Improvement effect and mechanism of Xiaoyao San on metabolic dysfunction-associated fatty liver disease rats based on gut microbiota and TLR4/MyD88/NF-κB pathway
Objective To investigate the protective effects of Xiaoyao San(XYS)on metabolically-related fatty liver disease(MAFLD)induced by high-fat diet in rats and its underlying mechanism.Methods SD rats were randomly divided into five groups:control group,model group,phlorizin(PH,positive drug,5 mg·kg-1),XYS low-dose group(19 gk·g-1),and high-dose group(38 g·kg-1).The control group was fed a basal diet,while the remaining groups were fed a high-fat and high-fructose diet for 12 weeks.From week 13,the rats were ig administered the drugs once daily for four weeks.Serum triglycerides(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),aspartate aminotransferase(AST),and endotoxin(LPS)levels,and interleukin-6(IL-6),IL-1β,and tumor necrosis factor-α(TNF-α)in the liver were determined by kits.The liver,colon,jejunum,and ileum were stained with hematoxylin and eosin(HE)and scored,and the liver was stained with oil red O to detect lipid accumulation.The integrity of the intestinal mucosal barrier was evaluated by measuring the excretion of diethylene triamine pentaacetic acid(DTPA).Western blotting was used to detect the expression of Toll-like receptor 2(TLR2),Toll-like receptor 4(TLR4),MyD88,nuclear factor-κB(NF-κB),ZO-1,Claudin-1,Occuludin-1,and PV-1 proteins in colon tissue;immunohistochemistry was used to analyze the expression of TLR4,MyD88,and NF-κB proteins in colon and liver tissue;and immunofluorescence was used to detect the expression of ZO-1,Claudin-1,and E-cadherin proteins.16S rRNA gene sequencing was used to determine the changes in the types and structure of the intestinal microbiome.Results Compared with the model group,the levels of TG,TC,LDL-C,ALT,AST,and LPS in the serum of the XYS and PH groups were significantly lower(P＜0.05,0.01,0.001),and the levels of IL-1β,IL-6,and TNF-α in the liver were significantly lower(P＜0.01,0.001).The level of HDL-C in the serum of the XYS high-dose group was significantly higher(P＜0.01).The scores of liver,colon,ileum,jejunum,and liver pathological grades were significantly lower in the XYS group,and the area stained with oil red O in the liver was significantly lower(P＜0.01,0.001).The immunohistochemistry and Western blotting results showed that the expression of TLR2,TLR4,MyD88,and NF-κB proteins was significantly lower in the XYS and PH groups(P＜0.01,0.001).The excretion rate of DTPA in 24 h urine of the rats in the XYS high-dose group and PH group was significantly lower(P＜0.05).In the high-dose group of XYS and the PH group,the expression of ZO-1,Claudin-1 and Occludin-1 proteins was significantly increased(P＜0.001),while the expression of PV-1 protein was significantly decreased(P＜0.001).Immunofluorescence showed that the expression of ZO-1,Claudin-1 and E-cadherin was significantly increased in the high-dose group of XYS and the PH group.Compared with the model group,XYS increased the richness and diversity of the intestinal microbiome,reduced the relative abundance of harmful bacteria such as Ruminococcus and increased the relative abundance of beneficial bacteria such as Bacteroides.Conclusion XYS has an ameliorative effect on MAFLD rats induced by high-fat diet,which can be achieved by alleviating liver inflammation,regulating liver function indicators.The mechanism may be related to repairing the intestinal mucosal barrier,regulating intestinal microbiota,and regulating the TLR4/MyD88/NF-κB pathway.
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