Accepted Manuscript

Protection of Quercetin against Triptolide-induced apoptosis by suppressing oxidativestress in rat Leydig cells

Jie Hu, Qinwei Yu, Fang Zhao, Jinzi Ji, Zhenzhou Jiang, Xin Chen, Peng Gao, YuranRen, Shuai Shao, Luyong Zhang, Ming Yan

PII: S0009-2797(15)30033-8

DOI: 10.1016/j.cbi.2015.08.004

Reference: CBI 7434

To appear in: Chemico-Biological Interactions

Received Date: 4 March 2015

Revised Date: 6 August 2015

Accepted Date: 7 August 2015

Please cite this article as: J. Hu, Q. Yu, F. Zhao, J. Ji, Z. Jiang, X. Chen, P. Gao, Y. Ren, S. Shao, L.Zhang, M. Yan, Protection of Quercetin against Triptolide-induced apoptosis by suppressing oxidativestress in rat Leydig cells, Chemico-Biological Interactions (2015), doi: 10.1016/j.cbi.2015.08.004.

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http://dx.doi.org/10.1016/j.cbi.2015.08.004

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Protection of Quercetin against Triptolide-induced apoptosis by

suppressing oxidative stress in rat Leydig cells

Jie Hua,1 , Qinwei Yua,1 , Fang Zhaoa, Jinzi Jia,c, Zhenzhou Jianga, b, Xin

Chena, Peng Gaoa, Yuran Rend , Shuai Shaod, Luyong Zhanga, b, *, Ming

Yana, *

a Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical

University, Nanjing 210009, China.

b Key Laboratory of Drug Quality Control and Pharmacovigilance (China

Pharmaceutical University), Ministry of Education, Nanjing 210009, PR

China

c Central Laboratory, General Clinical Research Center, Nanjing First

Hospital, Nanjing 210006, PR China

d School of Pharmacy, China Pharmaceutical University, Nanjing 210009,

PR China

1 These authors equally contributed to this work.

*Corresponding authors at: Jiangsu Key Laboratory of Drug Screening,

China Pharmaceutical University, Jiangsu Province, Nanjing 210009,

China; Tel: +86 025 83271142; Fax: +86 025 83271142.

E-mail addresses: lyzhang@cpu.edu.cn (Luyong Zhang), Abbreviations: TP, Triptolide; Que, Quercetin; ∆Ψm, mitochondrial membrane potential; GPx, glutathione peroxidase; SOD, superoxide dismutase; ROS, reactive oxygen Species; Nrf2, NF-E2-related factor; Cyt-C, cytochrome C; JNK, c-Jun Nterminal kinase.

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brookming@163.com (Ming Yan).

1. Introduction

Triptolide (TP), derived from the medicinal plant Triterygium wilfordii Hook.f. (TWHF), is a

diterpene triepoxide with variety biological activities, such as anti-inflammatory, anti-cancerogenic,

immunomodulatory and pro-apoptotic activities [1, 2]. However, its potential toxicity to circulatory

and reproductive system limits its clinical application [3]. A recent study on male reproductive

toxicity showed that TP could induce decrease of testis and epididymis weights and apparent

changes of seminiferous tubules and epididymides [4]. In addition, it was demonstrated that the

sperm viability and motility in canda epididymal fluid could be reduced by TP, but the mechanism

has been unknown [5]. One major disruptive factor of reproductive function in Leydig cells is

oxidative stress, which is also the main reason of male infertility in pathology [6]. Similarly,

spermatogenesis is also vulnerable to oxidative stress because of a low oxygen demand in

physiology [7]. All of these damage are resulted from over-accumulation of reactive oxygen species

(ROS). Therefore, anti-oxidant enzymes, such as superoxide dismutase (SOD) and glutathione

peroxidase (GPx), and other free radical scavengers are demanded for protecting testis from free

radical damage to maintain its normal function [8-10]. NF-E2-related factor (Nrf2) is a

redox-sensitive transcription factor. With the increase of ROS, the dissociation of Nrf2 and Keap1

causes Nrf2 translocating into nucleus and binding to antioxidant response elements (AREs), which

leads protection against oxidative stress [11].

