铅致大鼠心肌细胞损伤中蛋白激酶C对缝隙连接蛋白43表达的调控作用

Regulatory effect of protein kinase C on connexin 43 expression in lead-induced cardiomyocyte injury in rats

目的研究醋酸铅染毒大鼠心肌细胞株(H9c2)后蛋白激酶C(protein kinase C,PKC)、缝隙连接蛋白43(connexin 43,Cx43)及其磷酸化蛋白的表达变化,探讨心肌损伤过程中PKC对Cx43的调控作用。方法(1)将对数生长期H9c2随机分组,以醋酸铅0、5、10、20μmol/L梯度浓度处理细胞12 h。(2)将对数生长期H9c2细胞随机分为对照组、醋酸铅组、醋酸铅+佛波酯(phorbol 12⁃myristate 13⁃acetate,PMA)组和PMA组。PMA预处理后的醋酸铅+PMA组、醋酸铅组细胞均给予10μmol/L醋酸铅染毒12 h,PMA组不予染毒。(3)检测各组H9c2细胞培养上清乳酸脱氢酶(lactate dehydrogenase,LDH)活性;采用蛋白质印迹法(Western blot)检测各组H9c2细胞内Cx43、PKC蛋白相对表达含量及s368位点的Cx43磷酸化水平。结果10、20μmol/L醋酸铅染毒的H9c2细胞存活率较对照组明显降低(P<0.05);10、20μmol/L醋酸铅组H9c2细胞培养液LDH活性较对照组显著升高(P<0.05);5、10、20μmol/L醋酸铅组Cx43、p⁃Cx43^( s368)、PKC蛋白相对表达含量与对照组相比差异具有统计学意义(P<0.05),且呈浓度依赖性降低;20μmol/L醋酸铅组p⁃Cx43/Cx43显著低于对照组(P<0.05)。与对照组相比,醋酸铅组、醋酸铅+PMA组H9c2细胞培养液LDH活性显著升高(P<0.05),醋酸铅+PMA组细胞培养液LDH活性显著低于醋酸铅组(P<0.05)。与对照组相比,醋酸铅组、醋酸铅+PMA组细胞内Cx43蛋白相对表达含量降低,差异均有统计学意义(P<0.05);醋酸铅+PMA组Cx43蛋白相对表达含量显著高于醋酸铅组(P<0.01)。结论铅致心肌细胞损伤机制可能与PKC、Cx43及其磷酸化蛋白表达降低有关,PKC信号通路对Cx43表达具有调控作用。

Objective To investigate the expression changes of protein kinase C(PKC),connexin 43(Cx43)and their phosphorylated proteins in cardiomyocytes of rats exposed to lead acetate(H9 c2),thereby explore the regulatory effect of PKC on Cx43 during myocardial injury.Methods(1)H9 c2 cells at logarithmic growth stage were randomly divided into several groups and treated with 0,5,10,20 μmol/L of lead acetate, respectively, for 12 h.(2)H9 c2 cells at logarithmic growth stage were randomly divided into control group, lead acetate group, lead acetate+PMA(phorbol 12-myristate 13-acetate)group and PMA group.After pretreatment with PMA,cells in lead acetate group and lead acetate+PMA group were treated with 10 μmol/L of lead acetate for 12 h, PMA treatment group was not treated with lead acetate after PMA pretreatment.(3)Lactate dehydrogenase(LDH)activity in the culture medium supernatants of H9 c2 cells in each group was detected, and Western blot was used to detect the relative expression levels of Cx43 and PKC proteins in H9 c2 cells and Cx43 phosphorylation level of s368.Results The results showed that survival rates of H9 c2 cells exposed to lead acetate at 10 and 20 μmol/L levels were significantly lower than that of control group(P<0.05).The LDH activities of H9 c2 cell culture medium supernatants of 10 and 20 μmol/L of lead acetate groups were significantly higher than that of control group(P<0.05).The relative protein expression levels of Cx43,p-Cx43;and PKC in 5,10 and 20 μmol/L of lead acetate groups were significantly lowered compared with control group(P<0.05)in a concentration-dependent manner, and the p-Cx43/Cx43 in 20 μmol/L of lead acetate group was lower than that of control group(P<0.05).Meanwhile, compared with control group, the LDH activities in H9 c2 cell culture medium supernatants of lead acetate group and lead acetate+PMA group were all significantly increased(P<0.05),but the level in lead acetate+PMA group was lower than that in lead acetate group(P<0.05),and the relative expression levels of Cx43 protein in lead acetate group and lead acetate+PMA group were decreased(P<0.05),the relative expression level of Cx43 protein in lead acetate+PMA group was significantly higher than that in lead acetate group(P<0.01).Conclusion The results suggested that mechanism of cardiomyocyte injury induced by lead toxicity may be related to the decreased expression of PKC,Cx43 and their phosphorylated proteins, and the PKC signaling pathway may have some regulatory effect on the expression of Cx43.