Inc., Wi-

nooski, VT, USA) at excitation/emission = 380/460 nm.Total glutathione in each sample was calculated using GSH

standard curve.

Nitric oxide (NO) assay

The levels of NO in Leydig cells were estimated as previ-

ously described (Lyle et al., 2009). The assay is based on

the measurement of nitrites in the supernatant using Gri-

ess reagent. Briefly, the cells, treated and untreated, were

centrifuged at 10 000 g for 5 min, and 100 ll of thesupernatant was taken in triplicates in a microtitre plate.

Equal volume of Griess Reagent (1% sulphanilamide,

0.1% napthylethylenediamine dihydrochloride and 2.5%

hydrochloric acid) was added, and the plate was incubated

in dark for 15 min at room temperature following which

absorbance was measured at 550 nm using a microtitre

plate reader (BioTek, Inc.). The NO concentrations in the

treated samples were calculated as percentage of the

control.

[TdT]-mediated deoxyuridinetriphosphate nick end

labelling (TUNEL) assay

TUNEL was performed as per the manufacturers (R&D

System, Inc.) instructions. Briefly, treated or untreated

cells were washed with PBS, smeared on poly-L-lysine

coated slides and fixed with 4% formaldehyde for 5 min.

Cytonin treatment was given to the cells for 10 min

followed by quenching with H2O2. TdT was used to

incorporate biotinylated nucleotides into the 3-OH endsof the DNA fragments and detected by streptavidin-

horseradish peroxidase (HRP). DAB was used to develop

the colour in cells that were counterstained with methyl

green. The slides were examined using a Nikon micro-

scope, and the percentage of TUNEL-positive cells was

calculated.

Caspase-3, -8 and -9 activities

Caspase-3, -8 and -9 activities were assayed as per the

protocol supplied by the manufacturer (Biovision, San

Diego, CA, USA). Briefly, approximately 5 9 106 cells

were resuspended in cold lysis buffer and incubated for

10 min. The lysates were centrifuged at 10 000 g for

2 min at 4 C, and an aliquot of the supernatant (100 lgprotein per 50 ll) was added to 50 ll of the reaction buf-fer containing 200 lM of the chromogen (Ac-DEVD-pNA, Ac-IETD-pNA and Ac-LEHD-pNA for caspase-3, -8

and -9, respectively). The reaction mixture was incubated

at 37 C and terminated by addition of stop buffer after2 h. The release of pNA leads to a change in the absor-

bance that is measured at 405 nm using a microtitre plate

reader (BioTek, Inc.).

Western blot analysis

Primary antibodies (rabbit polyclonal) anti-MnSOD, anti-

poly-ADP-ribose polymerase (PARP), anti-caspase-3, anti-

caspase-9, anti-Fas, anti-FasL, anti-p53, anti-JNK,

anti-c-Jun, anti-Akt, anti-NF-jB, anti-I-jB, anti-c-Fos,anti-c-Flip and (mouse monoclonal) anti-catalase, anti-cas-

pase-8, anti-phosphorylated form of JNK (pJNK), anti-b-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA),

anti-GST (Bangalore Genei, Bangalore, India) were utilised.

Rabbit monoclonal anti-Cox-2 and anti-p-Akt were from

Cell Signaling Technology, Inc., Danvers, MA, USA, and

rabbit monoclonal anti-inducible nitric oxide synthase

(iNOS) was from Biomeda, Foster City, CA, USA. Goat

anti rabbit/mouse-HRP conjugate secondary antibody was

from Santa Cruz Biotechnology. Whole cell lysates were

prepared in 200 ll lysis buffer, and western blots were car-ried out as previously described (Maheshwari et al., 2011).

b-actin was used as an internal control to ensure equal pro-tein loading. Densitometric analysis was performed with

the help of Image analysis software (LAB WORKS IMAGE analy-

sis software 4.0; UVP, Upland, CA, USA).

