Protective Effects Of Citrus Flavonoids On Non-alcohol Fatty Liver Disease Based On Gut Microbiota And Liver Metabolomics

The high prevalence of Non-alcoholic fatty liver disease(NAFLD)worldwide has attracted great attention.Unhealthy diet is one of the important causes of NAFLD.High-fat and high fructose diet(HFFD)leads to disorders of lipid metabolism,lipid accumulation in liver and destruction of the "gut-liver axis",resulting in fatty liver disease.Hesperetin(HES)and naringenin(NAR)are the main citrus flavonoids,and previous studies have demonstrated that HES and NAR have a variety of anti-NAFLD biological activities,including anti-obesity,anti-inflammatory,lipid-lowering,etc.At present,knowledge on the effects of HES and NAR on the gut microbiota and hepatic metabolites are still limited,and whether there are differentiated effects of the two citrus flavonoids on the protection of NAFLD has not been reported.Targeting the protective effects and mechanisms of HES and NAR on NAFLD induced by HFFD,the main findings are as follows:(1)NAFLD model was established in mice induced by a HFFD for 9 weeks.The experiment was divided into normal control group(NCD),model control group(HFFD),Hesperetin intervention group(HFFD+HES,0.4g/kg·BW),and naringenin intervention group(HFFD+NAR,0.4g/kg·BW),the experiment was lasted 9 weeks.The results showed that HES and NAR interventions markedly reduced body weight,body weight gain,liver index,epididymal fat index,hepatic AST,ALT levels,total cholesterol and triglycerides in serum and liver and hepatic inflammatory factors levels(P<0.05).Hepatic pathological damages were also improved.At the same time,the hepatic levels of bacteria derived lipopolysaccharide(LPS)and intestinal permeability in the HES and NAR groups were significantly reduced(P<0.05).Comparation of the effects of the two citrus flavonoids on NAFLD-related injury factors showed that,the inhibitory effects of HES on serum TC and serum LPS levels were significantly prior to that of NAR(P<0.05),while the inhibitory effects of NAR on the epididymis fat index and TNF-α level was significantly prior to that of HES(P<0.05).These results suggested that HES and NAR can be used in dietary interventions to alleviate NAFLD.Meanwhile,HES and NAR have different emphases on the protective effects of NAFLD.(2)In order to explore the characteristics and potential differences of the gut microbiota of NAFLD after intervention with HES and NAR,and to reveal the correlations between the gut microbiota and NAFLD-related injury after the interventions,this study performed high-throughput sequencing of 16 S r RNA gene in mice feces.The results showed that HFFD significantly improved the ratio of Phylum Firmicutes and Bacteroides(P<0.05)compared with the NCD group.NAR interventions significantly altered gut microbial composition of HFFD,while HES interventions failed to reach such effects.Compared with the HFFD group,the NAR intervention significantly increased the diversity of gut microbiota,restored the ratio of Firmicutes/Bacteroidetes to NCD levels,improved the relative abundance of Bacteroidaceae 、 Muribaculaceae 、 Oscillospiraceae 、 Rikenellaceae and Lachnospiraceae,and decreased the relative abundance of Erysipelotrichaceae and Lactobacillaceae(P<0.05)."Pearson" correlation analysis showed that NAFLD-related injury factors(including body weight,body weight gain,serum and hepatic TC,TG levels,serum AST,ALT levels,hepatic inflammation levels)were positively correlated with Erysipelotrichaceae(P<0.05)and negatively correlated with Muribaculaceae,Oscillospiraceae,Lachnospiraceae,and Rikenellaceae(P<0.05).These findings suggested that NAR,rather than HES,can improve intestinal flora disorders caused by HFFD and alleviate NAFLD in mice.Meanwhile,the NAR-induced microbial changes play an important role in improving obesity,serum and hepatic lipid levels,serum AST,ALT levels,and hepatic inflammation in NAFLD mice.(3)The HPLC-Q-TOF/MS method was used to conduct metabolomics analysis of mice livers to explore the changes of liver metabolites.Orthogonal partial least squares discriminant analysis(OPLS-DA)analysis showed that HFFD significantly changed the hepatic metabolite phenotype,and the differential metabolites mainly involved changes in fatty acid synthesis,fatty acid oxidation,amino acid metabolism and glucose metabolism.Compared to the HFFD group,both HES and NAR interventions reduced oleic acid levels(P<0.05)and increased L-serine levels(P<0.05).There are significant differences in HES and NAR according to OPLS-DA analysis.HES interventions reduced maltotriose levels and increased glycine and tryptophan levels.The involved metabolism pathways include carbohydrate digestion and absorption,tryptophan metabolism and serine,glycine,and threonine metabolism.NAR interventions reduced levels of raffinose,galactose,and phenylalanine,and increased levels of L-acetyl carnitine(P<0.05),The involved metabolism pathways include galactose metabolism,phenylalanine metabolism,and β oxidation.Correlation analysis found that oleic acid and D-glucose in the liver were significantly positively correlated with Erysipelotrichaceae(P<0.05)and significantly negatively correlated with Erysipelotrichaceae,Muribaculaceae,Lachnospiraceae,and Rikenellaceae(P<0.05).L-Carnitine,dimethylglycine,glutaric acid,L-serine and glycine were significantly positively correlated with Muribaculaceae,Lachnospiraceae,and Rikenellaceae(P<0.05)and Erysipelotrichaceae(P<0.05).These results showed that HES and NAR intervention regulated liver fatty acid synthesis,fatty acid oxidation,amino acid metabolism and glucose metabolism in NAFLD mice,thus alleviating liver metabolic disorders,but there are differences in specific metabolites and metabolic pathways.In summary,both HES and NAR have protective effects on HFFD-induced mice NAFLD,and there are differences between the two citrus flavonoids.Among the improvements of NAFLD-related damage factors,HES and NAR interventions improve the growth status,biochemical indexes,lipid levels and liver injury of NAFLD mice.HES focuses on reducing the elevated serum lipid levels and intestinal permeability while NAR focuses on reducing epididymal fat index and liver inflammatory factor level;In terms of improvements on the gut microbiota of NAFLD mice,NAR intervention significantly changed the composition of intestinal microbial composition,while HES has no significant effect;On the effects of the hepatic metabolic profile of NAFLD mice,HES modulated tryptophan metabolism and serine,glycine,and threonine metabolism to exert a protective effect on NAFLD.NAR modulated galactose metabolism,phenylalanine metabolism,carbohydrate digestion and absorption,and β oxidation to exert a protective effect on NAFLD.