胡萝卜苷对H_(2)O_(2)诱导的HK-2细胞氧化应激的保护作用研究

Protection and Mechanism of Daucosterol Against Oxidative Stress Induced by Hydrogen Peroxide in Human Renal Tubular Epithelial Cells

目的分析胡萝卜苷干预对过氧化氢(H_(2)O_(2))诱导的人肾小管上皮细胞(HK-2细胞)氧化应激的保护作用及作用机制。方法体外培养HK-2细胞,对照组加入等体积0.9%氯化钠溶液,实验组中加入不同浓度的H_(2)O_(2)处理24h(1、5、10、50和100μmol/L),干预组中加入50μmol/L胡萝卜苷干预2h后再加入不同浓度H_(2)O_(2)处理,CCK-8实验检测各组细胞活力;Hochest33342/PI荧光染色和caspase-3免疫荧光实验分析各组细胞凋亡;DCFH-DA荧光探针染色分析各组细胞中的活性氧簇(reactive oxygen species,ROS)水平;Western blot法检测各组细胞中Bax、Bcl-2和NOX4蛋白的表达水平。结果50μmol/L和10μmol/L H_(2)O_(2)处理后实验组中的抑制率分别为20.74%±3.32%和53.90%±2.37%,胡萝卜苷干预组中各抑制率分别为10.29%±1.69%(t=4.859,P=0.008)和37.38%±3.95%(t=6.212,P=0.003),差异均有统计学意义;Hochest33342/PI荧光染色结果显示,实验组中Hochest33342荧光信号聚集细胞以及PI荧光阳性细胞明显多于干预组;免疫荧光实验结果显示实验组中caspase-3表达水平明显高于干预组;荧光探针染色结果显示实验组中ROS水平明显高于干预组;Western blot法结果显示,H_(2)O_(2)实验组中NOX4蛋白表达水平高于对照组,经干预后出现明显下调,干预组中Bax蛋白表达水平也低于H_(2)O_(2)实验组。结论胡萝卜苷干预可减轻H_(2)O_(2)诱导的HK-2细胞氧化应激,并降低细胞凋亡发挥保护作用。

Objective To investigate the protective effect and mechanism of Daucosterol on H_(2)O_(2)-induced oxidative stress in human renal duct epithelial cells(HK-2 cells).Methods HK-2 cells were cultured in vitro,and treated with different concentrations of H_(2)O_(2)(1μmol/L,5μmol/L,10μmol/L,50μmol/L and 100μmol/L)for 24h in H_(2)O_(2)treatment group;In Daucosterol intervention group,HK-2 cells were pretreated with 50μmol/L Daucosterol for 2h and then treated with different concentrations of H_(2)O_(2).CCK-8 assay was used to detect cell viability in each group.Hochest33342/PI fluorescence staining and caspase-3 immunofluorescence assay were used to analyze cell apoptosis.The levels of reactive oxygen species(ROS)were analyzed using DCFH-DA fluorescent probe staining.Western blot assay was used to analyze the expression levels of Bax,Bcl-2 and NOX4.Results The inhibition rates in 50μmol/L and 100μmol/L H_(2)O_(2)treatment groups were 20.74%±3.32%and 53.90%±2.37%;the inhibition rates in Daucosterol intervention group were 10.29%±1.69%(t=4.859,P=0.008)and 37.38%±3.95%(t=6.212,P=0.003),respectively,and the differences were statistically significant.The results of Hochest33342/PI fluorescence staining showed significantly more Hochest33342 fluorescence signal aggregation and PI fluorescence positive cells in H_(2)O_(2)treatment group than in Daucosterol intervention group.Immunofluorescence assay showed that the expression level of caspase-3 in H_(2)O_(2)treatment group was significantly higher than that in Daucosterol intervention group.ROS level in Daucosterol intervention group was significantly attenuated compared with H_(2)O_(2)treatment group.The results of Western blot showed that the expression level of NOX4 protein in H_(2)O_(2)treatment group was higher than that in the control group,but significantly decreased in Daucosterol intervention group;The expression level of Bax protein also decreased in Daucosterol intervention group,compared with H_(2)O_(2)treatment group.Conclusion Daucosterol intervention can alleviate H_(2)O_(2)-induced oxidative stress in HK-2 cells and reduce apoptosis to exert a protective effect.