内质网应激和线粒体通路交联介导微囊藻毒素致斑马鱼雄性生殖细胞凋亡中的作用

Microcystins (MCs) are potent hepatotoxins. But recently studies showed that gonads are the second important target organ of MCs. Previous study has demonstrated that MCs damaged the testes and was toxic to the reproductive system of male zebrafish mainly through causing oxidative stress and MCs could induce germ cell apoptosis in male mammals. In the present study, we will investigate the apoptosis of germ cells after long-term microcystin exposure at low doses and the effects of the Fas/FasL pathway, mitochondrial pathway, endoplasmic reticulum (ER) stress in the microcystin-induced apoptosis in gonads of male zebrafish. In this study, microcystin-induced ER stress will be inhibited by glucose regulated protein 78 (GRP78) overexpression and ER chemical chaperone. We will then observe whether inhibition of ER stress can alleviate microcystin-induced apoptosis and explore the role of ROS-mediated ER stress in the process of germ cell apoptosis induced by microcystin. Finally, we will observe whether inhibition intracellular calcium concentration under the ER stress can attenuate microcystin-induced cell apoptosis through mitochondria pathway. This study will provide theoretical basis for the prevention and control of microcystins and exploring molecular mechanism of microcystin-induced apoptosis in germ cells.

微囊藻毒素(MCs)是已知的肝毒素，最近发现生殖腺是微囊藻毒素作用下仅次于肝脏的第二靶器官。本课题拟在前期通过蛋白质组学证实急性微囊藻毒素暴露导致雄性斑马鱼生殖细胞氧化损伤和前人研究微囊藻毒素能够致雄性生殖细胞凋亡的基础上，研究低剂量长期微囊藻毒素暴露对斑马鱼雄性生殖细胞凋亡的影响；深入研究微囊藻毒素对参与凋亡过程的 Fas/FasL 通路、线粒体通路和内质网应激的调控作用，以探讨微囊藻毒素致生殖细胞凋亡的可能途径；通过观察内质网化学分子伴侣或 GRP78 过表达对微囊藻毒素调控生殖细胞参与凋亡相关信号通路的拮抗作用，阐明活性氧导的内质网应激诱发生殖细胞凋亡分子机制；通过抑制内质网应激下胞内钙离子水平，阐明内质网应激通过钙离子影响线粒体通路从而介导微囊藻毒素对细胞凋亡的作用机制。本课题为阐明微囊藻毒素对生殖细胞凋亡的机制和为防治微囊藻毒素产生的毒性提供理论依据。

微囊藻毒素是已知的肝毒素，最近发现生殖腺是微囊藻毒素作用下仅次于肝脏的第二靶器官。本项目研究了微囊藻毒素对线粒体通路和内质网应激的调控作用，以探讨微囊藻毒素致雄性生殖细胞凋亡的可能途径，具体表现为：微囊藻毒素MCLR损伤雄性性腺，激活性腺组织内质网应激相关蛋白（Caspase12,Grp78,Chop,IRE1和PERK）和凋亡相关蛋白(Caspase 3,Caspase8,Caspase 9,Bcl2和Bax)的表达造成生殖细胞凋亡；进一步的研究发现，抗氧化剂N-乙酰半胱氨酸（NAC）对微囊藻毒素诱发斑马鱼雄性生殖细胞非折叠蛋白反应具有拮抗效应，NAC可以显著缓解生殖细胞凋亡的发生和抑制caspase依赖性蛋白表达，证明活性氧介导的内质网应激在诱发生殖细胞凋亡中起到重要作用；内质网可能通过钙离子来影响线粒体通路从而导致细胞凋亡的发生，MCLR可以导致内质网应激的发生，随后胞浆中钙离子浓度升高，激活磷酸化钙调蛋白（pCaMKⅡ），pERK1/2和p-P38蛋白，进而导致凋亡蛋白和抗凋亡蛋白表达变化，细胞的通透性增大，细胞色素C蛋白明显上升，而这一过程在加入钙离子螯合剂BAPTA后可以明显改善，最终导致细胞凋亡率也出现明显下降。本课题为阐明微囊藻毒素对生殖细胞凋亡的机制和为防治微囊藻毒素产生的毒性提供理论依据。