RNA isolation and RT-PCR analysis

Total RNA was extracted using TRI-Reagent (Life Tech-

nologies Corp., Ambion , TX, USA). cDNA was synthes-

ised using 2 lg of total RNA by omniscript reversetranscriptase (RT) kit (Qiagen, Hilden, Germany). PCR

was carried out with 2 ll of RT reaction using the HotStarHiFidelity DNA polymerase (Qiagen). Specific primers

were obtained from Eurofins MWG Operon (Whitefield,

Bangalore, India). The following temperature profiles were

used for running the PCR reaction: (i) denaturation at

95 C for 15 min; (ii) 30 cycles of 95 C for 30 sec, 5567 C for 1 min, 72 C for 1 min; (iii) 72 C for 10 minfor final extension. Table 1 shows the sequences, source,

annealing temperatures, Mg2+ concentrations and product

sizes of various primers used. Post-amplification, the prod-

ucts were separated on a 1.5% agarose gel and documented

with the help of a gel documentation system (UVP).

b-actin was used as an internal as well as a nontemplate


H. Anand et al. Effects of Eugenia jambolana on rat Leydig cells

control, and densitometric analysis was performed as pre-

viously described.

Statistical analysis

All the figures (for Western Blotting and RT-PCR) are

representative of three independent experiments with

similar results. The results have been shown as the

mean standard deviation (SDs) of three experiments.Statistical analysis was performed using one-way ANOVA

followed by Tukeys test using GRAPH PAD PRISM Software.

P < 0.05 was considered statistically significant.

Results

Eugenia jambolana extract counters the decrease in

Leydig cell viability induced by H2O2

To determine the beneficial effects of EJE, a systematic

analysis of Leydig cell viability through trypan blue dye

exclusion was carried out with different doses of H2O2under incubation for 4 h and with or without EJE supple-

mentation. As compared to controls, a significant decline

in viability (~13%) was observed with 100 lM H2O2 expo-sure that increased further (~25%) with the increase inH2O2 (250 lM) concentration (Fig. 1a). The cell viabilitywas found unaffected when examined for 13 h evenwith the highest concentration of H2O2 used (data not

shown). Accordingly, the dose (100 lM) and duration(4 h) of H2O2 exposure was maintained for all subsequent

investigations. A significant (P < 0.001) improvement incell viability (88.51 2.023%) was seen following EJE(100 lg ml1) supplementation (Fig. 1b). The effect ofEJE (100 lg ml1) on cell survival was found comparablewith NAC (5 mM), a known but synthetic antioxidant

(Fig. 1c). Therefore, this particular dose of EJE

(100 lg ml1) was used in all subsequent experiments.

EJE co-treatment counteracts H2O2-induced oxidative

stress

H2O2 exposure induced a two-fold rise in lipid peroxida-

tion in the target cells as compared to unexposed controls.

EJE intervention, however, completely prevented this rise

(Fig. 2a). It also helped to restore the decline in the activi-

ties of antioxidant enzymes, SOD, catalase and GST to

normal levels (Fig. 2bd). When the protein and tran-script levels of these enzymes were further examined, iden-

tical up-regulation in the expressions was observed for

catalase (Fig. 2e,f). Up-regulation only in the transcript

(Fig. 2f) or protein levels (Fig. 2e) was seen for MnSOD,

glutathione peroxidase (GPx) and GST, respectively. A sig-

nificant improvement in the GSH levels (P < 0.001) andTAC (P < 0.001) was observed (Fig. 3a,b).

EJE supplementation counters H2O2-induced rise in

nitric oxide

A significant rise (P < 0.001) in NO levels was seen inLeydig cells following H2O2 exposure that was successfully

Table 1 Primer specific conditions used for PCR amplification of candidate genes

Name Primer sequence Reference/accession no.

Mg2+ conc.

