基于PKCβⅡ/NOX2/ROS信号通路探讨柴胡三参胶囊对心肌缺血再灌注损伤大鼠的保护作用

Chaihu Sanshen Capsules protect rats from myocardial ischemia reperfusion injury via PKCβⅡ/NOX2/ROS signaling pathway

基于氧化应激介导的程序性细胞死亡探讨心肌缺血再灌注损伤的发病机制,及基于PKCβⅡ/NOX2/ROS信号通路探讨柴胡三参胶囊调节心肌缺血再灌注损伤的机制及作用靶点。结扎左前降支建立心肌缺血再灌注损伤大鼠模型,随机分为6组:假手术组,模型组,柴胡三参胶囊临床等效、高剂量组,N-乙酰半胱氨酸(N-acetylcysteine)组,蛋白激酶C抑制剂(CGP53353)组。柴胡三参胶囊各剂量组连续给药7 d后,检测各组大鼠心肌梗死面积;苏木精-伊红(HE)染色观察大鼠心肌组织病理形态;TUNEL染色观察大鼠心脏组织凋亡情况;酶联免疫吸附(enzyme-linked immunosorbent assay,ELISA)法检测大鼠心肌损伤指标和氧化应激相关指标;流式细胞仪检测心脏组织中活性氧(ROS)水平;蛋白免疫印迹法(Western blot)及实时荧光定量聚合酶链式反应(real-time quantitative PCR,RT-qPCR)检测大鼠心脏组织相关蛋白及mRNA相对表达水平。结果显示,与假手术组比较,模型组大鼠心肌梗死面积明显,心肌结构紊乱、间质水肿及出血,可见大量空泡,心肌损伤标志物及心肌凋亡水平明显升高,ROS及丙二醛(MDA)水平明显升高,超氧化物歧化酶(SOD)水平明显下降,心肌组织中蛋白激酶C(PKCβⅡ)、烟酰胺腺嘌呤二核苷酸磷酸氧化酶2(NOX2)、半胱氨酸蛋白酶3(caspase-3)、长链脂酰辅酶A合成酶4(ACSL4)蛋白及mRNA表达水平明显升高。与模型组比较,柴胡三参胶囊可改善大鼠心肌梗死面积和心脏组织病理形态,改善心肌损伤标志物和氧化应激相关指标,减少心肌组织凋亡水平,降低心肌组织中PKCβⅡ、NOX2、caspase-3、ACSL4蛋白及mRNA的表达。结果表明,柴胡三参胶囊可通过调控PKCβⅡ/NOX2/ROS信号通路,抑制氧化应激和程序性细胞死亡(凋亡、铁死亡),从而减轻心肌缺血再灌注损伤。

This study aims to explore the pathogenesis of myocardial ischaemia reperfusion injury(MIRI)based on oxidative stressmediated programmed cell death and the mechanism and targets of Chaihu Sanshen Capsules in treating MIRI via the protein kinase Cβ(PKCβⅡ)/NADPH oxidase 2(NOX2)/reactive oxygen species(ROS)signaling pathway.The rat model of MIRI was established by the ligation of the left anterior descending branch.Rats were randomized into 6 groups:sham group,model group,clinically equivalent-,high-dose Chaihu Sanshen Capsules groups,N-acetylcysteine group,and CGP53353 group.After drug administration for 7 consecutive days,the area of myocardial infarction in each group was measured.The pathological morphology of the myocardial tissue was observed by hematoxylin-eosin(HE)staining.The apoptosis in the myocardial tissue was observed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL).Enzyme-linked immunosorbent assay(ELISA)was employed to measure the levels of indicators of myocardial injury and oxidative stress.The level of ROS was detected by flow cytometry.The protein and mRNA levels of the related proteins in the myocardial tissue were determined by Western blot and real-time quantitative PCR(RT-qPCR),respectively.Compared with the sham group,the model group showed obvious myocardial infarction,myocardial structural disorders,interstitial edema and hemorrhage,presence of a large number of vacuoles,elevated levels of myocardial injury markers,myocardial apoptosis,ROS,and malondialdehyde(MDA),lowered superoxide dismutase(SOD)level,and up-regulated protein and mRNA levels of PKCβⅡ,NOX2,cysteinyl aspartate specific proteinase-3(caspase-3),and acyl-CoA synthetase long-chain family member 4(ACSL4)in the myocardial tissue.Compared with the model group,Chaihu Sanshen Capsules reduced the area of myocardial infarction,alleviated the pathological changes in the myocardial tissue,lowered the levels of myocardial injury and oxidative stress indicators and apoptosis,and down-regulated the mRNA and protein levels of PKCβⅡ,NOX2,caspase-3,and ACSL4 in the myocardial tissue.Chaihu Sanshen Capsules can inhibit oxidative stress and programmed cell death(apoptosis,ferroptosis)by regulating the PKCβⅡ/NOX2/ROS signaling pathway,thus mitigating myocardial ischemia reperfusion injury.