| Objective:To study and predict the protective effect of clove on the heart of rats with myocardial ischemia-reperfusion injury(MIRI)based on the network pharmacology method,and to explore the role of clove in the heart of rats with energy metabolism signaling pathway AMPK/PGC-1α.The role of myocardial energy metabolism,based on this,to explore the mechanism of clove’s protective effect on MIRI.Methods:(1)The mechanism of action of clove on MIRI was analyzed and predicted by the method of network pharmacology,the word "Clove" was searched in the TCMSP database,and the OB value was greater than or equal to 30%and the DL value was greater than or equal to 0.18.6 active ingredients,and according to the research progress of clove at home and abroad,combined with relevant literature,eugenol was included in this study;"Myocardial Ischemia Reperfusion Injury" was entered in GeneCards and OMIM database to search for MIRI disease targets,and duplicates were deleted.Save the relevant targets after the value is obtained;input the obtained drug and disease targets into the online website VENNY 2.1.0 to predict the intersection targets and make Venn diagrams;input the intersection targets into the STRING database to establish protein-protein interaction(protein-protein interaction),PPI)network diagram,and save the protein interaction data,and import the obtained data into Cytoscape 3.7.2 software to draw the protein interaction network diagram.The drug-active ingredient-targets and MIRI disease targets were input into Cytoscape 3.7.2 software to construct the "drug ingredient target disease" network diagram.GO set analysis and KEGG pathway analysis of common targets were performed using Bioconductor bioinformatics software.(2)Using the improved method for the isolation and culture of primary neonatal rat cardiomyocytes,Wistar neonatal rat cardiomyocytes were extracted and cultured,the cell growth state was observed by microscope,and the viability of cardiomyocytes was detected by placental blue staining;the mixed gas method(95%N2+5%CO2+1%O2)treatment of cardiomyocytes,respectively,hypoxia(4,6,8,10,12,14h)/reoxygenation(CO2 5%+37℃)for 4h to determine the hypoxia-reoxygenation injury model(Ischemia-Reperfusion,I/R)conditions;choose different concentrations of eugenol(2μL·mL-1,4μL·mL-1,8μL·mL-1,12μ·mL-1,14μL·mL-1,16μL·mL-1)Pretreatment of cells to determine drug concentration;CCK-8 method was used to detect cell viability;automatic biochemical analyzer was used to detect phosphocreatine kinase(CK)and creatine kinase isoenzyme(CK)in cell culture medium-MB),lactate dehydrogenase(LDH),aspartate aminotransferase(AST)activities;colorimetric method was used to detect the oxidative stress markers superoxide dismutase(SOD),malondialdehyde(MDA)activities and ELISA was used to detect the opening degree of mitochondrial permeable membrane transition(mPTP)in cardiomyocytes;Hoechst cell apoptosis assay and flow cytometry were used to detect the apoptosis of cardiomyocytes in each experimental group;Western Blot was used to detect the apoptosis of cardiomyocytes.The protein expression levels of Sirt-1,AMPK,P-AMPK,PGC-1α,GLUT4 and cTn1 in cardiomyocytes were detected by the method.(3)The eugenol/β-cyclodextrin inclusion complex was prepared by ultrasonic method;the content of eugenol in the inclusion complex and the properties of the inclusion complex were detected by HPLC,and the inclusion complex of eugenol β-cyclodextrin was calculated Rate.(4)70 Wistar male rats of SPF grade were randomly divided into sham operation group,ischemia-reperfusion model group,β-cyclodextrin matrix group,diltiazem group,and eugenol inclusion complex low group after adaptive feeding for one week.,middle and high dose groups,10 in each group.Animals in each group were given continuous intragastric administration for 15 days,β-cyclodextrin matrix group(0.5g·kg-1·d-1),diltiazem group(8.61 mg·kg-1·d-1),eugenol inclusion The low,medium and high dose groups(0.125g·kg-1·d-1,0.25g·kg-1·d-1,0.5g·kg-1·d-1)were suspended once a day.The sham operation group and the ischemia-reperfusion injury model group were given the same dose of 1%CMC-Na suspension every day.One hour after the last administration,the left anterior descending coronary artery of the rat heart was surgically ligated for 30 minutes,and the MIRI model was established by restoring the perfusion for 24 hours.The biological function experimental system was used to detect the ECG signal of the rat;The activities of CK,CK-MB,LDH and AST;colorimetric method was used to detect eugenol on superoxide dismutase(SOD)and malondialdehyde(MDA)activities in myocardial tissue of each experimental group;TTC staining method was used to detect eugenol The effect of myocardial infarction size in each group of experimental rats;Western Blot was used to detect the protein expression levels of Sirt-1,AMPK,P-AMPK,PGC-1α,GLUT4 and cTnl in cardiomyocytes;-Eosin method(HE staining method)to detect the cardiac pathological changes of rats in each group.