Hesperetin Attenuates Liver Fibrosis in Mice by Modulating Selected Gut Microbial Populations and SIRT2-Associated Inflammatory Signaling.

Abstract

Background: Chronic liver injury progressively induces fibrotic remodeling through persistent inflammatory activity and aberrant extracellular matrix accumulation. Growing evidence indicates that this process involves not only intrahepatic signaling but also gut-liver crosstalk and sirtuin-regulated inflammatory pathways. Hesperetin, a citrus-derived flavanone with reported antioxidant and anti-inflammatory properties, has not been adequately evaluated with respect to microbiota-associated changes and SIRT2-associated signaling during fibrotic liver injury.

Objectives: This study investigated whether hesperetin attenuates CCl₄-induced liver fibrosis in mice by modulating selected gut microbial populations and suppressing SIRT2-associated inflammatory signaling pathways.

Methods: Male mice were randomly assigned to the vehicle, CCl₄, and CCl₄ + hesperetin groups (n = 5 per group). Liver fibrosis was induced by intraperitoneal administration of CCl₄ twice weekly for six weeks, and hesperetin was administered orally at 100 mg/kg/day. Histological, biochemical, inflammatory, SIRT2-associated, and selected gut microbial endpoints were assessed. The expression levels of α-SMA, TGF-β1, TNF-α, IL-6, and SIRT2 were quantitatively analyzed using quantitative polymerase chain reaction (qPCR), Western blotting, and an enzyme-linked immunosorbent assay (ELISA). Levels of selected gut microflora were measured using targeted qPCR for Firmicutes, Bacteroidetes, Akkermansia, and Escherichia coli.

Results: CCl₄ exposure increased collagen deposition, collagen proportionate area (CPA), and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, indicating fibrotic remodeling and hepatocellular injury. Hesperetin treatment significantly decreased collagen deposition and CPA and partially normalized ALT and AST activities compared with CCl₄ treatment alone. Fibrosis biomarkers, including α-SMA and TGF-β1, and inflammatory cytokines, including TNF-α and IL-6, showed significant decreases in expression at both the mRNA and protein levels. Hesperetin reduced CCl₄-associated SIRT2 upregulation and restored acetyl-α-tubulin levels, suggesting attenuation of SIRT2-associated deacetylase activity.

Conclusions: These exploratory findings suggest that hesperetin attenuates CCl₄-induced liver fibrosis by reducing collagen accumulation and biochemical liver injury, suppressing inflammatory and fibrogenic signaling, modulating selected gut microbial populations, and regulating SIRT2-associated acetylation signaling.