(mM)

Annealing

temp. (C)

Product

size (bp)

Catalase F-CCGACGAGATGGCACACTTTGACA

R-CGCGAGCACGGTAGGGACAGTTC

Maheshwari et al. (2009) 2.5 62 972

Mn SOD F-CTTCAGCCTGCACTGAAGTTCAAT

R-CTGAAGATAGTAAGCGTGCTCCC


GPx F-CTCTCCGCGGTGGCACAG

R-CCACCACCGGGTCGGACATAC

Bhor et al. (2004) 3.5 62 290

Inducible nitric

oxide synthase

F-TTGGGTCTTGTTAGCCTAGTC

R-TGTGCAGTCCCAGTGAGGAAC

Hierholzer et al. (1998) 2.5 59 264

Caspase-8 F-CTGGGAAGGATCGACGATTA

R-CATGTCCTGCATTTTGATGG


Fas F-GCAATGCTTCTCTCTGTGACCACT

R-GCTGTTGTGCTCGATCTCATCG


FasL F-GAATGGGAAGACACATATGGAACTGC

R-CATATCTGGCCAGTAGTGCAGTAATTC


Caspase-9 F-AGCCAGATGCTGTCCCATAC

R-CAGGAGACAAAACCTGGGAA


p53 F-GGCCATCTACAAGAAGTCAC

R-CCAGAAGATTCCCACTGGAG


b-Actin F-CTGTGCCCATCTATGAGGGTTAC

R-AATCCACACAGAGTACTTGCGCT




brought down by EJE co-treatment (Fig. 4a). The pro-

tein/mRNA levels of iNOS found simultaneously

(P < 0.01; P < 0.05, respectively) up-regulated as a resultof H2O2 treatment were restored to original levels follow-

ing EJE supplementation (Fig. 4b,c).

Inhibition of H2O2-induced apoptosis in Leydig cells by

EJE

H2O2 as an oxidant is a recognised apoptosis inducer

as TUNEL positivity in the exposed cell population

(a)

(c)

(b)

Fig. 1 Leydig cell viability (%) as determined

by trypan blue dye exclusion. (a) Viability was

assessed after 4 h of incubation with different

doses of H2O2 (10250 lM). 100 lM H2O2was selected for further studies as it main-

tained the cell viability >75%. (b) Eugenia

jambolana extract (EJE) (100 lg ml1) inter-vention with 100 lM H2O2 significantly

improved cell viability comparable with that of

(c) 5 mM N-acetyl-L-cysteine (NAC). **P < 0.01

and ***P < 0.001 compared with untreated

controls. ###P < 0.001 compared with H2O2,DDDP < 0.001 compared with 100 lg ml1

EJE, $P < 0.05 compared with 100 lg ml1

EJE + 100 lM H2O2,aaaP < 0.001 compared

with 1 mM NAC, bbbP < 0.001 compared with

1 mM NAC + 100 lM H2O2 treatment.

(a) (b)

(c) (d)

(e) (f)

Fig. 2 Evaluation of oxidative stress in H2O2-

treated isolated rat Leydig cells with or with-

out Eugenia jambolana extract (EJE). (a) Lipid

peroxidation after H2O2 treatment as mea-

sured by thiobarbituric acid reactive sub-

stances formation was significantly brought

down with EJE co-incubation. Restoration in

the activities of the antioxidant enzymes, (b)

superoxide dismutase (SOD), (c) catalase and

(d) glutathione-s-transferase (GST) was also

evident. A similar trend was observed in (e)

western blot analysis for catalase, MnSOD and

GST, as well as (f) RT-PCR analysis for catalase,

MnSOD and glutathione peroxidase (GPx).

*P < 0.05 and ***P < 0.001 compared with

untreated controls. #P < 0.05 and ##P < 0.01

compared with H2O2 treatment.



demonstrated a significant (P < 0.001) increase (Fig. 5b,g). While EJE alone showed no adverse effect (Fig. 5c,g),

co-treatment with H2O2 prevented this apoptotic trans-

formation in the target cells (Fig. 5d,g). The inhibition of

apoptotic induction was very much comparable to the

effect when EJE was replaced with NAC (Fig. 5e,f,g). EJE

was also able to counter the rise in the expression and

activity of caspase-3 in H2O2-treated cells (Fig. 6a,b).

Similarly, it prevented the H2O2-induced over-expression

of PARP cleavage in the target cells (Fig. 6b).

