| Chapter 1: The Effect of resistant starch on non-alcoholic fatty liver disease mice and the change of gut microbiota.Objective: To investigate the improvement of resistant starch(RS)on non-alcoholic fatty liver disease(NAFLD)mice and the changes of gut microbiota.Method: NAFLD mouse model was induced by high-fat diet for 16 weeks,followed by intervention with or without RS for 4 weeks.As control groups,mice fed with control diet for 16 weeks followed by 4 weeks intervention with or without RS.Calorie intake,body weight,liver weight and liver index of mice were collected.Liver steatosis was determined by liver histomorphology.Serum alanine transaminase(ALT),aspartate aminotransferase(AST),total cholesterol(TC),triglyceride(TG),high-density lipids high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),fasting plasma glucose(FBG),fasting insulin(FINs)content were detected.Calculate the homeostasis model assessment for insulin resistance(HOMA-IR).The composition of gut microbiota from cecal content of mice was analyzed by 16 s amplicon sequencing.Result:(1)Mice in HFD+RS group had lower body weight,liver weight and liver index compared with mice in HFD group.and there was no significant difference in calorie intake.Compared with HFD group,hepatocyte steatosis was improved,Non-alcoholic fatty liver discase activity score(NAS)scores and hepatocyte steatosis scores were decreased,serum levels of ALT,TG,TC,LDL-C,FBG,FINS,HOMA-IR were decreased in HFD+RS group.(2)The alpha diversity of the gut microbiota increased and the ratio of Firmicutes/Bacteroidetes(F/B)decreased in HFD+RS group mice compared with mice in HFD group.The abundance of Akkermansia increased in HFD+RS group mice compared with mice in HFD group.LEf Se analysis suggested that Akkermansia could be a biomarker for NAFLD mice.Conclusion:RS can improve NAFLD mice by reducing hepatocyte steatosis,lowering serum lipid and glucose levels and improving insulin resistance,and increasing gut microbiota diversity.Akkermansia,as a biomarker of NAFLD mice,was significantly increased after RS intervention.Chapter 2: The Effect of RS and(or)Akkermansia on NAFLD mice and the change of gut microbiota.Objective: To investigate the improvement effect of RS and(or)Akkermansia on NAFLD mice and the change of gut microbiota.Method: The NAFLD mouse model was induced by high-fat diet for 16 weeks,followed by intervention with RS,live Akkermansia,pasteurized Akkermansia,culture supernatant of Akkermansia,RS combined with live Akkermansia for 4 weeks.The indexes of calorie intake,body weight,liver weight and liver index of mice were collected.Liver steatosis was determined by liver histomorphology.Detect the content of ALT,AST,TC,TG,HDL-C,LDLC,FBG,FINs,and calculate HOMA-IR.The composition of gut microbiota from cecal content of mice was analyzed by 16 s amplicon sequencing.Result:(1)Compared with HFD group,body weight,liver weight,liver index,NAS scores,hepatocyte steatosis scores,serum ALT,TC,TG,LDL-C,FBG,FINS,HOMA-IR and the intestinal F/B ratio of mice in HFD+RS group decreased.HDL-C,alpha diversity and Akkermansia abundance increased in HFD+RS group.(2)Compared with HFD group,body weight,liver weight,liver index,NAS scores,hepatocyte steatosis scores,serum TC,TG,LDL-C,FBG,FINS,HOMA-IR and the intestinal F/B ratio in HFD+LAKK(live Akkermansia)group decreased.HDL-C,alpha diversity and Akkermansia abundance increased in HFD+LAKK group.(3)Compared with HFD group,body weight,liver weight,liver index,serum FBG,FINS,HOMA-IR,alpha diversity,Akkermansia abundance were decreased in HFD+ PAKK(Pasteurized Akkermansia)group,and the intestinal F/B ratio was increased.HFD+ PAKK group and HFD group showed similar microflora aggregation.There were no significant differences in NAS scores,hepatocyte steatosis scores,serum TC,TG and LDL-C levels between the two groups.(4)Compared with HFD group,body weight,liver weight,liver index,serum FBG,FINS,HOMA-IR of mice in HFD+ supernatant group decreased.The alpha diversity of gut microbiota increased,and the intestinal F/B ratio decreased in HFD+ supernatant group.There were no significant differences in NAS scores,hepatocyte steatosis scores,serum TC,TG,LDL-C and Akkermansia abundance between the two groups.