Catalpol Promotes The Survival And VEGF Secretion Of Bone Marrow-Derived Stem Cells And Their Role In Myocardial Repair After Myocardial Infarction In Rats

Introduction:Bone mesenchymal stem cells?BMSCs?transplantation has been recognized as a safe and effective measure to regenerate cardiac muscle cells.Growing evidence has demonstrated the curative effect of BMSCs transplantation on Myocardial infarction?MI?.However,its efficacy is restricted by low survival rate of transplanted cells caused by ischemia,anoxia and inflammation.Therefore,promoting the proliferation and survival of BMSCs plays important roles in the improvement of efficacy of BMSCs transplantation.Vascular endothelial growth factor?VEGF?is a pro-angiogenesis factor that facilitates the proliferation,migration and survival of endothelial cells.It has been suggested that VEGF could increase blood flow and improve cardiac function after MI.Interestingly,VEGF also plays pivotal roles in BMSCs transplantation after MI.So regulation of the secretion of VEGF may affect the therapeutic effect of BMSCs transplantation.Catalpol is an active compound isolated from a Chinese medicine Radix rehmanniae,which has been reported to have wide biological activities,including hypoglycemic action,neuroprotection,anti-aging,and anti-tumor.In addition,a previous study has shown that catalpol could promote VEGF-mediated angiogenesis in rats'stroke model.Studies also indicated that catalpol could promote survival and inhibit apoptosis in various cell models.According to the above background,we speculate that catalpol may enhance the survival and VEGF secretion of the transplanted BMSCs in the treatment of MI.In the present study,we explored whether catalpol could protect BMSCs against hypoxic-ischemic damage and thereby promote their curative effect in a rat model of MI.Methods:1.Isolation and identification of BMSCsThe BMSCs were isolated from bone marrow derived from the tibias and femurs of 4-week old Wistar rats.Three passages later,BMSCs were identified by incubation with primary antibodies against CD44,CD90,CD45,CD34 via flow cytometry and used for the following experiments.2.Catalpol pre-treatment and oxygen glucose deprivation?OGD?The BMSCs were pre-treated with different concentrations?12.25?g/ml?L?,24.5?g/ml?M?,or 49?g/ml?H??of catalpol for 24 h.After that,the BMSCs were subjected to OGD for 12 h.To induce OGD,the BMSCs were cultured in glucose-,serum-and sodium pyruvate-free DMEM/F12 at an atmosphere of 95%N₂ and 5%CO₂ in an anoxia chamber.While the control cells were maintained in complete DMEM/F12 under normal conditions.3.CCK-8 assayThe viability of BMSCs was determined by CCK-8 assay.Briefly,BMSCs were seeded in96-well plates.After pre-treatment with catalpol for 24 h and OGD stimulation for 12 h,10?l of CCK-8 solution was added to cells.The results were measured at 450 nm by a microplate reader after incubation for 2 h at 37?.4.Annexin V/PI apoptosis assayThe apoptosis of BMSCs was assessed using an Annexin V-FITC/PI apoptosis detection kit.In short,BMSCs with different treatments were stained with 5?l of Annexin V-FITC and 10?l of PI for 15 min in the dark.The stained BMSCs were immediately detected on a flow cytometer.5.ELISAThe level of VEGF in the supernatant liquid of BMSCs or the heart tissues of rats was assessed by a commercial rat VEGF ELISA kit,according to the manufacturer's instructions.6.Western blot analysisThe BMSCs or heart tissues were lysed at 4?by RIPA containing 1%Phenylmethanesulfonyl fluoride.The protein concentration was determined by BCA Protein Assay Kit.The protein samples were loaded onto SDS-polyacrylamide gel electrophoresis,then transferred to polyvinylidene fluoride membranes.After blocking with 5%not-fat milk,the membranes were blotted with corresponding primary antibodies against VEGF-A?Bcl-2?Bax?and cleaved caspase-3,HIF-1?at 4?overnight,respectively.Thereafter,the membranes were incubated with goat anti-rabbit secondary antibody at 37?for 45 min.The results were visualized by BeyoECL Plus.The density of the bands was analyzed by Gel-Pro-Analyzer software.7.Animal experimentsHealthy male Wistar rats weighing 200-220 g were obtained from Liaoning changsheng biotechnology co.The rats were randomly divided into four groups?n=12 per group?:sham,MI,MI+BMSCs,MI+BMSCs+catalpol.The rats in BMSCs transplantation groups were injected with 2×10⁶ BMSCs?with or without pre-treatment with 49?g/ml catalpol for 24h?in the area of MI.While the rats in MI group were injected with equal volume vehicle.Four weeks after the transplantation,the rats were euthanatized and the heart tissues were collected for further experiments.8.Survival detection of the transplanted BMSCsTo determine the in vivo survival of BMSCs,before transplantation,the BMSCs were stained with PKH26,according to the manufacturer's instruction.The collected heart tissues at four weeks after transplantation were cut into 5?m sections and the nuclei were stained with DAPI.Then the survived BMSCs that expressed red fluorescence were detected by a fluorescence microscopy.9.EchocardiographyAt 4 weeks after the transplantation,the rats were anesthetized and the heart function was determined by two-dimensional targeted M-mode