EJE mediated regulation of apoptotic pathway

To explore the molecular mechanisms of EJE mediated

regulation of Leydig cell apoptosis induced by H2O2, the

expression of upstream markers in the extrinsic and

intrinsic pathway of apoptosis was examined. Raised cas-

pase-8 activity (Fig. 7a) and expression (Fig. 7b,c) in the

H2O2 exposed cells was found effectively checked by EJE

intervention. EJE modulation of expression of the associ-

ated markers Fas and FasL followed an identical trend

(Fig. 7b,c). In the intrinsic pathway, caspase-9 activity

(Fig. 8a) and expression (Fig. 8b,c) was similarly down-

regulated by EJE supplementation in the H2O2-treated

cells. However, EJE was seen to have very little influence

on the other intrinsic, apoptotic protein markers such as

Bax, Bid, Bak and Bad and similarly the anti-apoptotic

Bcl-2 (data not shown).

c-Jun NH2-terminal Kinase (JNK) modulation by EJE

Mitogen Activated Protein Kinase (MAPK) modulation

by EJE was also investigated in the H2O2 exposed Leydig

cells that demonstrated a significantly up-regulated

expression of JNK and its phosphorylated form (pJNK).

EJE co-treatment was able to down-regulate these expres-

sions back to the control levels (Fig. 9a). Protein blots

indicating c-Jun, c-Fos and pP38 expressions demon-

strated an identical pattern of regulation by EJE (Fig. 9a).

On the other hand, EJE induced up-regulation of the

expression of anti-apoptotic marker proteins like nuclear

factor-jB (NF-jB), cellular FLICE-inhibitory protein(c-Flip) and cyclooxygenase-2 (Cox-2) in the H2O2-

treated cells (Fig. 9b). The expression of I-jB, the inhibi-tor of NF-jB, followed an opposite trend and was seencompletely reversed by EJE treatment (Fig. 9b).

Regulation of p53 and Akt expression

While p53 was over-expressed in the H2O2-treated Leydig

cells, the anti-apoptotic Akt and pAkt were significantly

down-regulated. EJE was seen to modulate the expression

of these proteins favourably back to control levels

(Fig. 9c,d,e).

Discussion

The findings from this study establish that the active

ingredient from the fruit pulp of Eugenia jambolana

(EJE) supplementation significantly ameliorates the

adverse effects of H2O2 on rat Leydig cells in vitro and

promotes cell survival by mitigating oxidative stress and

apoptosis. The underlying molecular mechanism includes

favourable modulation of up- and down-stream marker

proteins in the extrinsic/intrinsic and other associated

pathways of metazoan apoptosis.

The onset of oxidative stress in any cellular system may

be mediated by an excessive production of ROS or expo-

sure to ROS/ROS stimulating agents that may eventually

affect its normal functioning. We had earlier reported

that hCG-stimulated testosterone production in Leydig

cells was severely impacted following exposure to H2O2even in low concentrations (Gautam et al., 2006). As

impaired Leydig cell function is closely associated with

testicular dysgenesis syndrome that includes a range of

(a)

(b)

Fig. 3 Assessment of (a) total glutathione (b) total antioxidant

capacity (TAC) in H2O2 exposed Leydig cells with or without Euge-

nia jambolana extract (EJE). EJE supplementation restored the cellu-

lar glutathione levels and improved the TAC by ~45% compared

with untreated controls. *P < 0.05, **P < 0.01, and ***P < 0.001

compared with untreated controls, ###P < 0.001 compared with

H2O2 treatment.



male reproductive disorders (hypospodiasis and cryptor-

chidism) leading to genital malformations, hypospermato-

genesis and even testicular cancer (Joensen et al., 2008),

the implication of oxidative stress in all these conditions

cannot be overemphasised. All these clinical problems are

the outcome of an irreversible developmental disorder

and Leydig cell dysfunction originating in early foetal life

(Anderson et al., 2004). An association between compro-

mised Leydig cell function and testicular cancer has been

indicated due to the fact that significantly increased

(a)

