芪参益气滴丸联合间充质干细胞移植改善小鼠心功能与巨噬细胞表型的相关性研究
|
摘要
目的:基于心肌梗死后巨噬细胞在损伤心肌修复过程中的主要效应及炎症影响干细胞的存活,探讨芪参益气滴丸是否可以影响巨噬细胞表型从而达到提高骨髓间充质干细胞的存活率、加强心肌细胞保护作用。方法:本研究采用eGFP+/-雄性实验小鼠,将小鼠随机分为PBS组(MI+PBS)、干细胞移植芪参益气滴丸组(MI+MSCs+QSYQ)和干细胞组(MI+MSCs),每组20只实验小鼠。心肌梗死模型造模前2周干细胞移植芪参益气滴丸组开始予芪参益气滴丸溶液灌胃(3.9 mg/mL,无菌水溶解),而其他两组则予等量无菌水灌胃作为对照。将实验前期分离培养所获得小鼠骨髓间充质干细胞计数后PBS重悬备用,采取开胸结扎心脏前降支建立小鼠心肌梗死模型,建立心肌梗死模型后运用30G进样针抽取20μL干细胞悬液,在心肌小鼠模型梗死区取4个点均匀注射,每个点注射量约5μL。空白组则予抽取等量的PBS,操作方法一致,术后撤除呼吸机,缝合术口。造模前、干细胞移植后2周、3周行超声心动图检测,评估心功能及左室重构情况,观察3周后处死实验小鼠,留取心脏标本予HE染色计算心肌梗死面积,并最终于荧光显微镜下观察巨噬细胞表型特征。结果:干细胞移植3周后心脏超声评估结果显示MI+MSCs+QSYQ组的LVIDd、LVIDs明显小于另外两组,而心脏FS和EF值均明显升高(P<0.05),心肌HE染色结果提示与单纯注射PBS相比较,移植MSCs能够明显减少梗死面积,且明芪参益气滴丸联合MSCs能够进一步减少心肌梗死面积。免疫荧光双标方提示间质充质干细胞移植3周后3组实验小鼠心肌梗死区均无M1型巨噬细胞亚群滞留,而存在大量M2型巨噬细胞浸润,并且MI+MSCs+QSYQ组的M2型巨噬细胞数量最多。结论:心肌梗死后骨髓间充质干细胞移植具有促进小鼠巨噬细胞向M2型表型转化的作用,而芪参益气滴丸联合骨髓间充质干细胞移植能够进一步增加巨噬细胞向M2型表型转化及炎症消退、心肌的修复效应。
Objective:Based on the main effect of macrophage in the repair of injured myocardium after myocardial infarction and the effect of inflammation on the survival of stem cells, it discussed whether Qishen Yiqi Pills can affect the phenotype of macrophage so as to improve the survival rate of bone marrow mesenchymal stem cells and strengthen the protective effect of cardiac myocytes. Methods:EGFP+/-male mice were used in this study. The mice were randomly divided into PBS group(MI+PBS), stem cell transplantation Qishen Yiqi Dropping Pills group(MI+MSCs+QSYQ) and stem cell group(MI+MSCs), 20 mice in each group. The Qishen Yiqi Dropping Pills groups were given Qishen Yiqi Dropping Pills for gastric perfusion(3.9 mg/mL, aseptic water dissolved) 2 weeks before the model of myocardial infarction model, while the other two groups were given the same amount of aseptic water as control. After the count of bone marrow mesenchymal stem cells obtained from the early stage of isolation and culture, PBS was suspended. A model of myocardial infarction was established by ligation of the anterior descending branch of the heart. After the establishment of myocardial infarction model, the 20 μL stem cell suspension was extracted with 30 G injection needle, and four points were injected evenly in the infarct area of the mice model of myocardial infarction, and the injection amount of each point was about 5 μL. The blank group was extracted with the same amount of PBS, and the operation method was the same. After operation, the ventilator was removed and the mouth was stitched. Cardiac function and left ventricular remodeling were evaluated by echocardiography before and two and three weeks after transplantation. After three weeks, the experimental mice were killed, the cardiac specimens were left to calculate the infarct size by HE staining, and the phenotypic characteristics of macrophages were observed under the fluorescence microscope. Results:After 3 weeks stem cell transplantation, the results of echocardiography showed that LVIDd and LVIDs in MI+MSCs+QSYQ group were significantly smaller than those in the other two groups, while the cardiac FS and EF values were significantly increased(P<0.05). There was no M1 type macrophage subgroup retention in the three groups' myocardial infarction areas after 3 weeks immunofluorescent labeling, and there was a large number of M2 macrophage infiltration in the myocardial infarction area, and the number of M2 macrophages in the MI+MSCs+QSYQ group was the most. Conclusion:After myocardial infarction, bone marrow mesenchymal stem cells transplantation can promote the transformation of macrophage to M2 phenotype, while Qishen Yiqi Dripping Pills combined bone marrow mesenchymal stem cell transplantation can further increase the effect of macrophage to M2 phenotype transformation, inflammatory regression and myocardial repair.