Quercetin (Que), as a plant phenolic compound, is a member of flavonoids discovered in many

dietary sources [12] with pharmacological activities involving anti-cancerogenic, antiviral,

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anti-ischemic, anti-inflammatory and antiallergenic [13]. It had been demonstrated that it had

protective effect on melphalan-mediated oxidative stress in kidney and liver [14]. Meanwhile, the

effect to protect embryonic chicken spermatogonial cells from oxidative damage was also be

confirmed, which suggests its availability in male reproductive protection as free radical scavenger

[15].

Interaction between apoptosis and mitochondria damage is revealed by accumulated data. As

one major source of ROS, mitochondria mediated an important signaling pathway in apoptosis [16].

The decrease of mitochondria membrane potential (∆Ψm) and the release of cytochrome C (Cyt-C)

are two major events in this mitochondrial pathway. Thereby, caspases, a family of cysteine

proteases, are activated, especially caspase-3 and caspase-9 [17, 18]. Bcl-2 family proteins include

both anti-apoptotic (BCL-2, BCL-XL) and pro-apoptotic (BAX, BAD, BAK, and BID) members,

could regulate Cyt-C releasing, thereby control apoptosis [19]. Yao et al. [20] showed TP-induced

cytotoxicity in human normal liver L-02 cells involved mitochondrial pathway.

In this study, we investigated the reproductive toxicity of TP and to assess whether these effect

can be ameliorated by pre-treatment with Que. The free radical damage and restoration were

assessed by measurement of ROS, SOD and GPx. The evaluation of mitochondrial function was

achieved by JC-1 assay. In addition, to identify the apoptotic action of TP and anti-apoptosis action

of Que, the activities of cytosolic CytC, Bcl-2 family proteins (BAX, Bcl-2), caspase-3 and

caspase-9 were measured. This study would guide a clinical approach to reduce the TP-mediated

productive toxicity by anti-oxidant candidates.

2. Materials and methods

2.1 Materials and reagents

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Triptolide and Quercetin (>98% purity) were purchased from the National Institute for the

Control of Pharmaceutical and Biological Products (Beijing, China). Antibodies to Nrf2 were

purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to caspase-9,

caspase-3 and bcl-2 were purchased from Cell Signaling Technology (CST, MA, USA).

Anti-Cytochrome c antibody was purchased from GeneTex (GeneTex, CA, USA). Anti-Bax

antibody was purchased from Signalway Antibody (SAB, CA, USA).

2.2 Animals

Sprague-Dawley (SD) rats (250±20g) were purchased from QingLongShan Laboratory

Animal Company (Nanjing, China).

2.3 Isolation and culture of Leydig cells

Leydig cells were isolated as previously described [21] with some modifications. Briefly, the

testis were dissociated in 1mg/ml collagenase II (SigmaAldrich, MO, USA) by shaking in an

orbital miser incubator at 1.16 g for 30 min at 37°C. The isolation procedure involved collagenase

digestion and Percoll (GE, CT, USA) density centrifugation according to a method described

previously. Following testis digestion, the collecting filtrate was then subjected to Percoll density

centrifugation and isolation of Leydig cells at a density between 1.070 and 1.088 g/ml. In general,

Leydig cells were cultured in 6-well plates (1.0×106 cells/well) at 37°C and 5% CO2 under

humidified conditions. The cells were cultured in DMEM/F12 medium (Invitrogen, CA, USA )

supplied with 5% fetal bovine serum (HyClone, UT, USA), 1 mM sodium pyruvate (Invitrogen,

CA, USA), antibiotics (Invitrogen, CA, USA), 10% BSA(Shengxing, Jiangsu, China ) and hCG

(0.075 IU/ ml) for 18 h.

The purity was assessed by 3β-HSD immune-staining. In a separate experiment, Leydig cells

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were pre-incubated with Que for 1 h and then treated with TP for 24 h.

2.4 AlamarBlue assay

Leydig cells were seeded in a 96-well black plate at a density of 2×104 cells/well.

