Protective Effect And Mechanism Of Methyl Eugenol On Liver Ischemia Reperfusion Injury In Mice

Objective: Liver ischemia reperfusion injury(LIRI)often occurs during liver transplantation,hepatectomy and various circulatory shock processes,leading to inflammatory immune response,oxidative stress response,severe metabolic disorders and apoptosis.Methyl Eugenol(ME)is similar to eugenol in structure and has anti-inflammatory and anti-apoptotic pharmacological effects.However,the protective effect of ME in liver ischemia reperfusion injury has not been reported.Therefore,the main purpose of this study is to explore the role of ME in mouse LIRI process and its potential mechanism.Methods: This study mainly involves in vivo animal experiments,in vitro cell experiments and network pharmacology related techniques.In animal experiments,male SPF C57BL/6 mice were grouped according to a random number table:There were 6 mice/group in sham operation group,sham +ME group,LIRI group,LY294002 group,LIRI+Vehicle group,LIRI+ME group,LIRI+LY294002 group and LIRI+ME+LY294002 group.Except for the sham operation group,mice in the other groups were subjected to 70% liver thermal ischemia-reperfusion treatment.In vitro cell experiments,the hypoxia/reoxygenation injury model of mouse normal liver cell line(AML12)was constructed,and the specific experimental groups were as follows:normal group,normal+ME group,H/R group,H/R+DMSO group,H/R+ME group,H/R+LY294002 group,H/R+ME+LY294002 group and H/R+ME+TST group,with at least 3 samples in each group.The liver function of mice in each group was evaluated by serum transaminase levels,and the degree of liver injury was determined by H&E staining.qRT-PCR was used to analyze the mRNA expression levels of inflammatory factors and the mRNA expression levels of core target moleculesof ME in the treatment of LIRI.MPO staining was used to evaluate the infiltration of liver neutrophils.TUNEL staining was used to determine the apoptosis of liver tissue cells,and flow cytometry was used to detect the apoptosis rate of each group.Apoptosis-related proteins,pathway-related proteinsand the expression of core target proteinsduring LIRI treatment with ME were detected and analyzed by Western blot.Network pharmacology and molecular docking were used to screen out the core target proteins that play a role in the treatment of LIRI by ME.In this process,Pharm Mapper,TCMSP and Swiss Target Prediction database were selected to screen potential active targets of ME,and Uni Prot KB database was used to standardize the official name of each Target gene.GeneCards and DisGeNET databases were used to obtain LIRI pathological targets,and VENNY was used to draw visual Venn diagrams of drug targets and disease proteins.Then,PPI network was constructed to screen core target genes,and core target molecules were analyzed for GO function enrichment and KEGG signal pathway enrichment.Finally,the best effector protein was obtained by molecular docking analysis.Results: Compared with sham group,LIRI+Vehicle group significantly increased serum transaminase,significantly aggravated liver tissue injury,Suzuki’s score significantly increased,neutrophil infiltration was serious,liver cell apoptosis increased,and the mRNA expression level of pro-inflammatory factor(IL-1β)increased.The mRNA expression level of anti-inflammatory factor(IL-10)decreased;At the same time,the expression of pro-apoptotic proteins(Bax and cleaved caspase 3)was increased,and the expression of anti-apoptotic proteins(Bcl-2)was decreased.After ME treatment,all the above conditions were significantly reversed.The results of in vitro cell experiments were consistent with those of in vivo experiments.Studies on the mechanism of ME in LIRI and the H/R process of liver cells found that,compared with the control group,the expression level of PI3K/Akt signaling pathway related proteins was significantly increased after ME application.After using the inhibitor LY294002 in vivo and in vitro,the liver inflammatory response,apoptosis rate and liver/hepatocyte injury were significantly increased.349 effective targets of ME and 1829 LIRI-related genes were identified by Multiple databases.Then,using Cytoscape software,46 therapeutic targets of ME were obtained by intersecting LIRI target genes.After the STRING treatment,46 potential target genes for LIRI were analyzed by GO and KEGG.KEGG analysis found that the PI3K/Akt signaling pathway was the main pathway involved in ME in the treatment of LIRI.Cytoscape was used to further analyze the network of LIRI target genes acting on ME.Two core topological networks were obtained and six core genes were screened out: Hsp90α,HDAC6,Rac1,KDR,EP300 and ERBB2.Finally,molecular docking and binding energy calculation showed that Hsp90α and HDAC6 had good binding affinity and high stability with ME.In vivo and in vitro liver ischemia reperfusion injury models,it was found that the mRNA and protein expression of Hsp90α in all treatment groups did not change significantly,but compared with the ischemia reperfusion injury group,the mRNA and protein expression level of HDAC6 were significantly increased,while the protein expression level of Acetyl-HSP90 was significantly decreased after ME treatment.Conclusion: Methyl eugenol can play a significant protective role in liver ischemia reperfusion injury in vivo and in vitro by alleviating liver function injury,improving inflammatory infiltration and reducing hepatocyte apoptosis.In addition,methyleugenol can promote the deacetylation of Hsp90α by increasing the expression of HDAC6 during LIRI in vivo and H/R in vitro,thus activating the PI3K/Akt signaling pathway and playing a protective role.