人参皂苷Rg1通过Sestrin2保护心肌细胞的作用

Mechanisms of Ginsenoside Rg1 in Protecting Cardiomyocytes by Sestrin2

目的研究人参皂苷Rg1(G-Rg1)对抗心肌缺血再灌注损伤中Sestrin2的作用及相关机制。方法体外培养大鼠H9c2心肌细胞,按实验要求随机分5组,分别为对照组,缺氧复氧组,G-Rg1组,缺氧诱导因子-1α(Hypoxia Inducible Factor-1,HIF-1,HIF-1α)抑制剂组,Sestrin2 siRNA干扰组。流式细胞术检测各组心肌细胞内活性氧簇(reactive oxygen species,ROS)含量。蛋白质免疫印迹法检测HIF-1α、Sestrin2、蛋白激酶R样内质网激酶(protein kinase R-like ER kinase,PERK)、活化转录因子6(the activating transcription factor6,ATF-6)和跨膜蛋白激酶1(inositol requiring enzyme 1,IRE-1)蛋白表达。免疫荧光法检测Sestrin2、HIF-1α、IRE-1蛋白表达。结果与对照组相比,缺氧复氧组细胞内ROS水平、HIF-1α、Sestrin2、ATF-6、PERK、IRE-1蛋白表达水平明显升高(P<0.05);与缺氧复氧组相比,G-Rg1组细胞内ROS水平明显降低(P<0.05),HIF-1α、Sestrin2、ATF-6、PERK、IRE-1蛋白表达水平明显升高(P<0.05);与G-Rg1组比较,HIF-1α抑制剂组心肌细胞内ROS水平升高,Sestrin2蛋白表达明显下调(P<0.05);与G-Rg1组比较,siRNA干扰组心肌细胞内ROS水平升高,IRE-1表达明显升高(P<0.05)。结论G-Rg1通过上调Sestrin2表达,保护缺氧复氧心肌细胞,并维持内质网应激平衡稳态。

Objective To explore the role and mechanisms of Sestrin2 in ameliorating hypoxiareoxygenation injury of rat’s cardiomyocytes by ginsenoside-rg1(G-Rg1).Methods Rat H9c2 cardiomyocytes cultured in vitro were randomly divided into 5 groups according to the experimental requirements:control group,Hypoxia reoxygenation group,G-Rg1 group,hypoxia Inducible Factor-1(HIF-1,HIF-1α)inhibitor group,and Sestrin2 siRNA interference group.Flow cytometry was used to detect the expression levels of ROS in cardiomyocytes.Western blot was used to detect the protein expression of HIF-1α,Sestrin2,ATF-6,PERK,IRE-1 in cardiomyocytes.Immunofluorescence was used to test the protein expression of Sestrin2,HIF-1α,IRE-1.Results Compared with control group,the level of ROS and protein expression of HIF-1α、Sestrin2、ATF-6、PERK、IRE-1 of hypoxiareoxygenation group were significantly increased(P<0.05).Compared with the hypoxiareoxygenation group,the ROS level in G-Rg1 group were significantly reduced and protein expression of HIF-1α、Sestrin2、ATF-6、PERK、IRE-1 in G-Rg1 group were significantly increased(P<0.05).Compared with G-Rg1 group,the level of ROS were significantly increased and the protein level of Sestrin2 were significantly reduced,in HIF-1αinhibition group(P<0.05).Compared with G-Rg1 group,the level of ROS were significantly increased and the protein level of IRE-1 were significantly upregulated in Sestrin2 siRNA group(P<0.05).Conclusions G-Rg1 protects rat cardiomyocytes and maintains endoplasmic reticulum homeostasis in vitroby upregulating expression of Sestrin2.