阿托伐他汀诱导EPC-MVs增多对STEMI患者心肌细胞的保护作用研究
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摘要
目的:观察阿托伐他汀诱导内皮祖细胞微囊泡(EPC-MVs)增多对ST段抬高型心肌梗死(STEMI)患者心肌细胞的保护作用,并探讨其可能机制。方法:选取2015年2月-2018年2月我院收治的STEMI患者168例,按阿伐他汀剂量分为A组(88例)和B组(94例)。所有患者均接受经皮冠脉介入治疗,术后予注射用比伐芦定、硫酸氢氯吡格雷片、阿托伐他钙片治疗。其中,A组予阿托伐他汀钙片20 mg,每日1次;B组予阿托伐他汀钙片20 mg,每日2次。30 d为1个疗程,两组患者均连续治疗至少3个疗程。观察两组患者血脂[总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)]水平(治疗前及治疗后第30、60、90天)和EPCs阳性细胞数(治疗后第30、60天);检测其EPC-MVs微RNA(miRNA)表达谱(治疗后第60天),验证差异表达miRNA的表达情况;分析表达差异最明显miRNA的靶基因及KEGG通路富集情况,并评价其对心肌HCM-a细胞增殖的影响;记录两组患者不良反应发生情况。结果:A组有8例患者脱落,B组有6例患者脱落。治疗前或治疗后30天,两组患者TC、LDL-C、HDL-C水平以及外周血EPCs阳性细胞数比较,差异均无统计学意义(P>0.05);治疗后,两组患者HDL-C水平(治疗后第60、90天)以及B组患者外周血EPCs阳性细胞数(治疗后第60天)均显著升高或增多,且B组显著高于或多于同时间点A组(P<0.05)。芯片检测结果显示,与A组比较,B组患者EPC-MVs中表达差异超过1.5倍的miRNA共有16个,其中7个上调、9个下调;差异表达前5位的分别为hsa-miR-126(上调)、hsa-miR-1275(上调)、hsa-miR-7704(下调)、hsa-miR-105-5p(下调)、hsa-miR-3180(下调)。荧光定量聚合酶链反应结果显示,B组患者hsa-miR-126、hsa-miR-1275的相对表达量均较A组显著升高,hsa-miR-7704、hsa-miR-105-5p、hsa-miR-3180的相对表达量均较A组显著降低(P<0.05)。表达差异最明显的为hsa-miR-126,其靶基因包括Ang-1、PDGF、p38 MAPK、Smad2/3、HIF-1、TGF-β等,参与调节的信号通路主要包括血管生成信号通路、慢性骨髓性白血病相关通路、肾上皮细胞癌相关通路等。CCK-8试验结果显示,hsa-miR-126特异性干扰物组细胞的光密度(OD)值较空白组显著降低,模拟物组细胞的OD值较空白组显著升高(P<0.05)。两组患者腹泻、恶心呕吐、皮疹等不良反应发生率比较,差异均无统计学意义(P>0.05)。结论:不同剂量的阿托伐他汀均可调节患者体内HDL-C水平,大剂量阿托伐他汀可显著提高EPCs数量,且不会影响用药的安全性。这种作用可能与上调EPC-MVs中hsa-miR-126等的表达从而促进心肌细胞增殖有关。
OBJECTIVE:To observe the protective effect of atorvastatin-induced increase of EPC-MVs on myocardial cells in ST-segment elevation myocardial infarction(STEMI)patients,and to investigate its potential mechanism. METHODS:Totally 168 STEMI patients was collected from our hospital during Feb. 2015-Feb. 2018,and then divided into group A(88 cases)and group B(94 cases) according to the dose of atorvastatin. All patients received percutaneous coronary intervention,and then given Bivaleridine for injection,Clopidogrel bisulfate tablets and Atorvastatin calcium tablets. Group A was given Atorvastatin calcium tablets 20 mg,once a day. Group B was given Atorvastatin calcium tablets 20 mg,twice a day. A treatment course lasted for 30 d,and two groups were treated for 3 courses at least. The levels of blood lipid(TC,LDL-C,HDL-C)(before treatment and30 th,60 th,90 th day after treatment)and the number of EPCs positive cells(30 th,60 th day after treatment) were observed in 2 groups. The expression of miRNA of EPC-MVs(60 th day after treatment)was detected,and the expression difference of miRNA were validated. Target gene and KEGG pathway enrichment of miRNA with most significant expression difference were analyzed,and the effects of it on the proliferation of cardiac HCM-a cells were evaluated. The occurrence of ADR was recorded in2 groups. RESULTS:Totally 8 patients withdrew from the study in group A,and 6 patients in group B. There was no statistical significance in the levels of TC,LDL-C and HDL-C or the number of EPCs positive cells in peripheral blood between 2 groups before treatment or 30 th day after treatment(P>0.05). After treatment,the level of HDL-C in 2 group(60 th and 90 th day after treatment) and the number of EPCs positive cells in peripheral blood in group B(60 th day after treatment) were increased significantly,and group B was significantly higher or more than group A at the same time point(P<0.05). Microarray analysis showed that compared with group A,16 miRNAs expressed more than 1.5 times differentially in EPC-MVs of group B,7 of which were up-regulated and 9 down-regulated. Top five differentially expressed genes were hsa-miR-126(up-regulated),hsa-miR-1275(up-regulated),hsa-miR-7704(down-regulated),hsa-miR-105-5 p(down-regulated),and hsa-miR-3180(down-regulated).Fluorescence quantitative polymerase chain reaction results showed that compared with group A,relative expression of hsa-miR-126 and hsa-miR-1275 in group B were increased significantly;and relative expression of hsa-miR-7704,hsa-miR-105-5 p and hsa-miR-3108 were decreased significantly(P<0.05). The expression difference of hsa-miR-126 was the most significant,and its target genes included Ang-1,PDGF,p38 MAPK,Smad2/3,HIF-1,TGF-β,etc. The signaling pathways involved in regulation mainly included angiogenesis signaling pathway,chronic myelogenous leukemia related pathway,renal epithelial cell carcinoma related pathway and so on. CCK-8 test showed that the optical density(OD)of cells in hsa-miR-126 specific interfering substance group was decreased significantly,and the OD value of cells in simulated substance group was increased significantly,compared with blank group(P<0.05). There was no statistical significance in the incidence of ADR as diarrhea,nausea and vomiting,rash,etc.