Study Of Bu Shen Huo Xue Formula On The Mobilization Of Bone Marrow Stem Cells Of Acute Myocardial Infarction

Acute myocardial infarction(AMI)is the most common cause of morbidity and mortality in coronary heart diseases.Current clinical therapy to minimize the devastating effects of myocardial infarction range from administration of drugs,acute percutaneous coronary intervention to coronary artery bypass grafting are usually not sufficient to prevent left ventricular remodeling and subsequent development of heart failure as they fall short of actual replacement of necrotic cardiac myocytes.By the time of end-stage heart failure,the effect of drug treatment is limited,and cardiac transplantation is the only viable alternative.However,this strategy is costly and severely limited by the availability of donor hearts.In addition,the use of immunosuppressive agents may diminish the patient' s quality of life.The recent progress in the area of stem cell research has led to the suggestion that stem cells might potentially be used to regenerate cardiomyocytes and improve impaired heart function.Current insights into stem cell plasticity have opened up new perspectives for regenerating the infarcted heart.There are several different types of stem cells containing embryonic stem cells(ESCs),skeletal muscle satellite cells and bone marrow-derived stem cells(BMSCs)being investigated for their capacity to proliferate and differentiate into cardiomyocytes.Embryonic stem cells are highly proliferative and totipotent,but they have some drawbacks,such as ethical issue,immunosuppressive therapy,the potential to form teratomas,etc. Skeletal muscle satellite cells and BMSCs,although limited in their differentiation capacity,provide a safer and ethically acceptable alternative to ESCs.For skeletal myoblasts,the question is that the cells differentiated from skeletal muscle satellite cell are different from cardiomyocytes because of the lack of the intercalated disks which are essential for mechanical activity in the myocardium.The bone marrow contains several reconstructing stem cell types,with overlapping phenotypes, including haematopoietic stem cells(HSCs),endothelial progenitor cells (EPCs),mesenchymal stem cells(MSCs),etc.Recent data suggest that BMSCs are the ideal sources because the cardiomyocyte-like cells induced from BMSCs might be able to form effective mechanoelectric coupling with host cardiomyocyte.Bone marrow stem cells can be harvested as autologous cells, avoiding the use of immunosuppressive drugs.Besides,BMSCs can be collected and expanded easily from adults without moral principles.The BMSCs mobilization is an attractive administration candidate because the treatment is noninvasive so that it is more convenient and acceptable for patients.There is increasing evidence that stem cell mobilization homing to the infarcted myocardium and differentiation into cardiac myocytes is a naturally occurring process.However,the amount of mobilized stem cells is too low to be sufficient to ameliorate the remodelling process after ischemia,preventing preexisting cardiomyocytes from undergoing apoptosis and improving heart function. Pharmacological mobilization of bone marrow stem cells is one of the strategies for cardiac repair.Granulocyte colony-stimulating factor(G-CSF)is an effective cytokine capable of mobilizing hematopoietic stems and progenitor cells from the bone marrow to the peripheral circulation.The previous studies demonstrated that G-CSF exert favorable effects on cardiac remodeling is enhancement of endogenous cardiac repair mechanisms that include both bone marrow stem cells mobilization,engraftment,and differentiation as well as proliferation of cardiomyocytes.Its efficacy and safety have been proved by many animal experiments and some small-sample clinic trials and G-CSF treatment was well tolerated despite of some adverse reaction,such as excessive instent restenosis,embolism,blood coagulation,ostalgia,fever, etc.It is also doubtful whether G-CSF therapy will benefit elderly patients because the increased age is associated with reduced angiogenic capacity and diminished cell cycling of cardiac stem cells.In the theories of traditional Chinese Medicine(TCM),kidney is in charge of the condition of the bone and marrow,heart and kidney is closely-related。