铁死亡在高糖诱导的视网膜色素上皮细胞损伤中的作用及机制

Effects and mechanisms of ferroptosis on high glucose-induced retinal pigment epithelial cells injury

目的:观察铁死亡在高糖(HG)诱导的视网膜色素上皮(RPE)细胞损伤中的作用及机制,为糖尿病视网膜病变(DR)的治疗提供新思路。方法:将体外培养的ARPE-19细胞分为正常对照组(NC组)、高糖组(HG组)、高糖+铁死亡抑制剂组(Fer-1组)。采用CCK-8法检测各组细胞活力;使用ELISA试剂盒检测白细胞介素6(IL-6)、IL-1β及单核细胞趋化蛋白-1(MCP-1)的表达水平;使用化验试剂盒检测丙二醛(MDA)、谷胱甘肽(GSH)、谷胱甘肽过氧化物酶4(GPX4)水平;使用铁离子检测试剂盒检测细胞铁含量;通过透射电子显微镜观察细胞线粒体变化;通过Western blotting和免疫荧光染色技术检测铁死亡相关蛋白长链脂酰CoA合成酶4(ACSL4)、GPX4以及血管内皮生长因子(VEGF)蛋白的表达。结果:与NC组相比,HG组细胞活力显著下降,细胞上清液中炎症因子表达水平升高,细胞中氧化应激指标MDA和铁含量显著升高,GSH含量和GPX4活性显著降低(均P<0.01),且线粒体皱缩,ACSL4及VEGF蛋白表达增加,GPX4蛋白表达降低(均P<0.01)。与HG组相比,Fer-1组细胞活力明显增加,细胞上清液中炎症因子表达水平下降,细胞中MDA和铁含量明显下降,GSH含量和GPX4活性显著提高(均P<0.05),且线粒体形态改善,ACSL4及VEGF蛋白表达减少,GPX4蛋白表达升高(均P<0.05)。结论:铁死亡参与高糖诱导的RPE细胞损伤,抑制铁死亡能改善细胞活性,降低炎症及氧化应激水平,减轻高糖诱导的RPE细胞损伤。

AIM:To observe the effects and mechanisms of ferroptosis on high glucose(HG)-induced retinal pigment epithelium(RPE)cells injury,and to provide new ideas for the treatment of diabetic retinopathy(DR).METHODS:The ARPE-19 cell lines cultured in vitro were divided into normal control group(NC group),high glucose group(HG group),and high glucose+Ferrostatin-1 group(Fer-1 group).The cell viability of each group was detected by CCK-8 assay.The expressions of interleukin 6(IL-6),IL-1βand monocyte chemotactic protein-1(MCP-1)were detected using ELISA kits.The levels of malondialdehyde(MDA),glutathione(GSH),glutathione peroxidase 4(GPX4)and iron content were detected using the corresponding assay kits.The mitochondrial changes in ARPE-19 cells were observed by transmission electron microscopy.The expressions of ferroptosis-related proteins including long-chain lipoyl CoA synthase 4(ACSL4)and GPX4,as well as vascular endothelial growth factor(VEGF)were detected by Western blotting and immunofluorescence staining.RESULTS:Compared with NC group,the cell viability of HG group decreased significantly,the expression levels of inflammatory factors in cell supernatant increased,the contents of MDA and iron significantly increased,GSH and GPX4 significantly decreased(all P<0.01),the mitochondria of ARPE-19 cells shrunk,the expression of proteins ACSL4 and VEGF increased,while the expression of GPX4 decreased(all P<0.01).Compared with HG group,the cell viability of Fer-1 group significantly increased,the expression levels of inflammatory factors in cell supernatant decreased,MDA and iron contents significantly decreased,GSH contents and GPX4 viability significantly increased(all P<0.05),the morphology of mitochondria in ARPE-19 cells improved,the expression of ACSL4 and VEGF decreased,while the expression of GPX4 increased(all P<0.05).CONCLUSION:Ferroptosis is involved in the injury of RPE induced by HG.Inhibiting ferroptosis can improve cell viability,reduce inflammation and oxidative stress,and alleviate HG-induced RPE cells injury.