Dihydromyricetin Ameliorates Metabolic Associated Fatty Liver Disease By Regulating Gut Barrier Integrity Through Gut Microbiota/ILC3 Pathway

BackgroundNon-alcoholic fatty liver disease(NAFLD)h affects more than 25%of the adult population worldwide,and its onset is gradually becoming younger.It has become the world’s leading chronic liver disease and public health problem,posing a significant burden on the healthcare system and socio-economic development of various countries.NAFLD is a complex,multifactorial disease closely related to metabolic syndrome,obesity,and type 2diabetes.With the continuous deepening of research on the pathogenesis,diagnosis,and treatment of NAFLD,NAFLD can no longer effectively reflect the current understanding of such diseases,and cannot fully include the existing liver diseases related to complex metabolic dysfunction.In 2020,an international panel consensus proposed a new definition of fatty liver,renaming NAFLD as metabolic associated fatty liver disease(MAFLD)and developing new diagnostic criteria.It is based on positive diagnostic criteria for fatty liver disease associated with metabolic dysfunction.The new definition can capture this complex multisystem metabolic disorder more accurately and help clinical doctors diagnose this kind of patient easier.In recent years,due to changes in diet and lifestyle,the incidence of MAFLD has also been increased rapidly in the Chinese population.MAFLD is a severe threat to human health.Despite some progress has been made in elucidating the pathogenesis of MAFLD,there are still many unresolved challenges remain.There are no approved drugs for the treatment of MAFLD(or NAFLD)currently.The pathogenesis of MAFLD is complex,and the"multiple parallel hits hypothesis"has become the leading model hypothesis to describe the pathogenesis of MAFLD.This hypothesis assumes that gut microbiota disorders,dietary factors,genetic factors,endoplasmic reticulum stress,and other signaling pathways are involved in the development of MAFLD.Recent studies have also confirmed that the gut microbiota and its metabolites are critical determinants in the development of MAFLD.Gut microbiota dysbiosis leads to impaired intestinal mucosal barrier function,altered intestinal permeability,and excessive production of intestinal-derived endotoxins,which enter the liver through the damaged intestinal mucosal barrier to induce the development of liver disease.The homeostasis and tolerance of the tissue at the intestinal mucosal barrier site are regulated by the gut microbiota and coordinated by the cells of the immune system,which strengthen the integrity of the epithelial barrier and dynamically crosstalk other lymphocytes and myeloid cells in the tissue microenvironment.Moreover,innate lymphoid cells(ILCs)are a critical component of the body’s innate immunity,which play an important role in maintaining tissue homeostasis,immunity and inflammation by responding rapidly to signals expressed by tissue-resident cells or inducing cytokines in the early stage of immune response.ILCs are divided into five subpopulations based on their development and function:natural killer cells,ILC1,ILC2,ILC3,and lymphoid tissue-inducing cells,of which ILC3 is the predominant subpopulation of ILCs in the gastrointestinal tract and is central to the maintenance of homeostasis and tolerance in the gastrointestinal tract.ILC3 mainly defends against pathogens by releasing effector cytokines,and is regulated by different factors such as diet,nervous system,circadian rhythm,microbiome,other immune cells,etc.Thus,gut microbiota and ILC3 play an essential role in maintaining the intestinal mucosal barrier and regulating the development of MAFLD through the"gut-liver"axis.Dihydromyricetin(DHM)is a flavonoid of the polyphenol family and is found in the natural plant vine tea in high concentrations.Previous studies have found that DHM has various antibacterial,anti-inflammatory,antioxidant,and anti-cancer properties and acts as a potential preventive or therapeutic agent in the prevention and treatment of various diseases such as diabetes,atherosclerosis,and osteoporosis.Therefore,DHM may be an ideal phytochemical for preventing the development of MAFLD.DHM was found to be rapidly distributed to various tissues after gavage administration in rats.However,due to its low bioavailability,it is present mainly in the gastrointestinal tract and excreted in the feces.DHM may be involved in regulating gut microbiota in the colon and may be involved in regulating ILC3 cell immune function,thereby maintaining the homeostasis of the intestinal mucosal barrier to prevent the development of MAFLD.Therefore,this study focuses on the intestinal mucosal barrier function and explores the role and mechanism of DHM in improving the development of MAFLD through the regulation of intestinal mucosal barrier function by gut microbiota/ILC3 pathway,aiming to provide a new target for the prevention and treatment of MAFLD,as well as a new experimental basis for the use of DHM in the prevention and treatment of chronic metabolic diseases.ObjectiveUsing high-fat diet(HFD)to induce C57BL/6J mice to establish an animal model of MAFLD and an indirect co-culture system for MNK3/Caco-2 cells in vitro,combined with flow cytometry,transmission electron microscopy,broad-spectrum antibiotic intervention,16S r RNA high-throughput sequencing and other research methods,to explore the role of gut microbiota and ILC3 cells in the occurrence and development of HFD-induced MAFLD,aiming to elucidate the role of gut microbiota/ILC3 in DHM regulation of intestinal mucosal barrier function to improve HFD-induced MAFLD.Methods1.C57BL/6J mice were fed with HFD for 12 weeks to establish the MAFLD animal model,and DHM was added to the diet for intervention by free feeding.Changes in body mass and food intake were monitored in all groups;serological and pathological parameters,level of fasting blood