Based On The "Gut-Liver Axis" Approach To Explore Effect And The Mechanism Of Radix Puerariae Thomsonii Polysaccharide Preventing And Treating Alcoholic Liver Disease

Objective Investigation of the protective effect and mechanism of Radix Puerariae thomsonii polysaccharide on liver injury in rats with alcoholic liver disease induced by gradient infusion of alcohol through the " gutliver axis".Methods 1.Hepatoprotective efficacy of Radix Puerariae thomsonii polysaccharide in alcoholic liver disease model rats 1.1.Animal grouping and drug administration A total of 60 SD rats(SPF grade)that were adaptively fed for one week were randomly divided into blank control group(control),model group(Model),positive group(BG)and Pueraria polysaccharide high,medium and low dose groups(GD-H,GD-M,GD-L),10 in each group.BG group was given 440 mg/kg Baogan Tablets,and Kudzuvine root polysaccharide high and low dose groups were intragastrically administered at 50,25,and 12.5 mg/kg.1.2.Establishment of chronic alcoholic liver disease(ALD)model in rats A gradient white wine gavage was used to establish the alcoholic liverdisease model.Firstly,7 ml/kg was used as the base modeling dose for gavage of 56° Hongxing Erpotou white wine,and then the dose was increased by 2 ml/kg every 2 weeks for a total of 8 weeks.The oral gavage was performed in two stages,each administration group was gavaged according to the above-mentioned doses,and the dummy control group and the model group were gavaged with the same amount of physiological saline instead.After 30 min of drug administration,the modeling was started according to the established dose gradient of white wine gavage,and the blank group was replaced by saline.Blood,liver tissue,ileum tissue,and feces were collected for the corresponding tests after the modeling.1.3.Effect of Radix Puerariae thomsonii polysaccharide on biochemical parameters related to liver injury in rats with chronic alcoholic liver injury The effects of Radix Puerariae thomsonii polysaccharide on liver injury biochemical indexes(AST,ALT,TG,TB,TP,ALB)in alcoholic liver disease model rats were detected by automatic biochemical analyzer,and the effects of Radix Puerariae thomsonii polysaccharide on liver fibrosis in chronic alcoholic liver injury model rats were detected by ELISA Kit.1.4.Histomorphological observation of liver Liver tissue sections from ALD model rats were stained with HE stain and observed to clarify the effect of Radix Puerariae thomsonii polysaccharide on liver histology in model rats with chronic alcoholic liver injury.Masson staining was used to stain the liver tissue sections of ALD model rats and to observe the effect of radix thomsonii puerariae polysaccharide on the fibrosis of liver tissue of rats in the model of alcoholic liver disease 2.Effects of Pueraria lobata polysaccharide on LPS and LPS-mediated liver inflammatory response in liver tissues of rats with chronic alcoholic liver disease model First,we tested Radix Puerariae thomsonii polymorphism for LPS levels and LPS-mediated pro-inflammatory cytokines(TNF-α,IL-1β and IL-18)in liver tissue from a rat model of chronic alcoholic liver disease using an ELISA kit.Effects demonstrated by Sugar.Western blot was used to detect the effect of Radix Puerariae thomsonii polysaccharide on LPS-mediated TLR4/NF-κB/NLRP3 signaling pathway in liver tissues of chronic alcoholic liver disease model rats and the effect of Radix Puerariae thomsonii polysaccharide on key proteins of pyroptosis in liver tissues of chronic alcoholic liver disease model rats.3.Effect of Radix Puerariae Thomsoni polysaccharide on gut mucosal barrier permeability and gut microbiota in chronic alcohol-induced liver-damaged rats HE staining was used to observe the effect of Radix Puerariae thomsonii polysaccharide on colonic histomorphology in rats with chronic alcoholic liver injury;immunohistochemical method was used to observe the effect of Radix Puerariae thomsonii polysaccharide on the expression levels of tight junction proteins ZO-1 and claudin-1 in colonic tissues of rats with chronic alcoholic liver injury;ELISA Kit was used to detect the effect of Radix Puerariae thomsonii polysaccharide on LPS levels in blood of rats with chronic alcoholic liver injury;Miseq high-throughput sequencing technology was used to detect the effect of Radix Puerariae thomsonii polysaccharide on intestinal microorganisms in rats with chronic alcoholic liver injury.High-throughput sequencing sequence analysis,annotation and evaluation of intestinal flora,analysis of intestinal microbial flora composition,Alpha diversity analysis Radix Puerariae thomsonii polysaccharide on intestinal microbial abundance and diversity changes