The Role Of "Gut Microbiota-endocannabinoid System" In The Prevention And Therpay Of Non-alcoholic Fatty Liver Disease By Resveratrol Supplementation

BackgroundNon-alcoholic fatty liver disease(NAFLD),including non-alcoholic simple fatty liver(NAFL),non-alcoholic steatohepatitis(NASH),fibrosis,hepatocirrhosis and hepatocellular carcinoma,is one of the most common types of chronic liver disease worldwide.The pathogenesis of NAFLD has not been entirely expounded and has been considered as a development from the "two-hit theory" to the "multiple-hit mode".The impaired gut barrier integrity and metabolic endotoxemia originating from gut microbial dysbiosis are responsible for the development of NAFLD.The “gut-liver” axis disorder(involving gut microbiome imbalance,gut-derived lipopolysaccharide(LPS)overgrowth,and alteration of mucosal permeability)was identified to be related to NAFLD progression.Moreover,LPS,derived from some gram-negative bacteria,can access the liver and even the systemic circulation through a dysfunctional intestinal barrier,triggering hepatitis and hepatic impairment.LPS regulates the intestinal permeability and inflammation through the toll-like receptor(TLR4)signaling pathway.As the understanding of “gut-liver” axis,intestinal mucosal barrier integrity can lead to a reduction of bacterial invasion and endotoxin translocation,which went by the name of "the first line of defence".In recent years,the researchers found that the endocannabinoid system(ECS)plays a key role in mediating the function of gut mucosal barrier,regulating the intestinal permeability and intestinal endotoxemia(IETM),which is closely associated with the development of NAFLD.The ECS consists of endocannabinoids,cannabinoid receptors(e.g.CB1 and CB2),as well as the relevant metabolic enzymes which modulate ligand biosynthesis and degradation.The ECS is not only comprises of the central nervous system but also the peripheral tissues,especially in liver and gut tissue.It has been demonstrated that ECS activity could be up-regulated or down-regulated by particular microbes(e.g.A.muciniphila),accompanied by an improvement of the intestinal mucosal barrier function and a reduction of IETM.It's indicated that "gut microbiota-ECS" plays a critical role in maintaining gut barrier integrity to prevent the progression of high-fat diet(HFD)-induced NAFLD.At present,no drug has been approved for the treatment of NAFLD.However,several studies indicate that genetic differences and lifestyle modifications,especially the inclusion of dietary supplements,may effect the progression of NAFLD.SIRT3 plays a crucial role in the progression of chronic metabolic diseases,such as NAFLD,diabetes and insulin resistance.SIRT3,an integral regulator of mitochondrial function,protect individuals from the development of NAFLD by regulating metabolism and energy balance.The mutant SIRT3-V208 I,which exhibits low enzyme efficiency,could offer partial explanations for why patients with rs11246020 have enhanced susceptibility to steatosis and NASH.SIRT3 knockout(SIRT3KO)mice fed a HFD have more hepatic steatosis,liver fibrosis and inflammation than wild-type(WT)mice.Despite advances in this field,the role of SIRT3 in the development of NAFLD remains obscure.To sum up,SIRT3 KO mice can be used as an outstanding animal model to simulate the pathogenesis of NAFLD,performed significantly liver steatosis and inflammation under the condition of HFD.Resveratrol(RSV)is a natural polyphenol present mainly in grapes,berries,and other plants.Several studies suggest that RSV is beneficial for the prevention and cure of multiple metabolic diseases,such as NASH.It's well established that RSV is primarily absorbed in the small intestine with high effectiveness.Nevertheless,it has an extremely poor bioavailability as a result of fast metabolism of the human body.Thereby,the dominant physiochemical benefits of RSV are quite impressive if we compare it to its low bioavailability in vivo.Recently,researchers have claimed that several polyphenols with low bioavailability probably act principally by reshaping the intestinal microflora.It has been discovered that a polyphenol-rich cranberry extract diminished the diet-induced metabolic syndrome in mice in an intestinal microflora-dependent