The Study Of Protective Effect And Mechanism Of Fucoidan On Mice With Alcoholic Liver Injury By Regulating Intestinal Microecology

Objective:Alcoholic liver disease(ALD)is a toxic liver damage caused by persistent excessive drinking,has attracted worldwide attention.It has been reported that ALD is associated with intestinal flora disorder and intestinal mucosal injury.Fucoidan can regulate the intestinal flora and improve the barrier function of the intestinal mucosa,which has become a hot spot in natural marine medicine research.Therefore,this experiment was to study the protective effect and mechanism of fucoidan on the alcoholic liver disease by regulating the intestinal microecology in mice.Methods:After an adaptation period of one week,eight-week-old male C57BL/6J mice were randomly divided into 5 groups(n=10): the normal control group,the ethanol model group,the diammonium glycyrrhizinate group(DG group,gavage: 200 mg/kg/d),the low-dose fucoidan group(F1 group,gavage: 150 mg/kg/d)and the high-dose fucoidan group(F2 group,gavage: 300 mg/kg/d).In the model group,DG group,F1 group and F2 group,50%(v/v)ethanol was given 8 m L/kg/d for 2 weeks and 12 m L/kg/d for 6 weeks to establish the animal model.After the last gavage,fresh feces,serum,liver and small intestine tissues of mice fasted for 12 hours were collected and stored at-80 ℃.The main indicators detected in this experiment: 1)Body weight was recorded weekly to analyze weight changes.2)The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected by automated biochemistry analyzer.3)Determination of endotoxin(LPS),D-lactic acid(D-LA)and diamine oxidase(DAO)activities by enzyme-linked immunosorbent assay(ELISA).4)Green fluorescent biotin was used to detect the permeability of ileum mucosal.5)HE staining was used to observe the pathological changes of liver in mice with alcoholic liver injury.6)Transmission electron microscopy(TEM)was used to observe the ultrastructural changes of the liver and small intestine.7)The expressions of ZO-1,Occludin and Claudin-1 were determined by western blot(WB)and immunofluorescence.8)The expressions of inflammatory factors were determined by ELISA.9)The structure of the intestinal flora was analyzed by 16 S r DNA gene high-throughput sequencing.Results:After 8 weeks of intervention,the results of HE staining and TEM observation showed that fucoidan could alleviate the pathological damage and cell structure damage of mice.At the same time,fucoidan could significantly reduce the expressions of IL-1β,IL-6 and TNF-α(P<0.05),and the anti-inflammatory effect of the F2 group was more significant.fucoidan could reverse the increase in ALT caused by alcohol exposure.The results of TEM showed that the fucoidan could improve the ultrastructure of ileum mucosa in mice;ELISA results showed that the level of DAO in the F1 group was12.19% lower than those in model group(P < 0.05),and the levels of DAO,D-LA and LPS in the F2 group were 21.26%,14.14% and 18.18% lower than those in model group(P < 0.05),respectively;WB results showed that the expression of ZO-1,Claudin-1 and Occludin in F1 and F2 groups were higher than those in model group(P<0.05).The above points indicated that fucoidan reduced the intestinal permeability of mice and alleviate endotoxemia by enhancing the expression of tight junction proteins,thus protecting intestinal microecology.16S rDNA high-throughput sequencing was used to analyze the structure of intestinal flora.β diversity analysis showed that there was a significant difference in the structure of gut flora between the control group,the model group and the fucoidan treatment group(R=0.366,P=0.001).α diversity analysis results showed that there was no significant difference in the diversity of intestinal flora between the model group and the fucoidan group.Heat map analysis showed that the distribution of intestinal flora in fucoidan group was similar to control group(P < 0.05).Analysis of the composition of gut flora at the phylum level showed that fucoidan reduced the abundance ratio of Firmicutes/Bacteroidetes(P < 0.05).At the genus level,fucoidan increased the abundance of Prevotella,and decreased the abundances of Paraprevotella,Romboutsia and Clostridium sensu stricto,which indicated that fucoidan could ameliorate ethanol-induced imbalance of the gut microbiota(P < 0.05).Conclusion:1.Fucoidan has a protective effect on liver damage in alcohol-exposed mice,and reduce the level of inflammation in mice.2.The mechanism of fucoidan in improving ALD may be related to regulating the balance of intestinal flora,improving the barrier function of intestinal mucosa and relieving endotoxemia.Reasonable supplementation of bioactive seaweed substances such as fucoidan may be a potential way to prevent ALD.