To Explore The Mechanism Of Gegen Qinlian Decoction In Improving Non-alcoholic Steatohepatitis Based On Multi-omics Technology

Objective:Application of proteomics,transcriptomics and metabonomics techniques to explore the mechanism of Gegen Qinlian Decoction in improving non-alcoholic steatohepatitis.Method:1.The proteomics and transcriptomics study of Gegen Qinlian Decoction on the liver tissues of non-alcoholic steatohepatitis rats:(1)The NASH model was established by feeding high-fat for 24 weeks,and giving high,medium,and low levels at the 18 th week.Doses of Gegen Qinlian Decoction and pioglitazone,gavage for 7 weeks,take out liver tissue,wash with ice PBS,add RIPA to extract liver tissue protein and pretreat,use LTQ-Orbitrap electrostatic field Orbitrap LC/MS to detect differential expression The protein was analyzed by KEGG and GO enrichment analysis to further analyze the potential targets and pathways of Gegen Qinlian Decoction in preventing and treating non-alcoholic steatohepatitis.(2)Take out liver tissue,wash with ice PBS,add Trizol,extract liver tissue RNA,perform KEGG analysis and GO enrichment analysis of differentially expressed genes,and further analyze the potential targets and pathways of Gegen Qinlian Decoction in preventing and treating non-alcoholic steatohepatitis.(3)Verify by RT-PCR.2.The proteomics and transcriptomics study of Gegen Qinlian Decoction-containing serum on free fatty acid-induced human liver cancer HepG2 cells:(1)Induced HEPG2 hepatocytes by 1m M free fatty acid(palmitic acid:oleic acid=1:2)to induce NASH in vitro The model was treated with high,medium,and low doses of Gegen Qinlian Decoction containing serum intervention,and the establishment of NASH was confirmed by oil red O staining and measuring the content of TG triglycerides.(2)The model was built for 24 hours,and RIPA was added to extract hepatocyte proteins and pretreated.The differentially expressed proteins were analyzed by KEGG and GO enrichment analysis to further analyze the potential targets of Gegen Qinlian Decoction in the prevention and treatment of non-alcoholic steatohepatitis.And access.(3)The model was built for 24 hours,Trizol was added,the liver cell RNA was extracted,the differentially expressed genes were analyzed by KEGG and GO enrichment analysis,and the potential targets and pathways of Gegen Qinlian Decoction-containing serum in the prevention and treatment of non-alcoholic steatohepatitis were further analyzed.(4)Verify by RT-PCR.3.Gegen Qinlian Decoction Study on Fecal Metabolomics of Rats with Non-alcoholic Steatohepatitis: Based on the preliminary experimental basis of this research group,take out animal feces,add deionized water,shake,add methanol,shake,add acetonitrile,shake,combine The supernatant is filtered and tested on the machine.Copy the data from LTQ-Orbitrap,use Xcalibur to obtain the secondary mass spectrum,retention time and proton number/charge number ratio of potential biomarkers,enter the database Human Metabolome Datebase for identification,and use Metabo Analyst3.0 for differential markers The KEGG PATHWAY metabolic pathway is enriched.Result:1.(1)Proteomic analysis of liver tissue: We found out the differentially expressed proteins related to non-alcoholic steatohepatitis are Aldob,Itpkc,Rab43,Mapk8,Gpx1,Gnmt,Ahcy,Ces1 d,Mat1a,Acadm,ACOX3,ACADL.Mainly involved in oxidative stress and inflammation-related metabolic pathways,glycolysis/gluconeogenesis,glycine,serine and threonine metabolism,fatty acid metabolism,arachidonic acid metabolism,glucagon signaling pathway,PPAR signaling pathway,etc.;Differentially expressed proteins participate in the metabolic process,the binding process of various molecules and compounds,and the activities of various parts of the cell.(2)Transcriptomics analysis of liver tissue: We identified the differentially expressed genes related to non-alcoholic steatohepatitis: Nhp2,Cd40,Junb,Nr4a1,Irs2,Lepr,Lect2,Myc,Ntf3,Map3k8,Itpkc,Mapk8,Rab43,Traf6,Hsp90a1.Mainly involved in non-alcoholic fatty liver disease,Toll-like receptor signaling pathway,MAPK signaling pathway,PI3K-Akt signaling pathway,NF-κB signaling pathway,arachidonic acid metabolism,PPAR signaling pathway,type II diabetes,insulin resistance,I Type diabetes,etc.;differentially expressed genes are involved in the process of metabolism and regulation of transport,the process of binding transcription factors and NADH dehydrogenase,and the activities of cell ribosomes and mitochondria.(3)RT-PCR analysis: Compared with the blank group,the MAPK8 m RNA and RAB43 m RNA in the model group were extremely significantly reduced(P<0.01),and the LEPR m RNA,IRS2 m RNA,Nr4a1 m RNA,CD40 m RNA and Itpkc m RNA in the model group were extremely significantly increased(P <0.01);Compared with the model group,MAPK8 m RNA and RAB43 m RNA in the high-dose group of Gegen Qinlian Decoction were significantly increased(P<0.01),LEPR m RNA,Nr4a1 m RNA,CD40 m RNA and Itpkc m RNA in the high-dose group of Gegen Qinlian Decoction Very significantly decreased(P<0.01),and IRS2 m RNA in the high-dose group of Gegen Qinlian Decoction significantly decreased(P<0.05).The change trend is consistent with the change trend of differentially expressed genes in liver tissue transcriptomics.2.