manner.Furthermore,previously studies and our preliminary experiment demonstrated that RSV administration could significantly regulate the abundance of spe`cified intestinal bacteria in vivo,and regulate the expression of CB1 and CB2 in rat colon.Endocannabinoids can modulate the intestinal permeability and inflammation through CB1 and CB2.Thus,we hypothesized that RSV attenuates HFD-induced NAFLD by maintaining gut barrier function through regulation of the "gut microbiota-ECS".Our findings uncover a novel role of CB1 and CB2 in the prevention of NAFLD by dietary RSV supplementation.ObjectiveThe aim of this study was to identify the correlations among gut microbiota,ECS and SIRT3 gene in the progression of HFD-induced NAFLD.To investigate the ameliorative effects of RSV in a HFD-induced NAFLD model,focusing on the "gut microbiota-ECS",through the maintenance of gut barrier integrity and inhibition of intestinal inflammation.Methods(1)Male wild-type(WT)129 mice and SIRT3 knockout(SIRT3KO)mice were under a chow diet or HFD treatment for 18-weeks.The body weight change and liver index were observed,and the degree of hepatic steatosis,liver inflammatory cell infiltration,pathological changes and plasma biomarker concentration were evaluated among groups.The mRNA expression levels of key genes including carbohydrate metabolism and lipogenesis metabolism in liver were assessed by qRT-PCR.Intestinal permeability,the expression of tight junction proteins,gut inflammation status,plasma LPS and liver LPS was measured.The mRNA expression levels of CB1 and CB2 in liver and colon of mice were assessed.We analysed the intestinal microbiota by 16 S rRNA gene sequencing,PCoA and phylogenetic reconstruction of unobserved states(PICRUSt)analysis.The correlation heatmap and redundancy analysis(RDA)were used to find the potential correlations between the bacterial community structure and the physiological biochemistry factor,further identifying the correlations among gut microbiota,ECS and SIRT3 in the progression of HFD-induced NAFLD.(2)Male Sprague-Dawley(SD)rats were fed HFD with or without RSV for 6 weeks.For the depletion of gut microbiota test,the rats were subjected to HFD with or without RSV of 100 mg/kg·bw per day for 6 weeks,followed by an administration of broad-spectrum antibiotic(Abx)cocktail solution in water for 4 weeks.For the study of the ECS function in vivo,the rats were fed HFD with RSV of 100 mg/kg·bw/day for 6 weeks,followed by the administration with or without the CB1 agonist ACEA(1 mg/kg·day),the CB2 antagonist AM630(1 mg/kg·day),or equal volume of the dissolved solution(vehicle)containing DMSO,cremophor and saline intraperitoneally for an additional 4 weeks.The body weight and food consumption were recorded weekly.Feces and cecal contents were collected at the beginning and the end of the administration.After sacrificing them,the samples of blood,liver tissues,as well as distal colon were collected for biomedical analysis.The degree of hepatic steatosis,liver inflammatory cell infiltration,pathological changes and plasma biomarker concentration were evaluated among groups.The experiments regarding bacterial translocation,the mRNA expression of tight junction-associated genes,intestinal permeability,intestinal inflammation status,and plasma LPS levels were performed according to the indicated protocols.The mRNA expression levels of CB1,CB2,ECS metabolic enzymes DAGL?,FAAH,MAGL and NAPE-PLD in colon of rats were assessed.The intestinal microbiota were analysed by 16 S rRNA gene sequencing,PCA,LEfSe analysis,PICRUSt analysis,correlation heatmap and RDA.To further identifying the role of "gut microbiota-endocannabinoid system" in the prevention of NAFLD by RSV supplementation through the maintenance of gut barrier integrity and inhibition of intestinal.Results(1)The gut microbiota,ECS and SIRT3 play an important role in the progression of HFD-induced NAFLD.HFD resulted in a significantly increased hepatic steatosis and inflammation,gut microbial dysbiosis,impaired intestinal barrier function and increased CB1 expression,which were exacerbated in SIRT3 KO mice.It's implied that gut microbiota and ECS may become the essential intervention targets for the prevention and treatment of NAFLD.