Sitagliptin ameliorates liver fibrosis through reducing inflammation and sestrin2 through Nrf2/SOD in cholestasis rats

Technical Summary: Sitagliptin Ameliorates Liver Fibrosis in Cholestasis Rats via Nrf2/SOD and Sestrin2 Pathways

Problem Statement
Cholestasis, characterized by impaired bile flow, leads to severe liver injury, progressive fibrogenesis, and potentially liver failure. The pathogenesis involves the accumulation of cytotoxic bile acids and bilirubin, which trigger oxidative stress and the release of pro-inflammatory cytokines (e.g., TNF-α, TGF-β). While the mechanisms are not fully defined, reactive oxygen species (ROS) and inflammatory signaling are identified as primary drivers of tissue damage. Current research seeks therapeutic agents that can mitigate these oxidative and inflammatory cascades to prevent fibrosis. Sitagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor primarily used for type II diabetes, has demonstrated antioxidant and anti-inflammatory properties in other models, but its efficacy in biliary obstruction-induced cholestasis requires investigation.

Methodology
The study utilized a rat model of extrahepatic cholestasis induced by bile duct ligation (BDL).

• Subjects: 35 male Wistar rats were randomized into five groups (n=7 per group): Sham, BDL (untreated), and three BDL groups treated with Sitagliptin at doses of 10, 50, or 100 mg/kg.
• Procedure: Following BDL surgery (or sham laparotomy), rats received oral gavage of Sitagliptin or saline for 14 days.
• Assessments:
  • Biochemical: Serum levels of glucose, total bilirubin (T-bil), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were measured.
  • Oxidative Stress Markers: Liver tissue was analyzed for Malondialdehyde (MDA), Superoxide Dismutase (SOD) activity, Total Antioxidant Capacity (TAC), and Nrf2 levels via ELISA.
  • Inflammation and Fibrosis: Hepatic levels of TNF-α, TGF-β, and Sestrin2 were quantified. Hydroxyproline content was measured to assess collagen deposition.
  • Histopathology: Liver tissues underwent H&E staining to evaluate necroinflammation and Masson's trichrome staining to grade fibrosis scores based on collagen deposition.
• Statistical Analysis: Data were analyzed using one-way ANOVA with Tukey's post hoc test or Kruskal-Wallis with Mann-Whitney U test, with significance set at P<0.05.

Key Results

• Liver Injury and Enzymes: BDL significantly increased liver weight, serum ALP, LDH, and total bilirubin compared to the sham group. Sitagliptin treatment at 10 mg/kg significantly reduced serum ALP, while all doses showed a dose-dependent improvement in LDH levels, with 100 mg/kg showing the best effect. Notably, Sitagliptin prevented the BDL-induced drop in serum glucose.
• Oxidative Stress and Antioxidant Defense: BDL induced a significant reduction in SOD activity, TAC, and Nrf2 levels, alongside an increase in MDA (lipid peroxidation). Sitagliptin treatment restored antioxidant defenses. The 10 mg/kg dose was identified as the most effective in restoring SOD activity, TAC, and reducing MDA levels.
• Sestrin2 and Inflammation: BDL increased hepatic Sestrin2 (a stress-induced protein), TNF-α, and TGF-β. Sitagliptin treatment at all doses significantly reduced Sestrin2, TNF-α, and TGF-β levels.
• Fibrosis and Histology: BDL resulted in significant fibrosis and collagen deposition (increased hydroxyproline). While all Sitagliptin doses reduced TGF-β and hydroxyproline content, only the 10 mg/kg dose significantly reduced the histological fibrosis score compared to the untreated BDL group.
• Dose-Response: The study observed that lower doses (specifically 10 mg/kg) were often more effective in modulating specific antioxidant and fibrotic markers than higher doses (50 and 100 mg/kg).

Significance and Claims
The paper concludes that Sitagliptin mitigates BDL-induced liver fibrosis in rats. The authors attribute this protective effect to the drug's ability to suppress oxidative stress and inflammatory signaling. Specifically, the study claims that Sitagliptin:

1. Enhances Antioxidant Defense: It restores the Nrf2/SOD pathway, thereby increasing the liver's capacity to neutralize ROS.
2. Reduces Stress Response: It downregulates Sestrin2, a marker of cellular stress.
3. Attenuates Fibrosis: By reducing pro-fibrotic cytokines (TGF-β) and inflammation (TNF-α), Sitagliptin decreases collagen deposition and improves liver histology.

The authors emphasize that the beneficial effects are particularly evident at lower doses (10 mg/kg), suggesting a potential therapeutic window where Sitagliptin acts as a potent modulator of the redox system and inflammatory pathways in cholestatic liver injury. The study positions Sitagliptin not merely as an anti-hyperglycemic agent but as a potential hepatoprotective agent capable of addressing the oxidative and inflammatory drivers of liver fibrosis.