(b)

(g)

(c)

(d)

(e)

(f)Fig. 5 TUNEL of H2O2-treated Leydig cellswith or without Eugenia jambolana extract

(EJE) supplementation. The rise in TUNEL-posi-

tive cells (?) after H2O2 treatment was signif-icantly brought down by EJE, (a, g) control,

(b, g) H2O2, (c, g) EJE only, (d, g) H2O2 + EJE,

(e, g) 5 mM N-acetyl-L-cysteine (NAC) only,

and (f, g) H2O2 + 5 mM NAC showing per-

centage of TUNEL-positive cells under differ-

ent incubating conditions. The data from the

graph were collected from four different

fields each from three separate experiments.

*P < 0.05, ***P < 0.001 compared with

untreated controls, ###P < 0.001 compared

with H2O2 treatment.

(a)

(b) (c)

Fig. 4 Estimation of (a) nitric oxide (NO) and

(b) protein and (c) transcript expression of

inducible nitric oxide synthase (iNOS) in Leydig

cells with or without Eugenia jambolana extract

(EJE) intervention. EJE supplementation helped

to reduce NO levels (expressed as percentage

of untreated control) significantly and the

over-expression of iNOS protein and mRNA.

*P < 0.05, and ***P < 0.001 compared with

untreated controls, ##P < 0.01 compared with

H2O2 treatment.



luteinising hormone (LH) levels and low testosterone

were found in men with carcinoma testis in situ (Petersen

et al., 1999). Besides, there are a host of other clinical

conditions in adult hood like liver disease, alcoholism,

chronic renal disease, metabolic cardiovascular syndrome,

diabetes and rheumatic disease, in which Leydig cell func-

tion is also found to be affected (Karagiannis & Harsou-

lis, 2005). However, interventions aimed to improve or

restore the functions of Leydig cells under such condi-

tions are not many. A recent finding indicates that oral

administration of sodium tungstate to adult male strepto-

zotocin-diabetic rats not only normalised serum levels of

glucose but also Leydig cell function leading to restored

LH levels in circulation and improved testosterone pro-

duction (Ballester et al., 2005). As all synthetic products

have the risk of bio-incompatibility leading to adverse

reactions, in this work, we preferred to use a natural

plant product, active ingredient from the fruit pulp of

Eugenia jambolana, under oxidant-induced stressed

conditions in Leydig cells in vitro to explore its beneficial

cytoprotective effects first before examining its potency

on cellular function in-vivo.

Testicular oxidative stress has long been implicated in

several conditions of male infertility including toxicant

exposure (Samanta & Chainy, 1997; Han et al., 2004;

McClusky et al., 2007), chemotherapy (Arnon et al.,

2001), ionising radiation (Sohal et al., 1995; Manda et al.,

2007), inflammation (Allen et al., 2004), varicocele

(Santoro & Romeo, 2001), cryptorchidism (Misro et al.,

2005; Li et al., 2006), ageing (Syntin et al., 2001; Cao

et al., 2004; Luo et al., 2006) and testicular torsion

(Lysiak et al., 2001, 2007). All these conditions whether

therapeutical or pathological end up generating more

(a)

(b)

Fig. 6 (a) Activity and expression (b) of caspase-3 was down-regu-

lated along with cleaved poly-ADP-ribose polymerase after Eugenia

jambolana extract co-treatment. **P < 0.01 and ***P < 0.001 com-

pared with untreated controls, ###P < 0.001 compared with H2O2treatment.

(a)

(b) (c)Fig. 7 Role of Eugenia jambolana extract (EJE)

modulation of extrinsic pathway of apoptosis.

EJE co-treatment with H2O2 significantly con-

tained the rise in (a) activity and (b, c) expres-

sion (protein and transcript levels) of caspase-8.

Identical down-regulation in (b, c) Fas, and (b,

c) FasL expression was also seen. **P < 0.01,

***P < 0.001 compared with untreated

controls, ###P < 0.001 compared with H2O2treatment.