(P>0.05). CONCLUSIONS:Different doses of atorvastatin can regulate the level of HDL-C,and large dose of atorvastatin can increase the number of EPCs significantly,but dose not influence the safety of drug use. This effect may be associated with upregulating the expression of hsa-miR-126 in EPC-MVs so as to promoting the proliferation of myocardial cells.
OBJECTIVE:To observe the protective effect of atorvastatin-induced increase of EPC-MVs on myocardial cells in ST-segment elevation myocardial infarction(STEMI)patients,and to investigate its potential mechanism. METHODS:Totally 168 STEMI patients was collected from our hospital during Feb. 2015-Feb. 2018,and then divided into group A(88 cases)and group B(94 cases) according to the dose of atorvastatin. All patients received percutaneous coronary intervention,and then given Bivaleridine for injection,Clopidogrel bisulfate tablets and Atorvastatin calcium tablets. Group A was given Atorvastatin calcium tablets 20 mg,once a day. Group B was given Atorvastatin calcium tablets 20 mg,twice a day. A treatment course lasted for 30 d,and two groups were treated for 3 courses at least. The levels of blood lipid(TC,LDL-C,HDL-C)(before treatment and30 th,60 th,90 th day after treatment)and the number of EPCs positive cells(30 th,60 th day after treatment) were observed in 2 groups. The expression of miRNA of EPC-MVs(60 th day after treatment)was detected,and the expression difference of miRNA were validated. Target gene and KEGG pathway enrichment of miRNA with most significant expression difference were analyzed,and the effects of it on the proliferation of cardiac HCM-a cells were evaluated. The occurrence of ADR was recorded in2 groups. RESULTS:Totally 8 patients withdrew from the study in group A,and 6 patients in group B. There was no statistical significance in the levels of TC,LDL-C and HDL-C or the number of EPCs positive cells in peripheral blood between 2 groups before treatment or 30 th day after treatment(P>0.05). After treatment,the level of HDL-C in 2 group(60 th and 90 th day after treatment) and the number of EPCs positive cells in peripheral blood in group B(60 th day after treatment) were increased significantly,and group B was significantly higher or more than group A at the same time point(P<0.05). Microarray analysis showed that compared with group A,16 miRNAs expressed more than 1.5 times differentially in EPC-MVs of group B,7 of which were up-regulated and 9 down-regulated. Top five differentially expressed genes were hsa-miR-126(up-regulated),hsa-miR-1275(up-regulated),hsa-miR-7704(down-regulated),hsa-miR-105-5 p(down-regulated),and hsa-miR-3180(down-regulated).Fluorescence quantitative polymerase chain reaction results showed that compared with group A,relative expression of hsa-miR-126 and hsa-miR-1275 in group B were increased significantly;and relative expression of hsa-miR-7704,hsa-miR-105-5 p and hsa-miR-3108 were decreased significantly(P<0.05). The expression difference of hsa-miR-126 was the most significant,and its target genes included Ang-1,PDGF,p38 MAPK,Smad2/3,HIF-1,TGF-β,etc. The signaling pathways involved in regulation mainly included angiogenesis signaling pathway,chronic myelogenous leukemia related pathway,renal epithelial cell carcinoma related pathway and so on. CCK-8 test showed that the optical density(OD)of cells in hsa-miR-126 specific interfering substance group was decreased significantly,and the OD value of cells in simulated substance group was increased significantly,compared with blank group(P<0.05). There was no statistical significance in the incidence of ADR as diarrhea,nausea and vomiting,rash,etc.(P>0.05). CONCLUSIONS:Different doses of atorvastatin can regulate the level of HDL-C,and large dose of atorvastatin can increase the number of EPCs significantly,but dose not influence the safety of drug use. This effect may be associated with upregulating the expression of hsa-miR-126 in EPC-MVs so as to promoting the proliferation of myocardial cells.
