Abstract

Background: Emerging evidence demonstrates microbial metabolites play pivotal roles in onset and progression of liver fibrosis and imidazole propionate (ImP) as a microbially produced histidine-derived metabolite was significantly increased in patients with chronic hepatitis B/cirrhosis compared to healthy controls. However, whether ImP is involved in hepatic fibrosis remains unclear. The present study aimed to explore the effects of ImP on hepatic fibrosis and its potential mechanisms.

Methods: ImP (100μg) was administered i.p. twice daily for late 3 weeks in CCl₄-induced model (i.p. 9 weeks, twice weekly). Phosphorylated H2AX (γH2AX) DNA damage was detected by Western blot. M1 macrophage polarization was determined by gene expression of inducible nitric oxide synthase (iNOS), IL1β and IL6.

Results: Although ImP administration alone did not cause liver fibrosis compared to controls, but it significantly exacerbated the progression of CCl₄-induced hepatic injury as documented by serum levels of ALT (452.5±103.7 vs. 170.0±27.5U/L, p<0.05), AST (545.8±102 vs. 237.0±39.8U/L, p<0.05). Collagen deposition was dramatically increased in CCl₄+ImP-treated compared to CCl₄-treated mice as indicated by Masson’s staining and Sirius red staining. Gene expression of smooth muscle α-actin (α-SMA) (3.35±0.86 vs. 0.90±0.09), procollagen 1α1 (COL1α1) (2.72±0.58 vs. 1.00±0.16) and procollagen 3α1 (COL3α1) (2.94±0.78 vs. 0.98±0.013, p<0.05) was upregulated in CCl₄+ImP-treated mice. Additionally, liver showed more inflammation in CCl₄+ImP-treated mice, as demonstrated by enhanced infiltration of macrophages with CD11b immunofluorescence. To further analyze how ImP regulates macrophages in vitro, bone marrow-derived macrophages (BMDMs) were differentiated from WT mice. ImP increased expression of M1 polarization markers, iNOS (3.06±0.45 vs. 1.03±0.17, p<0.05), IL1β (1.82±0.09 vs. 0.98±0.04, p<0.005) and IL6 (3.17±0.67 vs. 1.03±0.19, p<0.05) in BMDMs following LPS treatment. Furthermore, ImP elevated γH2AX protein in CCl₄-induced mouse liver and mRNA expression of iNOS in LPS-stimulated primary hepatocytes. Based on Hoechst staining, percentage of apoptotic primary hepatocytes in the LPS+ImP group was increased compared to LPS control. The supernatant of hepatocytes treated with LPS+ImP further potentiated proinflammatory polarization in BMDM than only LPS-stimulated hepatocytes, as evidenced by increased mRNA levels of iNOS. The supernatant of BMDM derived from hepatocytes treated by LPS+ImP further facilitated hepatic stellate cell (HSC) activation.

Conclusion: Gut microbe-generated ImP potentiated fibrotic progression induced by CCl₄ intoxication in mice. Pronounced hepatocellular apoptosis, recruitment of inflammatory cells to damaged liver, release of pro-inflammatory cytokines (IL1β, IL6) may account for enhanced activation of HSCs into collagen type I-producing myofibroblast-like cells.