Objective To analyze the structural characteristics of water-eluted fraction of Sanghuangporus vaninii polysaccharides (SHP-W) and explore the mechanism of SHP-W on improving carbon tetrachloride (CCl₄)-induced liver fibrosis in mice by regulating gut microbiota. Methods SHP-W was extracted from S. vaninii using water extraction and ethanol precipitation, followed by purification through weak anion-exchange chromatography. The total sugar, uronic acid and protein contents of SHP-W were determined by phenol sulfuric acid method, m-hydroxybiphenyl method and BCA method. The structural characterization of SHP-W was carried out by high performance size exclusion chromatography (HPSEC), Fourier transform infrared spectrophotometer (FT-IR), ion chromatography (IC) and methylation combined gas chromatography-mass spectrometry (GC-MS). C57BL/6J mice were prepared with a 15% CCl₄ olive oil solution via ip for six weeks to establish a liver fibrosis model. SHP-W (25, 50, 100 mg/kg) or sorafenib (10 mg/kg) were administered for three weeks, and the pathological morphological changes in liver tissues were observed using hematoxylin-eosin (HE), sirius red and Masson staining. A fully automated biochemical analyzer was used to analyze the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bile acids (TBA), total bilirubin (TBIL) and direct bilirubin (DBIL) in serum. Immunohistochemistry was used to detect the expressions of collagen-I (Col-I) and α-smooth muscle actin (α-SMA) in liver tissue. Western blotting was used to detect the expressions of Col-I, α-SMA and transforming growth factor-β1 (TGF-β1) proteins in liver tissue. 16S rDNA sequencing was used to analyze the gut microbiota, and antibiotic interference was used to verify the role of the microbiota. Results SHP-W was a refined polysaccharide composed of two polysaccharides with different relative molecular weights (1.818 × 10⁴, 2.040 × 10³), with a total sugar content of (88.27 ± 3.76)%, and without uronic acid and protein. The monosaccharide composition results showed that SHP-W was composed of galactose, mannose, fucose and glucose, with a molar ratio of 34.6∶23.0∶22.7∶19.7, and also contained 3-O-methylgalactose. Methylation analysis showed that SHP-W was composed of 14 glycosidic bonds, including 1,3,6-Glcp.The animal experiment results showed that compared with control group, the levels of AST, ALT, TBIL, DBIL and TBA in serum of mice in model group were significantly increased (P < 0.001), the arrangement of liver cell cords was disordered, inflammatory cell infiltration was obvious, and a large amount of fibrous tissue proliferation was appeared, the expressions of Col-I, α-SMA and TGF-β1 protein in liver tissue was significantly up-regulated (P < 0.05, 0.001). Compared with model group, the collagen fiber deposition in pseudo lobules and portal area of liver tissue of mice in each treatment group was improved to varying degrees, and the serum liver function level was significantly reduced (P < 0.05, 0.01, 0.001), the expressions of Col-I, α-SMA and TGF-β1 protein in liver tissue was down-regulated (P < 0.05, 0.01, 0.001). After antibiotic interference, the effect of SHP-W on improving liver fibrosis was significantly inhibited. Conclusion SHP-W were prepared from S. vaninii fruiting bodies, the determination of uronic acid content, infrared spectroscopy and monosaccharide composition showed that SHP-W were neutral polysaccharide. SHP-W could significantly improve serum liver function, inhibit HSCs activation, reduce collagen fiber deposition, and thus improve CCl₄-induced liver fibrosis in mice. Its anti-fibrotic effect depends on the regulation of gut microbiota.

neutral polysaccharides from Sanghuangporus vaninii (Ljub) L. W. Zhou et Y. C. Dai  /  structural characteristics  /  monosaccharide composition  /  liver fibrosis  /  gut microbiota  /  activation of hepatic stellate cells  /  collagen fiber deposition