Investigation Of The Anti-HF Mechanism Of Taraxacum Mongo Licum Polysaccharide With Astragalus Polysaccharide Via Gut–liver Axis

Objective The paper investigates the pharmacodynamic activity of Taraxacum mongolicum polysaccharide with Astragalus polysaccharide for hepatic fibrosis（HF）and the synergistic effect of their combination through in vivo pharmacological experiments,explores the anti-HF effect of combined polysaccharide based on the"gut-liver axis",and provides a theoretical reference for the development of combined polysaccharide to resist HF.Method（1）The crude Taraxacum mongolicum polysaccharide and crude Astragalus polysaccharide were extracted by water extraction and alcohol precipitation.The two crude polysaccharides were deproteinized by the trichloroacetic acid method,and the purified Taraxacum mongolicum polysaccharide（TMP）and Astragalus polysaccharide（APS）were obtained after decolorization with macroporous adsorption resins;High performance gel permeation chromatography（HPGPC）and pre-column PMP derivatization were used to determine the molecular weight and monosaccharide composition of TMP and APS,respectively;Modern analytical instruments were used to detect their physical and chemical properties.（2）The CCl₄-induced HF mouse model was used to evaluate the pharmacodynamics of TMP+APS through the changes of body weight and liver index and pathological observation.（3）ELISA method was used to detect ALT,AST,HA,PCⅢ,ColⅣand other indicators in mouse serum,as well as the content of inflammatory factors LPS,IL-6 and TNF-αin mouse liver tissue and s Ig A in ileum tissue;Western blot and RT-PCR techniques were used to detect the expression of TLR4,My D88,NF-κB p65protein and m RNA in mouse liver;High performance liquid chromatography（HPLC）was used to determine the content of short chain fatty acids（SCFAs）in mouse feces;16S r DNA amplicon sequencing technology was used to detect the structural changes of mouse intestinal flora.Results（1）The extraction rates of crude Taraxacum mongolicum polysaccharide and crude Astragalus polysaccharide were 8.32±0.07%and 4.96±0.12%,respectively;The total sugar contents of TMP and APS were 69.72±0.45%and 79.01±0.81%,respectively.The molecular weight distribution of TMP was between 2.30×10⁶Da and 3.26×10³Da,concentrated in two components,with molecular weights of 4.85×10⁵Da and 7.08×10⁴Da,respectively,and was composed of Man,Rha,Gal A,Gal,Xyl,Fuc,with a molar ratio of1.25:1.75:2.83:8.33:4.83:1;APS was distributed in two components with molecular weights of 8.18×10⁶Da and 2.21×10⁴Da,respectively.It consisted of Man,Rib,Rha,Glc A,Gal A,Glc,Gal,Xyl,and Ara,and the molar ratio was 1:1.36:1.43:2.93:2.64:2.08:4.21:3.36:7.14;The results of FI-IR method showed that both TMP and ASP had characteristic absorption peaks of polysaccharides;The appearance characteristics of polysaccharides were observed by SEM,and it was found that TMP and APS had different morphologies;XRD results showed that TMP and APS had no obvious sharp peaks,indicating that these two polysaccharides had low crystallinity.（2）The results of mouse body weight and liver index showed that TMP+APS could alleviate the weight loss of HF mice induced by CCl₄and significantly reduce the liver index（P<0.05）;The pathological observation results showed that TMP+APS could reduce the damage of liver and ileum tissue cells in HF mice,lower the deposition of collagen fibers in HF mice,and significantly reduce the proportion of fibrosis（P<0.05）,and the effects were better than those of the TMP group and APS group.（3）Compared with the Mod group,the levels of ALT,AST,PCIII,Co IIIV and HA in mouse serum in the TMP+APS group were significantly decreased（P<0.05）,and the levels of inflammatory factors LPS,IL-6 and TNF-αin the liver tissue were significantly decreased（P<0.05）;The content of s Ig A in ileum tissue was significantly increased（P<0.05）;The protein and m RNA expression of TLR4,My D88,NF-κB p65 were significantly decreased（P<0.05）;The expression of SCFAs increased in TMP+APS group;The results of 16S r DNA analysis showed that the richness and diversity of intestinal flora of mice in the TMP+APS group increased,which could regulate the intestinal flora,reduce the abundance of Firmicutes,and increase the relative abundance of Bacteroidetes.Conclusions The molecular weight range,monosaccharide composition,molar ratio and other parameters,as well as the infrared spectrum characteristic peak,morphology and appearance characteristics of TMP and APS were determined.The combination of TMP and APS can synergistically exert anti-HF effect.The anti-HF mechanism of TMP+APS may be achieved through the immune system of the"gut-liver axis"and the TLR4/My D88/NF-κB signaling pathway.By regulating the composition of the intestinal flora,it affects the richness of each bacterial phylum,promotes the expression of SCFAs,inhibits the expression of TLR4/My D88/NF-κB signaling pathway,reduces the overexpression of inflammatory factors in vivo,restores the intestinal mucosal barrier,and relieves liver fibrosis.