摘要
目的在缺氧条件下探讨和阐明葫芦巴碱(TRG)对ARPE-19细胞铁死亡信号通路的影响。方法常规培养ARPE-19细胞,用不同浓度的TRG干预ARPE-19细胞,确认TRG干预ARPE-19细胞的最佳浓度为40 mg·L^(-1),随后根据此浓度进行分组:NC组(细胞正常培养24 h)、CoCl2组(150 mmol·L^(-1)CoCl2干预细胞24 h)、Fer-1组(1μmol·L^(-1)Fer-1处理细胞12 h,然后150 mmol·L^(-1)CoCl2干预细胞24 h)、TRG组(40 mg·L^(-1)的TRG处理细胞24 h,然后150 mmol·L^(-1)CoCl2干预细胞24 h)。采用检测试剂盒检测各组细胞中谷胱甘肽(GSH)、丙二醛(MDA)及Fe2+表达水平;流式细胞术检测各组细胞中活性氧(ROS)表达水平;Western blot检测各组细胞中缺氧诱导因子-1(HIF-1α)、血管内皮生长因子(VEGF)、谷胱甘肽过氧化物酶4(GPX4)及酰基辅酶A合成酶长链家族成员4(ACSL4)的表达水平;免疫荧光染色观察各组细胞中VEGF与ACSL4蛋白表达水平。结果与CoCl2组相比,TRG组及Fer-1组细胞中ROS表达水平均下降,差异均有统计学意义(t=27.91、31.61,均为P<0.01)。各组细胞中MDA及Fe2+的表达水平变化情况与ROS变化趋势相似。与CoCl2组相比,Fer-1组及TRG组细胞中GSH表达水平均升高,差异均有统计学意义(t=15.95、12.76,均为P<0.01)。与CoCl2组相比,Fer-1组及TRG组细胞中HIF-1α与VEGF蛋白相对表达水平均减低,差异均有统计学意义(均为P<0.01)。与NC组相比,CoCl2组细胞中ACSL4蛋白相对表达水平升高,而GPX4蛋白相对表达水平减低,差异均有统计学意义(t=31.05、13.18,均为P<0.01)。与CoCl2组相比,Fer-1组及TRG组细胞中ACSL4蛋白相对表达水平均减低,而GPX4蛋白相对表达水平均升高,差异均有统计学意义(均为P<0.01)。免疫荧光染色结果显示,与NC组相比,CoCl2组细胞中VEGF与ACSL4蛋白表达水平均增高。与CoCl2组相比,Fer-1组和TRG组细胞中VEGF与ACSL4蛋白表达水平均减低。结论TRG保护ARPE-19细胞免于缺氧诱导的氧化应激损伤是通过抑制铁死亡实现的。
Objective To investigate and elucidate the effect of trigonelline(TRG)on the ARPE-19 ferroptosis signaling pathway under hypoxia conditions.Methods ARPE-19 cells were routinely cultured and intervened with different concentrations of TRG.According to the optimal concentration(40 mg·L^(-1))of TRG determined for ARPE-19 intervention,the cells were divided into an NC group(routinely cultured for 24 h),a CoCl 2 group(treated with 150 mmol·L^(-1)CoCl 2 for 24 h),a Fer-1 group(treated at first with 1μmol·L^(-1)Fer-1 for 12 h and then with 150 mmol·L^(-1)CoCl 2 for 12 h),and a TRG group(treated at first with 40 mg·L^(-1)TRG for 24 h and then with 150 mmol·L^(-1)CoCl 2 for 24 h).The levels of glutathione(GSH),malondialdehyde(MDA)and Fe 2+ions in each group of cells were evaluated by detection kits.The levels of reactive oxygen species(ROS)in each group of cells were detected by flow cytometry.Western blot was used to analyze the expression levels of hypoxia-induced factor-1α(HIF-1α),vascular endothelial growth factor(VEGF),glutathione peroxidase 4(GPX4)and acyl-CoA synthetase long-chain family member 4(ACSL4)in each group of cells.The expression levels of VEGF and ACSL4 proteins in each group of cells were measured by immunofluorescence staining.Results The ROS levels in TRG and Fer-1 groups were significantly lower than those in the CoCl 2 group(t=27.91,31.61;both P<0.01).The change trend of MDA and Fe 2+levels was the same with that of ROS levels in each group.Compared with those in the CoCl 2 group,the GSH levels were increased significantly(t=15.95,12.76;both P<0.01)and the relative expression levels of HIF-1αand VEGF proteins were decreased significantly in Fer-1 and TRG groups(all P<0.01).The CoCl 2 group had higher ACSL4 levels and lower GPX4 levels than the NC group(t=31.05,13.18;both P<0.01).Fer-1 and TRG groups had lower ACSL4 expression levels and higher GPX4 expression levels than the CoCl 2 group(all P<0.01).The results of immunofluorescence staining showed that the expression levels of VEGF and ACSL4 proteins in the CoCl 2 group were higher than those in the NC group.The expression levels of VEGF and ACSL4 proteins in Fer-1 and TRG groups were lower than those in the CoCl 2 group.Conclusion TRG protects ARPE-19 cells from hypoxia-induced oxidative stress damage by inhibiting ferroptosis.
作者
岳欣欣
付洋
尹晓燕
王敏
YUE Xinxin;FU Yang;YIN Xiaoyan;WANG Min(He University,Shenyang 110163,Liaoning Province,China;Department of Burns and Plastic Surgery,General Hospital of Northern Theater Command,Shenyang 110000,Liaoning Province,China)
出处
《眼科新进展》
北大核心
2025年第4期263-268,共6页
Recent Advances in Ophthalmology
基金
国家自然科学基金项目(编号:81372972)
辽宁省联合基金项目面上资助计划项目(编号:2023011577-JH3/4600)
辽宁省教育厅高校基本科研项目(编号:LJKMZ20221981)。
