Antifibrotic potential of reserpine (alkaloid) targeting Keap1/Nrf2; oxidative stress pathway in CCl4-induced liver fibrosis

Abstract

The death rate due to liver cancer approaches 2 million annually, the majority is attributed to fibrosis. Currently, there is no efficient, safe, non-toxic, and anti-fibrotic drug available, suggesting room for better drug discovery. The current study aims to evaluate the anti-fibrotic role of reserpine, an alkaloid plant compound against CCl₄-induced liver fibrosis. In-silico docking analysis showed the interaction of reserpine with keap1 protein with the binding energy −9.0 kcal/mol. In-vitro, biochemical analysis, anti-oxidative indexes, and inflammatory cytokines analysis were performed in HepG2 cells. The non-toxic nature of the compound (<100 μg/ml) was evaluated through MTT assay in HepG2 and Vero cell lines. The antifibrotic potential of the reserpine compound (dose of 0.5 mg/kg) was assessed in CCl₄-administered C57BL/6J mice models. Hematoxylin & Eosin and Masson staining were performed to study the morphological changes of liver tissues. Immune histochemistry (IHC) analysis was performed to evaluate the effect of reserpine on the liver fibrosis marker. The biochemical assay indicated a significant decrease in ALT, AST, and MDA levels and increased catalase enzyme post-6-week reserpine treatment in mice models. Gene expression analysis revealed that the reserpine targets oxidative stress Keap1/Nrf2 pathway and down-regulated Keap1 expression by 5-fold and up-regulated Nrf2 and Nqo1 expression by 6 and 4.5-fold respectively showing its antioxidant response. It suppressed the expression of Cyp2e1 by 2.2-fold, illustrating the compound's ability to block lipid peroxidation. Histological and immunostaining exhibited improved hepatocyte morphology and reduced collagen deposition in liver tissues due to reserpine. Reserpine treatment lowered the fibrotic markers α-SMA and Col-1 by 1.3 and 1.5 folds respectively as compared to the control group and increased the expression of miR-200a and miR-29b by 15.5 and 8.2 folds (p < 0.05) while decreased miR-128-1-5p expression by 5-fold. A comprehensive In-silico, In-vitro, and In-vivo analysis revealed that reserpine has a strong anti-fibrotic effect against the CCl₄-induced liver fibrosis in C57BL/6J mice model by targeting the Keap1/Nrf2 pathway.