Twenty-four hours later, the cells were treated with control (0.1% DMSO) or different

concentrations of TP. Meanwhile, the cells were pre-incubated with Que for 1 h and then treated

with different concentrations of TP for 24 h. The viability of Leydig cells was assessed by the

AlamarBlue Cell Viability Assay (Invitrogen, CA, USA) according to manufacturer’s instructions.

2.5 Detection of apoptosis

Leydig cells (1×106 cells/well) plated in each well of 6-well plates were treated with

concentrations of TP for 24 h after pre-incubating with 5 µM Que for 1 h. Cell apoptosis was

determined via FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA) using

Annexin V-FITC/PI Apoptosis detection kit (Vazyme, Jiangsu, China) according to the

manufacturer’s instructions.

2.6 Measurement of antioxidant enzymes activity (SOD and GPx)

Leydig cells were seeded in a 6-well plate at a density of 1×106 cells/well, and then exposed

to TP for 24 h after pre-incubating with 5 µM Que for 1 h. Cells were lysed in RIPA buffer

(Vazyme, Jiangsu, China) and centrifuged at 12,000 g for 10 min. Total protein of each sample

was determined by the BCA protein assay (Beyotime, Haimen, Jiangsu, China). SOD or GPx

activity was determined using a SOD or GPx Detection Kit (Beyotime, Haimen, Jiangsu, China)

according to the manufacturer’s instructions.

2.7 Measurement of Reactive Oxygen Species (ROS)

DCFH-DA (SigmaAldrich, MO, USA), a lipophilic dye was used to determine the

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intracellular accumulation of ROS. Leydig cells (2×104 cells/well) were seeded in 96-welll blank

plate and exposed to TP for 24 h following pre-incubating with Que for 1 h. After that, cells were

incubated with DCFH-DA (10 µM) for 30 min at 37 °C in dark, and then washed in 100 µL

ice-cold PBS. Fluorescence at Excitation: 488nm and Emission: 520nm were measured in a

microplate reader (safire2, Tecan, Switzerland).

2.8 Measurement of mitochondrial membrane potential (∆Ψm)

Changes of ∆Ψm were assessed by lipophilic and cationic probe JC-1 (JC-1 Detection Kit

(Beyotime Biotech, Nantong, China)) according to the manufacturer’s instructions. Briefly, after

different treatments, cells (2×104 cells/well) were cultured in 96-well blank plate and incubated

with 10 mM JC-1 for 30 min in a 5% CO2 incubator at 37 °C. Subsequently, cells were washed

twice with cold JC-1 Buffer. The cells were observed by a Fluorescence microscope (OLYMPUS,

Japan) with a single excitation (488nm) and dual emission (shift from 530 nm to 590 nm).

2.9 RNA isolation and RT-PCR analysis

Total RNA was extracted from the treated cells by the TRIzol® reagent (Vazyme, Jiangsu,

China). Genomic DNA contamination was removed by treatment of the total RNA with

RNase-free DNase (Vazyme, Jiangsu, China). The RNA concentration and integrity were

determined at 260 and 280 nm by a GeneQuant Pro spectrophotometer (Amersham Biosciences,

USA). The total RNA (2 µg) was reverse transcribed to cDNA with HiScript® Reverse

Transcription kit (Vazyme, Jiangsu, China) with Oligo-dT primers (Vazyme, Jiangsu, China)

according to the manufacturer’s instructions. The target fragments quantified by real-time PCR

using AceQTMSYBR Green® PCR Kit (Vazyme, Jiangsu, China) were performed on a iCycler

iQ™5 Multicolor Reai-Time PCR Detection System (Bio-Rad, USA). The specific primers are

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described in Table 1. For the quantification of real-time PCR results, the threshold cycle Ct was

determined for each reaction. Ct values for each gene were normalized to the housekeeping gene

(GAPDH), Relative expression levels of the target genes were calculated based on 2-∆∆Ct

according to the manufacture's specifications.

Table 1

Primer sequences for PCR amplification.

