The Effects Of Mussel Polysaccharide α-D-Glucan(MP-A) On Non-alcoholic Fatty Liver Disease Based On Gut Microbiota And Related Gut-liver Axis Signaling Pathways

Nonalcoholic fatty liver disease(NAFLD)is the major chronic liver disease in the world.,not only in Asian countries such as China,but also in most western developed countries,which is closely related to obesity,diabetes and other metabolic syndromes.Moreover,NAFLD may further develop into non-alcoholic steatohepatitis(NASH),liver fibrosis,and eventually progress into cirrhosis and hepatocellular carcinoma.However,the pathogenesis of NAFLD has not been well elucidated so far.Considering that intestinal lumen and liver are closely correlated by portal vein system,studies have confirmed that the gut microbiome plays a key role in the pathogenesis of NAFLD.In addition,diet can significantly regulate gut microbiome and its the metabolic pathways involved in the development of NAFLD,suggesting a close relationship among the gut,diet and liver.Reponse to different eating habits,the levels of intestinal metabolites such as ethanol,lipopolysaccharides(LPS),short-chain fatty acids(SCFAs)change significantly,directly or indirectly contributing to a series of pathological changes.This may provide a new strategy for the clinical prevention and treatment of NAFLD.Previously,it has been reported that mussel polysaccharides(MPs)extracted from mussel have various biological activities,such as immunity-regulating,blood lipids-regulating,anti-atherosclerosis,anti-oxidation,anti-tumor and so on.In the preliminary experiments,we isolated a new mussel polysaccharide "a-D-glucan"(MP-A)from Mytilus coruscus and found its effects on liver TG level,indicating that it may have alleviative effects on NAFLD.However,no report has been demonstrated any kind of MP in this respect.Therefore,this study aims to evaluate the effects of MP-A on NAFLD and its possible action mechanisms.We theorized that it may alleviate NAFLD by modulating gut microbiota and related gut-liver axis pathways.1 Hepatoprotective effects of MP-A on rats fed with high-fat diet(HFD)Objective:To evaluate the hepatoprotective effects of MP-A on NAFLD rats.Methods:NAFLD model rats were induced by high fat diet(HFD).42 rats were randomly divided into 7 groups,namely normal group,HFD group,MP-A(100 mg/kg)group,MP-A(300 mg/kg)group,MP-A(600 mg/kg)group and Xuezhikang(216 mg/kg)group.Except for the normal group,the other rats were fed with HFD.Meanwhile,body weight and food intake were measured weekly.From the 3rd week of HFD,the intervention groups were intragastrically administered,while the nonnal group and the model group were treated with the same volume of solvent by gavage.At the end of 8th week,all animals were sacrificed after blood collection.The liver weights,blood lipid levels,hepatic triglyceride(TG)levels and liver pathology of rats in each group were evaluated.Results:The results show that MP-A can significantly attenuated the rate of weight gain and blood lipid levels,which were positively correlated with the dose.Additionally,MP-A significantly reduced the relative liver weight and hepatic TG level in HFD rats,effectively alleviating lipid deposition in the liver of HFD rats.Combined with the macroscopic observation of gross specimens,we can further confirm that MP-A has mitigating effects on hepatic steatosis in HFD rats.Particularly,when MP-A was administered at a dose of 100 mg/kg,the TG level in the rat liver was comparable to that of Xuezhikang 216 mg/kg.Conclusions:MP-A has hepatoprotective effects on HFD rats positively correlated with the dosage.2 Regulating effects of MP-A on intestinal flora structure and abundance in NAFLD ratsObjective:To investigate the effects of MP-A on gut microbiota in NAFLD rats induced by HFD.Methods:NAFLD model rats were similarly induced by HFD.18 rats were randomly divided into three groups namely normal group,HFD group and MP-A(600 mg/kg)group.There were 6 rats in each group.Except for the normal group,the other rats were fed with HFD.Body weights and food intakes were recorded once a week.From the 3th week of HFD.MP-A intervention was carried out.The normal group and the model group were treated with the same volume of solvent by gavage.At the end of the 8th week,all animals were sacrificed after venous blood collection.The liver,portal vein blood and cecum contents were collected immediately.And cecum contents were used for macrogenome sequencing and multivariate analysis.Results:MP-A could observably slow down the body weight gain rates and relative liver weight.Also,MP-A could significantly decrease hepaticTG and blood lipids levels in HFD rats,and had no significant effect on the food intake of rats.A total of 511,812 effective reads(high-quality sequences)were obtained from 18 fecal samples(28,434 ± 8820 sequences per sample)through high-throughput sequencing and further delineated into 6115 OTUs at a similarity cut-off of 97%.Based on the OTUs statistical results,α and β diversities analysis were carried out and cluster analysis after species annotation was proceeded.The results of α and β diversities analysis showed that MP-A could maintain the basic ecological structure and distribution of microflora and improve the richness of microflora.Furthermore,from the phylum level cluster analysis,MP-A increased the abundance of Bacteroides reduced by HFD induction,and decrease the abundance of Firmicutes and Proteobacteria.At the genus level,63 genera were significantly altered by HFD induction,of which 26 were up-regulated and 37 were down-regulated.However,13 of the 63 genera were markedly changed by MP-A.Conclusion:MP-A can maintain the ecological equilibrium of gut microbiota in HFD rats by improving the distribution and abundance of microbiota.3 Regulating effects of MP-A on microbial metabolites and related "gut-liver"axis signal pathwaysObjective:To investigate the effects of MP-A on microbial metabolites and its signaling pathways related to "gut-liver" axis.Methods:The contents of the cecum collected as above were analyzed by gas chromatography to detect the concentration of SCFAs.Ethanol and LPS concentrations were detected.Meanwhile.TLR4,NF-κB p65,PPAR y,SREBP-lc in liver were detected and analyzed by RT-PCR and Western Blot.The levels of IL-1β IL-6 and TNF-a in liver were quantified by ELISA.Results:MP-A could significantly reduce the levels of ethanol and LPS in portal vein.Then,the TLR4 and NF-κB p65 levels in the liver were inhibited.Following the MP-A treatment,downstream inflammatory cytokines were significantly reduced.Additionally,the results showed that MP-A can also promote the production of SCFAs in the intestine.After transport to the liver,it can inhibit the expression and activity of PPAR y and SREBP-lc and thereby regulate intrahepatic lipid metabolism.Conclusions:MP-A has alleviative effects on NAFLD in HFD rats by regulating related "gut-liver" axis signal pathways.