通冠胶囊修复动脉内皮损伤的作用及机制研究
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摘要
研究目的:
1通过随机双盲安慰剂对照的方法,研究通冠胶囊在冠心病患者行冠脉介入(percutaneous coronary intervention,PCI)治疗后修复动脉内皮损伤的可能机制。
2建立血管成形术后颈动脉内皮损伤动物模型,观察通冠胶囊、VEGF和SDF-1在大鼠颈动脉损伤后内皮修复及内膜增生过程中的相互关系,探讨通冠胶囊修复损伤内皮的可能机制。
研究方法:
1临床研究:
本课题采用随机、双盲、安慰剂对照方法,选择符合入选条件的冠心病患者60例,根据随机数据表分为安慰剂对照组(冠心病基础用药+安慰剂)、治疗组(冠心病基础用药+通冠胶囊),观察(1)各组一般情况(姓名、性别、年龄、个人既往史及相关危险因素状况等)、冠脉造影结果(病变支数、病变程度、既往PCI史)、手术方法(放置支架位置及支架个数);(2)使用酶联免疫吸附法检测两组患者在支架术前、术后即刻和治疗后30天时血清VEGF、SDF-1浓度的变化;(3)心梗患者术后2h心电图ST段回落情况;(4)术前及术后1月行心脏超声检查,检测左室射血分数(leftventricular ejection fraction,LVEF)以及室壁运动指数,并观察NYHA心功能分级。(5)随访至术后1月,观察两组患者主要心血管事件发生率(死亡、非致死性心梗、靶血管血运重建术、新发心功能衰竭)。
2实验研究:
选用健康雄性Sprague-Dawley(SD)大鼠54只,清洁级,体重180-220g。普通饮食,适应性喂养1周后,予左侧颈动脉内皮损伤,动物造模后,随机分为辛伐他汀组(2mg/kg/d)、通冠胶囊组(600mg/kg/d)和空白对照组(生理盐水10ml/kg/d),每组均为18只。分别于4、8、12周腹腔内注入过量戊巴比妥纳处死大鼠(每次每组各6只)进行取材和病理切片,HE染色及弹力纤维染色后光镜下观察管壁增生情况,计算机图像分析内、中膜面积及其比值观察通冠胶囊对新生内膜形成的影响,免疫组织化学染色法测定局部损伤组织VEGF、eNOS的表达,并进行图象分析;药物干预前后,检测三组大鼠血清中基质细胞衍生因子-1(SDF-1)、血管内皮细胞生长因子(VEGF)的浓度,探讨通冠胶囊修复损伤动脉血管内皮的可能分子机制。
研究结果:
1.临床研究:
(1)两组术前比较,差异无统计学意义(P＞0.05)。两组术后30天较术前血清VEGF、SDF-1水平均减低,差异有显著性意义(P均＜0.05)。但是术后通冠胶囊治疗组血清VEGF、SDF-1水平均明显高于安慰剂对照组,差异有显著性意义(P均＜0.01)。
(2)术后2h,治疗组较对照组比较,对心梗患者的ST段回落情况无统计学差异(P＞0.05)。
(3)术后1月,两组LVEF及室壁运动积分均较术前改善(均P＜0.05),说明PCI术后可明显改善患者心功能,且通冠胶囊治疗组较对照组可进一步改善LVEF及室壁运动积分(P＜0.05)。
(4)对照组有1例因重症左心功能不全,于术后4周死亡;两组患者在观察时间内均无靶血管血运重建。对照组新发的心功能衰竭发生率明显高于治疗组(P=0.045);两组主要心血管事件发生率比较,治疗组为6.7%,对照组为33.3%,治疗组明显低于对照组(P=0.024)。
2.实验研究:
(1)组织学观察:球囊损伤后,可见动脉内皮剥脱,个别处内弹力板破裂,内弹力板由正常时的波浪状变平坦,失去弹性。球囊损伤后4周内膜增生明显,8周形成的增生内膜有少量减少,12周时形成的新生内膜减少明显。空白盐水组球囊损伤后4周内膜增生达到高峰。通冠胶囊使损伤后4周、8周、12周形成的IA较盐水对照组显著减少(均P＜0.01):IA/MA较盐水对照组明显降低(均P＜0.01):与4周、8周同时段辛伐他汀组内膜面积及IA/MA无统计学差异。与12周辛伐他汀组内膜面积及IA/MA比较,差异有显著性意义(均P＜0.05)。辛伐他汀组的变化趋势与通冠胶囊组相似,与同时段盐水对照组比较有显著性差异(均P＜0.01)。
