Abstract
Background: Intrahepatic cholestasis is characterized by impaired bile formation or bile flow, leading to the accumulation of bile acid, oxidative stress, inflammation, and hepatocellular injury. Despite its clinical significance, effective therapeutic options remain limited. This study investigated the hepatoprotective potential of TND1128, a redox-active 5-deazaflavin derivative with mitochondrial-modulating capabilities, in an α-naphthyl isothiocyanate (ANIT)-induced model of intrahepatic cholestasis in rats. Methods: Thirty-six male Sprague–Dawley rats were randomly assigned to six groups: control, ANIT, TND1128, ursodeoxycholic acid (UDCA), TND1128 + ANIT, and UDCA + ANIT. Cholestasis was induced by a single oral administration of ANIT (100 mg/kg). Biochemical, oxidative stress, inflammatory, apoptotic, and molecular parameters were subsequently evaluated, including serum liver enzymes (ALT, AST, ALP), bilirubin, bile acids, malondialdehyde (MDA), glutathione (GSH), interleukin-6 (IL-6), NF-κB, Nrf2, caspase-3, and FXR/BSEP gene expression, alongside histopathological evaluation. Results: ANIT administration induced significant hepatocellular injury, as evidenced by elevated levels of liver enzymes, bile acids, bilirubin, MDA, IL-6, NF-κB, and caspase-3. Concurrently, it caused reduced GSH levels, downregulated expression of Nrf2, FXR, and BSEP, along with marked histological damage. Pretreatment with TND1128 significantly ameliorated these changes by restoring biochemical parameters, reducing oxidative stress and inflammatory mediators, enhancing antioxidant defense via Nrf2 upregulation, suppressing apoptotic signaling, and substantially improving liver histoarchitecture. However, TND1128 did not significantly restore FXR or BSEP expression, suggesting that its hepatoprotective effect is mediated through FXR/BSEP-independent redox, inflammatory, and apoptotic pathways. Conclusion: These findings indicated that TND1128 protects against ANIT-induced cholestatic liver injury primarily through modulation of oxidative stress, inflammation, and apoptosis, highlighting its potential as a promising therapeutic candidate for further preclinical development and translational investigation.