黄芪多糖对大鼠缺氧/复氧诱导的心肌细胞自噬及凋亡抑制作用的机制探讨

目的：探究黄芪多糖（APS）通过调控高迁移率族蛋白1/Toll样受体4/核转录因子-κB(HMGB1/TLR4/NF-κB)信号通路对大鼠缺氧/复氧（H/R）诱导的心肌细胞自噬及凋亡的抑制作用。方法：建立H9C2心肌细胞H/R损伤模型并分为4组：对照组、H/R组、APS组和HMGB1抑制剂组。CCK-8法和EdU染色法检测细胞增殖能力；酶联免疫吸附试验检测细胞肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、IL-6含量；透射电镜观察细胞自噬小体的形成；膜联蛋白V-异硫氰酸荧光素/碘化丙啶（AnnexinV-FITC/PI）双染法检测细胞凋亡；实时定量PCR检测细胞HMGB1、TLR4、NF-κB p65 mRNA表达水平；蛋白免疫印迹法检测细胞HMGB1、TLR4、NF-κB p65、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白（Bax）、含半胱氨酸的天冬氨酸蛋白水解酶-3(caspase-3)、P62、微管相关蛋白1A/1B-轻链3(MAP1LC3，缩写为LC3)-Ⅱ蛋白表达水平。结果：与对照组相比，H/R组细胞增殖能力明显减弱，凋亡及自噬水平明显增加，细胞内可见大量自...

Mechanism of Astragalus Polysaccharides on the Inhibition of Cardiomyocyte Autophagy and Apoptosis Induced by Hypoxia/reoxygenation in Rats

Objectives:To explore the role of Astragalus Polysaccharide(APS)on autophagy and apoptosis of rat cardiomyocytes induced by hypoxia/reoxygenation(H/R)and related mechanism.Methods:The H/R injury model of H9C2 cardiomyocytes was constructed and cells were divided into 4 groups:control group,H/R group,APS group,and HMGB1 inhibitor group.CCK8 method and EdU staining method were used to detect cell proliferation ability.ELISA method was used to detect the content of tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6.Transmission electron microscopy was used to observe the formation of autophagosomes.AnnexinV-FITC/PI double staining method was used to detect cell apoptosis;qRT-PCR was used to detect mRNA expression of HMGB1,TLR4,NF-κB p65.Western blot was used to detect the protein expression of Bcl-2,Bax,caspase-3,P62,LC3-II of various groups.Results:Compared with the control group,the cell proliferation ability was significantly weakened,and the levels of apoptosis and autophagy were significantly increased in the H/R group.A large number of autophagosomes were formed in the cells in the H/R group.The expression levels of HMGB1,TLR4,NF-κB p65 mRNA,and TNF-α,IL-1β,IL-6 content,Bax,caspase-3,LC3-II protein were significantly increased,Bcl-2,P62 expression levels were significantly reduced in the H/R group(all P<0.05);Compared with the H/R group,the cell proliferation capacity of the APS group and HMGB1 Inhibitor group were significantly increased,the apoptosis and autophagy capacity were significantly reduced,the number of autophagosomes in the cell were decreased,the expression level of HMGB1,TLR4,NF-κB p65 mRNA,TNF-α,IL-1β,IL-6 content,Bax,caspase-3,LC3-Ⅱprotein were significantly reduced,Bcl-2,P62 expression levels were significantly increased(all P<0.05).Conclusions:APS can attenuate the H/R injury in H9C2 cardiomyocytes by reducing cell apoptosis and autophagy activity via inhibiting the HMGB1/TLR4/NF-κB signaling pathway.