(2)VEGF免疫组织化学检测:通冠胶囊组、辛伐他汀组分别与盐水对照组比较,术后4W、8W和12W时差异均有显著性(P＜0.01)。4周、8周通冠胶囊组与辛伐他汀组比较无统计学差异。12周时通冠胶囊组较辛伐他汀组VEGF表达增高明显。
(3)eNOS免疫组织化学检测:通冠胶囊组、辛伐他汀组分别与盐水对照组比较,术后4W、8W和12W时差异均有显著性(P＜0.01)。各时段通冠胶囊组与辛伐他汀组比较无统计学差异。
(4)VEGF酶联免疫吸附法检测:通冠胶囊组、辛伐他汀组分别与盐水对照组比较,术后4W、8W和12W时差异均有显著性(均P＜0.01,表2)。4周、8周通冠胶囊组与辛伐他汀组比较无统计学差异。12周时通冠胶囊组较辛伐他汀组VEGF表达增高明显(P＜0.05)。
(5)SDF-1酶联免疫吸附法检测:通冠胶囊组、辛伐他汀组分别与盐水对照组比较,术后4W、8W和12W时差异均有显著性(均P＜0.01)。4周通冠胶囊组与辛伐他汀组比较,差异无显著性意义,8周通冠胶囊组与辛伐他汀组比较,差异有显著性意义。而12周时通冠胶囊组SDF-1的表达与辛伐他汀组比较,差异无显著性意义,但二者均可促进SDF-1的表达,从数值上看,通冠胶囊组似乎略显突出(P=0.053)。
结论:
1.大鼠颈动脉球囊损伤模型能较好地模拟冠心病内皮损伤及PTCA术后再狭窄的病理变化过程,对临床研究有指导价值。
2.VEGF、SDF-1参与抑制大鼠颈动脉球囊损伤后内膜增生和管腔狭窄。
3.血清中VEGF、SDF-1水平在冠心病介入术后患者中下降与冠状动脉再通有关。而通冠胶囊可以上调PCI术后患者血清VEGF、SDF-1的表达,可能是通过动员EPCs归巢到损伤血管处修复血管内皮,建立侧支循环,在改善左室收缩功能方面起重要作用。
4.通冠胶囊可通过上调VEGF和SDF-1表达以及eNOS的表达参与抑制大鼠颈动脉球囊损伤后内膜增生和管腔狭窄,对PTCA术后再狭窄有防治作用。
Research Purposes:
1.Through applying the randomized,double-blind,placebo-controlled comparison method,to research the possible mechanism on repairing arterial endothelial injury of the Tongguan capsule with percutaneous coronary intervention(PCI) of coronary heart disease(CHD).
2.Based on the rat carotid artery balloon injury models,to obsere the correlation of the Tongguan capsule,VEGF and SDF-1 during endothelial repairing and intimal hyperplasia after carotid artery injury,to investigate the possible mechanism on repairing arterial endothelial injury of the Tongguan capsule.
Methodology:
1.Clinical research:
Taking the randomized,double-blind,placebo-controlled comparison method,sixty patients treated with percutaneous coronary intervention(PCI) were randomized to two groups:group A(n=30,treated with Tongguan Capsule and conventional western medicine) and group B(n=30,treated with placebo and conventional western medicine).