(1)HFD resulted in a significantly increased hepatic steatosis and inflammation,increased the levels of liver TG and T-CHO,increased the levels of plasma TG,T-CHO,ALT,AST,TNF-? and IL-6 in WT mice and SIRT3 KO mice.SIRT3 deficiency resulted in an impaired intestinal permeability(increased the intestinal permeability,decreased the expression of ZO-1,occludin and claudin1 in colon)and inflammation(increased TNF-?,IL-1? and IL-6,decreased IL-10 mRNA expression in colon)in HFD-fed mice.HFD facilitates intestinal microbiota dysbiosis in SIRT3 KO mice,with increased Desulfovibrio,Oscillibacter,Mucispirillum,and decreased Alloprevotella.SIRT3 KO HFD-fed mice was followed by an increased LPS into the circulation and dysregulated expressions of CB1 in colon and liver,which were significantly associated with the alterations of intestinal microbiota.(2)Desulfovibrio manifested a significantly positive correlation with plasma LPS and colon CB1 mRNA expression.Lachnospiraceae demonstrated a significantly positive correlation with colon ZO-1 and occludin mRNA expression.(2)"Gut microbiota-ECS" plays a crucial role in the prevention of HFD-induced NAFLD by resveratrol supplementation through maintaining gut barrier function.The ECS,particularly the expressions of CB1 and CB2,appears to play a crucial role in the anti-NAFLD effect of RSV by the maintenance of gut barrier integrity and inhibition of gut inflammation.(1)HFD caused increase in body weight,liver index,hepatic lipid accumulation,and inflammation,which was inhibited by RSV.Moreover,RSV led to a reduction of metabolic endotoxemia and colon inflammation(decreased TNF-?,IL-1? and IL-6,increased IL-10 mRNA expression in colon)in HFD-fed rats.This was indicated by a decrease in bacterial invasion and translocation along with up-regulation of the mRNA levels of occludin,ZO1,claudin1,and down-regulation of FAK,MyD88,and IRAK4 in the distal colon.RSV also attenuated gut microbial dysbiosis,with an increase in Akkermansia muciniphila,Ruminococcaceae and Lachnospiraceae,and a decrease in Desulfovibrio.(2)Microbiota depletion using a cocktail of antibiotics was sufficient to block RSV-induced reduction in intestinal permeability and gut inflammation.The Abx test block RSV-induced the altered mRNA expressions of CB1 and CB2 in the distal colon.(3)RSV inhibited HFD-induced elevation in the expression of CB1 mRNA and suppressed CB2 mRNA levels in the colon.The RSV-induced benefits regarding enhanced gut barrier integrity and reduced intestinal permeability were abrogated with a CB1 agonist,ACEA,whereas the inhibitory effect of RSV on the intestinal inflammation was abolished by a CB2 antagonist,AM630.(4)Desulfovibrio manifested a significantly positive correlation with intestinal permeability,in addition to plasma LPS and colon CB1 mRNA expression.The characteristic bacteria,including Akkermansia muciniphila,Ruminococcaceae,and Lachnospiraceae,demonstrated a significantly positive correlation with the cecal concentration of butyrate as well as the mRNA expressions of ZO1,occludin,and CB2 in the distal colon.ConclusionsThe gut microbiota,ECS and SIRT3 play an important role in the progression of HFD-induced NAFLD.HFD resulted in a significantly increased hepatic steatosis and inflammation,gut microbial dysbiosis,impaired intestinal barrier function and increased CB1 expression,which were exacerbated in SIRT3 KO mice.It's implied that gut microbiota and ECS may become the essential intervention targets for the prevention and treatment of NAFLD."Gut microbiota-ECS" plays a crucial role in the prevention of HFD-induced NAFLD by RSV supplement through maintaining gut barrier function.RSV attenuates HFD-induced NAFLD by maintaining gut barrier integrity and inhibiting gut inflammation through regulation of the "gut microbiota-ECS".The ECS,particularly the expressions of CB1 and CB2,appears to play a crucial role in the anti-NAFLD effect of RSV by the maintenance of gut barrier integrity and inhibition of gut inflammation.Ultimately,considering the fact that ECS imbalance,intestinal microbial dysbiosis,impaired intestinal barrier,as well as aggravated intestinal inflammation are frequently associated with chronic liver diseases,the effect of RSV on alleviating these conditions emphasizes the potential of RSV supplementation as a therapeutic strategy for preventing NAFLD.