| Objective:Our preliminary research showed that velvet antler could improve the cardiac function of rats with heart failure following myocardial infarction as well as prevent the cardiac matrix collagen fibrosis and the ventricular remodeling,it could regulate the expression of SERCA2a and improve myocardial systolic function.In addition,velvet antler proteins(VA-pro)could promote the proliferation of HUVECs.On the base of these previous studies,the purpose of this thesis is to further discuss the mechanisms of VA improving heart function after acute myocardial infarction.We intend to do the proteomics analysis of VA-pro,and reveal the influence of VA-pro on endothelial progenitor cells(EPCs),meanwhile,we establish ischemia hypoxia model of cardiomyocytes(CMs)and cardiac microvascular endothelial cells(CMECs)in vitro based on the pathogenesis of ischemic heart disease to study the influence of VA-pro on CMs and CMECs,and to reveal that VA-pro repair the cardiac microvascular by influencing EPCs vitality and protecting the damaged CMECs as well as protect demaged CMs.Methods:(1)VA-pro were extracted with water solvent,the ultrasonic wave method,freeze-drying technology,and then analyzed by Nano LC-MS/MS.VA-pro was annotated based on the Gene Ontology database and Kyoto Encyclopedia of Genes and Genomes database.(2)The CMECs and CMs were isolated from SD rat pup hearts and identified by fluorescence immunoassay with CD31 antibody,vWF antibody and MHC antibody,respectively.CMECs were cultured at 37°C with IH condition for 46 h and CMs were cultured for 36 h.The role of VA-pro on cell viability,proliferation,migration,apoptosis,and mitochondrial membrane potential(MMP)were evaluated with MTS assay,EdU assay,scratch assay,transwell assay,Annexin V-FITC/PI double-staining assay,and JC-1 assay,respectively.The expression of Akt,p-Akt,Bcl-2,Bax,Caspase3 were detected using Western Blot,tube formation in Matrigel of CMECs and CMs contraction were also detected.(3)EPCs were isolated from 2-week-old male rats' femur by the density gradient centrifugation method and incubated in EBM-2 medium.The cells were identified by fluorescence immunoassay with CD 133 and VEGFR2 antibody.EPCs viability was detected with MTS assay,migration ability were detected with scratch assay and transwell assay,tube formation ability were detected with Matrigel,the expression of NICD,Hesl1 p-Akt,p-mTOR were detected using Western Blot.Result:(1)The VA-pro mass concentration was 465.56 |jpg/mg,the total number of VA-pro was 386 which identified by the MASCOT search(the taxonomy Ruminantia),and there were 145 uncharacterized proteins.The 386 proteins isoelectric point ranged from 4.14 to 11.57,and molecular weight ranged from 6 to 800 kDa.As for the biological processes of GO annotation,211 proteins were involved in the metabolic process,102 proteins were involved in metabolic regulation,151 proteins were involved in the biological process,110 proteins were involved in the localized biological processes,and 14 proteins involved in the growth of related biological processes.Further,38 proteins were involved in the cell apoptosis process,27 proteins were related to cell migration.Molecular function analysis showed that 323 proteins are involved,228 proteins associated with the process of binding,most of them belong to the binding functions of proteins responsible for ionic bonds,nucleotide,RNA binding process.53 proteins were Responsible for the regulation of molecular function,121 proteins participated in the catalytic activity,49 proteins involved in regulating enzymes activity,28 protein participated in the activities of transferase activity.(2)IH reduced CMECs viability,DNA activity,mitochondrial membrane potential,migration and tube formation ability,VA-pro could reverse the influence,especially the 0.5 and 1 mg/ml VA-pro.IH induced CMECs apoptosis,while 0.5 mg/ml,1 mg/ml,2 mg/ml VA-pro reversed it;Western Blot results showed that IH decreased Bcl-2 and p-Akt expression level,rose Bax and cleaved-caspase3 level;VA-pro intervention changed this trend of those proteins,raising the expression of Bcl-2 and p-Akt,and decreaseing Bax and cleaved-caspase3 levels;After applied LY294002,VA-pro effects was suppressed.(3)IH reduced CMs viability,DNA activity,mitochondrial membrane potential and contraction,VA-pro could reverse the influence to some degree,especially 1 mg/ml VA-pro.IH induced CMs apoptosis,0.5 mg/ml,1 mg/ml,2 mg/ml VA-pro reversed it and there is no statistical difference between them;Western Blot results were similar with(2).(4)0.25 mg/ml and 1 mg/ml of VA-pro significantly promoted the viability of EPCs,but had no obvious effect on tube formation ability of EPCs;1 mg/ml VA-pro promoted the EPCs migration ability,but 0.0625 mg/ml and 0.25 mg/ml VA-pro had no effect on it;VA-pro increased NICD and Akt activity levels of EPCs concentration dependently,at the same time,0.25.mg/ml and I mg/ml VA-pro rose Hes1 and mTOR activation le'vels.After applied DAPT and LY294002,the VA-pro effects was suppressed.Conclusions:I.VA-pro contains were complex and were shown to be involved in various molecular function and biological processes.2.VA-pro protected CMECs and CMs subjected with ischemia hypoxia as well as regulated p-Ak:t,Bcl-2,Bax and cleaved-caspase3 expression levels,maintained cells activity.3.VA-pro promoted the proliferation and migration of EPCs,and increased the Notch signaling pathways related proteins NICD and HesI activation levels,and the Akt/mTOR signaling pathway related proteins Akt and mTOR phosphorylation levels.Significance:1.This study provides a deeper theoretical support for our previous findings,the conclusions remind us that VA-pro repair the cardiac microvascular by protecting the damaged CMECs and influencing EPCs vitality as well as protect demaged CMs.2.Our study on VA were designed to explore its pharmacological effects and offer a new thought and method for the treatment of ischemic heart disease,meanwhile we want to standardize the use of velvet antler pharmaceutical and to reduce the using of deer antler with synthetic drugs. |
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