维生素C对LPS诱导脓毒症肾损伤的保护作用及其机制研究

Study of protective effect and mechanism of vitamin C in lipopolysaccharide-induced septic renal injury

目的探讨维生素C对脂多糖(LPS)所致脓毒症肾损伤的保护作用及机制。方法采用10 mg/L LPS分别诱导肾小管上皮HK-2细胞8 h和12 h,之后分别加入0.5 mmol/L与1 mmol/L维生素C继续作用,使用细胞增殖与毒性检测试剂(CCK-8)检测细胞生存率,以确定后续的合适实验条件。将细胞分为空白对照组、LPS组和LPS+维生素C组(LPS+VC组)。采用蛋白质免疫印迹试验(Western blotting)检测各组HK-2细胞坏死关键蛋白〔磷酸化混合谱系激酶结构域样蛋白(p-MLKL)、磷酸化受体相互作用蛋白激酶3(p-RIPK3)〕的蛋白表达,采用酶联免疫吸附试验(ELISA)检测各组HK-2细胞炎症因子〔白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)〕水平,比较各组间上述指标的差异。结果CCK-8检测结果显示,1 mmol/L维生素C能使LPS诱导12 h后的HK-2细胞生存率提高至86%,故选用该条件进行后续实验。LPS诱导HK-2细胞12 h后,p-MLKL和p-RIPK3的蛋白表达均显著高于空白对照组,IL-1β、TNF-α水平也均显著高于空白对照组〔IL-1β(ng/L):23.2±1.4比12.8±3.9,TNF-α(ng/L):36.4±3.9比11.6±1.8,均P<0.05〕,提示细胞坏死与炎症反应同时存在;而与LPS组比较,1 mmol/L维生素C可明显降低p-MLKL和p-RIPK3的蛋白表达,也能明显降低IL-1β与TNF-α水平〔IL-1β(ng/L):19.8±0.7比23.2±1.4,TNF-α(ng/L):17.4±5.8比36.4±3.9,均P<0.05〕。结论维生素C可缓解LPS诱导的HK-2细胞损伤,降低坏死关键蛋白与炎症因子的表达。

Objective To explore the protective effect and its mechanism of vitamin C on septic renal injury induced by lipopolysaccharide(LPS).Methods Renal tubular epithelial cells HK-2 were induced with 10 mg/L LPS for 8 hours and 12 hours,respectively,and then 0.5 mmol/L and 1 mmol/L vitamin C were added,respectively.Cell viability was measured using cell proliferation and toxicity assay cell counting kit-8(CCK-8)to determine suitable condition for subsequent experiments.HK-2 cells were divided into control group,LPS group and LPS+vitamin C group(LPS+VC group).The contents of necrosis factors phosphorylated mixed lineage kinase domain-like protein(p-MLKL)and phosphorylated receptor-interacting protein kinase 3(p-RIPK3)were measured by Western blotting.The contents of inflammatory factors interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were determined by enzyme linked immunosorbent assay(ELISA)in each group.Differences among the groups were compared.Results CCK-8 showed that 1 mmol/L vitamin C improved the survival rate of HK-2 cells to 86%after 12 hours of LPS induction,so this condition was selected for subsequent experiments.After 12 hours LPS induction in HK-2 cells,the expressions of p-MLKL and p-RIPK3 were significantly higher than those of the control group,and the levels of IL-1βand TNF-αwere also significantly higher than those of the control group[IL-1β(ng/L):23.2±1.4 vs.12.8±3.9,TNF-α(ng/L):36.4±3.9 vs.11.6±1.8,both P<0.05],indicating the co-existence of cell necrosis and inflammation.Compared with LPS group,1 mmol/L vitamin C significantly decreased the protein expression of p-MLKL and p-RIPK3,and also significantly decreased the levels of IL-1βand TNF-α[IL-1β(ng/L):19.8±0.7 vs.23.2±1.4,TNF-α(ng/L):17.4±5.8 vs.36.4±3.9,both P<0.05].Conclusion Vitamin C can alleviate LPS-induced HK-2 cell damage,and reduce the expressions of necrotic factors and inflammatory factors.