| Objective:To analyze the potential molecular biological mechanism of clove in the treatment of myocardial ischemia-reperfusion injury by network pharmacology.To study the protective effect of eugenol on myocardial ischemia-reperfusion injury.To explore the mechanism of NFκB signaling pathway in regulating myocardial ischemia-reperfusion injury.To provide a scientific theoretical basis for eugenol in the treatment of myocardial ischemia-reperfusion injury.Methods:(1)To find the components and targets of clove from tcmsp;Genes related to Miri were collected from genecards and OMIM;Draw Venn diagram was used to show the relationship between disease targets and drug prediction targets;Protein interaction platform(string)was used to construct PPI network;Bioconductor database was used for gene ontology(go)enrichment analysis and Kyoto Encyclopedia of genes and genomes(KEGG)pathway analysis.The network of "active component target pathway" was constructed by Cytoscape 3.7.2.(2)Primary cardiomyocytes were isolated and cultured from neonatal Wistar rats,The model of myocardial cell injury was established by mixed gas method and pre intervened by eugenol.The effect of eugenol on cardiomyocyte morphology was observed under inverted microscope;Cell counting kit-8(CCK-8)method was used to detect the viability of cardiomyocytes;The activities of LDH,CK and AST in cell culture medium were detected by automatic biochemical analyzer;Superoxide dismutase(SOD)and malondialdehyde(MDA)in cardiomyocytes were detected by kit method;The levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were detected by enzyme-linked immunosorbent assay(ELISA);Real time quantitative polymerase chain reaction(RT q PCR)was used to detect the m RNA expression of NFκB,IKB,Bcl-2 and Bax in cardiomyocytes;The protein expression levels of NFκB,IκB,P-IκB,Bcl-2 and Bax were detected by Western Blotting;Hoechst staining was used to detect the degree of apoptosis of primary neonatal rat cardiomyocytes;The apoptosis rate of primary cultured neonatal rat cardiomyocytes was detected by flow cytometry.(3)Naked oats starch was extracted by defatted alkali extraction method;Single factor and orthogonal experiments were used to determine the best extraction process of naked oat starch;Eugenol starch inclusion compound lyophilized powder was prepared by ultrasonic method;The content of eugenol in the inclusion complex was determined by HPLC;Scanning electron microscopy(SEM),Fourier transform infrared spectroscopy(FI-TR)and nuclear magnetic resonance(NMR)were used to identify the lyophilized powder of eugenol/starch inclusion complex.(4)Wistar rats were randomly divided into normal control group,sham model group,starch matrix group(0.96 g/kg),eugenol inclusion complex low,medium and high dose groups(0.24 g/kg,0.48 g/kg,0.96 g/kg),compound Danshen dripping pill group(85 mg/kg).The animals in each group were treated for 10 days,Miri model was established by ligating left anterior descending coronary artery for 30 minutes and reperfusion for 24 hours,The electrocardiographic signals of rats were measured by the method of bioelectrical signals detected by the biological function experimental system;The activities of LDH,CK and AST were detected by automatic biochemical analyzer;The activities of MDA and SOD in rat myocardium were determined by kit method;The levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were measured by enzyme-linked immunosorbent assay(ELISA);The m RNA expression levels of NFκB,IKB,Bcl-2 and Bax were detected by RT-q PCR;The protein expression levels of NFκB,IκB,P-IκB,Bcl-2 and Bax were detected by Western Blotting.Results:(1)93 potential targets of clove against myocardial ischemia-reperfusion injury were screened,The key targets include interleukin-6(IL-6),NFκB and apoptosis regulator Bax;The biological process of go enrichment mainly involves DNA binding transcription factor binding,cytokine receptor binding and receptor ligand activity;KEGG pathway enrichment results showed that IL-17 signaling pathway,NFκB signaling pathway and TNF signaling pathway were related to the anti myocardial ischemia-reperfusion injury mechanism of clove.(2)Compared with the model group,the activity of cardiomyocytes in eugenol concentration groups,positive drug groups and inhibitor groups increased,The activities of LDH,CK and AST in cell culture medium decreased significantly(P < 0.05),The activity of SOD was significantly increased(P < 0.05),and the activity of MDA was significantly decreased(P < 0.05),The activities of IL-6 and TNF-α were significantly decreased(P < 0.05).The apoptosis rate was significantly decreased(P < 0.05)in a concentration dependent manner,In addition,NFκB nuclear translocation and P-IκB/IκB ratio were significantly decreased and Bcl-2/Bax ratio was significantly increased in eugenol concentration groups,positive drug groups and inhibitor groups(P < 0.05),The m RNA expression levels of IκB and Bcl-2 were significantly increased(P < 0.05),while the m RNA expression levels of NFκB and Bax were significantly decreased(P < 0.05).(3)The optimum extraction conditions of naked oats starch were as follows: solid-liquid ratio was 1:10,shaking temperature was 50 ℃,shaking time was 2 h,The extraction rate of naked oat starch was 73.26%.Under the optimal conditions,the moisture content,ash content,fat content and protein residue of naked oat starch were7.56g/100 g,0.13g/100 g,0.17g/100 g and 0.56g/100 g respectively;The optimum technological conditions of eugenol starch inclusion compound lyophilized powder were as follows: the ratio of eugenol to starch was 10:0.8,the ultrasonic temperature was 50 ℃,the ultrasonic time was 50 min,and the inclusion rate was 44.54%.The order of influencing factors on inclusion rate was ultrasonic temperature > feed ratio > ultrasonic time.Scanning electron microscope(SEM)showed that naked oats starch granules were small,and their sizes were not consistent.Naked oats starch granules were irregular,polygonal or oval in shape,and belonged to small granule starch.Some starch granules had depressions on the surface.Gelatinized starch was lamellar network structure,and inclusion compound was lamellar network structure with many round small granules;Fourier transform infrared spectroscopy(FI-TR)showed that the inclusion complex still maintained the parent structure,and its absorption peak had a slight blue shift or red shift;Nuclear magnetic resonance(NMR)showed that the chemical shifts of the c-skeleton of the inclusion complex were partly shifted and some new chemical shifts appeared.The above changes can prove the formation of eugenol / starch inclusion complex.(4)Compared with the model group,the activities of LDH,CK,AST,IL-6 and TNF-α in serum of eugenol/starch inclusion complex groups were significantly decreased(P < 0.05),The activity of SOD in myocardium increased significantly(P < 0.05),MDA activity decreased significantly(P < 0.05)in a dose-dependent manner,NFκB nuclear translocation and P-IκB/IκB ratio were significantly decreased,and Bcl-2/Bax ratio was significantly increased(P < 0.05).The m RNA expression levels of IκB and Bcl-2 were significantly increased(P < 0.05),The expression levels of NFκB and Bax m RNA were significantly decreased(P < 0.05).Conclusion: Eugenol has a certain protective effect on myocardial injury,which is related to its inhibition of NFκB signal pathway and mitochondrial apoptosis signal pathway... |
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