glucose and oral glucose tolerance,level of liver lipid accumulation,and damage of liver inflammation were compared in all groups to evaluate the effect of DHM on the development of MAFLD in HFD-fed mice.The intestinal permeability,serum endotoxin level,intestinal ultrastructure,intestinal inflammatory injury,antimicrobial peptide,and tight junction protein levels,as well as the proportion of ILC3 cells in the lamina propria of the colon and the level of IL-22 production in each group of mice,were examined to investigate the ameliorative effect of DHM on the intestinal mucosal barrier during the development of MAFLD in HFD-fed mice.2.To evaluate the effect of DHM on the diversity of the gut microbiota during the development of MAFLD in HFD-fed mice by 16S r RNA high-throughput sequencing,Alpha diversity analysis,Beta diversity analysis,PCo A analysis and LEf Se analysis of the structural composition and functional differences of the gut microbiota.The content of various short-chain fatty acids in the cecum contents was analyzed to reveal the modulating effect of DHM on the intestinal short-chain fatty acid profile in HFD-induced mice.The intestinal permeability,serum endotoxin level,intestinal inflammatory factor level,and tight junction protein expression level of mice were detected and the intestinal ultrastructure was observed by transmission electron microscopy after broad-spectrum antibiotic intervention,to clarify the vital role of gut microbiota in the improvement of intestinal mucosal barrier function by DHM.3.Palmitic acid(PA)was used to induce intestinal epithelial cell line Caco-2 cells.And the expression of cell viability,inflammatory factors(IL-1β,IL-6,and TNF-α),and tight junction proteins(ZO-1 and Occludin)were measured.An indirect co-culture model was established using the ILC3 in vitro cell line MNK3 cells and intestinal epithelial cells line Caco-2 to investigate the effect of conditioned cultures generated after DHM stimulation of MNK3 cells on PA-induced Caco-2 cell viability,inflammatory factors,and tight junction protein expression levels.The effect of DHM on the level of IL-22 produced by MNK3 was examined to reveal the mechanism of DHM in inhibiting PA-induced Caco-2 cell injury by stimulating IL-22 production by MNK3 cells.Results1.DHM effectively delayed the development of HFD-induced MAFLD in mice and inhibited the impairment of intestinal mucosal barrier function in HFD-induced mice.(1)After DHM intervention,the increase of body weight of mice slowed down,the impaired state of oral glucose tolerance improved,the degree of liver lipid accumulation and inflammation were improved,and the levels of TG and TC in serum were reduced,suggesting that the occurrence and development of HFD-induced MAFLD in mice was effectively improved after DHM intervention.(2)DHM intervention reduced intestinal permeability,decreased serum levels of intestinal-derived endotoxin LPS,decreased levels of colonic pro-inflammatory factors,increased levels of antimicrobial peptides(Reg IIIβ,Reg IIIγ,and Muc2),and expression of tight junction proteins(ZO-1 and Occludin),suggesting that DHM could improve the barrier function of the intestinal mucosa(including epithelial and chemical barriers)in HFD-induced mice.(3)DHM intervention also increased the proportion of ILC3 cells(CD45~+CD90.2~+CD3~-RORγt~+)and IL-22~+ILC3(CD45~+CD90.2~+CD3~-RORγt~+IL-22~+)cells in the lamina propria of the colon.It promoted the expression level of IL-22 in ILC3 cells,suggesting that DHM could enhance the intestinal mucosal immune barrier function in HFD-induced mice.2.Gut microbiota and ILC3 cells in the lamina propria of intestinal mucosa played essential roles in DHM to ameliorate HFD-induced intestinal mucosal barrier damage.(1)DHM modulated HFD-induced changes in the structural diversity of the gut microbiota and intestinal short-chain fatty acid content in mice;DHM intervention inhibited HFD-induced increases in the relative abundance of Firmicutes and decreases in the relative abundance of Bacteroidota;DHM improved the HFD-induced reduction in the relative abundance of Lachnospiraceae and Oscillibacter.(2)DHM regulated HFD-induced intestinal mucosal barrier function through gut microbiota.After broad-spectrum antibiotic intervention and cleared most of the gut microbiota in mice,the protective effect of DHM on the HFD-induced intestinal mucosal barrier decreased,as evidenced by increased intestinal permeability,increased serum LPS levels,intestinal ultrastructural damage,increased expression levels of colonic inflammatory factors(IL-1β,IL-6,TNF-α),decreased expression levels of tight junction proteins(ZO-1 and Occludin).The proportion of ILC3 cells and IL-22 in the lamina propria of the colon was significantly reduced.3.DHM attenuated PA-induced intestinal epithelial cell injury by activating IL-22expression in MNK3 cells.(1)PA induced an increase in the expression levels of inflammatory factors(IL-1β,IL-6,TNF-α)and a decrease in the expression levels of tight junction proteins(ZO-1 and Occludin)in Caco-2 cells in vitro.(2)By the indirect co-culture system of MNK3 and Caco-2 cells,it was found that the conditional culture medium obtained after DHM stimulation of MNK3 cells could inhibit the increase in the expression of inflammatory factors and the decrease in the expression of tight junction proteins in Caco-2 cells induced by PA.(3)DHM promoted the production of IL-22 by MNK3 cells in vitro.ConclusionDihydromyricetin may improve the structural diversity of gut microbiota,improve the proportion and function of ILC3 cells,inhibit intestinal epithelial cell damage,reduce intestinal mucosal permeability,and maintain intestinal barrier function,and ameliorate hepatic lipid accumulation and inflammatory level,thereby delay the development of MAFLD induced by HFD.This study provides a new experimental basis for the use of DHM in the prevention and treatment of chronic metabolic diseases.