echocardiography using an ultrasonic echocardiographic system.The following parameters including left ventricular end-systolic diameter?LVESD?,left ventricular end-diastolic diameter?LVEDD?were measured from three consecutive cardiac cycles,and then calculate LVEF?LVFS.10.Histological examinationsThe heart tissues sections were subjected to routine hematoxylin-eosin?HE?and masson staining to assess pathological changes and myocardial fibrosis.The images were photographed under a light microscope.11.TUNELTo evaluate apoptosis of heart tissues,TUNEL staining was performed using In Situ Cell Death Detection Kit according to the manufacturer's protocols.The nuclei of the apoptotic cells were stained with green fluorescence.12.Immunofluorescence stainingThe expression of CD31 in heart tissues was detected by immunofluorescence staining.After stained with DAPI,the sections were observed under a fluorescence microscope.13.Statistical analysisStatistical analysis was determined by one-way ANOVA using GraphPad Prism 5 software.The results were presented as mean±standard deviation?SD?.Statistical difference was considered as P value less than 0.05.Results:1.Identification of BMSCsThe expressions of surface markers of BMSCs were assessed by flow cytometry.The percentages of CD44 and CD90 positive BMSCs were 92.1%and 75.7%,and CD34 and CD45 positive cells were 1.7%and 2.3%.Thus,BMSCs were successfully isolated and identified.2.Effect of catalpol on VEGF secretion in OGD-treated BMSCs in vitroThe secretion of VEGF in BMSCs was determined by ELISA.The VEGF level in the BMSCs culture supernatant was slightly increased by OGD stimulation,although there was no statistical difference.Pre-treatment with catalpol could dose-dependently enhance the VEGF secretion in BMSCs under OGD condition.In addition,the protein levels of VEGF-A and HIF-1?in BMSCs was evaluated by western blot analysis.The levels of VEGF-A,nuclear and total HIF-1?appeared to be on the rise under OGD condition,which was significantly up-regulated by catalpol pre-treatment.3.Effect of catalpol on survival of BMSCs under OGD condition in vitroTo assess the effect of catalpol on survival of OGD-treated BMSCs,CCK8 assay was performed.The viability of BMSCs was obviously decreased by stimulation of OGD,which could be effectively improved by pre-treatment with catalpol.Moreover,the results suggested that OGD-induced the apoptosis of BMSCs was strikingly restrained by catalpol.To further evaluate the mechanisms through which catalpol regulated the apoptosis of BMSCs,the expressions of apoptosis-related proteins were assessed.The level of anti-apoptotic protein Bcl-2 was down-regulated,while the pro-apoptotic Bax and cleaved caspase-3 levels were up-regulated in BMSCs under OGD condition.As expected,pre-treatment with catalpol could remarkably reverse the above changes.4.Effect of catalpol on survival of BMSCs after in vivo transplantationThe survival of BMSCs after 4 weeks of transplantation was determined.The surviving transplanted BMSCs in heart tissues were labeled with PKH26,a red fluorescent dye.According to the result,catalpol pre-treatment obviously improved the survival of BMSCs after in vivo transplantation.5.Effect of catalpol on curative effect of BMSCs transplantation on myocardial infarction in ratsThe pathological changes and myocardial fibrosis was obvious in MI group as assayed by HE and masson staining.BMSCs transplantation effectively alleviated MI-induced pathological changes and myocardial fibrosis,which could be enhanced by catalpol pre-treatment.Furthermore,TUNEL and myosin staining results indicated that MI caused increased apoptosis in myocardial cells in the border area.However,BMSCs transplantation inhibited MI-induced apoptosis and this inhibitory effect was more pronounced when BMSCs were pre-treated with catalpol.The cardiac function was detected by echocardiography.LVEF and LVFS was remarkably decreased in MI group.Whereas,BMSCs transplantation raised LVEF and LVFS,which could be further enhanced when BMSCs were pre-treated with catalpol.In addition,the increased LVESD and LVEDD induced by MI were restrained by BMSCs transplantation.As could be expected,catalpol pre-treated BMSCs could further decrease LVESD and LVEDD.These results suggested that catalpol pre-treatment further improved the efficacy of BMSCs in MI.6.Effect of catalpol on VEGF secretion in ischemic myocardium after BMSCs transplantationThe expression of CD31 was significantly enhanced in ischemic myocardium by BMSCs transplantation,which was strengthened by pre-treatment with catalpol.Moreover,the protein level of VEGF-A in heart tissues was evaluated by western blot.Catalpol pre-treatment promoted BMSCs-induced increase in VEGF-A level in heart tissues.The result of ELISA further demonstrated the enhancement of VEGF level by transplanting BMSCs that were pre-treated with catalpol.Conclusion:Our results suggest that catalpol contributes to the survival and VEGF secretion of BMSCs and therefore enhances the therapeutic effect of BMSCs transplantation in MI.Although further experiments are needed to elucidate the detailed mechanisms,our study provides evidence for better efficacy of BMSCs pre-treated with catalpol to protect against MI.