| Background After acute myocardial infarction(AMI)occurs,the left ventricle changes in size,structure and function,resulting in ventricular remodeling.Ventricular remodeling(VR)is the main course of heart failure development.Bone marrow mesenchymal stem cells(BMSCs)can alleviate post-AMI myocardial injury and improve ventricular remodeling through paracrine secretion,homing,and transformation,but due to the altered internal environment after AMI,the migration efficiency of BMSCs is low.The team’s previous study found that Yiqi Huoxue recipe could improve the post-AMI inflammatory environment and improve myocardial injury in AMI rats.However,it is worthwhile to investigate whether the formula can promote the migration of BMSCs to the infarct site in AMI rats.Objective To investigate the intervention of Yiqi Huoxue recipe combined with bone marrow mesenchymal stem cells on ventricular remodeling after myocardial infarction.mi RNA-210/BNIP3 was used as an entry point to investigate the possible mechanism of Yiqi Huoxue recipe in synergy with stem cells on ventricular remodeling from the autophagy-apoptosis pathway.Methods 1.BMSCs were extracted from the bone marrow of SD rats,and the cell morphology was observed under microscope and the cell surface markers were detected by flow-through.The rat myocardial infarction model was established by LAD ligation and divided into shamoperated group,model group,YQHX group,BMSCs group and YQHX+BMSCs group.Two hours into the modeling,myocardial ischemia was detected by electrocardiogram.After 4 weeks of drug intervention,rats were evaluated by cardiac ultrasound to assess the cardiac structure and function,and TTC staining for myocardial infarct size in rats,HE staining to observe the infiltration of inflammatory cells in the myocardium and Masson staining to observe the histopathological changes of myocardial fibers.2.BMSCs were stained for cell membrane markers using CM-DIL dye.The rats weretransplanted into the tail vein on the next day after moulding.After four weeks of intervention,BMSCs were enriched to the infarct site using immunofluorescence and SDF-1/CXCR4 protein expression in myocardial tissue was measured by immunohistochemistry.3.TUNEL was used to detect apoptosis in rat cardiomyocytes.The expression of autophagyrelated proteins LC3-II/LC3-Ⅰ,Beclin-1 and p62 was detected by Western Blot in all groups of rats after modeling.The expression of apoptosis-related proteins Bax,Bcl-2 and Caspase-3,and the expression of target protein BNIP3.RT-q PCR to detect mi R-210,BNIP3 mRNA expression.Resμlts 1.Compared with the sham group,LVESD and LVEDD increased(P<0.01)and LVEF and LVFS decreased(P<0.01)in the model group,suggesting that the rats in the model group had enlarged left ventricle,decreased cardiac function and altered cardiac structure;compared with the model group,LVEDD and LVESD decreased(P< 0.05 or P<0.01)and LVEF and LVFS increased(P<0.05 or P<0.01),suggesting that these two groups could reduce myocardial infarction-induced cardiac injury and cardiac structural alterations in rats.Compared with the model group,LVESD decreased(P<0.01),LVFS and LVEF increased and LVEDD decreased in the BMSCs group,but the differences were not statistically significant(P>0.05).Compared with the YQHX+BMSCs group,LVESD and LVEDD increased(P<0.05 or P<0.01),and LVEF and LVFS decreased(P<0.05 or P<0.01)in the YQHX group and BMSCs group.It is suggested that the protective effect of YQHX+BMSCs group on infarcted myocardium was the most significant.TTC staining results showed.,the infarct area was significantly increased in the model group compared to the sham group(P<0.01).Compared with the model group,the infarct area decreased in each administration group(P<0.05 or P<0.01).The infarct area in the remaining two dosing groups increased compared to that in the YQHX+BMSCs group(P<0.05 or P<0.01).This shows that the myocardial protection effect was the most significant in the YQHX+BMSCs group The HE staining results showed that the rats in the sham group had normal myocardial cell morphology,clear and centrally located nuclei,uniformly coloured myofilaments,neat and regular arrangement,and normal myocardial cell morphology;compared with the sham,the rats in the model group had severe lesions,irregular myocardial cell morphology,disorganized arrangement,deeply stained nuclei and increased inflammatory cell infiltration;compared with the model group,the rats in each administration group had reduced infarct extent In comparison with the model group,the extent of infarction in the rats was reduced,the structural changes of myocardium were reduced,the arrangement of myofilaments was more regular,and the inflammatory cell infiltration and edema were reduced.Masson staining showed that the myocardial cells in the sham group were neatly arranged with very little fibrous tissue deposition;the most