Objective:Based on the main effect of macrophage in the repair of injured myocardium after myocardial infarction and the effect of inflammation on the survival of stem cells, it discussed whether Qishen Yiqi Pills can affect the phenotype of macrophage so as to improve the survival rate of bone marrow mesenchymal stem cells and strengthen the protective effect of cardiac myocytes. Methods:EGFP+/-male mice were used in this study. The mice were randomly divided into PBS group(MI+PBS), stem cell transplantation Qishen Yiqi Dropping Pills group(MI+MSCs+QSYQ) and stem cell group(MI+MSCs), 20 mice in each group. The Qishen Yiqi Dropping Pills groups were given Qishen Yiqi Dropping Pills for gastric perfusion(3.9 mg/mL, aseptic water dissolved) 2 weeks before the model of myocardial infarction model, while the other two groups were given the same amount of aseptic water as control. After the count of bone marrow mesenchymal stem cells obtained from the early stage of isolation and culture, PBS was suspended. A model of myocardial infarction was established by ligation of the anterior descending branch of the heart. After the establishment of myocardial infarction model, the 20 μL stem cell suspension was extracted with 30 G injection needle, and four points were injected evenly in the infarct area of the mice model of myocardial infarction, and the injection amount of each point was about 5 μL. The blank group was extracted with the same amount of PBS, and the operation method was the same. After operation, the ventilator was removed and the mouth was stitched. Cardiac function and left ventricular remodeling were evaluated by echocardiography before and two and three weeks after transplantation. After three weeks, the experimental mice were killed, the cardiac specimens were left to calculate the infarct size by HE staining, and the phenotypic characteristics of macrophages were observed under the fluorescence microscope. Results:After 3 weeks stem cell transplantation, the results of echocardiography showed that LVIDd and LVIDs in MI+MSCs+QSYQ group were significantly smaller than those in the other two groups, while the cardiac FS and EF values were significantly increased(P<0.05). There was no M1 type macrophage subgroup retention in the three groups' myocardial infarction areas after 3 weeks immunofluorescent labeling, and there was a large number of M2 macrophage infiltration in the myocardial infarction area, and the number of M2 macrophages in the MI+MSCs+QSYQ group was the most. Conclusion:After myocardial infarction, bone marrow mesenchymal stem cells transplantation can promote the transformation of macrophage to M2 phenotype, while Qishen Yiqi Dripping Pills combined bone marrow mesenchymal stem cell transplantation can further increase the effect of macrophage to M2 phenotype transformation, inflammatory regression and myocardial repair.
引文
[1] Zhao ZQ,Corvera JS,Halkos ME,et al.Inhibition of myocardial injury by ischemic postconditioning during reperfusion:comparison with ischemic preconditioning[J].American Journal of Physiology Heart & Circulatory Physiology,2003,285(2):H579.
