Structural And Functional Disorders Of Gut Microbiota During NASH Progression And The Protective Role Of Butyrate

Backgrouds:Nonalcoholic fatty liver disease(NAFLD)has become the world’s major chronic liver disease and public health problem,but the pathogenesis of NAFLD is still poorly understood.The widely accepted "two hits" hypothesis is not enough to explain the pathogenesis of this heterogeneous disease.Nowadays,gut microbiota is considered to be a large metabolic organs,which has important physiological functions such as maintaining energy homeostasis.The role of gut microbiota in the development of NAFLD and nonalcoholic steatohepatitis(NASH)has been gradually revealed.However,dynamic alterations in structure and function of gut microbiota during the development of NASH is still poorly studied.Butyrate,an important member of short-chain fatty acids,has been found to have some beneficial effect in anti-inflammatory,regulating lipid metabolism,promoting apoptosis,regulating immune homeostasis,and improving intestinal barrier function.However,whether butyrate can protect NASH by regulating the structure and function of gut microbiota is still unknown.Therefore,based on the above backgrounds,our aim is to study the dynamic alterations in structure and function of gut microbiota during the development of nonalcoholic steatohepatitis and the protective role of butyrate.Methods:The study was divided into two parts.The first part was to dynamically monitor mice from simple steatosis(feeding for 2 weeks)to NASH(feeding for 4 weeks)by constructing a methionine-choline-deficient(MCD)diet induced NASH model.During the 4 weeks of feeding,structure and function of gut microbiota was studied,and the combined analysis mode of gut microbiome and metabolome was constructed.There were four groups in this part,Control 2w group,MCD 2w group,Control 4w group and MCD 4w group.In the second part,based on the constructed NASH mouse model,sodium butyrate(SoB)was administered daily for 6 weeks to study the protective effect of SoB on NASH and the reshaping function towards structure and function of gut microbiota.There were four groups in this section,Control group,MCD group,Control+SoB group,MCD+SoB group.Results:Part 1:1.Mice fed with MCD diet for 2 weeks showed simple steatosis,and NASH appeared after 4 weeks of feeding.During the progression of simple steatosis to NASH,alanine aminotransferase(ALT),aspartate aminotransferase(AST),intrahepatic triglyceride(TG)content,NAFLD activity score(NAS)were increased,macrophage infiltration was increased,and ZO-1 immunofluorescence staining showed an increasing degree of intestinal barrier damage.NASH mice were accompanied by liver fibrosis.2.The structure of gut microbiota in mice with simple steatosis was not significantly changed,but gut microbiota dysbiosis began to appear in NASH mice.Relative abundance of opportunistic pathogens:Bilophila,Anaerrotruncus,Clostridium,and Helicobacteraceae were significantly increased in NASH mice.Abundance of probiotics Sutterella and Akkermansia were significantly reduced in NASH mice.The metabolic function of gut microbiota kept changing during the progression of NASH.The four groups were clearly distinguished in the principal component analysis(PCA)and the orthogonal partial least-squares-discriminant analysis(OPLS-DA)analysis.3.Arachidonic acid was stable present in the normal group,and tetracosane was always exist during the progression of NASH.Finally,103 and 93 differential metabolites were identified after 2 and 4 weeks of MCD diet feeding,most of which were involved in pathways related to lipid,amino acid,carbohydrate,nucleotide,cofactor and vitamin metabolism.Part 2:1.Butyrate can improve liver damage,inflammation and lipid metabolism.Hepatic HE staining showed that butyrate reduced MCD diet induced hepatic steatosis and inflammation,decreased NAS score,improved liver function(ALT,AST),decreased triglyceride content,and regulated mRNA expression of inflammation and lipid metabolism related factors.2.Butyrate can delay the progression of liver fibrosis and regulate Toll-like receptor expression.Expression of liver fibrosis markers:TGF-β1 and α-SMA were decreased after butyrate intervention.MASSON staining and α-SMA immunohistochemical staining confirmed that liver fibrosis was improved.The mRNA expression levels of TLR4 and CD 14 were decreased.3.Butyrate improved serum cytokines and reduced endotoxin levels.Butyrate intervention not only significantly down-regulated the levels of pro-inflammatory cytokines(IL-1α,IL-1β,IL-2,IL-3,IL-6,IL-12 p70,IL-17a,TNF-α,Eotaxin).The levels of the anti-inflammatory cytokines IL-4 and IL-10 were also significantly up-regulated by butyrate.The level of serum endotoxin-binding protein(LBP)in the intervention group was significantly lower than that in the MCD group.4.Butyrate stabilized the colon barrier function.Expression levels of intestinal barrier marker ZO-1 and claudin-1 mRNA were up-regulated in the intervention group.Immunofluorescence staining of ZO-1 confirmed that the intestinal barrier function of this group was improved.5.Butyrate reshaped the microbiome dysbiosis induced by the MCD diet.Principal coordinates analysis(PCoA)results showed that the microbial structure of the four experimental groups can be well distinguished,and the linear discriminant analysis effect size(LEfSe)analysis showed that butyrate can reshape structure of gut microbiota with significantly increased abundance of some probiotics such as Akkermansia and Roseburia.6.Butyrate improved dysfunction of gut microbiota induced by the MCD diet.OPLS-DA model showed that the MCD group and the MCD+SoB group can be clearly distinguished.98 different metabolites were finally screened between the two groups,saturated fatty acids(such as stearic acid and behenic acid),unsaturated fatty acids(such as oleic acid and linoleic acid)and squalene(a natural 30 carbon triterpene)were significantly increased in the feces of MCD+SoB group,and the content of arachidonic acid(an unsaturated fatty acid)was significantly decreased in this group.7.Butyrate-modified structure and function of gut microbiota played an important role in improving NASH.Significantly increased abundance of Bilophila and Rikenellaceae in NASH mice were both positively correlated with ALT,IL-17α,LBP,TLR2,TLR4,and TNF-a levels.Significantly increased abundance of Akkemansia after butyrate intervention was significantly negatively correlated with ALT,NAS,TG,F4/80,IL-12 p70,IL-17α,IL-1β,IL-6,TNF-α,TGF-β1,TLR2,TLR4 and LBP levels.The abundance of squalene and stearic acid,which were also increased after butyrate intervention,may be related to the improvement of liver damage,they were negatively associated with ALT,IL-1β,IL-17α,LBP,F4/80.Conclusions:1.The MCD diet induced substantial alteration in structure and function of gut microbiota.Fundamental understanding of these alterations will provide new insights into NASH-associated intestinal disorder and gut-targeted therapies for NASH.This part of study provides model and technical foundation for the second part of butyrate intervention study.2.Butyrate induced a protective shift in structure and function of gut microbiota,thereby effectively preventing the MCD diet-induced liver and gut impairments associated with NASH.Thus,the current study indicates that oral butyrate administration is a promising strategy for treating NASH.