Abstract：
Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.
摘要：
五子-延宗丸（WZYZW）是男性不育的经典处方。我们以前的研究表明，它可以通过影响线粒体来抑制精子凋亡，但潜在的机制尚不清楚。本研究的目的是探讨WZYZW对白术苷（ATR）打开的小鼠精母细胞系（GC-2细胞）线粒体通透性转换孔（mPTP）的作用。起初,在口服施用WZYZW7天后,从大鼠制备WZYZW含药血清。将GC-2细胞分为对照组，模型组,阳性组，以及5%,10%,15%WZYZW含药血清组。环孢菌素A(CsA)用作阳性对照。药物孵育后加入50μmol·L-1ATR。使用CCK-8评估细胞活力。采用流式细胞术和TUNEL法检测细胞凋亡。分别用钙黄绿素AM和JC-1荧光探针检测mPTP和线粒体膜电位(MMP)的开放。电压依赖性阴离子通道1（VDAC1）的mRNA和蛋白水平，亲环蛋白D（CypD），腺嘌呤核苷酸转运蛋白(ANT),细胞色素C(CytC),分别通过RT-PCR（实时定量PCR）和Western印迹检测caspase3，9。结果表明，ATR处理24小时后，GC-2细胞的mPTP被打开，导致MMP减少和细胞凋亡增加。WZYZ含药血清和CsA的预保护抑制ATR诱导的GC-2细胞mPTP的开放，与MMP增加和凋亡减少有关。此外，RT-qPCR和WB结果表明，WZYZW含药血清能显著降低VDAC1和CypD的mRNA和蛋白水平，Caspase-3,9和CytC,以及增加的Bcl/Bax比率。然而,ANT没有显著影响。因此,这些发现表明WZYZW通过减弱GC-2细胞中mPTP的开放来抑制线粒体介导的凋亡。WZYZW含药血清抑制VDAC1和CypD的表达，增加Bcl-2的表达，从而影响mPTP的开放，对ATR诱导的GC-2细胞具有保护和抗凋亡作用。
