芍药苷通过调控硫氧还蛋白结合蛋白表达对脂多糖诱导的肾小球系膜细胞氧化应激及凋亡的影响

Effects of paeoniflorin on oxidative stress and apoptosis induced by lipopolysaccharide in glomerular mesangial cells by regulating TXNIP gene expression

目的探讨芍药苷对脂多糖(LPS)诱导的肾小球系膜细胞氧化应激及凋亡的影响及其作用机制。方法研究起止时间为2018年1月至2019年7月,体外培养人肾小球系膜细胞(HMCL),用不同浓度的(5、10、20μmol/L)芍药苷处理LPS诱导的HMCL细胞。采用流式细胞仪检测细胞凋亡率;应用试剂盒检测细胞中丙二醛(MDA)量及超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性;实时荧光定量聚合酶链反应(qRT-PCR)与蛋白质迹法(Western blotting)分别检测硫氧还蛋白结合蛋白(Thioredoxin-interacting protein,TXNIP)表达;观察干扰TXNIP的表达对LPS诱导的HMCL细胞氧化应激与凋亡的影响。结果与对照组(Con)比较,LPS组细胞凋亡率升高[(6.58±0.66)%比(28.41±2.83)%](P<0.05),MDA含量增加[(1.26±0.13)nmol/L比(5.06±0.49)nmol/L](P<0.05),SOD[(26.41±2.31)U/mL比(12.46±1.21)U/mL]、GSH-Px[(45.61±4.13)U/mL比(8.12±0.83)U/mL]活性下降(P<0.05),TXNIP信使RNA(mRNA)及蛋白表达升高[(1.02±0.09)vs(2.54±0.25);(0.42±0.04)比(0.87±0.05)](P<0.05);与LPS组比较,芍药苷不同剂量组细胞凋亡率降低[(28.41±2.83)%比(22.16±2.17)/(16.48±1.03)/(11.25±1.12)%](P<0.05),MDA含量下降[(5.06±0.49)nmol/L比(4.12±0.41)/(2.94±0.29)/(1.68±0.17)nmol/L](P<0.05),SOD[(12.46±1.21)U/mL比(15.46±1.52)/(18.24±1.63)/(22.54±2.03)U/mL]、GSH-Px[(8.12±0.83)U/mL比(17.62±1.74)/(26.14±2.63)/(39.44±3.25)U/mL]活性升高(P<0.05),TXNIP mRNA及蛋白表达降低[(2.54±0.25)比(2.13±0.21)/(1.84±0.17)/(1.46±0.15);(0.87±0.05)比(0.72±0.05)/(0.61±0.04)/(0.49±0.03)](P<0.05);干扰TXNIP的表达可减轻LPS诱导的HMCL细胞氧化应激损伤,降低细胞凋亡率;TXNIP过表达可逆转芍药苷对LPS诱导的HMCL细胞氧化应激及凋亡的作用。结论芍药苷可通过抑制TXNIP的表达对LPS诱导的HMCL细胞氧化应激发挥保护作用,抑制细胞凋亡。

Objective To investigate the effects of paeoniflorin on oxidative stress and apoptosis induced by lipopolysaccharide(LPS)in glomerular mesangial cells.Methods The research was carried out from January 2018 to July 2019.LPS was used as a stimulating factor to stimulate HMCL cells,and then LPS-induced HMCL cells were treated with different concentrations of(5,10,20μmol/L)paeoniflorin.Apoptosis rate was measured by flow cytometry.The content of malondialdehyde(MDA)and superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in cells were detected by using a kit.Real-time quantitative polymerase chain reaction(qRT-PCR)and Western blotting were used to detect the expression of thioredoxin-interacting protein(TXNIP)in LPS-induced HMCL cells.The effect of interference with TXNIP expression on oxidative stress and apoptosis induced by LPS in HMCL cells was observed.Results Compared with the Con group,the apoptosis rate and the content of MDA were increased[(6.58±0.66)%vs(28.41±2.83)%;(1.26±0.13)nmol/L vs(5.06±0.49)nmol/L;both P<0.05],the activities of SOD[(26.41±2.31)U/mL vs(12.46±1.21)U/mL]and GSH-Px[(45.61±4.13)U/mL vs(8.12±0.83)U/mL]were decreased(P<0.05),and the expressions of TXNIP mRNA and protein were increased[(1.02±0.09)vs(2.54±0.25);(0.42±0.04)vs(0.87±0.05)](P<0.05)in the LPS group.Compared with the LPS group,the apoptosis rates of paeoniflorin with different concentrations were decreased[(28.41±2.83)%vs(22.16±2.17)/(16.48±1.03)/(11.25±1.12)%](P<0.05),the content of MDA was decreased[(5.06±0.49)nmol/L vs(4.12±0.41)/(2.94±0.29)/(1.68±0.17)nmol/L](P<0.05),and the activities of SOD[(12.46±1.21)U/mL vs(15.46±1.52)/(18.24±1.63)/(22.54±2.03)U/mL]and GSH-Px[(8.12±0.83)U/mL vs(17.62±1.74)/(26.14±2.63)/(39.44±3.25)U/mL]were increased(P<0.05),but the expressions of TXNIP mRNA and protein were decreased[(2.54±0.25)vs(2.13±0.21)/(1.84±0.17)/(1.46±0.15);(0.87±0.05)vs(0.72±0.05)/(0.61±0.04)/(0.49±0.03)](P<0.05).Interference with TXNIP expression could attenuate LPS-induced oxidative stress damage in HMCL cells and reduce apoptosis rate.Overexpression of TXNIP reversed the effect of paeoniflorin on oxidative stress and apoptosis induced by LPS in HMCL cells.Conclusion Paeoniflorin inhibits apoptosis and protects LPS-induced oxidative stress in HMCL cells by inhibiting the expression of TXNIP.