Wu De-Ling, Wang Tong-Sheng, Liu Hong-Juan, Zhang Wei, Tong Xiao-Hui, Peng Dai-Yin, Kong Ling-Yi
State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Bioactive Natural Product Research, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, China; School of Pharmacy, Anhui University of Chinese Medicine, Anhui Province Key Laboratory of Chinese Medical Formula, Hefei 230012, China; School of Integrated Traditional Chinese and Western Medicine, Anhui University of Chinese Medicine, Hefei 230012, China.
School of Pharmacy, Anhui University of Chinese Medicine, Anhui Province Key Laboratory of Chinese Medical Formula, Hefei 230012, China.
Chin J Nat Med. 2022 Apr;20(4):282-289. doi: 10.1016/S1875-5364(22)60153-5.
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 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.
五子衍宗丸是治疗男性不育症的经典方剂。我们之前的研究表明，它可通过影响线粒体来抑制精子凋亡，但其潜在机制尚不清楚。本研究旨在探讨五子衍宗丸对由苍术苷（ATR）开启的小鼠精母细胞系（GC - 2细胞）中线粒体通透性转换孔（mPTP）的作用。首先，将五子衍宗丸给大鼠连续灌胃7天制备含药血清。GC - 2细胞分为对照组、模型组、阳性组以及5%、10%、15%五子衍宗丸含药血清组。环孢素A（CsA）用作阳性对照。药物孵育后加入50 μmol·L ATR。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Calcein AM和JC - 1荧光探针检测mPTP的开放和线粒体膜电位（MMP）。分别通过RT - PCR（实时定量PCR）和蛋白质印迹法检测电压依赖性阴离子通道1（VDAC1）、亲环蛋白D（CypD）、腺嘌呤核苷酸转位酶（ANT）、细胞色素C（Cyt C）、半胱天冬酶3、9的mRNA和蛋白水平。结果表明，ATR处理24小时后GC - 2细胞的mPTP开放，导致MMP降低和细胞凋亡增加。五子衍宗丸含药血清和CsA预保护抑制了ATR诱导的GC - 2细胞mPTP的开放，伴随MMP增加和细胞凋亡减少。此外，RT - qPCR和WB结果表明，五子衍宗丸含药血清可显著降低VDAC1、CypD、Caspase - 3、9和CytC的mRNA和蛋白水平，以及增加Bcl/Bax比值。然而，ANT未受到显著影响。因此，这些研究结果表明，五子衍宗丸通过减弱GC - 2细胞中mPTP的开放来抑制线粒体介导的细胞凋亡。五子衍宗丸含药血清抑制VDAC1和CypD的表达并增加Bcl - 2的表达，这影响了mPTP的开放，并对ATR诱导的GC - 2细胞发挥保护和抗凋亡作用。