If we tonify the kidney,the function of bone and marrow will be strengthened. Plenty of evidences showed that Chinese medicines of invigorating kidney and promoting blood circulation can facilitate the differentiation of bone marrow stem cells and improve the microenviroment of bone marrow.Besides,the Chinese medicines are effective and safe in treating the ischemic heart diseases.If the Chinese herbs could be used for the mobilization of BMSC to cure the AMI, the disadvantages of G-CSF may be overcome and it may offer a new therapeutic strategy for ischemic heart diseases.So,in this present study,we hypothesized that bone marrow stem cells could be mobilized by Bu Shen Huo Xue Formula,which is composed of invigorating kidney and promoting blood circulation Chinese medicines,and the mobilized stem cells would colonize and differentiate at the site of myocardial injury,limit the size of damage incurred by the infarction,and thereby improve myocardial function.The present study compared the BMSCs mobilization effects of the different-doses of Bu Shen Huo Xue.Formula and G-CSF on AMI in experimental rats with flow cytometer,pathohistology and immunohistochemical staining technique.On the base of the animal experiment,the further clinic study was carried out to observe the contribution of Bu Shen Huo Xue Formula to the percentages of CD34 positive cells expression in peripheral blood of AMI patients,the area of myocardial infarction,the left ventricular structure and ejection function.We explored that Bu Shen Huo Xue Formula might improve cardiac performance by increasing the number of bone marrow stem cell in the peripheral circulation.PartⅠStudy of Bu Shen Huo Xue Formula on the mobilization of bone marrow stem cells of Acute Myocardial Infarction experimental RatMethods AMI was induced in rat by permanent ligation of the left descending coronary artery.The rats were divided into four groups at random:CSF group, BS low-dose group,BS high-dose group and control group.Three hours after left anterior coronary artery ligation,the rats in CSF group were assigned to receive a first subcutaneous injection of recombinant human granulocyte colony-stimulating factor 10μg/kg of body weight followed by daily injections for 5 consecutive days.The rats in BS low-dose group and BS high-dose group took the different concentration of Bu Shen Huo Xue Formula separately daily for 5 consecutive days.Control group received equal volumes of isotonic saline. The percentages of CD34 positive cells expression in peripheral blood were measured with flow cytometer on day 1,day 5 and day 14 after model established separately.The histological changes of morphological characteristics of myocardium were observed with the haematine-eosin staining on day 1,day 5 and day 14 after MI.Immunohistochemistry for CD34 positive cells were performed in the tissues of myocardial infarcted area and marginal area.Results(1)The percentages of CD34 positive cells expression in peripheral blood of all groups had no difference on day lafter MI.On day 5 after MI, the values of CD34positive cells expression in BS low-dose group,BS high-dose group and CSF group increased significantly from(0.53±0.15),(0.55±0.19)% and(0.59±0.14)%at baseline to(1.77±0.46),(2.49±0.62)%and (3.31±0.87)%(p＜0.01 versus control).The values of BS high-dose group and CSF group had no significantly difference(p＞0.05).On day 14 after models established the percentages of CD34 positive cells expression in G-CSF groups, BS low-dose group,and control group showed a similar decline,except the value of BS high-dose group keep in high level(p＜0.05 versus other three groups). (2)It was observed in histological changes that there were CD34 positive infiltrative monocytes in the infarcted area and CD34 positive myocardium-like cells in the marginal area on day 5 and day 14 after mobilization in the CSF group,BS high-dose group and BS low-dose group separately.The vascular densities of above three groups were significantly greater(p＜0.01 versus control).In control group,it is showed that there were a few newborn vessels and more fibroblasts on day 5 after model established.A few CD34 positive infiltrative monocytes were observed in the marginal infractedarea.On day 14 after MI,there were larger myocardial scar tissues distributed in patch. The well-arranged basic structure of myocardial tissues was destroyed and the formation of scars could be observed.Conclusion In this study,we induced MI in rats via ligation of the LAD and applied rhG-CSF and different-doses Bu Shen Huo Xue Formula three hours after MI daily for 5 consecutive days.Our main findings were as follows:(1)AMI is followed by enhanced spontaneous mobilization of BMSCs;(2)Bu Shen Huo Xue Formula in different doses might both have mobilization effects on bone marrow stem cells,which could increase the percentages of CD34 positive cells