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目的探讨苦参碱对柯萨奇B3型病毒(CVB3)感染心肌细胞的保护作用及其与磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/AKT)信号传导通路的关系。方法通过CVB3感染原代培养的Wistar新生大鼠心肌细胞的方法,制备病毒性心肌炎体外实验模型。实验分为5组,正常对照组:不感染病毒、不加苦参碱及PI3K抑制剂;病毒组:感染病毒、不加苦参碱及抑制剂Wort;病毒+Wort组:加入抑制剂Wort(200 nmol.L-1)后感染病毒;苦参碱治疗组:感染病毒后再加入苦参碱(0.3 g.L-1);苦参碱治疗+Wort组加入抑制剂Wort后感染病毒,再加入苦参碱。倒置显微镜下观察各组心肌细胞病变效应(CPE)变化,流式细胞仪检测心肌细胞凋亡情况,免疫细胞化学法及Western blot法检测心肌细胞内磷酸化AKTSer-473蛋白的表达。结果与正常对照组比较,病毒组、苦参碱治疗组心肌细胞CPE变化明显,细胞凋亡率增加,磷酸化AKTSer-473蛋白表达上调(Pa<0.05)。与病毒组比较,苦参碱治疗组心肌细胞CPE变化不明显,细胞凋亡率降低,磷酸化AKTSer-473蛋白表达明显增强(Pa<0.05);病毒+Wort组心肌细胞CPE变化明显,心肌细胞凋亡率增多(Pa<0.05),磷酸化AKTSer-473蛋白的表达下调(P<0.05)。与苦参碱治疗组比较,苦参碱治疗+Wort组CPE变化明显及细胞凋亡率增多,磷酸化AKTSer-473蛋白表达减少(Pa<0.05)。结论苦参碱对CVB3感染心肌细胞具有保护作用,这种保护作用至少是部分通过PI3K/AKT信号通路介导的。
Objective To investigate the protective effect of matrine on CVB3-infected cardiomyocytes and its relationship with phosphatidylinositol 3-kinase / protein kinase B (PI3K / AKT) signaling pathway. Methods CVB3 infected primary cultured Wistar neonatal rat cardiomyocytes, in vitro experimental model of viral myocarditis. The experiment was divided into 5 groups, normal control group: no virus infection, no matrine and PI3K inhibitor; virus group: infected virus without matrine and inhibitor Wort; virus + Wort group: inhibitor Wort ( 200 nmol.L-1). The matrine group was treated with matrine (0.3 gL-1) after infection with the virus; the matrine-treated + Wort group was infected with the inhibitor Wort, Alkali. The changes of cardiomyocyte lesions (CPE) in each group were observed under inverted microscope. The apoptosis of cardiomyocytes was detected by flow cytometry. The phosphorylation of AKTSer-473 protein in myocardium was detected by immunocytochemistry and Western blot. Results Compared with the normal control group, the changes of CPE in the virus group and the matrine group were significant, and the apoptosis rate was increased. The phosphorylated AKTSer-473 protein was up-regulated (Pa <0.05). Compared with the virus group, the changes of CPE in myocardial cells in matrine group were not obvious, the apoptosis rate was decreased and the phosphorylation of AKTSer-473 protein was significantly increased (P <0.05); the CPE in virus + Wort group was significantly changed, (P <0.05). The phosphorylation of AKTSer-473 protein was down-regulated (P <0.05). Compared with matrine treatment group, the changes of CPE and the apoptosis rate of mastine + Wort group were significantly increased, and the phosphorylation of AKTSer-473 protein was decreased (Pa <0.05). Conclusion Matrine has a protective effect on CVB3-infected cardiomyocytes, and this protective effect is at least partly mediated by the PI3K / AKT signaling pathway.