Gene Primer sequence

Gpx1 Forward: 5'-CGGACATCAGGAGAATGGCA-3'

Gpx1 Reverse: 5'-GTAAAGAGCGGGTGAGCCTT-3'

Gpx4 Forward: 5'-GCCGTCTGAGCCGCTTATT-3'

Gpx4 Reverse: 5'-CGATGTCCTTGGCTGCGAAT-3'

SOD1 Forward: 5'-AGGGCGTCATTCACTTCGAG-3'

SOD1 Reverse: 5'-TCTGCAAGTGCATCATCGTT-3'

SOD2 Forward: 5'-GCCTCAGCAATGTTGTGTCG-3'

SOD2 Reverse: 5'-ATTGTTCACGTAGGTCGCGT-3'

GAPDH Forward: 5'-AGAACATCATCCCTGCATCCA-3'

GAPDH Reverse: 5'-CCGTTCAGCTCTGGGATGAC-3'

2.10 Western blot analysis

Cells were lysed in RIPA buffer (Vazyme, Jiangsu, China) containing protease inhibitors

(Vazyme, Jiangsu, China) to prepare the total protein fractions. The nuclear extracts were prepared

using a Nuclear Extract kit (Vazyme, Jiangsu, China), and the mitochondrial proteins were

prepared using a Cell Mitochondria Isolation Kit (Beyotime, Jiangsu, China) following the

manufacturer’s instructions. Protein concentration was measured by the BCA protein assay. 20 µg

protein sample was separated by 10% resolving SDS-PAGE and transferred to nitrocellulose

membranes. The membranes were incubated with the appropriate primary antibodies at 4°C

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overnight following incubated with 5% non-fat milk for 1h at room temperature. After incubating

with HRP-conjugated secondary antibodies for 1 h at room temperature, the membranes were

visualized using chemiluminescence HRP substrate in ChemiDoc XRS imaging system (Bio-Rad,

USA).

2.11 Statistical analysis

The data were expressed as mean ± standard deviation (SD). The statistical value p < 0.05

was considered statistically significant. Statistical analysis was done by two-way ANOVA using

GraphPad Prism 5 software. Each experiment was performed in triplicate and replicated

independently.

3. Results

3.1. Cytotoxicity of TP and Que pre-treatment countered the decrease in TP-induced Leydig cell

viability and inhibited TP-induced apoptosis in Leydig cell

To determine the cytotoxicity of TP in vitro, Leydig cells were treated with various

concentrations of TP for 24 h, the cell viability was measured using AlamarBlueTM assay. As

shown in Fig.1A, TP increased the viability of Leydig cells in a dose-dependent manner. Loss of

cell viability showed 30% by 60 nM TP and reached up to 95% by 640 nM. Meanwhile, to

determine the optimal dose of Que that countered the TP-induced decrease in Leydig cell viability,

Leydig cells were co-incubated with different doses of Que and 60 nM TP (final 2.5, 5, 10, 20 µM)

for 24 h. As shown in Fig.1B, compared with TP-treated cells, Que at 2.5 µM was not able to

maintain a significant rise in cell viability. On the other hand, high doses (10 and 20 µM) of Que

were not able to sustain the cell viability. Only 5 µM Que demonstrated the significant effect on

cell survival. Based on this observation, 20, 40 and 60 nM TP concentration were selected for

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general toxicity assessment impact on Leydig cells (Fig.1C). Further, 5 µM Que was used in

subsequent experiments. The annexin V-FITC/PI double staining assay results showed that TP

induced apoptosis in Leydig cells in a dose-dependent manner (Fig.1D). The TP induced apoptosis

was significantly restored by pre-treatment with 5 µM Que (Fig.1E and F).

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Fig.1. Inhibition of Que on TP-induced cytotoxicity in Leydig cell. Leydig cells were treated with

TP at various concentrations or DMSO (0.1%) as control for 24 h (A). Leydig cells were

co-incubated with 60 nM TP and various concentrations of Que (final 2.5, 5, 10, 20 µM) or

DMSO (0.1%) as control for 24 h (B). Leydig cells were treated with various concentrations of TP

(final 20, 40, 60 nM) with or without 5 µM Que for 24 h (C). Cell apoptotic was determined by

annexin V-FITC/PI double staining assay. Leydig cells were treated with various concentrations of

TP (final 20, 40, 60 nM) with or without 5 µM Que for 24 h (D and E). The percentage of cell

apoptosis is shown in (F). Data are presented as the mean ± SD. “*” indicates significant

difference between normal control and TP treated groups, “#” indicates significant difference

between TP treated and Que pre-treated groups (*** P < 0.001, #P < 0.05, ###P < 0.001), n=3.