ROS associated with reduced intracellular antioxidant

activity unable to counter the ROS mediated detrimental

effect. ROS, in general, include superoxide anions and

hydroxyl radicals, reactive oxygen, as well as species of

H2O2, NO and peroxynitrite anion that are not radicals

characteristically but possess the ability of forming

radicals in cellular environments (Chang et al., 2008).

Either produced indigenously or following exposure to

extracellular sources, these oxidants can cause tissue dam-

age by a variety of mechanisms including DNA damage,

lipid peroxidation, protein oxidation, depletion of cellular

thiols and activation of pro-inflammatory cytokine

release. Significant increase in Leydig cell death due to

apoptosis was reported from this laboratory following

H2O2 exposure at 100 lM concentrations althoughhCG-induced testosterone production was affected at

(a)

(b) (c)Fig. 8 Role of Eugenia jambolana extract(EJE) modulation of intrinsic pathway of apop-

tosis. EJE co-treatment restored the (a) activity

and expression of (b) protein and (c) transcript

levels of caspase-9 back to control levels.

**P < 0.01 compared with untreated control,###P < 0.001 compared with H2O2 treatment.

(a) (b)

(c)

(e)

(d)

Fig. 9 Role of Eugenia jambolana extract

(EJE) modulation of other pathways of apop-

tosis. EJE supplementation was seen favour-

ably modulating (a) JNK, p-JNK, c-Jun, c-Fos,

pP38 and (b) NF-jB, I-jB, c-Flip and Cox-2

expression in the target cells. A significant

down-regulation of p53 (c) protein and (d)

transcripts along with up-regulation of anti-

apoptotic (e) Akt and p-Akt expression was

also observed.



concentrations even less than 30 lM (Gautam et al.,2006). Simultaneous supplementation with antioxidants is

the only solution to salvage the situation that at present

is limited to the use of recognised antioxidants like NAC;

the exact molecular mechanism of its counteraction

against apoptotic cell death is still very much debated

(Lum et al., 2002). This opens up the quest for identify-

ing other sources or ingredients that could be used as

antioxidants and cyto-protectants under identical condi-

tions.

As plants possess natural antioxidant properties to

counter these toxic oxygen derivatives, in this work we

utilised a plant product, the active ingredient from the

fruit pulp of Eugenia jambolana, the seeds of which have

been recognised to possess antioxidant properties (Vasi &

Austin, 2009). This is for the first time we tested the anti-

oxidant potential of the fruit pulp preparation under the

oxidant-exposed conditions in Leydig cells in vitro and

found that the preparation has remarkable cytoprotective

effects that promote cell survival under adverse condi-

tions. Simultaneous exposure of EJE (100 lg ml1)+ H2O2 (100 lM) significantly improved the Leydig cellviability over a period of 4 h comparable only to similar

supplementation with 5 mM of NAC (Fig. 1c). It helped

in reducing the formation of thiobarbituric acid reactive

substances while improving the activities of antioxidant

enzymes, SOD, catalase and GST (Fig. 2ad). Even thegene expressions of these antioxidant enzymes are found

favourably augmented (Fig. 2e,f). Increase in NO levels

has been recognised as an indication of oxidative stress

leading to inhibition of testosterone production (Mehta

et al., 2002). There is evidence that H2O2 and NO besides

acting as independent signalling molecules may inter-

relate to form an oxidative death cycle. Addition of

10 mM H2O2 to a leaf protein preparation of mung bean

caused an 8.3-fold increase in the NO activity suggesting

the role of H2O2 as an upstream signal leading to NO

production (Turner & Lysiak, 2008). H2O2 exposure, in

this study, does increase the NO levels, but EJE was able

to regulate iNOS expression leading to NO depletion in

the target cells (Fig. 4); which further confirmed the anti-

oxidant potency of the preparation utilised.