[2] Orlic D,Kajstura J,Chimenti S,et al.Bone marrow cells regenerate infarcted myocardium [J].Nature,2001,7(s3):86-88.
[3] Chang W,Song BW,Hwang KC.Mesenchymal Stem Cell Survival in Infarcted Myocardium:Adhesion and Anti-death Signals[M]// Stem Cells and Cancer Stem Cells.Springer Netherlands,2013:35-43.
[4] Nagaya N,Ohgushi H,Shimizu W,et al.Abstract 2091:Clinical Trial of Autologous Bone Marrow Mesenchymal Stem Cell Transplantation for Severe Chronic Heart Failure[J].Circulation,2007(16):II(453).
[5] Fukuda K.Application of mesenchymal stem cells for the regeneration of cardiomyocyte and its use for cell transplantation therapy[J].Human Cell,2010,16(3):83-94.
[6] Swirski FK,Nahrendorf M.Leukocyte Behavior in Atherosclerosis,Myocardial Infarction,and Heart Failure[J].Science,2013,339(6116):161-166.
[7] Frangogiannis NG.Regulation of the inflammatory response in cardiac repair[J].Circulation Research,2012,110(1):159-173.
[8] 邵正斌,戴小华,毛静远,等.芪参益气滴丸对慢性心力衰竭患者心功能及超敏C-反应蛋白的影响[J].中国实验方剂学杂志,2015(15):152-155.
[9] Wu HY,Sun ZH,Cao DP,et al.Cardiovascular health status in Chinese adults in urban areas:analysis of the Chinese Health Examination Database 2010[J].Int J Cardiol,2013,168:760-764.
[10] Yellon DM,Hausenloy DJ.Myocardial reperfusion injury[J].N Engl J Med,2007,357:1121-1135.
[11] Frieler RA,Mortensen RM.Immune cell and other noncardiomyocyte regulation of cardiac hypertrophy and remodeling[J].Circulation,2015,131:1019-1030.
[12] Hasan AS,Luo L,Yan C,et al.Correction:Cardiosphere-Derived Cells Facilitate Heart Repair by Modulating M1/M2 Macrophage Polarization and Neutrophil Recruitment[J].PLoS One,2017;12:e0171892.
[13] Yan X,Anzai A,Katsumata Y,et al.Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction[J].Journal of Molecular & Cellular Cardiology,2013,62(3):24-35.
[14] Frangogiannis NG.The inflammatory response in myocardial injury,repair and remodeling[J].Nature Reviews Cardiology,2014,11(5):255-265.
[15] Zuylen VLV,Haan MCD,Geutskens SB,et al.Post-myocardial Infarct Inflammation and the Potential Role of Cell Therapy[J].Cardiovascular Drugs & Therapy,2015,29(1):59-73.
[16] Robey TE,Saiget MK,Reinecke H,et al.Systems Approaches to Preventing Transplanted Cell Death in Cardiac Repair[J].Journal of Molecular & Cellular Cardiology,2008,45(4):567-581.
[17] Yu B,Sondag GR,Malcuit C,et al.Macrophage-Associated Osteoactivin/GPNMB Mediates Mesenchymal Stem Cell Survival,Proliferation,and Migration Via a CD44-Dependent Mechanism[J].Journal of Cellular Biochemistry,2016,117(7):1511-1521.
[18] Benmordechai T,Holbova R,Landarouben N,et al.Macrophage subpopulations are essential for infarct repair with and without stem cell therapy[J].Journal of the American College of Cardiology,2013,62(20):1890-1901.
[19] 傅俊曾,宋生有,姜民,等.液相色谱-质谱联用同时测定芪参益气滴丸中黄芪甲苷、丹参素、原儿茶醛、人参皂苷Rg_1和Rb_1含量[J].中国药学杂志,2012,47(1):61-64.
[20] 张腾,张密霞,张艳军.芪参益气滴丸抗血管新生大鼠心肌缺血动态观察及机制探讨[J].中国实验方剂学杂志,2017(1):134-139.