3.2 Effects of Que on TP-induced decrease of antioxidant enzymes activities and generation of

intracellular ROS

In oxidative stress-induced organ pathophysiology, intracellular antioxidant enzymes are

considered to be the first line of cellular defense as these enzymes protect biological

macromolecules like DNA, proteins etc. from oxidative damage. In our study, we determined the

activities of SOD and GPx in Leydig cells which treated with various concentrations of TP and

pre-incubated with 5 µM Que separately. We observed that TP intoxication significantly decreased

the SOD activity at concentration of 40 and 60 nM and GPx activity at concentration of 20, 40 and

60 nM. However, compared to the TP-treated group, there were significantly increased the SOD

and GPx activity in the Que pre-treated group (Fig. 2A and B). The results of gene expression

were shown in Fig.2C to F. Meanwhile, TP exposure increased the accumulation of intracellular

ROS and the increase was significantly restored by pre-treatment with 5 µM Que (Fig. 2G).

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Fig.2. Effects of Que on TP-induced decrease of antioxidant enzymes activities and generation of

intracellular ROS. The activities (A and B) and expression (C to F) of GPx and SOD were

measured. Leydig cells were treated with various concentrations of TP (final 20, 40, 60 nM) with

or without 5 µM Que for 24 h. The ROS were detected using the fluorescent probe DCFH-DA (G).

Data are presented as the mean ± SD. “*” indicates significant difference between normal control

and TP treated groups, “#” indicates significant difference between TP treated and Que pre-treated

groups (*P < 0.05, *** P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001), n=3.

3.3. Effects of Que on TP-induced loss of mitochondrial membrane potential and release of Cyt

C

To elucidate the direct effect of TP on Leydig cell mitochondrial membrane potential (∆Ψm),

the ∆Ψm was measured after incubating with JC-1. As showed in Fig. 3A, TP induced ∆Ψm loss as

a dose-dependent manner compared to control, which was confirmed by High Content Screening.

Only 40 and 60 nM of TP significantly inhibited ∆Ψm. Meanwhile, TP caused the release of Cyt C

from mitochondria into cytosol as a dose-dependent manner, which was regarded as a key step of

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the mitochondria-mediated pathway in apoptosis (Fig. 3B and C). Treatment with 5 µM que prior

to the TP exposure however could significantly inhibit TP induced alterations of these parameters.

Fig.3. Effects of Que on TP-induced loss of mitochondrial membrane potential and release of

Cyt-C. Leydig cells were treated with various concentrations of TP with or without 5 µM Que for

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24 h. Representative pictures of JC-1 staining and quantitative analysis are shown. In control

non-apoptotic cells, the dye stains the mitochondria in red. Otherwise the cytosol was shown green

(A). CCCP was used as a positive control. Cyt-C protein expressed in the mitochondria and

cytosol were determined (B and C). Data are presented as the mean ± SD. “*” indicates significant

difference between normal control and TP treated groups, “#” indicates significant difference

between TP treated and Que pre-treated groups (** P < 0.01, *** P < 0.001, ##P < 0.01, ###P <

0.001), n=3.

3.4 Que counteracts TP induced alteration mitochondrion-dependent apoptosis pathway related

protein expression in Leydig cells

To investigate the effects of Que on TP-induced changes of apoptosis-related proteins

expression. The expression of BAX, Bcl-2, caspase-9 and caspase-3 were measured in Leydig

cells (Fig.4A). TP decreased protein levels of the apoptosis inhibitory protein Bcl-2, and increased

the expression levels of Bax, caspase-9 and caspase-3 (Fig.4B to E). In addition, pre-treatment

with Que down-regulated the expression of Bax, caspase-9 and caspase-3 and up-regulated the

Bcl-2 induced by TP.

