比索洛尔通过ASK1-JNK/p38 MAPK信号通路改善缺血再灌注诱导的H9C2心肌细胞损伤的研究

Bisoprolol Ameliorates H9C2 Cardiomyocyte Injury Induced by Ischemia Reperfusion Through ASK1-JNK/p38 MAPK Signaling Pathway

目的:探讨比索洛尔对缺血再灌注(I/R)诱导的H9C2心肌细胞损伤的影响及其作用机制。方法:体外培养H9C2心肌细胞,将H9C2心肌细胞分为对照组(Control组)、缺氧/复氧(H/R)模型组(Model组)、比索洛尔组(Bisoprolol组)、比索洛尔+凋亡信号调节激酶1(ASK1)重组蛋白组(Bisoprolol+ASK1组)。除Control组外,其余各组H9C2心肌细胞均进行H/R损伤处理。采用细胞计数试剂盒(CCK8)法检测细胞活力;采用流失细胞术检测细胞凋亡;采用酶联免疫吸附试验检测细胞中心肌损伤标志物[乳酸脱氢酶(LDH)、心肌肌钙蛋白T(cTnT)]和炎症因子[白细胞介素6(IL-6)、白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)]水平;采用激光共聚焦检测细胞中钙离子浓度;采用蛋白质印迹法检测细胞中ASK1-c-Jun氨基末端激酶(JNK)/p38丝分裂原活化蛋白激酶(p38 MAPK)信号通路相关蛋白表达水平。结果:与Control组比较,Model组H9C2细胞活力、LDH和cTnT水平均降低,细胞凋亡率、IL-6、IL-1β和TNF-α水平均升高,细胞内钙离子浓度、p-ASK1/ASK1、p-JNK/JNK和p-p38 MAPK/p38 MAPK比值均升高,差异均有统计学意义(P<0.05)。与Model组比较,Bisoprolol组和Bisoprolol+ASK1组H9C2细胞活力、LDH和cTnT水平均升高,细胞凋亡率、IL-6、IL-1β和TNF-α水平均降低,细胞内钙离子浓度、p-ASK1/ASK1、p-JNK/JNK和p-p38 MAPK/p38 MAPK比值均降低,差异均有统计学意义(P<0.05)。与Bisoprolol组比较,Bisoprolol+ASK1组H9C2细胞活力、LDH和cTnT水平均降低,细胞凋亡率、IL-6、IL-1β和TNF-α水平均升高,细胞内钙离子浓度、p-ASK1/ASK1、p-JNK/JNK和p-p38 MAPK/p38 MAPK比值均升高,差异均有统计学意义(P<0.05)。结论:比索洛尔能够通过抑制炎症反应和钙超载,改善I/R诱导的H9C2心肌细胞损伤,其作用机制可能与抑制ASK1-JNK/p38 MAPK信号通路激活有关。

OBJECTIVE:To probe into the effect of bisoprolol on H9C2 cardiomyocyte injury induced by ischemia reperfusion(I/R)and its mechanism.METHODS:H9C2 cardiomyocytes were cultured in vitro and divided into the control group(Control group),hypoxia/reoxygenation(H/R)model group(Model group),bisoprolol group(Bisoprolol group),bisoprolol+apoptosis signal-regulated kinase 1(ASK1)recombinant protein group(Bisoprolol+ASK1 group).Except for the Control group,the H9C2 cardiomyocytes were treated with H/R injury in the rest groups.The cell viability was detected by cell counting kit(CCK8)method;the cell apoptosis was detected by flow cytometry;the myocardial injury markers[lactate dehydrogenase(LDH),cardiac troponin T(cTnT)]and inflammatory factors[interleukin 6(IL-6),interleukin 1β(IL-1β)and tumor necrosis factorα(TNF-α)]levels were measured by enzyme-linked immunosorbent assay;the calcium ion concentration in cells was measured by laser confocal assay;the ASK1-c-Jun amino-terminal kinase(JNK)/p38 mitogen-activated protein kinase(p38 MAPK)signaling pathway-related protein expression was detected by Western blotting.RESULTS:Compared with the Control group,the H9C2 cell viability,LDH and cTnT levels were lower,the cell apoptosis rate,IL-6,IL-1βand TNF-αlevels were higher,the calcium ion concentration,p-ASK1/ASK1,p-JNK/JNK and p-p38 MAPK/p38 MAPK ratio were higher in the Model group,with statistically significant differences(P<0.05).Compared with the Model group,the H9C2 cell viability,LDH and cTnT levels were higher,the cell apoptosis rate,IL-6,IL-1βand TNF-αlevels were lower,the calcium ion concentration,p-ASK1/ASK1,p-JNK/JNK and p-p38 MAPK/p38 MAPK ratio were lower in the Bisoprolol group and Bisoprolol+ASK1 group,with statistically significant differences(P<0.05).Compared with the Bisoprolol group,the H9C2 cell viability,LDH and cTnT levels were lower,the cell apoptosis rate,IL-6,IL-1βand TNF-αlevels were higher,the calcium ion concentration,p-ASK1/ASK1,p-JNK/JNK and p-p38 MAPK/p38 MAPK ratio were higher in the Bisoprolol+ASK1 group,with statistically significant differences(P<0.05).CONCLUSIONS:Bisoprolol can improve H9C2 cardiomyocyte injury induced by I/R through inhibiting inflammatory response and calcium overload,its mechanism may related to the inhibition of ASK1-JNK/p38 MAPK signaling pathway activation.