| Ischemic heart disease such as acute myocardial infarction (AMI) remainsa significant threat to human life and health. The patients with chronic cardiacfailure after AMI have low long-term survival rate and poor prognosis.Traditional therapy methods could not fundamentally recover the amount ofcardiomyocytes and improve cardiac diastolic and systolic function. Celltherapy using stem cells to transdifferentiate into viable cardiomyocytes andregenerate scar tissue is an attractive prospect, with the aim of reversingventricular remodeling, preventing heart failure. Mesenchymal Stem Cells(MSCs) become the most potential candidate in stem cell transplantation field due to their peculiar character. However, a significant barrier to the effectiveimplementation of MSC therapy is the inability to target these cells to tissues ofinterest with high efficiency and engraftment. Combinatorial treatments, such ascombinations of medicine or genetic modification have been proposed topromote the effectiveness of transplanted MSCs to migrate toward damagedmyocardium by many researchers. Traditional Chinese medicine has a set ofintegrated theory system in treating ischemic heart disease. Chinese herbalprescription of replenishing qi and activating blood has evident effect onischemic heart disease in clinic. Our previous studies showed that thecombination MSCs with a Chinese herbal prescription, primarily consisting of S.miltiorrhiza Bunge and A. membranaceus could significantly improve cardiacfunction after acute myocardial infarction, compared with that of MSCs alone.However, it is unknown whether the beneficial effects of Chinese traditionalherbs on MSC therapy are related to the promotion of MSC migration to injuredmyocardium？Therefore，we carried out the experimental research as follow.Aims1. To observe the effect of Chinese herb extracts on the migration ofMesenchymal stem cells in vitro.2. To explore the effect of a Chinese herbal prescription of replenishing qi andactivating blood on the migration of MSCs toward sites of ischemiamyocardium.3. From the level of CXCR4expression, to demonstrate the mechanismsinvolved in effect of replenishing qi and activating blood prescription on themigration of MSCs.4. From the regulation of Notch signaling on CXCR4expression, to demonstrate the mechanisms involved in effect of replenishing qi andactivating blood formula on the migration of MSCs.Material and Methods1. We utilized bone marrow adherence to isolate and culture mouse bonemarrow mesenchymal stem cells, and then utilized flow cytometry toidentify the specific surface CD markers. Mouse bone marrowmesenchymal stem cells were induced to differentiate to osteoplasts, whichwere determined by alizarin red stain.2. The third generation of MSCs was exposed to different concentration oftanshinone IIA and astragaloside IV for72h. Subsequently, MSCs werestained with carboxyfluorescein diacetate succinimidyl ester, and then theproliferations of MSCs were analyzed by flow cytometry to screen theoptimal concentration of extracts.3. The third genetation of MSCs was exposed to the optimal concentration oftanshinone IIA、astragaloside IV and the plus of tanshinone IIA andastragaloside IV for72h. Subsequently, the transwell system was used tocarry out the experiment of MSCs migration to stromal cell-derivedfactor-1α (SDF-1α) in vitro.4. SD rats were divided into four groups randomly, the transplantation only(control) group, replenishing qi group, activating blood group, andQidantongmai tablet (formula) group. SD rats of each group wereadministered intragastrically with traditional Chinese medicine onceeveryday，respectively. It was lasted for14days. The model of myocardialinfarction was built by ligation of the left anterior descending artery. TheDio-labeling MSCs were injected to animals through tail vein. After three days, Flow Cytometry and fluorescence microscope were used to observeand analyze the numbers of Dio-labeling cells in ischemic myocardium.After4weeks, cardiac functions of SD rats in every group were recorded bymultichannel physiologic recorder.5. We first assayed CXCR4expression in MSCs by immunofluorescence. Thethird genetation of MSCs was exposed to the optimal concentration oftanshinone IIA、astragaloside IV and the plus of tanshinone IIA andastragaloside IV for72h. Furthermore, flow cytometry, real-time PCR andwestern blot were carried out to compare CXCR4expression on the MSCsamong groups. The CXCR4inhibitor AMD3100was added2h prior to theend of culture, and then the transwell experiment was repeated.6. We performed PCR agarose gel electrophoresis to investigate the express ofNotch receptors and ligands on MSCs. After that, we isolated and culturedthe MSCs from the RBP-J knock out mouse to block up the