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Fig.4. Resist of Que to TP-induced alteration of apoptosis-related protein expression in Leydig

cells. Cells were treated with various concentrations of TP (final 20, 40, 60 nM) with or without 5

µM Que for 24 h. The levels of the apoptotic-related proteins Bcl-2, Bax, caspase-9 and caspase-3

were analyzed (A to E). Data are presented as the mean ± SD. “*” indicates significant difference

between normal control and TP treated groups, “#” indicates significant difference between TP

treated and Que pre-treated groups (** P < 0.01, *** P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001),

n=3.

3.5 Effect of TP on the level of Nrf2

To confirm the role of Nrf2 in the TP-induced oxidative stress, the expression of Nrf2 were

measured in Leydig cells. TP decreased protein levels of Nrf2, and pre-treatment with Que resisted

this effect (Fig.5).

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Fig.5. Effect of TP on the regulation of Nrf2. Leydig cells were treated with various

concentrations of TP (final 20, 40, 60nM) with or without 5 µM Que for 24h. The levels of the

Nrf2 protein was analyzed. Data are presented as the mean ± SD. “*” indicates significant

difference between normal control and TP treated groups, “#” indicates significant difference

between TP treated and Que pre-treated groups (*** P < 0.001, ###P < 0.001), n=3.

4. Discussions

TP is a diterpene triepoxide with variety biological activities. Generally, tripterygium and

tripterygium glycosides pieces are the common forms for clinical application, in which TP is the

major component. In addition of anti-rheumatoid, TP also has pharmacological activities like

anti-cancerogenesis and anti-inflammatory [22]. However, the side effects of TP, especially its

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reproduction toxicity, have not been well illustrated. In the present study, we used Leydig cell as a

cellular model to explore the toxic effect of TP and the role of Que in defense against TP-induced

male reproductive injury. In Leydig cells, TP induced testosterone secretion decline by lowering

the expression of hormone synthesis enzyme and reproductive toxicity, which was caused by

accumulating of ROS and cell apoptosis. Meanwhile, Que is proved to protect Leydig cells from

TP-induced toxicity by the counteraction of oxidative damage.

The role of ROS and its metabolites in cellular physiology and pathogenesis of number of

diseases is the subject of today's research [23]. SOD is a scavenger of superoxide, is the most

important to resist the toxic effects of ROS defense mechanism. SOD accelerates the disproportion

of hydrogen peroxide, prevents further producing free radicals. GPx is antioxidant enzyme

containing selenium, existing in the cell cytoplasm or plasma. The main function of these enzymes

is to remove soluble hydrogen peroxide and alkyl peroxide by using of glutathione as the substrate

[24]. Decline in SOD activity showed superoxide surplus accumulation. Reduced GPx activity

showed H2O2 accumulation. TP had been reported its inhibition of the activity of SOD and GPx in

various tissues of rats [25]. In the present study, we observed that the content of intracellular ROS

significantly increased under TP exposure (Fig.2G), and the activity of the SOD and GPx both

reduced (Fig.2A and B). Meanwhile, the transcription of SOD1, SOD2, GPx1 and GPx4 genes

also showed a decrease (Fig.2C to F). The whole results performed as a dose-dependent manner.

Thus, we demonstrated that TP could induce oxidative stress in Leydig cells in vitro.

The interaction between oxidative stress and mitochondrial damage was revealed by large

amounts of data [8]. Loss of ∆Ψm is a common index of mitochondrial damage which was

resulted from the surplus generation of ROS. This phenomenon was shown in the present study

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(Fig.3A), which confirmed the dysfunction of mitochondria. As an important enzyme inducing

apoptosis, Cyt-C was released from mitochondria to cytoplasm due to the ROS-induced

mitochondrial damage. The substantial increase of Cyt-C and loss of ∆Ψm (Fig.3) suggested that

mitochondria played a critical role in TP-induced apoptosis.

Various motivators including oxidative stress foment the mitochondrial apoptosis pathway.

