Attenuation of tacrolimus induced oxidative stress, mitochondrial damage, and cell cycle arrest by Boerhavia diffusa root fraction in mdck cell lines

Abstract

Objective(s) The protective effect of ethyl acetate fraction (EAF) of Boerhavia diffusa roots against Tacrolimus (TAC) induced nephrotoxicity was studied using MDCK cell lines.Materials and Methods Ethanolic root extract of B. diffusa was fractionated using the liquid-liquid partition method. The cytotoxic effect of TAC and protective effect of EAF co-treatment were studied in MDCK cell lines by measuring ROS, LPO, and NO levels; collagen accumulation, effect on mitochondrial membrane integrity and cell cycle analysis were studied. The active component in EAF was quantified by HPLC analysis.Results TAC induced toxicity, leading to apoptosis and necrosis, was significantly reduced (p <0.001) in EAF co-treatment, with reversal of cell cycle arrest and reduced cell population at sub G0/G1 phase. Further, ROS (p <0.05), LPO and NO (p <0.001), were significantly reduced with EAF co-treatment compared with TAC individually treated cells. TAC induced mitochondrial membrane integrity loss was found to be significantly reduced in co-treated cells, as measured by rhodamine123 (p <0.05) and translocation of cytochrome c (p <0.001) from nucleus to cytoplasm, and caspase 3 release (p <0.001). The same was confirmed through annexin-FITC and PI staining (p <0.05) with reduced apoptotic and necrotic death in co-treated population. Interestingly, EAF co-treatment decreased collagen accumulation (p <0.001) with significant increase in the cell survival of tubular epithelial cells. HPLC analysis showed the presence of Quercetin (87.5 mg/g) in EAF, which may be responsible for the nephroprotective role. Conclusion Thus, these results provide sound evidence that EAF may be an effective adjuvant therapy to prevent nephrotoxicity induced by TAC.