山茱萸总苷抑制高糖诱导的人肾小管上皮细胞系HK-2凋亡和氧化损伤

Total cornus officinalis glycosides inhibit high glucose-induced cell apoptosis and oxidative damage of human renal tubular epithelial cell line HK-2

目的探究山茱萸总苷(COG)调节E2相关因子2(Nrf2)/血红素加氧酶1(HO-1)通路对高糖诱导的人肾小管上皮细胞系HK-2的凋亡和氧化损伤的影响。方法将人肾小管上皮细胞系HK-2分为对照组(control,5.5 mmol/L)、模型组(HG,30 mmol/L)、山茱萸总苷低、中和高剂量组(L-COG、M-COG、H-COG),加入Nrf2通路抑制剂后,HK-2细胞分为H-COG组、ML385组。CCK8法检测细胞活性,流式细胞术检测细胞凋亡,Western blot法检测Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤/白血病-2(Bcl-2)、活化的胱天蛋白酶(cleaved-caspase3)、Nrf2/HO-1通路蛋白表达,DCFH-DA法检测活性氧(ROS)含量,ELISA法检测谷胱甘肽过氧化物酶(GSH-Px)活性、超氧化物歧化酶(SOD)活性、白介素-6(IL-6)及肿瘤坏死因子α(TNF-α)含量。结果与对照组比较,模型组细胞活性、Bcl-2蛋白表达、GSH-Px及SOD活性明显降低,凋亡率、Bax、Nrf2、HO-1及cleaved-caspase3蛋白表达、ROS、IL-6、TNF-α含量明显增加(P<0.05);与模型组比较,L-COG、M-COG、H-COG组细胞活性、Bcl-2蛋白表达、GSH-Px、SOD活性明显增加,凋亡率、Bax、Nrf2、HO-1及cleaved-caspase3蛋白表达、ROS、IL-6、TNF-α含量明显降低(P<0.05)。与H-COG组比较,ML385组细胞活性、Bcl-2蛋白表达、GSH-Px活性、SOD活性明显增加,凋亡率、Bax及cleaved-caspase3蛋白表达、ROS、IL-6、TNF-α含量明显降低(P<0.05)。结论山茱萸总苷可能是通过抑制Nrf2/HO-1通路减轻高糖诱导的肾小管上皮细胞氧化应激和炎性反应,进而减轻细胞凋亡。

Objective To investigate the effect of cornus officinalis glycosides on apoptosis and oxidative damage of human renal tubular epithelial cells line induced by high glucose by regulating E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)pathway.Methods Human renal tubule epithelial cell line HK-2 was divided into control group(control,5.5 mmol/L glucose),model group(HG,30 mmol/L glucose),and total cornus officinalis glycosides groups with low,medium and high dose(L-COG,M-COG,H-COG).After Nrf2 pathway inhibitors were added,HK-2 cells were divided into H-COG group and ML385 group.CCK8 method was used to detect cell activity.Apoptosis was detected by flow cytometry.The expressions of Bcl-2 associated X protein(Bax),B-cell lymphoma/leukemia 2(Bcl-2),activated caspase3(cleaved caspase3),Nrf2/HO-1 pathway protein were detected by Western blot.Reactive oxygen species(ROS)content was detected by DCFH-DA,glutathione peroxidase(GSH-Px)activity,superoxide dismutase(SOD)activity,interleukin-6(IL-6)content and tumor necrosis factorα(TNF-α)content were detected by ELISA.Results Compared with control group,the cell activity,Bcl-2 protein expression,GSH-Px and SOD activity in model group were significantly decreased,and the apoptosis rate,Bax,Nrf2,HO-1 and cleaved caspase3 protein expression,ROS,IL-6 and TNF-αcontent were significantly increased(P<0.05).Compared with model group,the cell activity,Bcl-2 protein expression,GSH-Px and SOD activity of L-COG,M-COG,and H-COG were significantly increased.Apoptosis rate,Bax,Nrf2,HO-1,cleave-caspase3 protein expression,ROS,IL-6,TNF-αcontent were significantly decreased(P<0.05).Compared with H-COG,the cell activity,Bcl-2 protein expression,GSH-Px and SOD activity in ML385 group were significantly increased.The apoptosis rate,Bax and cleaved caspase3 protein expression,ROS,IL-6,TNF-αcontent were significantly decreased(P<0.05).Conclusions The total cornus officinis glucoside may reduce the oxidative stress and inflammatory response of renal tubular epithelial cells induced by high glucose through inhibiting Nrf2/HO-1 pathway and then reduce cell apoptosis.