The Bcl-2 family proteins, including pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2,

are involved in the apoptotic pathway. Following the expose to TP, the expressions of Bax and

Bcl-2 were increased and decreased separately (Fig.4B and C). In addition, the loss of Bax/Bcl-2

ratio demonstrated that mitochondrial permeability transition might be a reason of Cyt-C release

[26]. Caspase family, including caspase-3 and caspase-9 were significant elements mediated

apoptosis [17]. The release of Cyt-C which would bind to Apaf-1, an apoptosis-related protein,

could mediate activation of caspase-9, and then lead to cleavage of procaspase-3. Evidently, the

treatment of TP increased the expression of caspase-9 and caspase-3 (Fig.4D and E). These results

confirmed the effect of TP on apoptosis in Leydig cells.

Exogenous antioxidants, such as flavonoids, could resist oxidative stress by several ways,

major of which is scavenging or inhibiting the generation of ROS [27]. Que is one of the most

common flavonoids in the diet, distributed in many vegetables, fruits and other foods [28]. Que is

an effective antioxidant which can directly remove oxide free radicals, inhibit lipid peroxidation

and change in vivo and in vitro antioxidant defense pathways [12]. Zhang [29] and Robaszkiewicz

et al. [30] showed that Que reduced reactive oxygen species in human non-small cell lung cancer

cell lines A549 and chickens testicular germ cells. Kalender et al.[31] and Payne and Halles[32]

reported that Que could protect SOD, CAT, GPx activities from chlorpyrifos-induced the toxic

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effect of ROS in rat testis tissue. However, the protective effect of Que on TP toxicity is

little-known. The present study shows that a certain dose of Que had no adverse effects in Leydig

cells (Fig.1B). We observed the effect of TP exposure on Leydig cells could be restored by Que.

The protective effect of Que was achieved by treating prior to TP. Compared to TP group, ROS

generation was inhibited significantly (Fig.2G) and the activities (Fig.2A and B) and expressions

(Fig.2C to F) of SOD and GPx were up-regulated comparatively. Ascent of ∆Ψm was observed

(Fig.3A) and the content of Cyt-C was down-regulated to the physiological level (Fig.3B and C).

Apoptosis (Fig.1D and E) and apoptosis-related proteins expression (Fig.4) were also inhibited.

All of these results could be explained based on the antioxidant effect of Que. In that, Que could

protect Leydig cells from TP-induced oxidant damage.

Nrf2 is anti-oxidant transcription factor leading a protection against oxidative stress by

translocating into the nucleus to associate with AREs. The effect of TP to inhibit Nrf2 pathway

has been demonstrated in heart tissues [33]. We hypothesized it is also the target of TP in Leydig

cells. As shown in Fig.5, the expression of Nrf2 decreased obviously. This finding indicates a

promising strategy to protect reproduction from TP toxicity in clinical treatment.

The results of the present study suggest that TP induces mitochondrial apoptosis by

decreasing ∆Ψm and the expression of related protein (bcl-2, caspase-9 and caspase-3) which is

partly due to oxidative stress. As an effective antioxidant, Pre-treatment of Que can effectively

alleviate the above toxic reaction caused by TP in Leydig cells.

5. Conclusions

To the best of our knowledge, this is the first report demonstrating that TP-induced oxidative

stress is the cause of its toxic effect in Leydig cells and the impact of TP on the expression of Nrf2

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was first observed in this experiment. On the other hand, we found that Que provides protection

against TP-induces reproductive toxicity through its Antioxidant properties. Furthermore, Que acts

an anti-apoptotic activity by down-regulating the expression of the mitochondrial apoptotic

pathways related proteins. However, reproductive toxicity caused by TP involves not only single

factor but multiple signaling pathways including JNK and Nrf2 pathway.

Disclosure

None.

Conflict of interest

The authors declare that are no Conflict of interest.

Acknowledgements

The work was supported by the Major Scientific and Technological Special Project for

Significant New Drugs Creation (No. 2012ZX09504001-001), the National Natural Science

Foundation of China (NO.81102876, 81430082),the Fundamental Research Funds for the Central

Universities (ZL15005,YD2014SK0002), 333 high level project of Jiangsu Province.

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HIGHLIGHTS

• Reproductive toxicity induced by Triptolide in vivo were due to

oxidative stress in Leydig cells.

• Protective effect of Quercetin on oxidative stress and Apoptosis

induced by Triptolide in Leydig cells.

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