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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。
Objective: To investigate the role of reactive oxygen species (ROS) in necroptosis of renal tubular epithelial cells. Methods: The necroptosis model of human renal tubular epithelial cells HK-2 cells was established to detect the level of ROS. The NADPH inhibitor Apocynin was used to inhibit the ROS production in HK-2 cells necroptosis model. The changes of necroptosis in HK-2 cells were observed by flow cytometry and detection of key proteins of necroptosis. RESULTS: Necropsy models of HK-2 cells were successfully established using tumor necrosis factor alpha, benzyloxycarbonyl-valyl-alanyl-aspartyl-fluoromethylketone and antimycin A, and HK- (P <0.001). However, ROS production was inhibited by necrostatin-1 (35.58 ± 1.08 vs 43.29 ± 2.49, P = 0.002) after necroptosis. The ROS of HK-2 cells was significantly decreased (30.71 ± 2.82 vs 43.29 ± 2.49, P <0.001) when Apocynin intervention was used on the necroptosis model, and the proportion of necrotic cells was decreased by flow cytometry (2.00% ± 0.30% vs 6.99% ± 2.79%, P <0.001), while phosphorylation of receptor-associated protein 3 and mixed-family protein kinase-like domains decreased. CONCLUSION: ROS participates in the necroptosis of HK-2 cells and can reduce necroptosis by inhibiting the production of ROS, increase the survival rate of HK-2 cells in injured state and reduce acute tubular necrosis.