Observation:
(1) The clinical characteristics were evaluated;
(2) The level of the serum VEGF and SDF-1 with enzyme-linked immunosorbent assay(ELISA) 30 days before and after the operation;
(3) The returning of ECG ST-segment at 2h time point after the operation;
(4) Inspect TEE,LVEF(left ventricular ejection fraction) and ventricular wall motion parameters at 30 days before and after the operation,and observe the cardiac function classification of NYHA.
(5)Major adverse cardiac events(MACE) including death,nonfatal myocardial infarction(MI),target vessel revascularization(TVR) and new heart failure were followed in 1 month after PCI.
2.Experimental research:
A total of 60 male Sprague-Dawley rats which the left common carotid arteries injured were randomly divided into3 groups(n=18):Group 1(10mg/kg/d of PS was given after injury),group 2(2mg/kg/d of Simvastatin was given after injury)and group 3(600mg/kg/d of Tongguan capsule was given after injury). 18 rats from each group were euthanized by exsanguination at week 4,week 8 and week 12 after injury.The injuried left common carotid arteries were carefully removed for HE and elastic fibers staining,intima area,media area and IA / MA were performed in IPP6.0.which is a automatic computerized imaging analysis system.To observe the reparation after injury of blood vessel endothelium through detection of VEGF and eNOS with immunohistochemistry.
Research Results:
1.Clinical research:
(1)There had no difference between the treatment group and the control group before the operation.The level of VEGF and SDF-1 all decreased significantly at 30 days in the the treatment group and the control group,at the same time, The level of VEGF and SDF-1 at 30 days after the operation in the treatment group and which had significant differences compared with the control group(P＜0.01,P＜0.01)。
(2) ST-segment resolution all decreased significantly at 2 hours in the treatment group and the control group,but there had no differences between the treatment group and the control group at 2 hours after the operation(P＞0.05).
(3)Ate the 1 month time point after the operation,the LVEF and ventricular wall motion parameters of the both groups are improved(average P＜0.05), indicating that the PCI treatment can significantly improve the cardiac functions of the CHD patients.Meanwhile,compared with the control group, the LVEF ventricular wall motion scores of the Tongguan capsule treatment group are further improved.
(4)Main Adverse Cardiac Events:At 30 days follow-up,there didn't exist statistics difference in the incidence of death,non-fatal MI and TVR between two groups.The incidence of new heart failure of the treatment group was statistically lower than the control group(P=0.045),thus,the incidence of MACE in the treatment group was statistically lower than the control group (P=0.024).
2.Experimental research on animals:
(1) Histological Observation:Artery endothelium after balloon denudation, internal elastic lamina become flat from wavy and lost its elasticity.4 weeks after the operation,morphological analysis demonstrated that there were markedly differences in the area of neointima proliferation among all the groups.In comparison with Group 1,intima area(IA) of tunica intima in rats after balloon injury in Group 2 and group 3 decreased obviously(average P＜0.01) at 4 weeks,8 weeks and 12 weeks.The trends of ratio of IA/MA in rats were similar to which of IA.
(2) VEGF detected by immunohistochemical assay:the expression of VEGF of Group 3 increased obviously at week 12(P＜0.05).
(3) eNOS detected by immunohistochemical assay:In comparison with Group 1, the expression of eNOS in tunica intima in rats after balloon injury in Group 3 increased obviously(P＜0.01),The changes of the expression of eNOS of Croup 2 were similar to Group 3.There had no difference between Group 2 and Group 3.
(4) detection of VEGF and SDF-1 with enzyme-linked immunosorbent assay(ELISA):In comparison with Group 1,the expression of VEGF and SDF-1 in blood serum in rats after balloon injury in Group 3 increased obviously(P＜0.01),The changes of the expression of VEGF and SDF-1 of Group 2 were similar to Group 3.In comparison with Group 2,the expression of SDF-1 of Group 3 increased obviously at week 8(P＜0.05),however the expression of VEGF of Group 3 increased obviously at week 12(P＜0.05).
Conclusion:
1.Rat carotid balloon-injured model can simulates the process of pathological changes of endothelial injury and restenosis after percutaneous coronary intervention(PCI).
