微囊藻毒素-LR致大鼠睾丸生殖细胞凋亡的表观遗传调控机制

Microcystin-LR(MC-LR), a naturally occurring toxin produced by cyanobacteria, is a microcystins isomer and considered the most abundant and toxic compound. Therefore, many studies have been focused on reproductive toxicity of MC-LR in mammals including humans. Our previous results showed that the expression of apoptosis-related genes can be regulated by MC-LR, indicating that apoptosis plays an important role in the reproductive toxicity of MC-LR. Up to now, molecular mechanism of reproductive toxicity of MC-LR is still unclear. It was reported that epigenetic modification can regulate the expression of apoptosis related gene, suggesting that epigenetic modification may be involved in the cell apoptosis induced by MC-LR. In this project, MC-LR will be used as test sample, the co-culture of sperm cells with testis sertoli cells will be used as cell model, and prepubertal and pubertal rats will be used as animal model. The testicular damage caused by MC-LR in rats will be explored with Chromatin Immunoprecipitation-chip (ChIP-chip), Laser scanning confocal microscopy, and so on. The cell apoptosis induced by MC-LR through mitochondrial mediated caspase dependent pathway will be surveyed at the anmial, cell and molecular level. The regulation function of H3K4me3, SIRT1 and acetyl-Histone in cell apoptosis will also be anaylazed, revealing molecular mechanism of testicular germ cell apoptosis caused by MC-LR at the epigenetic level. The study will provide a new train of idea for comprehensive understanding reproductive toxicity of MC-LR, and lay the foundation for preventing people health from algal toxins.

微囊藻毒素LR（MC-LR）是污染严重、人群接触广、危害大的微囊藻毒素异构体，其生殖毒性受到了广泛关注和重视。本课题组研究表明MC-LR可改变凋亡相关基因表达，诱导细胞凋亡，但其分子机制尚不明确。表观遗传改变调控凋亡相关基因的表达，可能在MC-LR致睾丸生殖细胞凋亡中起着调控作用。为此，本项目以MC-LR为受试物，以体外共培养的睾丸支持细胞和生精细胞为细胞模型，以青春前期、青春期SD大鼠为动物模型，应用染色质免疫共沉淀联合芯片技术、激光扫描共聚焦显微技术等方法，从动物、细胞和分子水平检测MC-LR通过线粒体介导的caspase依赖通路诱导的睾丸生殖细胞凋亡，分析H3K4me3、SIRT1和组蛋白乙酰化在MC-LR致睾丸生殖细胞凋亡中的调控作用，从表观遗传学角度探索MC-LR致睾丸生殖细胞凋亡的分子机制，为全面深入认识和理解MC-LR的生殖毒性机制提供新思路，为保护人群生殖健康提供理论基础。

微囊藻毒素-LR（Microcystin-LR, MC-LR）是一类广泛存在于水体中的、危害较大的细胞内毒素，性腺是其主要靶器官。本项目以MC-LR为受试物，以体外共培养的睾丸支持细胞-生精细胞为细胞模型，以SD大鼠为动物模型，应用染色质免疫共沉淀联合芯片技术和激光扫描共聚焦显微技术等方法，从动物、细胞和分子水平对MC-LR导致雄性生殖细胞凋亡的机制进行了研究。结果显示MC-LR能够抑制SD大鼠生殖细胞增殖，刺激细胞发生氧化应激，降低线粒体膜电位，改变线粒体通透性，从而促使细胞色素c从线粒体释放到胞质中，激活Caspase-9，引起Caspase蛋白级联反应，最终通过线粒体介导的Caspase依赖通路引起生殖细胞凋亡。氧化应激在MC-LR诱导的雄性生殖细胞凋亡中发挥重要作用，是MC-LR发挥生殖毒性的始发因素，抑制MC-LR诱导的氧化应激水平可以明显改善通过线粒体介导的Caspase依赖通路引起的生殖细胞凋亡。MC-LR可降低组织和细胞中SIRT1的含量，增加p53和Ku70乙酰化水平，诱导生殖细胞发生凋亡，而白藜芦醇则可以通过上调SIRT1的表达，降低p53和Ku70乙酰化水平，抑制Bax表达来拮抗MC-LR引起的大鼠睾丸生殖细胞凋亡。p53依赖途径和mPTP开放在MC-LR诱导的大鼠睾丸生殖凋亡过程也发挥重要作用，P53抑制剂PFT-α和线粒体损伤抑制剂CsA可以明显改善共培养细胞线粒体膜电位水平，阻止Cyt-c从线粒体向细胞质移动，减少凋亡发生。这为MC-LR致大鼠生殖毒性的研究提供了新的思路和机制靶点。同时发现表观遗传参与了MC-LR诱导的雄性生殖细胞凋亡的过程，MC-LR可以通过增加H3K4me3水平，干扰MAPK通路凋亡相关因子表达，进而通过线粒体Caspase通路导致细胞凋亡的发生；MC-LR可上调SD大鼠睾丸组织和SD大鼠睾丸支持-生精共培养细胞组蛋白去乙酰化酶活性，降低组蛋白乙酰化水平，进而通过激活线粒体Caspase通路、扰乱细胞周期，导致细胞凋亡的发生。总之，本研究从线粒体介导的Caspase依赖凋亡通路和表观遗传学角度，阐明了MC-LR致雄性生殖毒性的作用过程，丰富了微囊藻毒素的生物学作用及生殖毒性理论，为相关疾病的防治提供理论依据和治疗靶点。