Anti-Inflammatory Effects of Vardenafil Against Cholestatic Liver Damage in Mice: a Mechanistic Study

Abstract

Background/Aims: Phosphodiesterase-5 inhibitors have beneficial effects in multiple liver diseases possibly through the reduction of oxidative stress and inflammatory response. However, these effects have not yet been examined in cholestatic liver dysfunction. Hence, this study aimed to explore the ability of vardenafil, a known phosphodiesterase-5 inhibitor, to repress lithocholic acid (LCA)-induced cholestatic liver injury and investigate the possible molecular pathways. Methods: Male Swiss albino mice were treated with LCA (0.125 mg/g) twice daily for 7 days to induce cholestatic liver damage. Vardenafil was administered 3 days before and throughout the administration of LCA. Serum markers of hepatotoxicity and hepatic nitro-oxidative stress along with antioxidant parameters were measured, and the histopathology of liver tissues was assessed. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target genes was examined using PCR. The activation of nuclear factor kappa-B (NF-κB) and the levels of inflammatory cytokines were determined. NLRP3 inflammasome and its components were studied by PCR and western blot. Results: LCA induced marked cholestatic liver damage as demonstrated by increased serum transaminases, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin, and bile acids. Examination of liver specimens confirmed the biochemical results. Nitro-oxidative stress parameters were significantly elevated along with reduced antioxidant capacity in hepatic tissue following LCA administration. LCA suppressed Nrf2 and its target genes and decreased the mRNA expression and binding capacity of Nrf2 as well as the mRNA expression of GCLm, GCLc, Nqo1, and HO-1. Additionally, LCA enhanced the activation of NF-κB, which was accompanied by elevations of inflammatory cytokines. Importantly, LCA induced the activation of NLRP3 inflammasome. LCA increased the expression of NLRP3, ASC, caspase-1, and IL-1β genes and proteins in hepatic tissue. The activities of IL-1β and caspase-1 were increased in the LCA group. Interestingly, vardenafil ameliorated LCA-induced hepatic injury and alleviated all biochemical, histopathological, and inflammatory parameters. Conclusions: These data elucidated the effects of Nrf2 inhibition and NLRP3 inflammasome activation in LCA-induced liver injury. The hepatoprotective activity of vardenafil in LCA-induced cholestatic damage may result from the drug’s ability to activate Nrf2 signaling and prevent the activation of NLRP3, which could suppress the inflammatory responses in hepatic tissue. Thus, vardenafil can be considered a novel anti-inflammatory remedy for cholestatic liver damage.

Inflammasome Is Activated in the Liver of Cholestatic Patients and Aggravates Hepatic Injury in Bile Duct–Ligated Mouse

“…Previous studies that have supported this hypothesis have used hydrophobic bile acid species such as chenodeoxycholic acid and lithocholic acid at high concentrations that are not seen in cholestatic patients or animal models and thus are likely to be artifacts of these in vitro conditions. 7,8,23 The major species of endogenous bile acids are conjugated bile acids that require bile acid transport proteins to enter cells. These transporters are only seen in hepatic parenchymal cells and are absent in macrophages and other hepatic cells.…”

“…7 However, it must be stressed that the cells in all of these studies were treated with bile acids that either were not the major endogenous bile acid in mouse or used concentrations that were not physiologically or pathophysiologically relevant. Nevertheless, the Nlrp3 inflammasome could be activated in liver tissue in a mouse model of lithocholic acid induced cholestasis, 8 while elevated messenger RNA (mRNA) expression of IL1R1, NLRP3, and caspase-1 was also observed in the liver of patients with biliary atresia. 9 Loss of Il-1r1 or Nlrp3, but not of the Caspase-1 gene, reduced bile duct injury in a mouse model of biliary atresia induced by rotavirus infection.…”

Agmatine protects against sodium valproate–induced hepatic injury in mice via modulation of nuclear factor‐κB/inducible nitric oxide synthetase pathway

“…The formalin‐fixed paraffin blocks of liver and heart of all animals were used to construct a tissue miniarray (TmA). Immunohistochemical (IHC) staining was performed on 4‐µm slices of paraffin‐embedded TmA tissue sections using primary antibodies against NF‐κB (p65) and iNOS (Santa Cruz Biotechnology, Santa Cruz, CA) automatically using Ventana Bench Mark XT system (Ventana Medical Systems, Tucson, AZ) as previously described …”

“…The excised livers were rinsed in ice-cold physiological saline and automatically using Ventana Bench Mark XT system (Ventana Medical Systems, Tucson, AZ) as previously described. [19]…”

Gut–Liver Axis and Inflammasome Activation in Cholangiocyte Pathophysiology

“…Interestingly, treatment of mice with Davanat, a registered inhibitor of Galectin-3 that is currently being studied in clinical trials, determined a significant reduction in inflammasome activation and biliary damage in a model of autoimmune cholangitis [99]. Vardenafil, a phosphodiesterase-5 inhibitor, has been shown to significantly reduce the expression of Nlrp3 and inflammasome components in a cholestatic murine model induced by lithocolic acid administration [100]. Finally, a significant reduction in Nlrp3 inflammasome activation has been demonstrated in the liver tissue of BDL rats subjected to treatment with a saline solution enriched with methane, which has been previously demonstrated to possess anti-oxidative and anti-inflammatory properties [101,102].…”