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目的:观察雌激素膜受体GPER1对心肌细胞氧化损伤的保护作用,并探讨其通过PI3K/Akt信号通路上调Nrf2,减轻心肌氧化损伤的机制。方法:H_2O_2处理原代培养的新生大鼠心肌细胞建立氧化损伤模型,分为对照组、H_2O_2处理组,GPER1受体激动剂G1预处理+H_2O_2处理组和GPER1拮抗剂G15+G1预处理+H_2O_2处理组,MTT检测细胞活性,Hoechst33342染色和cleaved caspase-3免疫荧光染色观察细胞凋亡,并检测细胞内氧自由基,总抗氧化能力,超氧化物歧化酶(SOD)和丙二醛(MDA)的水平。Western blot测定细胞中p-Akt和细胞核内Nrf2的水平。结果:G1显著抑制H_2O_2导致细胞活性下降和细胞凋亡,并降低细胞内氧自由基水平,提高总抗氧化能力,增加SOD活性,减少MDA含量,但G15能拮抗G1的上述效应。同时G1能增加细胞内Akt磷酸化水平和细胞核内Nrf2的表达,这些效应可被G15和LY-294002阻断。结论:GPER1通过PI3K/Akt信号通路,调节Nrf2的表达,抑制氧化应激导致的心肌细胞损伤。GPER1可以作为开发心肌缺血损伤保护剂的一个潜在靶点。
OBJECTIVE: To observe the protective effect of estrogen receptor GPER1 on cardiomyocyte oxidative damage and to explore its mechanism of up-regulating Nrf2 through PI3K / Akt signaling pathway to reduce myocardial oxidative damage. Methods: Oxidative injury model was established in neonatal rat cardiomyocytes treated with H 2 O 2 and divided into control group, H 2 O 2 treatment group, GPER 1 receptor agonist G1 pretreatment + H 2 O 2 treatment group and GPER 1 antagonist G 15 + G1 pretreatment + H 2 O 2 treatment The cell viability was measured by MTT assay. Hoechst33342 staining and cleaved caspase-3 immunofluorescence staining were used to observe the cell apoptosis. The contents of oxygen free radicals, total antioxidant capacity, superoxide dismutase (SOD) and malondialdehyde (MDA) )s level. Western blot was used to determine the levels of p-Akt and Nrf2 in the nucleus. Results: G1 significantly inhibited H 2 O 2 -induced cell viability and apoptosis, reduced intracellular free radical levels, increased total antioxidant capacity, increased SOD activity and decreased MDA content, but G15 antagonized the above effects of G1. At the same time G1 can increase intracellular Akt phosphorylation and nuclear Nrf2 expression, these effects can be G15 and LY-294002 blocking. Conclusion: GPER1 regulates Nrf2 expression through PI3K / Akt signaling pathway and inhibits cardiomyocyte injury induced by oxidative stress. GPER1 can be used as a potential target for the development of protective agents against myocardial ischemia.