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

Abstract

To explore the protective effect and its mechanism of vitamin C on septic renal injury induced by lipopolysaccharide (LPS). 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. 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-1β and TNF-α were 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-1β and 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]. Vitamin C can alleviate LPS-induced HK-2 cell damage, and reduce the expressions of necrotic factors and inflammatory factors.