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.

Keywords

• apoptosis
• boerhavia diffusa
• caspase 3
• cell cycle
• hplc
• nephrotoxicity
• quercetin
• reactive oxygen species