咪达唑仑调节河马/Yes相关蛋白信号通路缓解心肌缺血再灌注损伤

Midazolam alleviates myocardial ischemia-reperfusion injury by regulating hippopotamus/Yesassociated protein signaling pathway

目的探讨咪达唑仑(MDZ)通过调节河马(Hippo)/Yes相关蛋白(YAP)信号通路对大鼠心肌缺血再灌注损伤(MIRI)的影响。方法48只大鼠按随机数字表法分为4组,其中3组用结扎左冠状动脉前降支的方法构建MIRI大鼠模型,并随机分别给予生理盐水(10 mL/kg)、MDZ(0.1 mg/kg)、盐酸法舒地尔(10 mg/kg),为MIRI组、MDZ组、盐酸法舒地尔组,另外1组手术不结扎给予生理盐水(10 mL/kg)为sham组,每组12只。酶联免疫吸附测定法检测血清肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白I(cTnI)、乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)水平;氯化三苯基四氮唑、苏木精伊红、末端尿苷核苷酸末端标记染色检查心肌梗死面积、心肌组织病理变化及心肌细胞凋亡情况;qPCR检测结缔组织生长因子(CTGF)、富含半胱氨酸蛋白61(CYR61)mRNA的表达;免疫印迹法检测切割型半胱氨酸天冬氨酸蛋白酶1(cleaved Caspase-1)及磷酸化(p)-大肿瘤抑制激酶1(LATS1)/LATS1、p-YAP/YAP、p-哺乳动物不育系20样激酶1(MST1)/MST1的表达。结果与sham组比较,MIRI组CK-MB、cTnI、LDH、MDA、TNF-α、IL-1β、心肌梗死面积、心肌细胞凋亡率、cleaved Caspase-1、p-LATS1/LATS1、p-YAP/YAP均升高,CTGF、CYR61 mRNA表达、p-MST1/MST1、SOD水平降低(P<0.05);与MIRI组比较,MDZ组、盐酸法舒地尔组CK-MB、cTnI、LDH、MDA、TNF-α、IL-1β、心肌梗死面积、心肌细胞凋亡率、cleaved Caspase-1、p-LATS1/LATS1、p-YAP/YAP均降低,CTGF、CYR61 mRNA表达、p-MST1/MST1、SOD水平升高(P<0.05)。结论MDZ可能通过激活Hippo/YAP信号通路,抑制氧化应激与炎症反应,降低心肌细胞凋亡,改善MIRI大鼠的心肌损伤。

Objective To investigate the effects of midazolam(MDZ)on myocardial ischemiareperfusion injury(MIRI)in rats by regulating the hippopotamus(Hippo)/Yes-associated protein(YAP)signaling pathway.Methods Forty-eight rats were randomly divided into four groups with 12 rats in each group.The MIRI rat model was constructed by ligation of the anterior descending branch of the left coronary artery in three groups,and then the three groups were randomly assigned to receive normal saline(10 mL/kg),MDZ(0.1 mg/kg),Fasudil hydrochloride(10 mg/kg),this formed the MIRI group,MDZ group,and Fasudil hydrochloride group.The other group only underwent operation without ligation assigned to receive normal saline(10 mL/kg)formed the sham group.Serum levels of creatine kinase isoenzyme(CK-MB),cardiac troponin I(cTnI),lactate dehydrogenase(LDH),malondialdehyde(MDA),superoxide dismutase(SOD),tumor necrosis factor(TNF-α)and interleukin-1β(IL-1β)were detected by enzyme linked immunosorbent assay.Triphenyltetrazolium chloride staining,hematoxylin eosin staining,and terminal-deoxynucleoitidyl transferase mediated nick end labeling staining were used to examine myocardial infarction area,myocardial histopathological changes,and cardiomyocyte apoptosis.The expression of YAP target genes connective tissue growth factor(CTGF)and cysteinerich protein 61(CYR61)mRNA was detected by qPCR.Western blot was used to detect the expression of cleaved cysteine aspartate proteinase 1(cleaved Caspase-1)and ratios of phosphorylated(p)-large tumor suppressor kinase 1(LATS1)/LATS1,p-YAP/YAP,p-mammalian sterile 20-like kinase 1(MST1)/MST1.Results Compared with the sham group,CK-MB,cTnI,LDH,MDA,TNF-α,IL-1β,myocardial infarction area,cardiomyocyte apoptosis rate,cleaved Caspase-1,p-LATS1/LATS1,and p-YAP/YAP were increased in MIRI group,while CTGF,CYR61 mRNA expression,p-MST1/MST1,and SOD level were decreased(P<0.05).Compared with the MIRI group,the CK-MB,cTnI,LDH,MDA,TNF-α,IL-1β,myocardial infarction area,cardiomyocyte apoptosis rate,cleaved Caspase-1,p-LATS1/LATS1,and p-YAP/YAP in MDZ group and Fasudil hydrochloride group were decreased,CTGF,CYR61 mRNA expression,p-MST1/MST1,and SOD level were increased(P<0.05).Conclusion MDZ may activate the Hippo/YAP signaling pathway,inhibit oxidative stress and inflammatory responses,reduce apoptosis in cardiomyocyte,and improve myocardial injury in MIRI rats.