expression in peripheral blood after acute myocardial infarction.It was shown that Bu Shen Huo Xue Formula in high dose application increased peripheral blood stem cells after MI better than that in low dose did.The mobilized bone marrow stem cells may migrate to the site of myocardial infarction and differentiated into myocardium-like cells and vascular endothelials which might lead to neovascularisation and resulted in the reduced scar extension; (3)High-dose Bu Shen Huo Xue Formula has more continuous mobilization effect when compared to G-CSF.PartⅡProtective effect of Bu Shen Huo Xue Formula on acute myocardial infarction patientsMethods Twenty AMI patients were randomly assigned to receive treatment with Bu Shen Huo Xue Formula(BS group)and to receive placebo(control group). Two groups patients had comparable demographic and clinical and infracted-related characteristics.The standard care included the glyceryltrinitrate,anti-coagulation drugs,platelet aggregation inhibitors and angiotensin converting enzyme inhibitor,etc.Symptomatic treatments were practiced when the complications such as heart failure and arrhythmia occurred. Chinese medicines of invigorating kidney and promoting blood circulation(Bu Shen Huo Xue Formula)were administered daily after MI in BS group.The percentages of CD34 positive cells expression in peripheral blood were measured by flow cytometry on day 3,5,7,14 after treatment.The myocardial infarction sizes were estimated by QRS Scoring System before treatment and on day 7,28 after treatment.The left ventricular structure and function were determined with color doppler ultrasonography on day 7,28 after treatment.Results(1)The percentages of CD34 positive cells expression in peripheral blood in all AMI patients increased gradually after treatment and peaked on day 5(p＜0.01 versus baseline).BS group had significantly higher mean level of CD34 positive cells expression(p＜0.05 versus control).The CD34 positive cells expression of control group decreased obviously on day 14 while BS group keep in high level(p＜0.05 versus control).(2)According to QRS Scoring System, the score on day 28 after treatment in BS group decreased from 5.59±0.65 to 3.79±0.46(p＜0.05 versus baseline),and the left ventricular infarct size on day 28 after treatment reduced significantly from 37.98±3.45%to 28.00±2.26%(p＜0.01 versus baseline),while the score and infarct size in control group patients showed no significant decrease(p＞0.05).(3)The baseline values of left ventricular ejection fraction(LVEF),left ventricular end-diastole dimension(LVEDd)and left ventricular end-systole dimension (LVEDs)had no differences between BS group and control group.But on day 28 after treatment,the LVEF in BS group improved markedly from 52.67±4.69%to 60.11±4.57(p＜0.01 versus baseline)and LVEDd and LVEDs lowered obviously from 61.11±5.90 mm,40.67±5.72 mm to 54.11±2.62 and 34.67±4.12(p＜0.01 and p＜0.05 versus baseline),while in control group,LVEDd and LVEDs on day 28 after treatment had no significantly differences respectively(p＞0.05 versus baseline).Conclusion In this study,we examined the effect of Bu Shen Huo Xue Formula administration on AMI patients' peripheral blood stem cells expressions, infarction areas,cardiac structures and ejection functions at the base of animal experiment in different time set after treatment.Our main findings were as follows:(1)the spontaneous mobilization of BMSCs after AMI was proven to be existent;(2)our results showed beneficial effects of Bu Shen Huo Xue Formula treatment after MI on infarction areas,cardiac structure and function in a follow up of 4 weeks.The beneficial outcome on cardiac structure and function may related to the increasing number of bone marrow stem cells in the peripheral circulation because of bone marrow stem cells mobilization through which may attenuate left ventricular dilation,decrease infarction size and reversed a decline in cardiac function.In conclusion,this present investigation suggests that there may be a naturally occurring but inefficient repair process that attempts to regenerate myocardial tissue in response to a myocardial infarction.Furthermore,our results offered a discovery that Bu Shen Huo Xue Formula might have a mobilization effect on bone marrow stem cells.The mobilized bone marrow stem cells may migrate to the site of myocardial infarction and differentiated into cardiomyocytes and vascular endothelial so that myocardial perfusion could be improved and cardiomyocytes be promoted to regenerate which would be beneficial for left-ventricular remodeling after myocardial infarction.