干扰S100钙结合蛋白B表达对多柔比星诱导心肌细胞凋亡的影响及相关机制

Effect of interfering S100B expression on the doxorubicin induced cardiomyocyte apoptosis and its related mechanisms

目的探讨干扰S100钙结合蛋白B(S100B)表达对多柔比星诱导心肌细胞凋亡的影响及其相关机制。方法分离培养SD大鼠心肌细胞,共分4组:(1)空白对照组:正常培养,不处理;(2)多柔比星组:2μmol/L多柔比星处理2 h;(3)阴性对照组:2μmol/L多柔比星处理2 h+转染S100B小干扰RNA(siRNA)阴性对照;(4) S100B组:2μmol/L多柔比星处理2 h+转染S100B siRNA。采用噻唑蓝比色法检测各组心肌细胞存活率,采用TUNEL法检测各组心肌细胞凋亡率,采用Elisa法检测各组心肌细胞上清液中肿瘤坏死因子(TNF)-α、白介素(IL)-1β、IL-6水平,采用蛋白印记法检测各组心肌细胞S100B、Caspase-3、Bax、B淋巴细胞瘤(Bcl)-2表达。结果分离培养的大鼠心肌细胞状态良好,阳性率可达(92.33±5.16)%。与空白对照组比较,多柔比星组心肌细胞存活率和Bcl-2蛋白表达降低;心肌细胞凋亡率,TNF-α、IL-1β、IL-6水平,S100B、Caspase-3、Bax蛋白表达增加(P <0.01)。与多柔比星组比较,S100B组心肌细胞存活率和Bcl-2蛋白表达增加;心肌细胞凋亡率,TNF-α、IL-1β、IL-6水平,S100B、Caspase-3、Bax蛋白表达降低(P <0.01)。结论干扰S100B表达能够抑制多柔比星诱导的心肌细胞凋亡,其机制可能与抑制炎性损伤和调节凋亡相关蛋白有关。

Objective To investigate the effect of interfering S100 calcium-binding protein B(S100 B) expression on the doxorubicin induced cardiomyocyte apoptosis and its related mechanisms.Methods The cardiac myocytes cells were isolated and cultured from SD rats.The experiment was divided into 4 groups:(1)blank control group:without treated;(2)doxorubicin group:treated with doxorubicin(2 μmol/L) for 2 h;(3)negative control group:treated with doxorubicin(2 μmol/L) for 2 h,and transfected with S100 B small interference RNA(siRNA) negative control;(4)S100 B group:treated with doxorubicin(2 μmol/L) for 2 h,and transfected with S100 B siRNA.The survival rate of cardiomyocyte was detected by thiazolium colorimetry,the apoptosis rate of cardiomyocyte was detected by TUNEL,the levels of TNF-α,IL-1β and IL-6 in the supernatant were detected by Elisa method,and the expressions of S100 B,Caspase-3,Bax and Bcl-2 were detected by Western blot.Results The purity of cardiomyocyte reached(92.33±5.16)%.Compared with the blank control group,the survival rate and Bcl-2 expression of cardiomyocyte in doxorubicin group were decreased,the apoptotic rate of cardiomyocyte,the levels of TNF-α,IL-1β and IL-6,the expressions of S100 B,Caspase-3 and Bax were increased(P<0.01).Compared with the doxorubicin group,the survival rate of cardiomyocyte and the expression of Bcl-2 protein in S100 B group were increased.Apoptotic rate,TNF-α,IL-1β andIL-6,S100 B,Caspase-3,Bax expression were decreased(P<0.01).Conclusion Interfering S100 B expression can inhibit doxorubicin induced cardiomyocyte apoptosis,and its mechanism may be related to inhibiting inflammatory injury and regulating apoptosis related proteins.