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The Role And Mechanism Of Safranal In Isoproterenol-induced Myocardial Injury

Posted on:2023-11-21Degree:MasterType:Thesis
Country:ChinaCandidate:M J YanFull Text:PDF
GTID:2544306848485734Subject:Pathology and pathophysiology
Abstract/Summary:PDF Full Text Request
Objective: Cardiomyocyte apoptosis and inflammatory injury caused by Sympathetic hyperactivation are important reasons to promote the progression of cardiovascular disease.Safranal(SFR)has been shown to have good myocardial protection in recent studies,but the mechanism of its role in myocardial injury induced by sympathetic overactivation is not fully understood.In this study,the effect and mechanism of safranal on myocardial injury induced by isoproterenol were explored from multiple perspectives by using network pharmacology,molecular simulation docking and and in vivo,in vitro experimental verification,providing some new theoretical basis for drug development of myocardial injury and clinical application of safranal.Methods:1.In vivo pharmacodynamics study: The rat model of myocardial injury was established by subcutaneous administration of isoproterenol.The experiment was divided into Control group,ISO group(85mg/kg),ISO+SFR-L group(administration of safranal 0.025 m L/kg),ISO+SFR-M group(0.050 m L/kg)and ISO+SFR-H group(0.075 m L/kg).The enzyme activities of CK and CK-MB were analyzed by the detection of serum.HE staining was used to observe the morphological changes and inflammatory infiltration of myocardial tissue.Masson staining was used to evaluate the level of myocardial fibrosis,and TUNEL staining was used to analyze the level of myocardial apoptosis.The expression changes of caspase-3,Bax and Bcl-2 in rat myocardium were analyzed by immunohistochemical staining.Western blot was used to detect the protein expression changes of cleaved caspase3,Bax,Bcl-2 in rat myocardial tissue.2.In vitro cell experiment:(1)H9c2 cells were induced by ISO to establish myocardial cell apoptosis injury model,which was divided into Control group,ISO group,ISO+SFR(20 μM,40 μM,80 μM)group.CCK-8 was used to detect the effects of ISO and safranal on the survival rate of cells.LDH activity assay was used to observe cell damage.Hoechst 33342 staining was used to observe and calculate the positive rate of apoptotic nuclei,and flow cytometry was used to calculate and analyze the sum of apoptosis rates of cells.Western blot was used to detect the protein expression changes of cleaved caspase3,Bax,Bcl-2 in H9c2 cells.(2)Network pharmacology prediction:(1)Target analysis: To predict the potential targets of safranal in the treatment of myocardial injury caused by sympathetic overactivation and screen the common core targets;(2)Pathway prediction: By integrating common targets,the pathway related to myocardial injury induced by sympathetic hyperactivation was predicted;(3)Molecular docking: The core targets enriched in the pathway were docked with safranal,and the conformational changes and binding strength were analyzed.(3)Mechanism verification: H9c2 cells were used to validate the network pharmacology prediction of targets,pathways,and molecular docking related to safranal and sympathetic hyperactivation induced myocardial injury.Results:1.In vivo experimental results: Results of CK and CK-MB enzyme activities in serum showed that safranal alleviated myocardial injury induced by sympathetic hyperactivation(P < 0.01).HE staining,Masson staining,TUNEL staining and immunohistochemical staining showed that safranal could reduce cardiac inflammatory infiltration,apoptosis and fibrosis induced by ISO stimulation(P < 0.01),and reduced the expression levels of caspase-3 and Bax in myocardium,and it also increased the expression level of Bcl-2(P < 0.01).Western blot showed that ISO could promote myocardial apoptosis,up-regulate the expression of apoptotic proteins Bax and cleaved caspase-3,down-regulate the expression of anti-apoptotic protein Bcl-2(P < 0.001),and Pre-administration of safranal significantly down-regulated the expression of Bax and cleaved caspase-3(P < 0.05).Correspondingly,the expression level of Bcl-2 was up-regulated(P < 0.01).2.In vitro experimental results:(1)CCK-8 showed that safranal increased the cell viability decreased by ISO stimulation(P < 0.001);LDH results showed that safranal reduced the level of cell damage induced by ISO stimulation(P < 0.01).Western blot showed that ISO could promote apoptosis of H9c2 cells,up-regulate the expression of Bax and cleaved caspase-3,down-regulate the expression of Bcl-2(P < 0.001),while safranal could significantly down-regulate the expression of Bax and cleaved caspase-3(P < 0.01).Correspondingly,the expression level of Bcl-2 was up-regulated(P < 0.05).Hoechst 33342 and flow cytometry showed that ISO increased the apoptosis rate of H9c2 cardiomyocytes(P < 0.001),while safranal significantly reduced the apoptosis rate of H9c2 cardiomyocytes in all pretreatment groups(P < 0.01).(2)Potential candidate targets of safranal were predicted by TCMSP database,SWISS database,Pharm Mapper and QSAR database.Sympathetic hyperactivation and myocardial injury targets were obtained by Dis Ge NET and Gene Cards database respectively.A total of 26 targets were obtained by Venn diagram.The core targets are sorted according to PPI Degree in String database.KEGG enrichment analysis suggested that the mechanism of safranal against sympathetic hyperactivation induced myocardial injury may be closely related to the regulation of TNF signaling pathway,and it plays a protective role by regulating TNF signaling pathway related targets(TNF,CAS3,PTGS2,MMP9,RELA).At the same time,molecular docking results showed that safranal could interact with TNF pathway-related targets to form dense complexes.(3)Cell experiments verified that ISO induced up-regulation of TNF,PTGS2,MMP9 and p RELA protein expressions in H9c2cells(P < 0.001),and safranal significantly down-regulated the protein expressions of TNF,PTGS2,MMP9 and p RELA(P < 0.01).Conclusion:1.Safranal ameliorated myocardial injury induced by isoproterenol.2.In myocardial injury caused by sympathetic overactivation,safranal may play a protective role in myocardium by regulating TNF signaling pathway and related targets.
Keywords/Search Tags:Sympathetic hyperactivation, Safranal, Myocardial injury, Network Pharmacology, TNF signaling pathway
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