表观遗传调控在正己烷暴露对大鼠卵巢颗粒细胞凋亡中的作用机制

Mechanism of epigenetic regulation in apoptosis of rat ovarian granulosa cells exposed to n-hexane

目的 了解表观遗传调控在正己烷暴露对大鼠卵巢颗粒细胞凋亡中的作用机制。方法 成年雌性Wistar大鼠48只分为对照组、正己烷低剂量组、中剂量组和高剂量组,其染毒剂量分别为0.00、3.01、9.03、27.09 g/m3;提取各组大鼠卵巢颗粒细胞分为卵巢颗粒细胞组、正己烷低剂量组、正己烷中剂量组和正己烷高剂量组,每组12只,以上各组每孔设6个平行样,培养72 h。培养结束后,采用细胞计数试剂盒-8测定细胞增殖,流式细胞仪测定细胞凋亡,透射电镜技术观察卵巢颗粒细胞超微结构,逆转录聚合酶链反应及蛋白印迹法测定细胞中Bax、Bcl-2、Caspase 3和Caspase 9 m RNA与蛋白水平,甲基化DNA免疫沉淀芯片法测定Bax、Bcl-2、Caspase 3和Caspase 9 CPG位点甲基化水平。结果 与卵巢颗粒细胞组比较,正己烷低、中、高剂量组光密度值、存活率降低,凋亡率、Bax、Caspase 3、Caspase 9 m RNA和蛋白表达、Bcl-2 CPG位点甲基化水平升高(均P<0.05)。各组Bax、Caspase 3和Caspase 9 CPG位点甲基化水平差异均无统计学意义(均P>0.05)。卵巢颗粒细胞组卵巢颗粒细胞结构正常;正己烷各剂量组可见线粒体肿胀,线粒体嵴稀疏,细胞质浓缩,细胞核边集,明显可见凋亡小体。结论 正己烷抑制Bcl-2、促进Bax表达,诱导卵巢颗粒细胞凋亡,正己烷可能是通过增加Bcl-2启动子的甲基化状态来降低Bcl-2的表达。

Objective To investigate the mechanism of epigenetic regulation in the apoptosis of rat ovarian granulosa cells exposed to n-hexane. Methods 48 adult female Wistar rats were divided into control group,n-hexane low-dose group,middle-dose group,and high-dose group,and the exposure doses were 0.00,3.01,9.03 and 27.09 g/m3,respectively. The ovaries granulosa cells extracted from all groups were divided into ovarian granulosa cell group,n-hexane low-dose group,n-hexane medium-dose group,and n-hexane high-dose group,12 animals in each group. Each of the above groups had 6 parallel samples per well and cultured for 72 hours. After the culture,CCK-8 was used to measure cell proliferation,flow cytometry was used to measure cell apoptosis,transmission electron microscopy was used to observe the ultrastructure of ovarian granulosa cells,and Bax,Bcl-2,and Caspase 3,Caspase 9 mRNA and protein levels in cells were measured by reverse transcription polymerase chain reaction and Western blotting. Methylated DNA immunoprecipitation chip method was used to determine the methylation levels of Bax,Bcl-2,Caspase3,Caspase 9 CPG sites. Results Compared with the ovarian granulosa cell group,the optical density value and survival rate of the n-hexane low,medium,and high dose groups decreased,and the apoptosis rate,Bax,Caspase 3,Caspase 9 mRNA and protein expression,and Bcl-2 CPG site methylation level increased(all P<0.05). There were no statistically significant differences in the methylation levels of Bax,Caspase 3,Caspase 9 CPG sites among groups(all P>0.05). The structure of ovarian granulosa cells in the ovarian granulosa cell group was normal,and mitochondria were swollen,mitochondrial cristae were sparse,cytoplasm were concentrated,nuclei were gathered at each dose group of n-hexane,and apoptotic bodies were clearly seen. Conclusion N-hexane can inhibit the expression of Bcl-2,promote the expression of Bax and induce apoptosis of ovarian granulosa cells,and n-hexane may decrease the expression of Bcl-2 by increasing the methylation status of the Bcl-2 promoter.