铁死亡抑制剂Ferrostatin-1对高糖缺氧复氧过程中心肌细胞的保护机制

Mechanism of ferrostatin-1 attenuating myocardial cell injury during high glucose hypoxia and reoxygenation by ferroptosis pathway

目的:探讨铁死亡抑制剂ferrostatin-1对高糖(HG)缺氧复氧(H/R)过程中心肌细胞的保护机制。方法:正常培养的H9C2心肌细胞随机分为3组:高糖常氧组(HG)、高糖缺氧复氧组(高糖H/R)和高糖H/R+ferrostatin-1组。高糖培养基作用于3组细胞使其糖浓度达到33 mmol/L,放置于普通培养箱(体积分数90%O2+体积分数10%CO2)培养24 h制备高糖模型。高糖H/R组细胞在三气培养箱(体积分数95%N2+体积分数5%CO2)培养4 h后,于普通培养箱培养2 h。高糖H/R+ferrostatin-1组细胞在进行上述高糖和缺氧复氧处理前24 h提前给予ferrostatin-1刺激。运用乳酸脱氢酶(LDH)试剂盒测定上清培养液中LDH的含量,CCK-8试剂盒测定细胞的活性,超氧化物歧化酶(SOD)试剂盒测定细胞氧化应激水平,Western Blot检测铁死亡标志蛋白酯酰辅酶A合成酶长链家族成员4(ACSL4)、谷胱甘肽过氧化物酶-4(GPX4)和凋亡指示蛋白cleaved-caspase 3(c-caspase-3)的表达。结果:与HG组比较,高糖H/R组细胞活性和SOD水平降低(P<0.05),细胞上清液中LDH含量增加(P<0.05),ACSL4和c-caspase-3蛋白表达量增加,GPX4蛋白表达量降低(P<0.05)。与高糖H/R组比较,高糖H/R+ferrostatin-1组细胞活性和SOD水平升高(P<0.05),细胞上清LDH含量降低(P<0.05),ACSL4和c-caspase-3蛋白表达量降低(P<0.05),GPX4蛋白表达量升高(P<0.05)。结论:Ferrostatin-1减轻铁死亡对高糖缺氧复氧诱导H9C2心肌细胞损伤具有一定的保护作用。

Objective:To elucidate the protective mechanism of ferroptosis inhibitor ferrostatin-1 on cardiomyocytes during high glucose(HG)hypoxia/reoxygenation(H/R).Methods:Normally cultured H9C2 cardiomyocytes were randomly divided into three groups:high glucose normoxia group(HG),high glucose hypoxia reoxygenation group(high glucose H/R)and high glucose H/R+ferrostatin-1 group.The high-sugar medium was applied to three groups of cells to achieve a sugar concentration of 33 mmol/L,and the cells were placed in a common incubator(volume fraction 90%O2+volume fraction 10%CO2)for 24 hours to prepare a high glucose model.The high glucose H/R cells were cultured for 4 hours in a three-gas incubator(volume fraction 95%N2+volume fraction 5%CO2),and the were cultured in a common incubator for 2 hours.The high glucose H/R+ferrostatin-1 cells were stimulated with ferrostatin-124 hours prior to the above high glucose and anoxic reoxygenation treatment.The lactate dehydrogenase(LDH)kit was used to measure the LDH content in the supernatant culture medium,and the cell activity was measured by the CCK-8 kit.Superoxide dismutase(SOD)kit was adopted to measure the level of oxidative stress in cells,and Western Blot was used to detect the expression of iron death marker protein acyl-CoA synthetase long-chain family member 4(ACSL4),glutathione peroxidase-4(GPX4),and cleaved-caspase 3(c-caspase-3).Results:Compared with that respectively in HG group,cell activity and SOD level in high glucose H/R group decreased(P<0.05),LDH content in cell supernatant increased(P<0.05),ACSL4 and c-caspase-3 protein expression increased,and GPX4 protein expression decreased(P<0.05).Compared with that in high glucose H/R group,the cell activity and SOD level of the high glucose H/R+ferrostatin-1 group were increased(P<0.05),the LDH content of the cell supernatant decreased(P<0.05),ACSL4 and c-caspase-3 proteins were decreased(P<0.05),and GPX4 protein expression increased(P<0.05).Conclusion:Ferrostatin-1 attenuates ferroptosis and has protective effect on H9C2 cardiomyocyte injury induced by high glucose hypoxia-reoxygenation.