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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: [email protected] (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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[email protected] (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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