(5)Compared with HFD group,body weight,liver weight,liver index,NAS scores,hepatocyte steatosis scores,serum levels of ALT,TC,TG,LDL-C,FBG,FINS,HOMA-IR and the intestinal F/B ratio were decreased in HFD+RS+LAKK group.The alpha diversity and Akkermansia abundance of gut microbiota increased in HFD+RS+LAKK group.Compared with HFD+RS group,liver weight,liver index,NAS scores,hepatocyte steatosis scores,serum TC,TG,LDL-C,FINS,HOMA-IR were decreased in HFD+RS+LAKK group.Compared with HFD+LAKK group,liver weight,liver index,NAS scores,serum levels of ALT,TC,TG decreased and HDL-C increased in HFD+RS+LAKK group.HFD+RS group,HFD+LAKK group and HFD+RS+LAKK group showed similar aggregation.Conclusion:RS,live Akkermansia or the combination of RS and live Akkermansia can treat NAFLD mice by reducing hepatocyte steatosis,reducing serum lipid and serum glucose contents,improving insulin resistance,increasing gut microbiota alpha diversity and intestinal Akkermansia abundance.RS combined with live Akkermansia has a better therapeutic effect.Chapter 3: The mechanism of RS and(or)live Akkermansia bacteria in the treatment of NAFLD miceObjective: To investigate and verify the mechanism of RS and(or)live Akkermansia in treating NAFLD mice.Method: Short-chain fatty acids(SCFA)in portal serum of mice in HFD group,HFD+RS group,HFD+LAKK group and HFD+RS+LAKK group were detected by Gas Chromatography-Mass Spectrometry(GC-MS).The association between propionic acid and gut microbiota was analyzed by WGCNA analysis,and the bacterial modules were clustered.PICRUSt2 was used for gut microbiota function prediction analysis.RNA-seq was performed to evaluate the gene expression level of the liver in mice above.Multi-omics correlation analysis was performed to study the effect and mechanism of RS and(or)live Akkermansia intervening NAFLD mice,subsequently confirmed by quantitative real-time PCR(q PCR)and western blot(WB).The m RNA relative levels of IRS-1,PI3 K,Akt1,Akt2,and GSK-3β were detected by q PCR,and the changes of AKT,p-AKT and PI3 K were detected by WB.The effect of propionic acid on NAFLD cell model was investigated by in vitro cell experiment,and the gene changes were investigated by q PCR and WB.Result:(1)Compared with HFD group,the level of portal vein serum propionic acid in HFD+RS group and HFD+RS+LAKK group increased and the level of valerate decreased.Correlation analysis of propionic acid and valeric acid with bacteria showed that 40 bacteria were positively correlated with the level of portal serum propionic acid,and these bacteria showed clustering phenomenon.Functional prediction of bacteria was found to be associated with energy metabolism and lipid metabolism.(2)The NAFLD pathway and PI3K-AKT pathway enriched in the liver genes corelated with clinical phenotypes such as HFD,RS intervention,LAKK intervention,serum lipid,HOMAIR.Compared with HFD group,the expression level of IRS-1,PI3 K,Akt1,and Akt2 in liver of HFD+RS+LAKK group were increased,and the expression level of GSK-3β were decreased in HFD+RS+LAKK group.The protein level of p-AKT and PI3 K increased in mice liver in the HFD+RS+LAKK group compared with the HFD group.(3)The result from the experiment of NAFLD cell model treated by propionic acid showed that,cells had less lipid droplets and less TG in the propionic acid treated group compared with untreated group.the gene expression level of IRS-1,PI3 K,AKT1,AKT2 increased and GSK-3β decreased in propionic acid treated group compared with untreated group.The protein level of PI3 K and p-AKT increased in cells in propionic acid treated group compared with untreated group.Conclusion:The propionic acid in portal vein serum of NAFLD mice increased by RS,RS and live Akkermansia intervention.The bacteria positively correlated with propionic acid functioned as lipid metabolism.PI3K/ Akt pathway was activated in HFD+RS+LAKK group.Propionic acid can reduce the lipid droplets and the accumulation of TG in NAFLD cell model.PI3K/AKT pathway was activated after the intervention of propionic acid. |
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