Study On The Effect And Mechanism Of Latifolin From Dalbergiae Odoriferae On Bone Marrow Mesenchymal Stem Cells For Myocardial Repair Of Ischemic Heart Disease

Object:Bone marrow mesenchymal stem cells transplantation for treating myocardial infarction is considered to be an effective treatment.However,the inflammatory microenvironment leads to low survival rate and differentiation rate of transplanted BM-MSCs,which limits its efficacy.improving survival and differentiation ability is very important to improve the efficacy of BM-MSCs transplantation in the treatment of myocardial infarction.Latifolin is isolated from Dalbergiae Odoriferae.Research reports show that latifolin has anti-inflammatory effects.Previous studies found that latifolin has protective effects on myocardial cells,which can reduce myocardial tissue inflammation cell infiltration and improve cardiac function.Therefore,it is assumed that latifolin can improve the survival rate of BM-MSCs by improving the inflammatory microenvironment and improve the ability of BM-MSCs to differentiate,and then improve the efficacy of BM-MSCs transplantation for myocardial infarction.This study explores effects of the newflavonoid compound latifolin from Dalbergiae Odoriferae on the improvment of the survival rate of BM-MSCs by changing the inflammatory microenvironment and promoting the myocardial differentiation of BM-MSCs,repairing damaged myocardium of ischemic heart disease,laying a foundation for cells treatment for myocardial infarction.Methods:1.The primary BM-MSCs were collected by whole bone marrow extraction method,and the cells were purified by digestion.The morphology of the BM-MSCs was observed in the inverted microscope.The surface antigens CD29,CD90,CD34 and CD45 of BM-MSCs were detected by flow cytometry.2.MTT assay and Annexin V-FITC/PI were used to determine the effect of ischemia,hypoxia and inflammatory environment on the survival rate of BM-MSCs.RT-PCR and ELISA were used to detect the expression and secretion of inflammatory factors of BM-MSCs in ischemia,hypoxia and inflammatory environment.MTT assay and Annexin V-FITC/PI assay for the protective effect of latifolin on BM-MSCs;and the effect of latifolin on the secretion of inflammatory factors in BM-MSCs by ELISA.3.The morphology was observed weekly.RT-PCR were used to measure the expression of GATA4,NKX2.5 and MEF2C.Immunofluorescence staining was done to investigate the cardiac-specific proteins.Transmission electron microscopy?TEM?were performed to observe the ultrastructure.4.?1?BM-MSCs and H9c2 co-culture systems was established in vitro.The experimental groups were:H9c2 normal group,H9C₂ model group,H9c2+MSC co-culture group;H9c2+MSC co-culture+drug?2.5?g/mL,5?g/mL,10?g/mL?.Determination of apoptosis of H9c2 by Annexin V/PI after ischemia,hypoxia and inflammation and latifolin intervention.?2?The model of myocardial infarction was established by ligating left anterior descending coronary artery.The experiment was divided into 9 groups:1)sham group;2)MI model group;3)MSCs transplantation group;4)MSCs+latifolin transplantation group?25,50,100mg/kg?;5)latifolin group?25,50,100 mg/kg?.PowerLab detected ST segment changes;LDH and CK-MB levels was detected in serum to evaluate myocardial damage;cardiac function was evaluated by cardiac ultrasound;heart infarct size was measured by TTC staining;HE staining was used to evaluate myocardial injury and inflammatory condition;Masson staining was used to evaluate myocardial collagen fibrosis;immunofluorescence was used to detect survival and differentiation of BM-MSCs;immunohistochemistry was used to detect inflammatory cell infiltration in myocardial tissue;WB was used to detect the target protein expression in myocardial tissue.Results:1.The spindle BM-MSCs were observed in spindle-shaped,radioactive and spiral growth in inverted microscope.The expression rate of surface antigens CD29,CD90,CD34 and CD45 in BM-MSCs by flow cytometry were 99.8%,99.5%,0.2%,4.7%,respectively.2.?1?The results of MTT and flow cytometry showed that the cell viability was significantly enhanced after 6 h and 12 h of oxygen-free glucose deprivation,but the cell viability decreased significantly at 24 h.The cell viability was decreased at 6,12,24 h after oxygen glucose deprivation+TNF-?and LPS.?2?The results of ELISA and RT-PCR showed that IL-6,IL-10 levels in the supernatant and IL-6,IL-10,TNF-?and NF-?B genes expression were decreased after 6 and 12 h of oxygen glucose deprivation.Under the conditions of oxygen glucose deprivation+TNF-?and LPS,IL-6,TNF-?levels in the supernatant and IL-6,TNF-?and NF-?B genes expression were significantly increased after 6 and 12 h.?3?MTT and Annexin V/PI results showed that lafifolin at the dose of 10?g/mL significantly increased the viability,and significantly decreased the apoptosis of BM-MSCs under the condition of oxygen glucose deprivation+TNF-?.Latifolin significantly reduced IL-6 and TNF-?levels secreted by BM-MSCs under oxygen glucose deprivation+TNF-?conditions.3.The bone marrow mesenchymal stem cells were divided into normal group,5Aza induction group,latifolin combined with 5Aza induction group and latifolin induction group.The morphological changes of BM-MSCs differentiation into cardiomyocytes were observed under microscope.The cardiac specific gene GATA4 was detected by RT-PCR.Expression of NKX2.5 and MEF2C;immunofluorescence staining for detection of cardiac specific protein a-actinin and cTnI expression;transmission electron microscopy observation of ultrastructure of differentiation of bone marrow mesenchymal stem cells.4.?1?The Annexin V/PI assay showed that the percentage of apoptosis in model group was significantly higher than that in the normal group.Compared with the model group,H9c2+MSC co-culture group,H9c2 and MSC co-culture+drug?2.5?g/mL,5?g/mL,10?g/mL?group can significantly reduce the proportion of H9c2 apoptosis.Compared with H9c2+MSC co-culture group,H9c2+MSC+10?g/ml latifolin group can significantly reduce H9c2 apoptosis.?2?The results of PowerLab test showed that the ST segment si significantly elevated after myocardial infarction.After 7 days of administration,the high dose of MSC+latifolin significantly decreased LDH and CK-MB content.Ultrasound results showed that MSC+latifolin with high dose group significantly increased LVEF,LVFS and CO;TTC staining showed that the infarct size reached 39%in the model group,and the MSC group and the MSC+latifolin group significantly reduced the infarct size;HE staining and immunohistochemistry results showed that MSC+latifolin group significantly decreased the infiltration of inflammatory cells and myofilament dissolvement in the infarcted area.Masson staining showed that MSC+latifolin group had less fibrosis and no obvious myofilament dissolvement.The immunofluorescence showed that the number of red fluorescent cells in MSC+latifolin group was significantly higher than that in the MSC group,there was significant expression of cTnI protein observed in MSC+latifolin group.Immunohistochemical results showed that the MSC transplantation group+latifolin groups and latifolin groups can significantly reduce the number of positive CD68expression.Latifolin significantly reduced the protein expressions of IL-6,p-NF-?B and HIF-1?,and significantly increased the expression of?-catenin in myocardial tissue.Conclusions:The new flavonoid compound latifolin can improve the survival of BM-MSCs by changing the inflammatory microenvironment.Latifolin can directly promote the differentiation of BM-MSCs into myocardial differentiation.In vivo latifolin can improve the survival and myocardial differentiation of BM-MSCs,and can effectively promote treatment effect of transplantation of BM-MSCs for myocardial infarction.Its mechanism may be related to the HIF-1?/NF-?B pathway and the Wnt/?-catenin pathway.