(1)Oil red O staining and triglyceride content: The red lipid droplet content in the cells showed a decreasing trend.Compared with the normal group,the triglycerides in the model group HepG2 liver cancer cells were extremely significantly increased(P<0.01);and Compared with the model group,Gegen Qinlian Decoction contained high,medium and low serum triglycerides in HepG2 liver cancer cells extremely significantly(P<0.01),and the positive drug pioglitazone HepG2 liver cancer cells significantly reduced triglycerides(P<0.01)).(2)Proteomics analysis of hepatocytes: We identified the differentially expressed proteins related to non-alcoholic steatohepatitis including YWHAB,YWHAG,YWHAZ,YWHAE,PKM,HSP90B1,PRDX4,ALDOC,and YWHAH.Mainly involved in glycolysis/gluconeogenesis,steroid hormone synthesis,carbon metabolism PI3K-Akt signaling pathway,etc.;differentially expressed proteins are involved in metabolic processes,various molecular and compound activity binding processes,and activities of various parts of cells.(3)Transcriptomics analysis of liver tissue: We identified differentially expressed genes related to non-alcoholic steatohepatitis:MAP2K6,FOSL1,CTSL,DUSP5,DUSP1,JUN,HSPA6,IL1 A,IL11,RELB.Mainly involved in MAPK signaling pathway,PI3K-Akt signaling pathway,NF-κB signaling pathway,Toll-like receptor signaling pathway,insulin signaling pathway,PPAR signaling pathway,non-alcoholic fatty liver disease,etc.;differentially expressed genes Participate in the process of mitosis and chromosome separation,the combination of Rho GTP dehydrogenase,the process of MAP kinase phosphatase activity and cell chromosome activity.(4)RT-PCR analysis: Compared with the blank group,the MAP2K6 m RNA of the model group was extremely significantly reduced(P<0.01),and the model group FOSL1 m RNA,CTSL m RNA,DUSP5 m RNA,DUSP1 m RNA,IL1 A m RNA,IL11 m RNA and RELB m RNA were extremely significantly increased.High(P<0.01),JUN m RNA in the model group was significantly increased(P<0.05);Compared with the model group,MAP2K6 m RNA in the high-dose group of Gegen Qinlian Decoction-containing serum was extremely significantly increased(P<0.01),and Pueraria lobata Qinlian FOSL1 m RNA,CTSL m RNA,DUSP5 m RNA,DUSP1 m RNA,JUN m RNA,HSPA6 m RNA,IL1 A m RNA,IL11 m RNA and RELB m RNA were extremely significantly reduced in the high-dose serum of the decoction-containing serum group(P<0.01),and the trend of change was consistent with the transcription of HepG2 liver cancer cells The change trend of omics differentially expressed genes is consistent.3.After being fed with high-fat diet,the metabolic phenotypes of the rats in the blank group and the model group changed significantly,and there was a metabolic difference;after giving Gegen Qinlian Decoction,compared with the model group,the fecal metabolism profile of the rats occurred Changed.Through analysis,a total of 24 differential marker metabolites were found,o-phenylalanine,2-(3-phenylpropyl)tetrahydrofuran,hydroxyprolylmethionine,4,6,7-trihydroxy-1,2,3,4-Tetrahydroisoquinoline,valproic acid,catechol,alanylvaline,leucyl asparagine,4-(3,7-dimethyloctyl-2,6-di En-1-yl)benzene-1,2,3,5-tetraol,octadecenoic acid,N5-acetyl-N2-γ-L-glutamyl-,L-ornithine,MG(13 :0/0:0/0:0),arginine methionine,trihexyl ethyl,24,25,26,27-Tetranor-23-oxo-hydroxy vitamin D3,sorbic acid laurate,fumonis B3,saxagliptin,12-deoxycholic acid,juteoside A,CE(22:1(13Z)),4a-carboxy-4b-methyl-5a-cholesta-8,24-dien-3b-ol,DG(16:1n7/0:0/18:1n7),CL(i-14:0 / i-12:0 / i-12:0 / i-18:0);involved in steroid biosynthetic metabolic pathways.Conclusion:1.In the in vivo liver tissue experiment,Gegen Qinlian Decoction may down-regulate the differentially expressed proteins of Aldob and up-regulate the differentially expressed proteins of Gpx1,Gnmt,Ahcy,Ces1 d,Mat1a,Acadm,ACOX3,ACADL,and down-regulate the differentially expressed proteins of Nhp2,Cd40,Junb,Nr4a1.Irs2,Lepr,Lect2,Myc,Ntf3,Map3k8 differentially expressed genes and up-regulated Traf6,Hsp90a1 differentially expressed genes,through oxidative stress,glucagon signaling pathway,PPAR signaling pathway,MAPK signaling pathway,TOLL-like receptor signaling pathway,TNF signaling pathway,NAFLD signaling pathway,type 2 diabetes and PI3K-AKT signaling pathway to achieve non-alcoholic resistance Steatohepatitis.2.In the in vitro liver cell experiment,Gegen Qinlian Decoction may regulate differentially expressed proteins such as YWHAB,YWHAG,YWHAZ,YWHAE,PKM,HSP90B1,PRDX4,ALDOC,YWHAH and differentially expressed genes such as FOSL1,CTSL,DUSP5,DUSP1 JUN,HSPA6,IL1 A,IL11,RELB,MAP2K6,through MAPK signaling pathway,PI3K-Akt signaling pathway,NF-κ B signaling pathway,Toll-like receptor signaling pathway and non-alcoholic fatty liver disease signaling pathway to achieve resistance Non-alcoholic steatohepatitis.3.According to metabolomics,Gegen Qinlian Decoction has a significant improvement mechanism on non-alcoholic steatohepatitis rats,by regulating the differential marker metabolites4,6,7-trihydroxy-1,2,3,4-tetra Hydroisoquinoline,valproic acid,and arginine methionine are involved in steroid biosynthesis and amino acid anabolism to participate in energy metabolism to improve non-alcoholic steatohepatitis.