[2] Orlic D,Kajstura J,Chimenti S,et al.Bone marrow cells regenerate infarcted myocardium [J].Nature,2001,7(s3):86-88.
[3] Chang W,Song BW,Hwang KC.Mesenchymal Stem Cell Survival in Infarcted Myocardium:Adhesion and Anti-death Signals[M]// Stem Cells and Cancer Stem Cells.Springer Netherlands,2013:35-43.
[4] Nagaya N,Ohgushi H,Shimizu W,et al.Abstract 2091:Clinical Trial of Autologous Bone Marrow Mesenchymal Stem Cell Transplantation for Severe Chronic Heart Failure[J].Circulation,2007(16):II(453).
[5] Fukuda K.Application of mesenchymal stem cells for the regeneration of cardiomyocyte and its use for cell transplantation therapy[J].Human Cell,2010,16(3):83-94.
[6] Swirski FK,Nahrendorf M.Leukocyte Behavior in Atherosclerosis,Myocardial Infarction,and Heart Failure[J].Science,2013,339(6116):161-166.
[7] Frangogiannis NG.Regulation of the inflammatory response in cardiac repair[J].Circulation Research,2012,110(1):159-173.
[8] 邵正斌,戴小华,毛静远,等.芪参益气滴丸对慢性心力衰竭患者心功能及超敏C-反应蛋白的影响[J].中国实验方剂学杂志,2015(15):152-155.
[9] Wu HY,Sun ZH,Cao DP,et al.Cardiovascular health status in Chinese adults in urban areas:analysis of the Chinese Health Examination Database 2010[J].Int J Cardiol,2013,168:760-764.
[10] Yellon DM,Hausenloy DJ.Myocardial reperfusion injury[J].N Engl J Med,2007,357:1121-1135.
[11] Frieler RA,Mortensen RM.Immune cell and other noncardiomyocyte regulation of cardiac hypertrophy and remodeling[J].Circulation,2015,131:1019-1030.
[12] Hasan AS,Luo L,Yan C,et al.Correction:Cardiosphere-Derived Cells Facilitate Heart Repair by Modulating M1/M2 Macrophage Polarization and Neutrophil Recruitment[J].PLoS One,2017;12:e0171892.
[13] Yan X,Anzai A,Katsumata Y,et al.Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction[J].Journal of Molecular & Cellular Cardiology,2013,62(3):24-35.
[14] Frangogiannis NG.The inflammatory response in myocardial injury,repair and remodeling[J].Nature Reviews Cardiology,2014,11(5):255-265.
[15] Zuylen VLV,Haan MCD,Geutskens SB,et al.Post-myocardial Infarct Inflammation and the Potential Role of Cell Therapy[J].Cardiovascular Drugs & Therapy,2015,29(1):59-73.
[16] Robey TE,Saiget MK,Reinecke H,et al.Systems Approaches to Preventing Transplanted Cell Death in Cardiac Repair[J].Journal of Molecular & Cellular Cardiology,2008,45(4):567-581.
[17] Yu B,Sondag GR,Malcuit C,et al.Macrophage-Associated Osteoactivin/GPNMB Mediates Mesenchymal Stem Cell Survival,Proliferation,and Migration Via a CD44-Dependent Mechanism[J].Journal of Cellular Biochemistry,2016,117(7):1511-1521.
[18] Benmordechai T,Holbova R,Landarouben N,et al.Macrophage subpopulations are essential for infarct repair with and without stem cell therapy[J].Journal of the American College of Cardiology,2013,62(20):1890-1901.
[19] 傅俊曾,宋生有,姜民,等.液相色谱-质谱联用同时测定芪参益气滴丸中黄芪甲苷、丹参素、原儿茶醛、人参皂苷Rg_1和Rb_1含量[J].中国药学杂志,2012,47(1):61-64.
[20] 张腾,张密霞,张艳军.芪参益气滴丸抗血管新生大鼠心肌缺血动态观察及机制探讨[J].中国实验方剂学杂志,2017(1):134-139.