H2O2对大鼠阴茎海绵体平滑肌细胞connexin43蛋白表达的影响及红景天苷的干预作用

Inhibitory effect of salidroside on H2O2-induced down-regulation of Cx43 expression in corpus cavernosum smooth muscle cells in rats

目的:探讨红景天苷对H2O2所致大鼠阴茎海绵体平滑肌细胞(CCSMC)缝隙连接蛋白43(Cx43)表达下调的干预作用。方法:体外分离、培养SD大鼠CCSMC,并利用免疫细胞荧光技术进行细胞鉴定。MTT法检测H2O2对CCSMC存活率的影响,以确定最佳干预浓度。利用最佳浓度H2O2于体外对CCSMC进行干预,并设立干预时间梯度;利用H2O2处理CCSMC的同时,加入低(16μg/ml)、高浓度(64μg/ml)红景天苷进行干预,最终提取获得各组别蛋白,并通过Western印迹检测Cx43表达。结果:原代培养的CCSMC生长状态正常,细胞鉴定结果显示阳性率达90%以上。MTT试验确定用于体外实验的H2O2最佳浓度为200μmol/L。利用200μmol/L H2O2处理CCSMC0、1、2、4h,与空白组相比,1、2、4h组CCSMC中Cx43蛋白水平显著下降(P<0.01);与1h和2h组相比,4h组CCSMC中Cx43蛋白水平显著下降(P<0.01)。利用200μmol/L H2O2处理CCSMC的同时,分别加入低剂量和高剂量红景天苷进行干预,与正常对照组相比,H2O2处理组CCSMC中Cx43蛋白表达明显下降(P<0.05),高剂量红景天苷处理后可抑制该现象(P<0.01),且高剂量红景天苷组CCSMC中Cx43蛋白水平与正常对照组相比无统计学差异(P=0.3222)。结论:红景天苷在体外具有调节H2O2所致大鼠CCSMC中Cx43蛋白表达下降的作用。

Objective:To explore the regulatory effect of salidroside on H2O2-induced decrease in the expression of the connexin43 (Cx43) protein in corpus cavernosum smooth muscle cells (CCSMC).Methods:Rat CCSMCs were isolated,primarily cultured in vitro and identified by immunocytochemical assay.The optimum concentration of H2O2 for intervention was determined by detecting its effect on the viability of the CCSMCs and used in the treatment of the CCSMCs for different lengths of time,and meanwhile salidroside was applied at 16 μg /ml (low dose) or 64 μg /ml (high dose) for intervention.Finally,the expressions of the Cx43 protein in the CCSMCs of different groups of rats were determined by Western blot.Results:The CCSMCs grew normally,with a positive rate of over 90%.At 1,2 and 4 hours of treatment with H2O2 at the optimum concentration of 200 μmol /L,the expression of Cx43 in the CCSMCs was significantly decreased as compared with that in the blank control group (P < 0.01),even more significantly at 4 hours than at 1 and 2 (P < 0.01).Intervention with high-dose salidroside,however,markedly inhibited the down-regulation of the Cx43 expression (P < 0.05),which showed no statistically significant difference from that in the normal control group (P = 0.322 2). Conclusion:Salidroside can suppress H2O2-induced decrease in the expression of the Cx43 protein in rat CCSMCs.