| Objective:To explore the metabolic disorders in patients with non-alcoholic fatty liver disease(NAFLD),to evaluate the effect of glucagon-like peptide-1(GLP-1)on NAFLD,the influence of inflammatory indicators and the correlation.To explore the mechanism of GLP-1 on NAFLD at the level of in vitro cell models and human intestinal microecology.Provide more theoretical basis for NAFLD prevention and treatment.Methods:Part I: By comparing the biochemical indexes and inflammatory indexes between 121 NAFLD patients and 121 normal population,the changes of inflammatory indexes and their correlation with IR resistance were analyzed.The improvement of biochemical indicators and inflammatory indicators in NAFLD patients before and after GLP-1treatment was further analyzed,and the correlation between inflammatory indicators and clinical indicators was analyzed.Part II: The fatty liver chip data were downloaded from the public database,namely the Gene Expression Database(GEO),and the differential target genes and pathways were found through bioinformatics analysis.HepG2 cells were treated with different concentrations of free fatty acid(FFA)and 10% fetal bovine serum(FBS)low glucose DMEM medium for 24h.Cell activity was determined by MTT,combined with oil red O staining and intracellular triglyceride determination,and the establishment of NAFLD model in vitro was determined.After the model was established,different concentrations of GLP-1 were given for 24h intervention.After drug intervention,total RNA was extracted and reverse transcripted.The m RNA expressions of APN,adiponectin receptor,AMPK and PPARα in the differentially gene-related pathways were detected by real-time quantitative PCR.Total cell proteins were extracted and the expressions of lipid APN,adiponectin receptor,AMPK and PPARα were detected by western blot.At the same time,intracellular triglyceride(TG)content and oil red O staining were detected to observe the accumulation of intracellular lipid droplets.The effects of GLP-1on the above indexes were analyzed.Part III: Using high-throughput sequencing,the differences in intestinal bacterial community diversity,structure and function between 30 NAFLD patients and 30 healthy people were firstly compared.Further,60 patients with NAFLD were randomly divided into liraglutide treatment group and metformin treatment group.The co-change mechanism of inflammatory indexes,biochemical indexes and intestinal bacterial community of patients before and after liraglutide treatment was further investigated,and the therapeutic effect of liraglutide was evaluated and verified by functional prediction.Results:Part I:(1)the NAFLD patients in the study of body mass index(BMI),systolic blood pressure(SBP),diastolic blood pressure(DBP),waist circumference(WC),hip circumference(HC),waist hip ratio(WHR),alanine aminotransferase(ALT),fasting blood glucose(FBG),triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)is significantly higher than healthy people,but the high-density lipoprotein cholesterol(HDL-C)was significantly lower than that of healthy people(P<0.05).The results showed that FBG,fasting insulin(FINS),insulin resistance index(HOMA-IR),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)in NAFLD patients were significantly higher than those in the healthy population(P<0.05),and IL-6,TNF-αwere significantly positively correlated with HOMA-IR(P<0.001).(2)There were significant correlations between the changes of various indexes before and after treatment with liraglutide in patients with NAFLD(P<0.05).General information on the patients(BMI,WHR,DBP and SBP),islet beta cell function(FPG,2hPG and FINS,HbA1 c,HOMA-IR),inflammation,related indicators(IL-6,TNF-α and APN),lipid metabolism(TC,TG,LDL-C),liver function((ALT),aspartate amino transferase(AST),gamma glutamyl transpeptidase(GGT))and fatty deposits(CAP)are extremely significant effect(P<0.001),no significant effect on HDL-C,LSM(P>0.05).Part II:(1)We found that the 5 ’-adenosine phosphate activated protein kinase(AMPK)pathway and peroxisome proliferator-activated receptor α(PPARα)signaling pathway were the downstream pathways of APN and adiponectin receptor(Adipor)through bioinformatics analysis.(2)The results of oil red O staining,TG determination and MTT showed that the concentration of(oleic acid)molded solution was 1mM,and the molded time was 24h,and there was no significant effect on the cell activity,and the intracellular TG was significantly increased,and the accumulation of intracellular lipid droplets was obvious,which was the best condition for inducing cell steatosis.(3)Determination of TG content:Compared with model group,TG content in GLP-1 treatment group was significantly decreased,and was positively correlated with concentration(P<0.01).(4)The results of oil red O staining showed that different concentrations of GLP-1 treated for 48hours could reduce the accumulation of lipid droplets in different degrees,especially at 1000n M.(5)Real-time quantitative PCR results showed that compared with model group,m RNA expressions of adiponectin(APN),adiponectin receptor(Adipor),5 ’-adenosine phosphate activated protein kinase(AMPK)and peroxisome proliferator-activated receptor α(PPARα)were significantly increased after 48h treatment with different concentrations of GLP-1.(6)Western blot results showed that compared with model group,the protein expressions of APN,Adipor,AMPK and PPARα of lipid metabolism genes were significantly increased after 48h treatment with different concentrations of GLP-1.Part III(1)The intestinal bacterial community of patients with NAFLD was significantly different from that of healthy people.The abundance of typical dominant beneficial bacteria such as Firmicutes and Bacteroidetes in the intestines of NAFLD patients was lower than that of healthy people,while the abundance of Proteobacteria containing a variety of opportunistic pathogens was significantly higher than that of healthy people.(2)Liraglutide is better than metformin in the treatment of NAFLD.The effect of liraglutide on intestinal bacterial community was relatively small.After treatment with liraglutid,changes in intestinal bacterial abundance were closely related to islet β-cell function,lipid metabolism,inflammation-related indexes,liver function,fat deposition and other indicators.In addition,after treatment with liraglutide,there were significant differences in the abundance of several genes in the intestinal tract,including significantly decreased bile acid biosynthesis,insulin resistance,glucagon signaling pathway,and significantly increased propionic acid metabolism and butyric acid metabolism(P<0.05).Conclusion:(1)Patients with NAFLD have disorders of blood pressure,blood glucose and lipid metabolism,as well as high inflammatory indexes and insulin resistance.Therefore,early prevention and treatment of NAFLD should be paid attention to,and glucose and lipid metabolism disorders,hypertension,etc.,should be actively treated.GLP-1 can significantly improve the clinical indicators of patients with NAFLD,reduce inflammatory indicators,improve liver fat deposition,and has a significant effect on the treatment of fatty liver.(2)APN gene and downstream pathway genes were differentially expressed in fatty liver.HepG2 cells were cultured with 1mMFFA for 24hours,which could induce adipose degeneration and could be used as a model of fatty liver cells.GLP-1 alleviates steatosis and reduces intracellular lipid droplets in HepG2 cells by activating the expressions of lipid-metabolizing genes APN,Adipor,AMPK and PPARα.Therefore,GLP-1has a certain mitigating effect on fatty liver cell model.(3)Liraglutid has a better effect on NAFLD than metformin,and has a relatively small effect on the intestinal bacterial community of patients,which is related to inflammatory indicators,and the intestinal bacterial community structure and metabolomics may be an important breakthrough in improving fatty liver. |
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