目的 表征桑黄水部位多糖（water-eluted fraction of Sanghuangporus vaninii polysaccharides，SHP-W）的结构特征，并探讨其调节肠道菌群改善四氯化碳（carbon tetrachloride，CCl₄）诱导的小鼠肝纤维化的作用机制。方法 采用水提醇沉、弱阴离子交换色谱法从桑黄Sanghuangporus vaninii子实体中制备得到SHP-W。采用苯酚硫酸法、间羟基联苯法和BCA法测定SHP-W的总糖、糖醛酸和蛋白含量，通过高效尺寸排阻色谱法（high performance size exclusion chromatography，HPSEC）、傅里叶红外光谱法（fourier transform infrared spectrophotometer，FT-IR）、离子色谱法（ion chromatography，IC）和甲基化结合气相色谱-质谱联用（gas chromatography and mass spectrometry，GC-MS）等对SHP-W的结构特征进行表征。C57BL/6J小鼠ip 15% CCl₄-橄榄油溶液6周构建肝纤维化模型，给予SHP-W（25、50、100 mg/kg）或索拉非尼（10 mg/kg）干预3周，采用苏木素-伊红（hematoxylin-eosin，HE）、天狼星红和Masson染色观察小鼠肝脏的病理形态学改变；采用全自动生化分析仪分析血清丙氨酸氨基转移酶（alanine aminotransferase，ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase，AST）、总胆汁酸（total bile acids，TBA）、总胆红素（total bilirubin，TBIL）和直接胆红素（direct bilirubin，DBIL）水平；采用免疫组化法检测肝组织I型胶原（collagen-I，Col-I）和α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）的表达；采用Western blotting检测肝组织Col-I、α-SMA和转化生长因子-β1（transforming growth factor-β1，TGF-β1）的蛋白表达；采用16S rDNA测序分析肠道菌群，并采用抗生素干扰验证菌群作用。结果 SHP-W是由2个不同相对分子质量（1.818×10⁴、2.040×10³）的多糖组成的部位多糖，其总糖质量分数为（88.27±3.76）%，不含糖醛酸和蛋白质。单糖组成结果显示SHP-W由半乳糖、甘露糖和岩藻糖以及葡萄糖组成，物质的量比为34.6∶23.0∶22.7∶19.7，同时含有3-O-甲基半乳糖；甲基化分析显示SHP-W由1,3,6-Glcp等14种糖苷键组成。动物实验结果显示，与对照组比较，模型组小鼠血清AST、ALT、TBIL、DBIL和TBA水平显著升高（P＜0.001），肝细胞索排列紊乱，炎性细胞浸润明显，出现大量的纤维组织增生，肝组织中Col-I、α-SMA和TGF-β1蛋白表达显著上调（P＜0.05、0.001）；与模型组比较，各给药组小鼠肝组织的假小叶及汇管区胶原纤维沉积均有不同程度改善，血清肝功能水平显著降低（P＜0.05、0.01、0.001），肝组织Col-I、α-SMA和TGF-β1蛋白表达下调（P＜0.05、0.01、0.001）。抗生素干扰后，SHP-W改善肝纤维化的效果被显著抑制。结论 从桑黄子实体中制备得到SHP-W，糖醛酸含量测定、红外光谱以及单糖组成显示其为中性多糖。SHP-W可显著改善血清肝功能、抑制HSCs活化、减少胶原纤维沉积从而改善CCl₄诱导的小鼠肝纤维化，且其抗肝纤维化作用依赖于对肠道微生物的调节。

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.

R285.5

浙江省自然科学基金项目（ZCLY24H2801）；浙江中医药大学“5151远志人才工程”（112123A12201/001/004/019）；浙江中医药大学科研项目（2023RCZXZK20）