| Objective: To study the protective mechanism of Forsythia-4 on pyloric ligated liver injury by using network pharmacology and metabonomics,and to explore the etiology of pyloric ligated liver injury and the material basis of forsythia-4’s liver-protecting effect by studying the effect of drug-containing serum on liver cells.Methods:1.Network pharmacological study: The chemical constituents of Forsythia-4were searched and collected by TCMSP database(oral availability(OB)and drug class(DL)were used as the screening conditions for active constituents).The target of active ingredients was predicted by Swis Starget Prediction data platform.The related targets of liver injury were collected by Disgenet database,and the related targets of forsythia-4 for the treatment of liver injury were obtained by using the intersection points of compound targets and disease targets.Finally,the GO function enrichment analysis and KEGG signal pathway enrichment analysis were performed using DVIDA online software.2.Study on the effect of forsythia-4 on pyloric ligated liver injury: 60 SD rats(half male and half female)were randomly divided into 5 groups: normal group,model group,and forsythia-4 low-dose,medium-dose and high-dose(1.3,2.6,3.9g/kg)groups,with 12 rats in each group.The normal group and the model group were given0.5% CMC-NA solution,and each administration group was given corresponding dose of Forsythia-40.5% CMC-NA suspension for 15 days.After the last administration,fasting and water could not be stopped for 24 hours.Rats in the model group and each administration group received pyloric ligation operation,and were fed in a single cage after surgery,fasting and water abstaining for 16-18 h.Liver,abdominal aorta and portal venous blood were collected,serum was separated and stored at-80 ℃ for later use.3.Metabonomics study: UPLC-MS was used to analyze the effect of forsythia-4 on pyloric ligated liver injury.T-test,Foldchange(FC)analysis and orthogonal partial least square discriminant analysis(OPLS-DA)were used to analyze the pathway enrichment of metabolites with different hepatoprotective effects of Forsythia-4.4.Verification experiment: Westernblotting was used to verify the differential metabolites(IGFBP1)obtained by metabonomics.5.Effect of portal vein serum and forsythia-4-containing serum on hepatocytes in rats with pyloric ligation liver injury: The effects of portal vein serum and drug containing serum(forsythia-4)on hepatocytes in rats with pyloric ligation liver injury were observed by the LO2 hepatocyte culture experiment in vitro.Results: 1.The results of network pharmacological analysis showed that 924 active compounds screened from Forsythia-4 could directly or indirectly act on 83 key targets of liver injury diseases.Through GO functional enrichment analysis and KEGG signal pathway enrichment,940 BP,28 CC,58 MF and 34 KEGG signal pathways were obtained(P<0.05).In addition,core components such as stigmasterol,beta-sitosterol,Kaempferol,quercetin and other core targets such as PTGS2,PPARA,MMP9,MMP2NR1H3,PPARG,CDC25 B,TNF,CYP2C19,Serpina6,CES2,and IL6 were found.2.The protective effect of forsythia-4 on pyloric ligation of liver injury showed that the liver injury model induced by pyloric ligation was successfully copied,and the comprehensive analysis of each administration group of forsythia-4 showed that the high-dose group had the best protective effect on the liver injury model.3.Metabolomics experiment results showed that 25 metabolic differences were obtained between the normal group and the model group;The model group was compared with forsythia-4 group,and 39 metabolic differences were obtained.Differences metabolites metabolic pathway analysis results show that Arachidonicacidmetabolism metabolic pathways(the metabolic pathways regulating phospholipid metabolism and bile acid metabolism in the body,and through the function of arachidonic acid IGBP1 adjustment factor)with the regulation(based on the screening conditions of vip>1,difference multiple fc≥2 or P value <0.05).4.Westernblotting experiment: Compared with the normal group,IGFBP1 factor was up-regulated in the model group;Compared with model group,forsythia-4 had down-regulation effect on IGFBP1 factor.5.The results of cell experiment showed that compared with the normal control group,the AST and ALT levels in the supernatant of LO2 hepatocytes in portal vein serum of the model group were significantly increased(P<0.01),and the cells in the normal control group were in good growth state,uniform size and clear boundary under microscope.The number of cells in the model group was significantly reduced and the cells were fused.There were no significant changes in AST and ALT in the cell supernatant between model group and forsythia-4 drug containing serum.Conclusion: The protective effect of forsythia-4 on pyloric ligated liver injury in rats is characterized by multi-component,multi-target and multi-pathway.Among them,stigmasterol,beta-sitosterol,Kaempferol and quercetin were the core components;PTGS2,PPARA,MMP9,MMP2NR1H3,PPARG,CDC25 B,TNF,CYP2C19,Serpin A6,CES2 and IL6 were the core targets,and the hepatoprotective effect was achieved through the regulation of IGFBP1 factor.In addition,through this study,it was preliminarily determined that the pathological process of liver injury caused by pyloric ligation may be caused by the disorder of "essence and dregs decomposition" in the stomach,which resulted in the delivery of the affected food essence to the liver through the portal vein,leading to abnormal liver function.However,due to the experimental methods and measurement errors,no effective material basis for protecting liver was found in the serum of Forsythia-4 abdominal aorta.Therefore,further exploration is needed on the effective material basis of this formula for protecting pyloric ligated liver injury. |
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