Study Of The Protective Effect Of Polygonatum Sibiricum Aqueous Extract On CCl₄-induced Chronic Liver Fibrosis In Mice Based On TLR4/MyD88/NF-κB Pathway

Objective:To analyze the bioactive component content and structural information of the main component polysaccharide of Polygonatum sibiricum aqueous extract and to explore the protective effect of PSAE against CCl₄-induced chronic liver fibrosis in BALB/C mice and its potential molecular mechanism.Methods:PSAE was obtained by hot water-assisted ultrasonic extraction of one steamed Jiuhua Huangjing.The content of bioactive components in PSAE was determined by a colorimetric method.Information on the monosaccharide composition and functional groups of the main bioactive components of PSP was analyzed by IC and FTIR.In animal experiments,sixty BALB/c mice were randomly divided into 6 groups of 10 mice each.The PSAE intervention group and the positive control group were given different doses of PSAE（0.2 g/kg,0.4 g/kg,0.8 g/kg）and SIL（0.2 g/kg）by gavage,while the rest of groups were given the same volume of saline by gavage.One week later,except for the control group,which was injected intraperitoneally with peanut oil,the remaining groups were injected intraperitoneally with 2 m L/kg of peanut oil containing 25%CCl₄twice a week for 4 weeks,and the whole process of gavage intervention was continued until the day before the mice were sampled.Changes in liver tissue structure and immune cell distribution were observed by HE staining,collagen distribution in liver tissue was observed by Masson staining and Sirius red staining.Serum levels of ALT and AST activity,HA and PIIINP,TNF-αand IL-6 were measured by ELISA.The expression levels of inflammatory IL-1β,INOS and TLR4/My D88/NF-κB pathway-related proteins and phosphorylated proteins in tissues were analyzed by immunohistochemistry and Western blot,and finally,the mRNA expression levels ofα-SMA,TGFβ1,Collagen IV and TLR4/My D88/NF-κB pathway-related genes in liver tissues were further analyzed by qPCR.Results:PSAE contained 64.28 g/100 g of polysaccharides,1.75 g/100 g of flavonoids,3.05 g/100 g of saponins and 0.17 g/100 g of polyphenols,indicating that PSAE mainly contained polysaccharides,and the polysaccharide was mainly composed of glucose,accounting for 89.9%.Combined with FTIR analysis,the molecule was found to exist mainly in the form ofβ-D-glucopyranose.The results of animal experiments showed that compared with the control group,the liver of mice in the model group underwent significant fibrotic injury,as evidenced by a significant increase in liver weight and liver index（P<0.0001）,a significant increase in serum ALT,AST,hyaluronic acid and type III procollagen peptide content（P<0.0001）,and a significant increase in the levels of inflammatory factors and intra-tissue cytokinesα-SMA and TGF-β1 in vivo（P<0.001,P<0.0001）,and the staining results showed that the structure of liver lobules and confluent area was not obvious,immune cell cell infiltration,and collagen was significantly increased.Compared with the model group,PSAE could reduce liver index（P<0.01）,alleviate hepatomegaly,lower serum ALT and AST activity（P<0.0001）,reduce the level of inflammatory factors in vivo（P<0.01,P<0.001）,inhibit the expression ofα-SMA,TGF-β1 and type IV collagen mRNA in liver tissue（P<0.05,P<0.01）,and reduce CCl₄-induced liver fibrosis damage.Immunohistochemistry,WB and qPCR results further confirmed that PSAE inhibited the expression of key proteins and mRNAs of TLR4/My D88/NF-κB signaling pathway.Conclusion:PSAE has the highest polysaccharide content,and structural analysis revealed that the molecules in the polysaccharide are mainly in the form ofβ-D-glucopyranose.The synergistic mechanism of action of multiple bioactive components in PSAE may be through the regulation of TLR4/Myd88/NF-κB pathway to inhibit the expression of hepatic inflammatory factors and cytokinesα-SMA and TGF-β1,reduce the activation of hepatic stellate cells,and exert a protective effect against CCl₄-induced liver fibrosis injury.This suggests that PSAE is a potential natural plant-based drug for the effective prevention of chronic liver fibrosis.