Analysis Of Potential Lipid-Lowering Pathways And Targets Of Yiguanjian In Preventing And Treating Alcoholic Fatty Liver Disease Based On RNA-Seq Transcriptomic Assay And Network Pharmacology

[Objective] Alcoholic fatty liver disease(AFLD)is the initial stage of alcoholic liver disease.Prevention and therapy of AFLD is of great significance for blocking the malignant progression of alcoholic liver disease.In traditional Chinese medicine,Yiguanjian was applied in the treatment of a variety of liver diseases,and it also had a perfect effect on alcoholic liver disease,nevertheless the molecular mechanism has not been illustrated.In this study,RNA-Seq transcriptome and network pharmacology was adopted to analyze and speculate the critical lipid-lowering pathway and dominant target of Yiguanjian in the prevention and treatment of AFLD.[Methods] 1.In vivo experiment: AFLD mice model was established by chronic alcohol feeding and single alcohol gavage,and the intervention group was set up by intragastric administration of Yiguanjian.Firstly,the function of liver from AFLD mice was evaluated by H&E pathological staining of liver tissue and colorimetric assay of serum ALT level;Then the level of TG in serum from mice was detected by colorimetric method,and liver tissue oil red O staining was combined to evaluate liver steatosis.Finally,the differential genes expression in the liver tissues of AFLD model group vs normal control group and Yiguanjian group vs AFLD model group were analysed by RNA-Seq.Combined with protein-protein interaction(PPI)analysis and KEGG pathway enrichment analysis,the pharmacological pathway and dominant target of Yiguanjian in reducing alcoholic steatosis in liver tissue of mice were explored.2.In vitro experiment: 1.AML12 cells were cultured with alcohol combined with palmitic acid(PA)to establish an in vitro cell model of alcoholic fatty liver disease(AFLD).The changes of intracellular steatosis were assessed by oil red O staining.The differential genes expression in the cells of the composite AFLD model group compared with the normal control group were analysed by RNA-Seq technology.Then,the genes set was overlapped with the genes set of differential genes in the liver tissue of mouse AFLD.The critical signaling pathways and dominant genes of differential genes directly related to hepatocyte steatosis in mouse AFLD were analyzed and discussed by bioinformatics methods.2.The effect of Yiguanjian on the viability of AML12 cells was detected by CCK-8 assay in the AFLD cell model of alcohol combined with PA.The changes of intracellular lipid synthesis were assessed by oil red O staining.RNA-Seq for transcriptome was used to explore the differentially expressed genes between Yiguanjian group and AFLD model group,and then the genes set was overlapped with the differentially expressed genes set of liver tissue in mice with AFLD treated by Yiguanjian.The critical signaling pathways and central target proteins of Yiguanjian in preventing and treating fatty degeneration of mouse AFLD hepatocytes were analyzed by bioinformatics methods.3.Network pharmacological analysis: The(AFLD)target genes of human alcoholic fatty liver disease were obtained from Gene Cards online database,and the target genes of mouse AFLD in vivo model and mouse hepatocyte AFLD cell model obtained by RNA-Seq transcriptome sequencing were analyzed by overlapping analysis.The potential pharmacological pathways and targets of Yiguanjian in clinical prevention and treatment of AFLD were analyzed and discussed by bioinformatics methods.[Results] 1.In vivo experiment:(1)the results of H&E staining of liver tissue and serum ALT level determination showed that the liver function defect of AFLD model group was more obvious than that of normal control group,and the results of oil red O staining for liver tissue and serum TG level quantification showed that the fatty degeneration of liver tissue from AFLD model group was significantly higher than that of normal control group.(2)Yiguanjian significantly decreased the level of ALT in serum of AFLD model mice and alleviated liver function defect,and meanwhile,it also significantly reduced the level of TG in serum from AFLD model mice and reduced steatosis of liver tissue.(3)The results of RNA-Seq for transcriptome and bioinformatics analysis revealed that Yiguanjian attenuated alcohol-induced hepatic steatosis by affecting MAPK signaling pathway,insulin signaling pathway,PPAR signaling pathway and regulating the expression of SRC,HGF,SIRT1,PPARG,IL1 B,IRS1,HO-1,FASN,CYP7A1,ACLY and other genes.2.In vitro experiment:(1)The results showed that 100 m M alcohol combined with ≤900 μg/m L palmitic acid had no cytotoxicity to AML12 cells,and ≤ 900 μg/m L Yiguanjian had no cytotoxicity to AML12 cells of the AFLD cell model induced by alcohol combined with PA.(2)Oil red O staining showed that 100 m M alcohol combined with PA at various concentrations could significantly increase liver lipid,and the increase of 6.25 μmol PA was the most obvious.At the same time,11-900 μg / m L Yiguanjian can significantly reduce the increase of liver cell lipid induced by alcohol combined with PA,and 300 μg / m L Yiguanjian has the most obvious effect.(2)The results of RNA-Seq for transcriptome combined with bioinformatics analysis showed that Yiguanjian may play a pharmacological role in preventing and treating hepatocyte steatosis in mice with AFLD by affecting Bile secretion signal pathways and regulating the expression of AQP1,ATP1B1,EPHX1 and other genes.3.Network pharmacological analysis: Yiguanjian may affect the process of lipid metabolism in hepatocytes by regulating the expression of AQP1 and EPHX1 genes from Bile secretion signal pathway,and functions the effect of prevention and treatment of human alcoholic fatty liver disease.[Conclusion]1.100 m M ethanol combined with 6.25 μM PA could induce the composite AFLD hepatocyte model,which is suitable for studying the pharmacological mechanism of AFLD hepatocyte steatosis.2.In vitro and in vivo experiments showed that Yiguanjian mainly reduced alcohol-induced fatty degeneration of mouse AFLD hepatocytes by regulating the expression of core target genes such as UGT2B35,SULT1D1 and NR1I2 and affecting the expression of AQP1,ATP1B1 and EPHX1 in the Bile secretion signaling pathway;Further network pharmacology analysis found that the Bile secretion signaling pathway and the AQP1 and EPHX1 genes in the pathway may be the key pathway and target for Yiguanjian to prevent and treat human AFLD liver cell steatosis.