锌离子对脂多糖作用下大鼠腹膜间皮细胞凋亡的影响及机制

目的 研究锌离子对脂多糖（LPS）诱导的大鼠腹膜间皮细胞（RPMC）凋亡的影响及机制的探索,从而为锌离子在腹膜透析中的应用提供实验基础.方法 胰蛋白酶消化法培养原代大鼠腹膜间皮细胞、传代、经鉴定后分组：①对照组;②LPS组;③ZnSO4组;④ZnSO4/LPS组.应用AnnexinV-FITC凋亡检测试剂盒及流式细胞仪检测RPMC凋亡率,并采用Westen Blot方法检测细胞外信号调节激酶（P-ERK）,BAX,凋亡诱导因子（AIF）,天冬氨酸特异性半胧氨酸蛋白酶3（caspase3）,天冬氨酸特异性半胧氨酸蛋白酶9 （caspase9）蛋白表达.应用流式细胞术,采用活性氧自由基（ROS）检测试剂盒检测各组别的ROS蛋白表达情况.结果 与对照组相比,LPS组RPMC凋亡细胞数量和相关凋亡蛋白（BAX,AIF,caspase3,caspase9）表达升高（P＜0.01）.与LPS组相比,ZnSO4作用组RPMC凋亡细胞数量和相关凋亡蛋白表达明显降低（P＜0.01）.LPS组ROS显著高于对照组而ZnSO4作用组ROS显著低于LPS组（P＜0.01,P＜0.01）.与LPS组相比,ZnSO4作用组P-ERK的表达明显升高（P＜0.01）.结论 ZnSO4可能可以逆转LPS所致的RPMC凋亡,对腹膜透析相关腹膜炎过程中所导致的RPMC损伤具有保护作用,其作用机制可能是通过ERK通路而发挥作用.

The effect of zinc ion on LPS-induced cell apoptosis in rat peritoneal mesothelial cells and its mechanism

Objective To explore whether Zn＋2 can be used in peritoneal dialysis, we investigated the effect of Zn＋ 2 on lipopolysaccharide （LPS）-induced cell apoptosis in rat peritoneal mesothelial cells （RPMCs） and its underlying mechanism. Methods RPMCs were isolated by enzyme disaggregation, cul- tured, and then identified by phase contrast microscopy, transmission electron microscopy, and immunocyto- chemistry methods. RPMCs were incubated with 100 μg/ml LPS for 24 hours, or stimulated by 100 μg/ml LPS after incubation with 50 μM ZnSO4 for 24 hours. RPMCs incubated in regular medium were used as the controls. The expressions ofp-ERK, BAX, AIF, caspase 3, and caspase 9 were assayed by western blot. Reac- tive oxygen species was measured by a reactive oxygen species assay kit. Results ZnSO4 significantly inhib- ited the LPS-induced RPMC apoptosis by attenuating ROS production, inhibiting LPS-induced BAX, AIF, and the activation of caspase 3 and caspase 9. Further studies revealed that ZnSO4 treatment facilitated cell sur- vival through the activation of ERK signaling pathway. Conclusion These results indicate that Zn＋2 inhib- its LPS-induced apoptosis in RPMCs by attenuating ROS production and inhibiting BAX, AIF, and the activa- tion of caspase 3 and caspase 9. The ERK signaling pathway may take part in these changes.