黄芪甲苷抑制缺血再灌注诱导的大鼠心肌细胞凋亡

摘要 目的：本研究旨在探究黄芪甲苷（Astragaloside IV，Ast-IV）在缺血再灌注（ischemia-reperfusion，I/R）大鼠模型中的保护作用，并讨论黄芪甲苷在抑制I/R诱导心肌细胞凋亡过程中的作用。方法：通过左冠状动脉前降支结扎构建I/R大鼠模型；将40只SD大鼠分为4组：假手术组（Sham组）、模型组（I/R组）、黄芪甲苷干预组（Ast组）和黄芪甲苷+LY294002干预组（Ast+LY组）。使用试剂盒测定血清中心脏功能障碍标记物CPK、ALT、LDH和tropornin-T的表达水平；通过HE染色和TUNEL分别检测心肌组织病理学变化和心肌细胞凋亡情况；通过超氧化物荧光探针染色检测细胞内ROS水平；通过ELISA试剂盒测定心肌组织MDA、GSH和GSH-PX含量；免疫组织化学检测SOD2和HO-1蛋白表达水平，分析心肌氧化应激状态；通过Western blot检测PI3K、p-Akt、Akt、p-eNOS、eNOS、caspase-3、Bax和Bcl-2蛋白表达水平变化。结果：Ast组大鼠血浆CPK、ALT、LDH和tropornin-T酶活性均明显低于I/R组（P<0.05）。Ast组大鼠心肌纤维断裂，心肌细胞坏死和炎性细胞浸润等病变程度均低于I/R组。Ast组大鼠TUNEL阳性细胞数低于I/R组（P<0.05)。相较于I/R组，Ast组大鼠Caspase3和Bax表达水平均明显下调，Bcl-2和PI3K蛋白表达水平上调，p-Akt/Akt和p-eNOS/eNOS比值均显著上调（P<0.05）。Ast组大鼠DHE荧光强度显著低于Ast+LY组（P<0.05）。与I/R组相比，Ast组大鼠心肌组织中MDA含量降低，GSH、GSH-PX、SOD2和HO-1表达水平升高（P<0.05）；与Ast组相比，Ast+LY组大鼠心肌组织中MDA含量升高，GSH、GSH-PX、SOD2和HO-1表达水平降低（P<0.05）。结论：黄芪甲苷通过激活PI3K/Akt信号通路，抑制心肌细胞氧化应激反应，从而减少I/R诱导大鼠心肌细胞凋亡，缓解I/R后大鼠心肌损伤。

Astragaloside IV Attenuates Myocardial Apoptosis Induced by Ischemia-reperfusion in Rats

ABSTRACT Objective: This study was to verify the protective effect of astragaloside IV (Ast-IV) in ischemia-reperfusion (I/R) rat model. And clarify the mechanism of astragaloside IV inhibiting I/R-induced cardiomyocyte apoptosis. Methods: I/R rat models were constructed by ligating the anterior descending coronary artery. 40 SD rats were randomly divided into four groups: sham group. I/R group, astragaloside IV intervention group (Ast group) and astragaloside IV+LY294002 intervention group (Ast+LY group). Serum cardiac dysfunction markers CPK, ALT, LDH and tropornin-T levels were measured by commercial kits. HE staining and TUNEL were used to detect myocardial histopathological changes and myocardial cell apoptosis, respectively. The intracellular ROS level was detected by superoxide fluorescent probe staining. The content of MDA, GSH and GSH-PX in myocardial tissue was determined by ELISA kit. Combined with immunohistochemistry to detect SOD2 and HO-1 protein expression level, and analyze the myocardial oxidative stress state. The protein expression levels of PI3K, p-Akt, Akt, p-eNOS, eNOS, caspase-3, Bax and Bcl-2 were detected by Western blot. Results: The plasma CPK, ALT, LDH and tropornin-T enzyme activities in the Ast group were significantly lower than those in the I/R group (P<0.05). Myocardial fiber rupture, myocardial cell necrosis and inflammatory cell infiltration in Ast group were lower than those in I/R group. The number of TUNEL positive cells in the Ast group was lower than that in the I/R group (P<0.05). Compared with the I/R group, the expression levels of Caspase3 and Bax protein were significantly down-regulated, the expression levels of Bcl-2 and PI3K protein were significantly up-regulated, and the ratios of p-Akt/Akt and p-eNOS/eNOS were significantly increased (P<0.05). The fluorescence intensity of DHE in Ast group was significantly lower than that in Ast+LY group (P<0.05). Compared with the I/R group, the MDA content of Ast group was decreased, and the expression levels of GSH, GSH-PX, SOD2 and HO-1 were increased (P<0.05). Compared with the Ast group, the MDA content was increased, and the expression levels of GSH, GSH-PX, SOD2 and HO-1 were decreased in Ast+LY group(P<0.05). Conclusion: Astragaloside IV inhibits oxidative stress response of cardiomyocytes by activating PI3K/Akt signaling pathway, thereby reducing I/R-induced cardiomyocyte apoptosis and alleviating myocardial injury in rats.