Notch signalingon MSCs. Furthermore, flow cytometry, real-time PCR and western blotmethods were carried out to compare the CXCR4expression between theRBP-J-/-group and RBP-J-/+group.7. The third genetation of MSCs was exposed to the optimal concentration oftanshinone IIA、astragaloside IV and the plus of tanshinone IIA andastragaloside IV for72h. Real-time PCR was applicated to assay theexpression of HES1mRNA and HES5mRNA, which are of downsteamtarget gene of Notch signaling.Results1. Flow cytometry of isolated cells showed that CD34, CD45andMHCⅡnegative, CD29, CD44, CD90, CD106and Sca-1positive, which is supposed to be specific characteristics of MSCs. Cultured cells could also beinduced into osteoplasts (alizarin red stain positive).2. CFSE proliferation experiments showed that the optimal concentration oftanshinone IIA to promote MSC proliferation was0.2μg/ml, compared to0.1μg/ml or0.4μg/ml, and0.4μg/ml was astragaloside IV, compared to0.2μg/ml or0.8μg/ml. These optimal concentrations were applied forsubsequent study.3. Cells migration analysis in vitro showed that there exist significantdifferences among groups (P<0.05). These MSCs treated with tanshinoneIIA plus astragaloside IV showed the strongest migration among that of allof the groups (P<0.05). MSCs treated with tanshinone IIA appeared to havestronger migration, compared with that of astragaloside IV treated-MSCs(P<0.05). MSCs treated with astragaloside IV showed higher migration thanthat of control MSCs (P<0.05).4. We successfully established the mouse acute myocardial infarction model.Three days afer MSCs transplantation, we utilized flow cytometry to assaythe amount of Dio positive MSCs migrating to sites of ischemic myocardium,which was in line with the observation by fluorescence microscope. Theresults demonstrated that the numbers of Dio-labeling MSCs in sites ofischemic myocardium of Qidantongmai tablet group were significantlygreater than that of other groups (P<0.05); Dio+cells of the activating bloodgroup were higher than that of replenishing qi group (P<0.05); Dio+cells ofthe replenishing qi group were increased compared with that of controlgroup, however, there was no significant difference (P>0.05). We performedimmunofluorescence staining of SDF-1α to show that there was nosignificant difference among groups. 5.4weeks after MSCs transplantation, we used multichannel physiologicrecorder to analyze cardiac function among groups. The result showed thatMAP、LVPSP、+LVdp／dtmax及-LVdp／dtmaxof Qidantongmai tablet groupwere significant higher than that of other group (P<0.05). However, therewere no significant differences among the group of control, replenishing qiand activating blood (P>0.05).6. We employed the laser scanning confocal microscope to observe theexpression of CXCR4in MSCs. The result showed that CXCR4wasexpressed both on the cell surface and intracellularly in MSCs, and the mostof them expressed intracellularly. We carried out flow cytometry, real-timePCR and western blot to detect the difference of CXCR4expression amongeach medicine-treated MSCs group. The result showed that the CXCR4expression in tanshinone IIA plus astragaloside IV treated-MSCs was thehighest among that of all of the groups (P<0.05). MSCs treated withtanshinone IIA appeared to have higher CXCR4expressions, compared withthat of astragaloside IV stimulated-MSCs (P<0.05). After SDF-1α/CXCR4axis was blocked by AMD3100, the enhanced migration of each group wasinhibited.7. The result of agarose gel electrophoresis showed that Notch receptors andligands were expressed on MSCs. The flow cytometry, real-time PCR andwestern blotting assay demonstrated that CXCR4expression of RBP-J-/-group was higher than RBP-J-/+group (P<0.05). MSCs were exposed to theChinese herb extracts, and than the expression of HES1mRNA andHES5mRNA of were detected by real-time PCR. The result showed that theexpression of HES5mRNA in each group was little; there was no significantdifference between groups. HES1mRNA expressions were down-regulated in medicine groups. HES1mRNA expressions in tanshinone IIA plusastragaloside IV treated-MSCs were the lowest among the medicine treatedgroup (P<0.05). HES1mRNA expressions in tanshinone IIA treated-MSCswere lower than that of the astragaloside IV-treated MSCs (P<0.05).Conclusion1. Replenishing qi and activating blood Chinese herbs could promote themigration of MSCs toward sites of ischemia myocardium. The migrationeffect of the formula is the best, and that of the activating blood herbs isbetter than that of the Replenishing qi herbs.2. The probable mechanisms of Chinese herbs promoting MSCs migration isthat the CXCR4expression is up-regulated through inhibiting the Notchsignaling. |
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