The onset of oxidative stress is potentially accompanied

by cell apoptosis in the testis that results from the activa-

tion of numerous molecular pathways (Mehta et al.,

2002; Maheshwari et al., 2009). Downstream caspase-3

up-regulation and the FasL activation were implicated in

the H2O2-mediated Leydig cell apoptosis (Gautam et al.,

2006). This work confirms the earlier findings and

extends to demonstrate the increase in PARP cleavage in

the target cells, which was successfully attenuated by EJE

supplementation (Fig. 6). EJE co-incubation also effec-

tively checked the increase in caspase-8 activity and

expression of caspase-8, Fas and FasL marker proteins of

the extrinsic pathway of metazoan apoptosis (Fig. 7).

However, upstream, intrinsic, Bcl-2 family of proteins

remained unaffected following H2O2 exposure to Leydig

cells (data not shown). Rise in activity and expression of

caspase-9 was noticed which was restored back to control

levels by EJE intervention (Fig. 8). The dichotomy in the

expression of the above-mentioned marker proteins may

be explained by the fact that pro-caspase-9 can act as a

substrate for caspase-12, and the activation of caspase-9

may be initiated independent of the cytochrome-c release

from the mitochondria, thereby, bypassing the involve-

ment of the Bcl-2 family of proteins (Morishima et al.,

2002). On the basis of these findings, it is difficult to ver-

ify the above-mentioned proposition that can only be

ascertained in future studies.

Exposure to H2O2 has also been implicated in the acti-

vation of MAP kinases (MAPK) that are important medi-

ators of stress-inducing signals (Maheshwari et al., 2009).

JNK that phosphorylates and regulates the activity of

transcription factors, c-Jun and p53, and the activation of

JNK, in turn, is regulated by scaffold proteins such as

NF-jB (Liu & Lin, 2005). JNK and c-Jun activation hasbeen recently shown to be involved in Burkitts Lym-

phoma cell line (BJAB) cell death after 2 h of exposure to

H2O2 (Son et al., 2009). c-Fos, a nuclear protein that

dimerises with c-Jun to form the transcription factor

complex activator protein-1 (AP-1), has been shown to

be regulated during c-Myc-induced apoptosis of serum-

deprived hepatoma cells involving the activation of P38

MAP kinase cascade (Kalra & Kumar, 2004). In this

work, H2O2-induced cell death in Leydig cells demon-

strated identical over-expression of JNK, pJNK, c-Jun,

c-Fos, pP38, I-jB and p53 (Fig. 9ad). Being anti-apop-totic, NF-jB, c-Flip, cox-2, Akt and pAkt were founddown-regulated (Fig. 9b,e). Introducing EJE along with

H2O2 favourably modulated the expression of all these

proteins promoting cell survival and inhibiting cell death

by apoptosis (Fig. 5). Based on the above-mentioned

findings, a model showing the molecular mechanisms of

EJE against H2O2-induced Leydig cell apoptosis is pro-

posed (Fig. 10).

The threat of the long-term side effects of synthetic

antioxidants has increasingly brought back the interest of

the use of natural resources for health and medicine

(Krishnaiah et al., 2007). The roots of Morinda officinalis

have been shown to have a cytoprotective effect on

H2O2-induced oxidative stress in Leydig TM3 cells

(Chang et al., 2008). Green tea polyphenols have been

found to have an anti-oxidative and anti-apoptotic effect

against azothioprine-induced liver injury in rats

(El-Beshbishy et al., 2011). Orthosiphon stamineus Benth

has anti-oxidative and anti-apoptotic effect on



H2O2-induced cellular damage in MDA-M231 cells (Ab-

delwahab et al., 2011). Identical to the above-mentioned

findings, the fruit pulp of Eugenia jambolana is shown

here for the first time to possess antioxidant properties

having the capacity to mitigate the oxidative stress in Ley-

dig cells induced by H2O2. The preparation is very much

cytoprotective and needs to be explored further for thera-

peutical use to alleviate Leydig cell dysfunction.

Acknowledgements

The generous gift of Eugenia jambolana extract (EJE)

from Dr Suman Bala Sharma, Professor, University Col-

lege of Medical Sciences, Delhi, is greatly acknowledged.

The study was funded by NIHFW.

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