The Mechanism Underlying The Regulation Of Gut Microbiota And Prevention Of Acute Alcoholic Liver Injury In Mice By Lycium Barbarum L.(Goji)

The liver and gut have a bidirectional relationship in function and metabolites,which is called“gut-liver axis”.Binge drinking can cause acute liver injury.Dietary intervention is an effective and healthy way to shape the composition of the gut microbiota and influence host metabolism,which is also a reliable strategy to prevent the onset and progression of alcoholic liver injury.The present study showed that Lycium barbarumL.(Goji)has beneficial effects on liver health,which may be due to antioxidant,anti-inflammatory as well as prebiotic properties.However,studies of Goji on the liver,gut microbiome,metabolites and their interactions are still very limited.Here,we evaluated the effect of Goji on the alcohol induced acute liver injury in mice by using RNA-Seq,16 S rRNA gene sequencing,metagenomic sequencing and cecal untargeted metabolomics,and performed antibiotic clearance treatment and fecal transplantation experiments to discover that the changes of gut microbiota induced by Goji feeding are important factors for its functions.Meanwhile,study has also found that the Goji-derived exosome like Nanovesicles(ELNa)also have the effects of intervening gut microtiota and preventing acute liver injury.The main results are as follows:(1)Goji improved the pathological symptoms of alcohol induced acute liver injury in mice,which was closely related to maintaining intestinal barrier function and regulating intestinal microecology.Goji can significantly alleviate the inflammation,oxidative stress injury induced by alcohol of mice,such as reducing the levels of ALT,AST,inflammatory cytokines in the serum and endotoxin(LPS)in the liver tissue,increasing the levels of GSH and GST in liver tissue,and maintaining the barrier function of colonic mucus layer.16 S rRNA gene sequencing of gut microbiota found that Goji significantly increased the relative abundance of microorganisms related to the production of short chain fatty acids,such as Lachnospiraceae＿NK4A136＿group,Roseburia,Bifidobacterium,Akkermansia,Ruminococcaceae＿UCG-014.Validation analysis confirmed that,compared with the model group,Goji increased the levels of acetic acid,butyric acid and isovaleric acid in the caecum of mice.Untargeted Metabolomics of the cecum showed that Goji significantly increased tryptophan metabolites related to intestinal barrier function,such as tryptophan,5-hydroxy-L-tryptophan,serotonin,indole,indolelactate,and indoleacetaldehyde.Furthermore,liverRNA Seq results showed that the molecular mechanism of Goji in preventing acute liver injury may be related to the MAPK,Fox O and TNF signaling pathways.(2)Goji plays a preventive role in alcohol induced acute liver injury mice by interfering with gut microbiota.Based on the antibiotic experiment and fecal microbiota transplantation(FMT),it is clarified that the microorganisms such as Akkermansia,Rikenella,Clostridiales＿vadin BB60＿group,and Ruminiclostridium Goji may play a key role in the Goji improvement of acute liver injury.Untargeted metabolomics analysis showed that Goji significantly changed the cecum metabolites of acute liver injury mice,such as reducing the levels of oleic acid synthesis substrate(R)-10-hydroxystearate,and the potential marker of liver injury phenylacetylglutamine;enhancing the levels of vanillic acid,retinoyl β-Glucuronide,pyroglutamate,and L-glutamate.It has been shown that these metabolites are closely related to antioxidant activity,intestinal mucosal barrier function and glutathione metabolism,indicating that Goji could maintain the cecum metabolic homeostasis of acute liver injury mice.(3)Intervention with Goji on mice without alcohol modeling,untargeted metabolomics analysis and 16 S rRNA sequencing analysis showed that Goji increased the levels of oleic acid,linoleic acid,docosahexaenoic acid(DHA),riboflavin,pantothenic acid,citrulline,tyrosol and other substances with the function of regulating intestinal barrier and antioxidant activity in the caecum of mice.And the level was significantly and positively correlated with the abundance of Ruminiclostridium,Ruminococcaceae UCG-009,Oscillibacter,Lactobacillus and Bifidobacterium.Metagenomic sequencing of cecal microbiota revealed that Goji increased the expression of alcohol dehydrogenase,as well as glutamate cysteine ligase in the first step of glutathione synthesis.RNA Seq analysis of liver showed that Goji could significantly reduce the expression of genes related to liver lipid accumulation(Fabp2,Fabp4,Fads2)and increase GST synthesis related genes(Gsta1,Gsta2,Gsta4,Gstm1,Gstm3),alcohol dehydrogenase related genes(Adh1,Adh4),aldehyde oxidase gene(Aox3)and retinol dehydrogenase gene(Aldh1a1)by regulating PPAR signaling pathway,PI3 K Akt signaling pathway,fatty acid degradation,and glutathione metabolism.(4)Goji derived exosome like nanovesicles(ELNs)can improve the symptoms of liver injury in ALI mice and maintain the gut microbiota and metabolic homeostasis.The particle size of ELNs was about 90-110 nm.In vitro experiments showed that ELNs(10 μg/mL)significantly reduced the production of NO induced by alcohol and LPS in Kupffer cells.In vivo experiments showed that ELNs could alleviate liver injury in alcohol acute liver injury mice,induced indicating by reducing the level of serum lactate dehydrogenase,improving the inflammation,steatosis and oxidative stress injury of liver.Moreover,ELNs increased the levels of nuclear factor E2 related transcription factor2(Nrf2)and other antioxidant related genes.The expression levels of Muc2,Muc3,tight junction protein 1(Tjp1),and claudin1(Cldn1)genes were also significantly increased by ELNs in the colon of mice.Administration of ELNs could alleviate the acute liver injury in mice,reflecting in reducing the levels of AST,ALT,LDH,ameliorating inflammation and steatosis in liver tissue.Moreover,ELNs significantly increased the m RNA levels of hepatic antioxidant related genes nuclear factor E2 related transcription factor 2(Nrf2),heme oxygenase 1(Ho-1),as well as glutamate cysteine ligase(Gclc),and elevated the m RNA levels of Muc2,Muc3 and tight junction protein 1(Tjp1),and Cldn1 in the colon.In addition,ELNs can increase the relative abundances of Akkermansia,Christensenellaceae＿R＿7＿group,Bifidobacterium,and Oscillibacter,Ruminiclostridium＿9,Ruminococcaceae＿UCG＿013 in the cecum of mice.Untargeted metabolomics showed that DHA,eicosapentaenoic acid ethyl ester,L-asparagine,L-serine,L-aspartic acid and D-glucuronic acid were the characteristic metabolites of ELNs.