Role Of Qishenyiqi Pills In Improving Effect Of Mesenchymal Stem Cells Transplantation On Cardiac Repair After Myocardial Infarction

BackgroundMyocardial infarction(MI)is a leading cause of cardiac injury.Various types of stem cells,including mesenchymal stem cells(MSCs),have been proposed to improve the healing process after MI.However,most of these cells undergo apoptosis in the first week.Therefore,the main limitation in the stem cell therapy is the small number of surviving stem cells.Anoikis,ischemia and inflammation are three main pathways have been proposed leading to cell death after transplantation.Strategies inducing intrinsic cytoprotective mechanism or targeting a specific molecular pathway,including heat shock,hypoxia,overexpression of anti-apoptotic factors(e.g.Akt or Bcl-2)or pro-survival factors(e.g.VEGF),eliminating free radical species,co-delivery of cells with matrix,depleting complement system or blocking leukocyte infiltration,have been employed to enhance survival of graft.However,the potential toxicity and side effect limit clinical use of these interventions.Qishenyiqi(QSYQ),a Chinese medicinal formula for coronary artery disease and heart failure,has been proved to protect acute ischemia-induced left ventricular remodeling by attenuating inflammation,anti oxidant and modulating energy metabolism in a multi-compound,multi-target,multi-pathway manner.These findings led to the speculation that QSYQ could facilitating the survival of MSCs after transplantation.The present study used an animal model to test whether QSYQ could promote MSC cell survival and accelerate infarct repair after acute MI.MethodsMSCs were isolated from green fluorescent protein-transgenic mice on a C57BL/6 background at 6-8 weeks.The MSCs were stained for surface antigen expression using the following antibodies:anti-CD90.2,anti-Ly6A,anti-CD31,anti-CD45,and anti-CD 117.Flow cytometry and immunofluorescence staining were performed to analyze the purity of MSCs.Wild-type C57BL/6 male mice were randomly assigned to MI+ PBS,MI+MSC,and MI+MSCs+QSYQ groups.QSYQ or PBS was administered 10 days preceding the induction of MI until sacrifice.The total volume of QSYQ or PBS administered was 0.1 mL/day.The concentration of QSYQ solution was 3.9 mg/mL.MI were induced by left anterior descending coronary artery ligation.5×105 GFP-MSCs(20?L)or 20?L PBS were transplanted into the infarcted hearts and border zone through intramyocardial injection.Sequential echocardiography were performed to evaluate cardiac structure and function before MI,14 days and 21 days after MI.After all echocardiographic evaluation,all mice were sacrificed and hearts were harvested.Infarction sizes,myocardial fibrosis,angiogenesis,cells apoptosis and proliferation were evaluated by histological analysis.GFP+MSCs apoptosis were determined by immunofluorescence co-staining for TUNEL.Immunofluorescence staining were performed to assess immune cells,including M1/M2 macrophages,neutrophils and regulatory T cells number in infarct heart.Genome-wide gene expression profiles were studied to determine genomic differences among the three groups.Pathway analysis of differentially expressed genes was performed using KEGG and Biocarta.ResultsFlow cytometry analysis confirmed that MSCs were positive for CD90.2 and Ly6A and negative for CD31,CD45,and CD117.A significant worsening of LVEF and LVFS and an enlargement of end-diastolic left ventricular inner diameter(LVID;d)and end-systolic left ventricular inner diameter(LVID;s)were observed in all groups at 14 days compared with the baseline.At 3 weeks after transplantation,the MI+MSCs+QSYQ group revealed better cardiac function compared with the other two groups with regard to all the echocardiographic parameters.Infarct size and collagen content inside myocardium within the border zone were significantly reduced in the MI+MSCs+ QSYQ group compared with the MI+ MSC and MI+PBS groups.In both the infarct area and border zone,capillary density and cell proliferation were increased in MI+MSCs+QSYQ group when compared to MI+MSC and MI+PBS groups.Within the infarct area,cell apoptosis was significantly decreased in MI+MSCs+QSYQ groups compared with the MI+PBS group and MI+MSC.The number of transplanted MSCs was higher in the MI+MSCs+QSYQ group than MI+MSC group accompanied with MSC apoptosis after transplantation.No co-staining of CD86 and F4/80,single staining of NIMP-R14 and Foxp3 were found within the infarct area.However,a large amount of co-localization of CD206 and F4/80 was observed in the infarcted myocardium.The prevalence of M2 cells in the scar tissue was significantly high in MI+MSCs+QSYQ group compared with those in the MI+MSC group.The differential expression of over 2700 genes was noticed in MI+MSCs+QSYQ group,while only 41 genes were changed in single MSCs transplanted mice.Pathway analysis indicated that these genes are closed related to "focal adhesion","ECM-receptor interaction","Calcium signaling pathway","MAPK signaling pathway","fatty acid metabolism","peroxisome","nuclear factor of activated T-cells(NFAT)" and "leukocyte transendothelial migration pathways",et al.ConclusionsQSYQ could enhance the cardiac-protecting effect of MSCs by reducing MSCs apoptosis after transplantation.The possible mechanism underlying this phenomenon was QSYQ increased M2 macrophage retention in infarcted myocardium.