in rats with chronic alcoholic liver injury model,Beta diversity analysis Radix Puerariae thomsonii polysaccharide on intestinal microbial colony composition diversity changes in rats with chronic alcoholic liver injury model,Radix Puerariae thomsonii polysaccharide on intestinal microbial community differences in rats with chronic alcoholic liver injury model,differential bacterial species screening between intestinal microbial groups and correlation analysis of intestinal flora and serum/liver biochemical indicators.Results 1.Radix Puerariae thomsonii polysaccharide can significantly reduce the biochemical indicators of liver injury and liver fibrosis caused by chronic alcoholic liver disease.Morphologically,Radix Puerariae thomsonii polysaccharide can ameliorate alcohol-induced structural degeneration of liver tissue and antagonize chronic alcohol-induced fibrosis of liver tissue.2.Radix Puerariae thomsonii polysaccharide can significantly decrease LPS levels and inhibit LPS-mediated production of pro-inflammatory factors in liver tissue of rats with chronic alcoholic liver disease.3.Radix Puerariae thomsonii polysaccharide can down-regulate LPSmediated TLR4/NF-κB/NLRP3 signaling pathway in ALD model rats.Following the Radix Puerariae thomsonii polysaccharide interventions,Compared with the model group,the TLR4 level of the treatment group was significantly reduced.Compared with blank group,NF-κB and NF-κB phosphorylation levels in ALD model group were significantly increased,and IκB phosphorylation level was significantly decreased.Following treatment with Radix Puerariae thomsonii polysaccharide,NF-κB and NF-κB phosphorylation were significantly inhibited in each treatment group compared with the model group.4.Radix Puerariae thomsonii polysaccharide can down-regulate key proteins of pyroptosis in liver tissue of ALD model rats.Compared to the blank group,caspase-1/GSDMD/NLRP3 levels were significantly increased in the ALD model group after pueraria polysaccharide treatment,and caspase-1/GSDMD/NLRP3 levels were significantly decreased in each treatment group.5.Radix Puerariae thomsonii polysaccharide can ameliorate reduced colonic permeability and increase expression of tight junction proteins ZO-1 and Claudin-1 in AD model rats.6.Detection of gut microbes revealed that the proportion of Ruminococcus in the model group was significantly reduced at the genus classification level and the proportion of gut flora was significantly increased in each treatment group.Actinomyces and Oscillospiraceae belong to Firmicutes and Staphylococcus.Compared with the blank group,the proportion of ALD model group was significantly increased,and the number of such flora was significantly decreased after low-dose Radix Puerariae thomsonii polysaccharide intervention.At the phylum classification level,the abundance ratio of Firmicutes and Bacteroidota in the intestine of rats in the ALD model group was significantly increased,and the abundance ratio of Actinobacteriota,Proteobacteria,and Patescibacteria was significantly decreased;after the treatment Radix Puerariae thomsonii polysaccharide,the abundance of Firmicutes and Proteoidota in each treatment group was significantly decreased,and the proportion of Proteobacteria and Patesciota was significantly increased.This suggests that Radix Puerariae thomsonii polysaccharide has a therapeutic effect on gut microbiota alterations in ALD model rats.Conclusion1.Hepatoprotective effect of Radix Puerariae thomsonii polysaccharide: Radix Puerariae thomsonii polysaccharide can improve biochemical index and decrease liver fibrosis index in alcoholic liver disease model rats.Morphologically,Radix Puerariae thomsonii polysaccharide inhibited liver fibrosis and necrosis in alcoholic liver disease model rats.2.Effect of Radix Puerariae thomsonii polysaccharide on LPS level in liver tissue and LPS-mediated hepatic inflammatory response: On the one hand,Radix Puerariae thomsonii polysaccharide can reduce LPS level in liver tissue,and also inhibit LPS-mediated expression of pro-inflammatory factors.On the other hand,Radix Puerariae thomsonii polysaccharide inhibits hepatic inflammatory reaction and pyroptosis by regulating the expression of TLR4/NF-κB pathwayrelated proteins and pyroptosis-related proteins in liver tissue.3.Radix Puerariae thomsonii polysaccharide plays a protective role in intestinal mucosal barrier damage caused by chronic repeated drinking,and can improve the diversity of intestinal flora.It reduces intestinal endocrine LPS and decreases LPS leakage into the liver via the intestine.Apparently,Radix Puerariae thomsonii polysaccharide can prevent alcoholic liver disease by modulating the gut-liver axis.