引文
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[2]贺甫威,叶红华,费晓红,等.经皮冠状动脉介入术前他汀再负荷对围术期循环内皮祖细胞及炎症因子的影响[J].中华病理生理杂志,2014,30(10):1772-1777.
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[9]TIAN Z,WANG R,ZHANG X,et al.Benzo a pyrene-7,8-diol-9,10-epoxide suppresses the migration and invasion of human extravillous trophoblast Swan 71 cells due to the inhibited filopodia formation and down-regulated PI3K/AKT/CDC42/PAK1 pathway mediated by the increased miR-194-3p[J].Toxicol Sci,2018,166(1):25-38.
[10]ABDEL HAMID M,BAKHOUM SW,SHARSF Y,et al.Circulating endothelial cells and endothelial function predict major adverse cardiac events and early adverse left ventricular remodeling in patients with st-segment elevation myocardial infarction[J].J Interv Cardiol,2016,29(1):89-98.
[11]ANTONIUCCI D,VALENTI R,MIGLIORINI A,et al.Comparison of rheolytic thrombectomy before direct infarct artery stenting versus direct stenting alone in patients undergoing percutaneous coronary intervention for acute myocardial infarction[J].Am J Cardiol,2004,93(8):1033-1035.
[12]Vasa M,FICHTLSCHERER S,Adler K,et al.Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease[J].Circ Res,2001,89(1):E1-E7.
[13]SCHMIDT-LUCKE C,FICHTLSCHERER S,AICHERA,et al.Quantification of circulating endothelial progenitor cells using the modified ishage protocol[J].PLoS One,2010.DOI:10.1371/journal.pone.0013790.
[14]CISZEWSKI M,PREGOWSKI J,TERESINSKA A,et al.Aspiration coronary thrombectomy for acute myocardial infarction improves myocardial salvage index:.single center randomized study[J].Circulation,2011,78(4):523-531.
[15]ZHOU J,CHENG M,LIAO YH,et al.Rosuvastatin enhances angiogenesis via eNOS-dependent mobilization of endothelial progenitor cells[J].PLoS One,2013.DOI:10.1371/journal.pone.0063126.
[16]HRISTOV M,FACH C,BECKER C,et al.Reduced numbers of circulating endothelial progenitor cells in patients with coronary artery disease associated with long-term statin treatment[J].Atherosclerosis,2007,192(2):413-420.
[17]SHAW SY,CHENG S,CUPPLES LA,et al.Genetic and clinical correlates of early-outgrowth colony-forming units[J].Circ Cardiovasc Genet,2011,4(3):296-304.
[18]SINGH N,HEGGERMONT W,FIEUWS S,et al.Endothelium-enriched microRNAs as diagnostic biomarkers for cardiac allograft vasculopathy[J].J Heart Lung Transpl,2015,34(11):1376-1384.
[19]JAKOB P,DOERRIES C,BRIAND S,et al.Loss of angiomiR-126 and 130a in angiogenic early outgrowth cells from patients with chronic heart failure role for impaired in vivo neovascularization and cardiac repair capacity[J].Circulation,2012,126(25):2962-2975.
[20]ZAMPETAKI A,WILLEIT P,TILLING L,et al.Prospective study on circulating microRNAs and risk of myocardial infarction[J].J Am Coll Cardiol,2012,60(4):290-299.