(5)The content of adenosine triphosphate(ATP),adenosine diphosphate(ADP),and adenosine monophosphate(AMP)in the myocardial tissue of each group of experimental rats was detected by high performance liquid chromatography.Results:(1)Network pharmacology data were available:A total of 7 effective compounds were screened out of cloves,and 304 targets were obtained from the 7 compounds,and a total of 1085 corresponding targets were obtained by deleting duplicate values in "Myocardial Ischemia Reperfusion Injury".304 drug-disease intersection targets were obtained,involving AKT,VEGFAA,TP53,CASP3,STAT3 and other intersection targets.The GO correlation analysis of the intersection targets showed that 1943 biological processes were enriched in the intersection targets,mainly including regulation of mitochondrial membrane potential and reactive oxygen species metabolism.metabolic process),regulation of inflammatory response(Rulategion of inflammatory response);72 cellular component expression processes,mainly involving membrane rafts,membrane microdomains,secretory granule lumen,etc.;118 processes related to molecular functions,mainly including Molecular functions such as nuclear receptor activity,ligand-activated transcription factor activity,and protein tyrosine kinase activity.KEGG pathway enrichment results found that tyrosine protein kinase/signal transducer and transcription activator(Janus activated kinase signal transducer and activator of transcriptions,JAK/STAT)JAK-STAT,interleukin 17(IL-17 signaling pathway)IL-7,VEGFA,phosphatidylinositol 3 kinase PI3K/AKT protein kinase B(PI3K-AKT),AMPdependent protein kinase(AMPK),nuclear transcription factor(NF-kappa B signaling pathway)NF-A variety of pathways such as κB are related to the mechanism of clove antimyocardial ischemia-reperfusion injury.(2)The experimental data showed that the optimal conditions for the IR model of primary neonatal rat cardiomyocytes were at 10 hours of hypoxia and 4 hours of reoxygenation.The survival rate of IR cardiomyocytes was significantly increased when 4 μL/mL was administered,and the highest survival rate of IR cardiomyocytes was reached when 14μL/mL was administered.(P<0.05 or P<0.01).Compared with the IR group,the activities of CK,CK-MB,LDH,and AST in the cardiomyocyte culture medium treated with each concentration group of eugenol gradually decreased with the increase of the dose,and the difference between the high-dose eugenol group and the IR group was as follows.Significant(P<0.01),the activities of CK,CK-MB,LDH and AST in the IR+inhibitor+drug group were lower than the values of IR+eugenol high dose.The activity of SOD in IR cardiomyocytes treated with high concentration of eugenol gradually increased(P<0.01),and the activity of MDA decreased significantly(P<0.01).The activity of MDA in IR+inhibitor+drug group was higher than that in high-dose eugenol group.Vitality(P<0.01),the activity of SOD in IR+inhibitor+drug group was lower than that of IR+eugenol high dose(P<0.01);Compared with the IR group,the content of mPTP in the IR+inhibitor+drug group was significantly higher than that in the IR+eugenol high dose(P<0.01);compared with the blank group,the apoptosis rate of cardiomyocytes in the IR group was significantly increased(P<0.01).0.01),eugenol could significantly inhibit the apoptosis rate of cardiomyocytes(P<0.05);compared with the blank group,the expression levels of P-AMPK/AMPK and cTnl in the IR group were significantly increased(P<0.01),Sirt-1,PGC The expression levels of-1α and GLUT4 decreased(P<0.05 or P<0.01).The high concentration group of eugenol could significantly up-regulate the expression levels of Sirt-1,PGC-1α and GLUT4 in primary neonatal rat cardiomyocytes(P<0.05 or P<0.01)down-regulated the expression levels of PAMPK/AMPK and cTnl(P<0.05 or P<0.01);(3)Preparation of eugenol/β-cyclodextrin inclusion complex:Eugenol(mL):β-cyclodextrin(g)=(1:10),ultrasonic time 60min,ultrasonic temperature 60℃,chromatographic conditions are Chromatography methanol:water(60:40),the inclusion rate of eugenol-starch inclusion complex was 76.2%.(4)The experimental data showed that compared with the sham operation group,the ECG of the rats in the model group showed ST segment elevation and the heart was damaged.Compared with the sham operation group,the activities of CK,CK-MB,LDH and AST in the serum of the model group were significantly increased(P<0.01);compared with the model group,the high dose of eugenol/β-cyclodextrin inclusion complex The activities of CK,CKMB,LDH and AST in serum of the two groups were significantly decreased(P<0.01);compared with the sham-operated group,the activities of SOD in myocardial tissue were significantly decreased(P<0.01),and the activities of MDA were significantly increased(P<0.01),compared with the model group,in the high-dose eugenol/β-cyclodextrin inclusion complex group,MDA was significantly decreased and SOD was significantly increased(P<0.05 or P<0.01).Compared with the sham operation group,the infarct size of the heart in the model group(P<0.01),the myocardial infarction size in each dose group of eugenol/βcyclodextrin was significantly reduced compared with the model group(P<0.01),with higher The dose was the most obvious;the results of pathological sections showed that compared with the sham-operated group,the rat heart tissue in the model group had a large area of necrosis,and the cells were arranged and disordered.Compared with the model group,the eugenol/β-cyclodextrin inclusion complex high-dose group Compared with the shamoperated group,the expression levels of P-AMPK/AMPK and cTnl in the model group were significantly increased(P<0.01),while the expression levels of Sirt-1,PGC-1α and GLUT4 were decreased in the model group(P<0.01).<0.05 or P<0.01)The high eugenol/βcyclodextrin inclusion complex group could significantly up-regulate the expression levels of Sirt-1,PGC-1α and GLUT4 in myocardial cells of MIRI rats(P<0.05 or P<0.01),downregulated the expression levels of P-AMPK/AMPK and cTnl(P<0.05 orP<0.01);(5)Detection of three adenosine triphosphates in rat myocardial tissue:chromatographic methanol-50 mmol/L potassium phosphate buffer solution(7:93),solution pH(6.88)to detect eugenol/β-cyclodextrin inclusion complex Compared with the IR group,the contents of ATP and ADP in the myocardial tissue of the rats in each treatment group increased with the increase of the dose(P<0.05,P<0.01),and the content of AMP decreased with the increase of the dose of eugenol(P<0.05)..Conclusion:Eugenol can regulate energy metabolism in myocardium through Sirt-1/AMPK/PGC-1α signaling pathway,thereby exerting its protective effect on myocardial injury. |
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