Objective:To investigate the role of reactive oxygen species on necroptosis in renal tubular epithelial cell.Methodology:The necroptosis model of HK-2 cell was constructed as our previous research.Apocynin,a specific inhibitor of NADPH oxidase was delivered in the necroptosis model.ROS production was detected by Dichlorodihydrofluorescein diacetate.The manner of cell death was identified by flow cytometry.Western Blot was used to determine phosphorylation of receptor-interacting protein kinase 3(RIP-3) and mixed lineage kinase domain-like (MLKL)which are essential to necroptosis.Results:Necroptosis model of HK-2 cell was established by TNF-α,benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and antimycin A.In this model,PI(+) HK-2 cells was increased and phosphorylation of RIP-3 and MLKL was augmented.ROS increased in necroptosis group(43.29±2.49 vs 25.90±1.27,P<0.001) and could be inhibited by Nec-1(35.58±1.08 vs 43.29±2.49,P=0.002).Apocynin not only decreased ROS production(30.71±2.82 vs 43.29±2.49,P<0.001),but also reduced the proportions of necrosis in the necroptosis model(2.00%±0.30% vs 6.99%±2.79%,P<0.001).Phosphorylated RIP-3 and MLKL was also decreased by Apocynin.Conclusion:ROS play an important role in necroptosis of HK-2 cell.Necroptosis could be ameliorated by inhibiting ROS production.%目的:探讨活性氧自由基(ROS)在肾小管上皮细胞necroptosis中的作用.方法:构建肾小管上皮细胞HK-2细胞necroptosis模型,检测其ROS升高程度.并使用NADPH酶抑制剂Apocynin 抑制HK-2细胞necroptosis模型中ROS的生成,通过流式细胞计数及检测necroptosis的关键蛋白观察HK-2细胞necroptosis的变化.结果:使用肿瘤坏死因子α、苄氧羰酰-缬氨酰-丙氨酰-天冬氨酰-氟甲基酮及抗霉素A成功建立了HK-2细胞necroptosis模型,并观察到HK-2细胞发生necroptosis时ROS显著升高(43.29±2.49 vs 25.90±1.27,P<0.001),而使用necrostatin-1抑制necroptosis后ROS生成受到抑制(35.58±1.08 vs 43.29±2.49,P=0.002).当对necroptosis模型使用Apocynin干预时,HK-2细胞ROS明显下降(30.71±2.82 vs 43.29±2.49,P<0.001),并且流式细胞计数结果显示坏死细胞比例减少(2.00%±0.30% vs 6.99%±2.79%,P<0.001),同时受体相关蛋白3和混合系列蛋白激酶样结构域的磷酸化水平降低.结论:ROS参与了HK-2细胞的necroptosis,并且通过抑制ROS的生成可减少necroptosis发生,提高损伤状态下HK-2细胞存活率,减轻急性肾小管坏死.
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