[2]贺甫威,叶红华,费晓红,等.经皮冠状动脉介入术前他汀再负荷对围术期循环内皮祖细胞及炎症因子的影响[J].中华病理生理杂志,2014,30(10):1772-1777.
[3]ROSSI F,BERTONE C,MONTANILE F,et al.HDL cholesterol is a strong determinant of endothelial progenitor cells in hypercholesterolemic subjects[J].Microvasc Res,2010,80(2):274-279.
[4]林小玲,刘婷婷,余梦.他汀类联合心血管药物治疗冠心病效果分析[J].临床合理用药杂志,2018,11(1B):36-37.
[5]GU S,ZHANG W,CHEN J,et al.EPC-derived microvesicles protect cardiomyocytes from AngⅡ-induced hypertrophy and apoptosis[J].PLoS One,2014.DOI:10.1371/journal.pone.0085396.
[6]李翠如,赵春霞.血浆miR-1、miR-133a以及miR-126:急性心肌梗死的新型生物学标记物[J].山西职工医学院学报,2017,27(3):4-8.
[7]PARADISI G,BRACAGLIA M,BASILE F,et al.Effect of pravastatin on endothelial function and endothelial progenitor cells in healthy postmenopausal women[J].Clin Exp Obstet Gynecol,2012,39(2):153-159.
[8]刘洁,陈祖平,储成顶,等.阿托伐他汀对急性ST段抬高型心肌梗死患者外周血内皮组细胞表面标志物表达的影响[J].中国病理生理杂志,2017,33(5):851-856.
[9]TIAN Z,WANG R,ZHANG X,et al.Benzo a pyrene-7,8-diol-9,10-epoxide suppresses the migration and invasion of human extravillous trophoblast Swan 71 cells due to the inhibited filopodia formation and down-regulated PI3K/AKT/CDC42/PAK1 pathway mediated by the increased miR-194-3p[J].Toxicol Sci,2018,166(1):25-38.
[10]ABDEL HAMID M,BAKHOUM SW,SHARSF Y,et al.Circulating endothelial cells and endothelial function predict major adverse cardiac events and early adverse left ventricular remodeling in patients with st-segment elevation myocardial infarction[J].J Interv Cardiol,2016,29(1):89-98.
[11]ANTONIUCCI D,VALENTI R,MIGLIORINI A,et al.Comparison of rheolytic thrombectomy before direct infarct artery stenting versus direct stenting alone in patients undergoing percutaneous coronary intervention for acute myocardial infarction[J].Am J Cardiol,2004,93(8):1033-1035.
[12]Vasa M,FICHTLSCHERER S,Adler K,et al.Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease[J].Circ Res,2001,89(1):E1-E7.
[13]SCHMIDT-LUCKE C,FICHTLSCHERER S,AICHERA,et al.Quantification of circulating endothelial progenitor cells using the modified ishage protocol[J].PLoS One,2010.DOI:10.1371/journal.pone.0013790.
[14]CISZEWSKI M,PREGOWSKI J,TERESINSKA A,et al.Aspiration coronary thrombectomy for acute myocardial infarction improves myocardial salvage index:.single center randomized study[J].Circulation,2011,78(4):523-531.
[15]ZHOU J,CHENG M,LIAO YH,et al.Rosuvastatin enhances angiogenesis via eNOS-dependent mobilization of endothelial progenitor cells[J].PLoS One,2013.DOI:10.1371/journal.pone.0063126.
[16]HRISTOV M,FACH C,BECKER C,et al.Reduced numbers of circulating endothelial progenitor cells in patients with coronary artery disease associated with long-term statin treatment[J].Atherosclerosis,2007,192(2):413-420.
[17]SHAW SY,CHENG S,CUPPLES LA,et al.Genetic and clinical correlates of early-outgrowth colony-forming units[J].Circ Cardiovasc Genet,2011,4(3):296-304.
[18]SINGH N,HEGGERMONT W,FIEUWS S,et al.Endothelium-enriched microRNAs as diagnostic biomarkers for cardiac allograft vasculopathy[J].J Heart Lung Transpl,2015,34(11):1376-1384.
[19]JAKOB P,DOERRIES C,BRIAND S,et al.Loss of angiomiR-126 and 130a in angiogenic early outgrowth cells from patients with chronic heart failure role for impaired in vivo neovascularization and cardiac repair capacity[J].Circulation,2012,126(25):2962-2975.
[20]ZAMPETAKI A,WILLEIT P,TILLING L,et al.Prospective study on circulating microRNAs and risk of myocardial infarction[J].J Am Coll Cardiol,2012,60(4):290-299.