severe lesion in the model group showed hypertrophy of myocardial cells in the marginal area of myocardial infarction with severe fibrous tissue deposition and disorganized arrangement,and large areas of blue fibrosis were visible;compared with the model group,each administration group showed reduced fibrous tissue deposition and neat myocardial arrangement compared with the model group.2.AMI rats were transplanted with BMSCs via tail vein on the next day after moulding,and their migration was observed under fluorescence microscope.Orange-red fluorescence was seen in myocardial tissue,and BMSCs were enriched towards the myocardial infarction site.Immunohistochemical results showed that the expression of SDF-1 protein was increased in the YQHX+BMSCs group compared with the BMSCs group(P<0.05),while the expression of CXCR4 was not statistically different.3.TUNEL staining showed no significant positive nuclear staining in the sham group,and positive nuclear staining was mostly seen in the model group.The number of positive nuclear staining was significantly reduced in the drug administration group compared to the model group.WB detection of apoptosis-related proteins showed that myocardial Bax protein expression was up-regulated(P<0.01),Bcl-2 protein expression was down-regulated(P<0.01)and Caspase-3 expression was up-regulated(P<0.01)in the model group compared with the sham-operated group,suggesting apoptosis activation.Compared with the model group,each administration group could down-regulate Bax protein expression(P<0.01),the YQHX group and YQHX+BMSCs group could down-regulate Caspase-3 protein expression(P<0.01)and up-regulate Bcl-2 protein expression(P<0.05),the BMSCs group had an up-regulation trend of Bcl-2 protein expression and Caspase-3 were not statistically different(P>0.05).Compared with the YQHX+BMSCs group,the Bax and Caspase-3 protein expressions were upregulated in the YQHX group and BMSCs group(P<0.01 or P<0.05),while Bcl-2 expression was downregulated(P>0.05),suggesting that the inhibitory effect of YQHX+BMSCs group was the most significant.WB detection of autophagy-related proteins showed that myocardial P62 protein expression was up-regulated(P<0.05)and LC3 B and Beclin-1 protein expression was down-regulated(P<0.01 or P<0.05)in the model group compared with the sham group,suggesting autophagy activation.Compared with the model group,each administration group could down-regulate P62 protein expression(P<0.01)and up-regulate Beclin-1 protein expression(P<0.01),the YQHX group and YQHX+BMSCs group could up-regulate LC3 B protein expression(P<0.01 or P<0.05),and there was no statistical difference in LC3 B protein expression in the BMSCs group(P>0.05).The expression of P62 protein was up-regulated(P<0.01 or P<0.05)and the expression of LC3 B and Beclin-1 was down-regulated(P<0.01 or P<0.05)in the YQHX group and BMSCs group compared to the YQHX+BMSCs group.It was suggested that the YQHX+BMSCs group induced the most significant effect of autophagy.BNIP3 protein WB assay showed that myocardial BNIP3 protein expression was up-regulated in the model group compared with the sham group(P<0.01).Compared with the model group,each administration group could down-regulate BNIP3 protein expression(P<0.01 or P<0.05).BNIP3 protein expression was upregulated in the YQHX group and the BMSCs group compared to the YQHX+BMSCs group(P<0.01).mi R-210 and BNIP3 mRNA PCR assay results showed that compared with the sham group: myocardial mi R-210 expression was downregulated(P<0.01)and BNIP3 mRNA expression level was up-regulated(P<0.01)in the model group of rats.Compared with the model group: mi R-210 expression could be up-regulated in the YQHX group and the YQHX+BMSCs group(P<0.01 or P<0.05),and there was no statistical difference in mi R-210 expression in the BMSCs group(P>0.05).BNIP3 mRNA expression was down-regulated in each administration group(P<0.01).Compared with the YQHX+BMSCs group : mi R-210 expression decreased in the YQHX group and the BMSCs group(P<0.01),BNIP3 mRNA expression increased in the BMSCs group(P>0.01),BNIP3 mRNA expression tended to increase in the YQHX group,but the difference was not statistically significant(P>0.05).It is suggested that Yiqi Huoxue recipe may work through the mi R-210/BNIP3 pathway,and the Yiqi Huoxue recipe combine with BMSCs has the most significant effect.Conclusion1.The Yiqi Huoxue recipe combine with BMSCs can increase the ejection fraction after heart infarction,improve the heart structure and reduce heart injury in rats.2.The Yiqi Huoxue recipe increases the enrichment efficiency of BMSCs to the infarct area by intervening in the SDF-1/CXCR4 axis.3.The Yiqi Huoxue recipe combine with BMSCs may exert an inhibitory mechanism on VR by regulating the mi RNA-210/BNIP3 axis and affecting cardiomyocyte autophagy-apoptosis. |
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