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目的:探讨次乌头碱(HA)配伍甘草次酸(GA)对缺氧缺糖(OGD)损伤H9c2心肌细胞的保护作用及其作用机制。创新点:首次在OGD模型中证明HA+GA对H9c2心肌细胞有明显的保护作用。此作用与减少细胞坏死和凋亡有关系,且其作用机制与磷脂酰肌醇-3-激酶/蛋白质丝氨酸-苏氨酸激酶(PI3K/Akt)信号通路有关。方法:采用H9c2心肌细胞为研究对象,将其分为七组:正常组、OGD模型组、OGD+HA组、OGD+GA组、OGD+HA+GA组、OGD+LY294002组、OGD+HA+GA+LY294002组。采用Hoechst 33342染色荧光显微镜及透射电镜观察前五组的H9c2心肌细胞的形态学改变;采用酶联免疫吸附测定法(ELISA)检测前五组细胞上清液中乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)以及天门冬氨酸氨基转移酶(AST)的释放量的改变;采用异硫氰酸荧光素-磷脂结合蛋白V/碘化丙啶(FITC-AV/PI)双染色法检测前五组细胞凋亡率的情况;采用蛋白质免疫印迹法(Western blot)检测加入抑制剂LY294002前后丝苏氨酸蛋白激酶(Akt)、磷酸化丝苏氨酸蛋白激酶(p-Akt)、B细胞淋巴瘤/白血病-2相关x蛋白(Bax)、B细胞淋巴瘤/白血病-2(Bcl-2)及半胱氨酸天冬氨酸蛋白酶-9(caspase-9)等细胞作用信号通路PI3K/Akt相关蛋白的情况。结论:(1)Hoechst 33342染色荧光显微镜显示OGD+HA+GA组抗凋亡作用最明显;(2)透射电镜观察OGD+HA+GA组凋亡现象改善最多;(3)LDH、CK-MB及AST的含量变化显示OGD+HA+GA组心肌细胞损伤指标降低最多(P<0.05);(4)Western blot法检测结果显示HA+GA可以减少OGD对H9c2心肌细胞的损伤,其作用机制与PI3K/Akt信号通路有关。
Objective: To investigate the protective effect and its mechanism of hypaconitine (HA) and glycyrrhetinic acid (GA) on H9c2 cardiomyocytes injured by oxygen and glucose deprivation (OGD). Innovative point: For the first time in the OGD model to prove HA + GA H9c2 cardiomyocytes significant protective effect. This effect is related to the reduction of cell necrosis and apoptosis, and its mechanism of action is related to the PI3K / Akt signaling pathway. Methods: The H9c2 cardiomyocytes were divided into seven groups: normal group, OGD + HA group, OGD + HA group, OGD + HA + GA group, OGD + LY294002 group, OGD + HA + GA + LY294002 group. The morphological changes of H9c2 cardiomyocytes in the first five groups were observed by Hoechst 33342 staining fluorescence microscope and transmission electron microscope. The levels of lactate dehydrogenase (LDH) and muscle in supernatants of the first five groups were detected by enzyme linked immunosorbent assay (ELISA) (CK-MB) and aspartate aminotransferase (AST) were measured. The effects of fluorescein isothiocyanate-phospholipid binding protein V / propidium iodide (FITC-AV / PI ) Double staining was used to detect the apoptosis rate of the first five groups. Western blot was used to detect the expression of Akt, p (p -Akt), B-cell lymphoma / Bax, Bcl-2, and caspase-9 Cell Signaling Pathway PI3K / Akt Related Proteins. Conclusion: (1) Hoechst 33342 staining fluorescence microscope showed that the anti-apoptotic effect of OGD + HA + GA group was the most obvious; (2) Transmission electron microscope showed the most improvement of apoptosis in OGD + HA + GA group; (3) (P <0.05). (4) The results of Western blot showed that HA + GA can reduce the injury of H9c2 cardiomyocytes by OGD and the mechanism of action is PI3K / Akt signaling pathway.