氯氰菊酯通过Ca2+-线粒体/内质网通路诱导Sertoli细胞凋亡

Sertoli cells play important roles in supporting spermatogenesis. In the preliminary studies, we found cypermethrin induced apoptosis in normal mouse Sertoli cell TM4 with increased calcium concentration and decreased mitochondrial membrane potential. Based on previous research，we propose cypermethrin induces apoptosis through calcium-regulated mitochondria and endoplasmic reticulum-associated pathway. In the project, calcium will be used as the starting points. The Fluo-3 AM fluorescent probe, Hoechst staining, JC-1 probe, flow cytometry assay, mammalian two-hybrid assay, and Western blot will be applied to study the effects of cypermethrin on calcium concentration, apoptosis, mitochondrial membrane potential, interaction between signaling molecules and molecular targets in the TM4 cells thoroughly. The project will indicate the effects of cypermethrin calcium-regulated mitochondria and endoplasmic reticulum-associated pathway, and to elucidate cypermethrin will cause Sertoli cell apoptosis through the pathway. The project will reveal the molecular mechanism of cypermethrin-induced cell apoptosis from a new perspective. The new theory will be formed that cypermethrin-induced cell apoptosis is related to the calcium-regulated mitochondria and endoplasmic reticulum-associated pathway. The study will supply important basic date for prevention and control of reroductive toxicity of endocrine disrupting chemicals (EDCs).

支持（Sertoli）细胞对精子发生意义重大。前期研究表明氯氰菊酯可诱导正常小鼠Sertoli细胞TM4凋亡，并伴胞质Ca2+浓度升高和线粒体膜电位下降。基于前期新发现，提出氯氰菊酯通过Ca2+-线粒体/内质网通路诱导Sertoli细胞凋亡。本项目拟以Ca2+为切入点，应用Fluo-3 AM探针、Hoechst染色、JC-1探针、流式细胞术、哺乳动物双杂交和Western blot等，从细胞和分子水平系统研究氯氰菊酯对TM4胞质Ca2+、细胞凋亡、线粒体膜电位、信号分子相互作用和标志分子靶标影响，明确氯氰菊酯对Ca2+-线粒体/内质网通路的作用，阐明氯氰菊酯通过Ca2+-线粒体/内质网通路调控Sertoli细胞凋亡。从新的视觉揭示氯氰菊酯诱导细胞凋亡的机制，形成氯氰菊酯致细胞凋亡与Ca2+-线粒体/内质网通路相关的新学说，为针对性地开展环境内分泌干扰物（EDCs）生殖毒性防控提供基础资料。

支持（Sertoli）细胞对精子发生意义重大。本项目研究了氯氰菊酯对Ca2+-线粒体/内质网通路介导的Sertoli细胞（TM4细胞）凋亡的影响。应用钙离子荧光探针、Western blot检测氯氰菊酯对TM4细胞内游离钙离子浓度及Ca2+信号通路相关蛋白的影响，发现氯氰菊酯诱导TM4细胞内游离钙离子浓度升高，促进CaM、CaMKⅡ表达及CaMKⅡ磷酸化水平。流式细胞术研究氯氰菊酯对TM4细胞凋亡的作用与Ca2+的相关性，表明氯氰菊酯致TM4细胞凋亡与Ca2+有关。JC-1探针检测氯氰菊酯对TM4细胞线粒体膜电位的影响，表明氯氰菊酯抑制TM4细胞线粒体膜电位。Western blot实验研究氯氰菊酯对TM4细胞内线粒体途径及内质网应激介导的凋亡相关蛋白的影响，发现氯氰菊酯抑制Bcl2、线粒体内cytochrome c表达，促进BAX、Cleaved Caspase-3、细胞浆cytochrome c、GRP78、PERK、p-PERK、eIF2α、p-eIF2α、ATF4、CHOP、Caspase-12、Caspase-9、Caspase-3表达。采用CaM活性抑制剂、CaMKⅡ活性抑制剂及内质网钙离子释放通道蛋白三磷酸肌醇受体拮抗剂，检测氯氰菊酯作用下TM4中Ca2+-线粒体/内质网通路中分子的变化，表明抑制Ca2+通路可以保护氯氰菊酯所致线粒体及内质网应激介导的TM4细胞凋亡。钙离子荧光探针检测氯氰菊酯对TM4细胞内线粒体Ca2+浓度的影响，发现氯氰菊酯能够提高线粒体内Ca2+浓度。透射电镜、共聚焦显微镜和Western blot实验研究氯氰菊酯对TM4细胞内质网-线粒体偶联及相关蛋白的影响，结果表明氯氰菊酯使TM4细胞中内质网-线粒体偶联增强，MCU、MICU1、Mfn2、IP3R、Grp75、VDAC1表达升高。使用MCU特异性抑制剂，检测氯氰菊酯所致TM4细胞凋亡、线粒体内Ca2+浓度及Grp75蛋白变化，发现阻断Ca2+转运能够在一定程度上保护氯氰菊酯引起的内质网-线粒体偶联介导的Sertoli细胞凋亡。本研究阐明了氯氰菊酯对Sertoli细胞Ca2+-线粒体/内质网通路的影响，证实了内质网-线粒体偶联与Ca2+介导的氯氰菊酯所致Sertoli细胞凋亡有关，形成氯氰菊酯雄性生殖毒作用机制与Ca2+相关的新学说，为其他环境抗雄激素的机制研究开辟新的思路。