2.After rat carotid artery balloon injury,VEGF and SDF-1 could restrain proliferation and restenosis.
3.The level of VEGF and SDF-1 decreased after the PCI operation is related to coronary artery reoanalization.At the same time the level of VEGF and SDF-1 increased in the Tongguan treatment group,Tongguan capsule played an important role in the improvement of left ventricular systolic function by promoting angiogenesis and reendothelialization after PCI through mobilization of EPCs.
4.Tongguan capsule inhibited intimal proliferation and restenosis by upregulating the VEGF,eNOS and SDF-1 expression.
1通过随机双盲安慰剂对照的方法,研究通冠胶囊在冠心病患者行冠脉介入(percutaneous coronary intervention,PCI)治疗后修复动脉内皮损伤的可能机制。
2建立血管成形术后颈动脉内皮损伤动物模型,观察通冠胶囊、VEGF和SDF-1在大鼠颈动脉损伤后内皮修复及内膜增生过程中的相互关系,探讨通冠胶囊修复损伤内皮的可能机制。
研究方法:
1临床研究:
本课题采用随机、双盲、安慰剂对照方法,选择符合入选条件的冠心病患者60例,根据随机数据表分为安慰剂对照组(冠心病基础用药+安慰剂)、治疗组(冠心病基础用药+通冠胶囊),观察(1)各组一般情况(姓名、性别、年龄、个人既往史及相关危险因素状况等)、冠脉造影结果(病变支数、病变程度、既往PCI史)、手术方法(放置支架位置及支架个数);(2)使用酶联免疫吸附法检测两组患者在支架术前、术后即刻和治疗后30天时血清VEGF、SDF-1浓度的变化;(3)心梗患者术后2h心电图ST段回落情况;(4)术前及术后1月行心脏超声检查,检测左室射血分数(leftventricular ejection fraction,LVEF)以及室壁运动指数,并观察NYHA心功能分级。(5)随访至术后1月,观察两组患者主要心血管事件发生率(死亡、非致死性心梗、靶血管血运重建术、新发心功能衰竭)。
2实验研究:
选用健康雄性Sprague-Dawley(SD)大鼠54只,清洁级,体重180-220g。普通饮食,适应性喂养1周后,予左侧颈动脉内皮损伤,动物造模后,随机分为辛伐他汀组(2mg/kg/d)、通冠胶囊组(600mg/kg/d)和空白对照组(生理盐水10ml/kg/d),每组均为18只。分别于4、8、12周腹腔内注入过量戊巴比妥纳处死大鼠(每次每组各6只)进行取材和病理切片,HE染色及弹力纤维染色后光镜下观察管壁增生情况,计算机图像分析内、中膜面积及其比值观察通冠胶囊对新生内膜形成的影响,免疫组织化学染色法测定局部损伤组织VEGF、eNOS的表达,并进行图象分析;药物干预前后,检测三组大鼠血清中基质细胞衍生因子-1(SDF-1)、血管内皮细胞生长因子(VEGF)的浓度,探讨通冠胶囊修复损伤动脉血管内皮的可能分子机制。
研究结果:
1.临床研究:
(1)两组术前比较,差异无统计学意义(P＞0.05)。两组术后30天较术前血清VEGF、SDF-1水平均减低,差异有显著性意义(P均＜0.05)。但是术后通冠胶囊治疗组血清VEGF、SDF-1水平均明显高于安慰剂对照组,差异有显著性意义(P均＜0.01)。
(2)术后2h,治疗组较对照组比较,对心梗患者的ST段回落情况无统计学差异(P＞0.05)。
(3)术后1月,两组LVEF及室壁运动积分均较术前改善(均P＜0.05),说明PCI术后可明显改善患者心功能,且通冠胶囊治疗组较对照组可进一步改善LVEF及室壁运动积分(P＜0.05)。
(4)对照组有1例因重症左心功能不全,于术后4周死亡;两组患者在观察时间内均无靶血管血运重建。对照组新发的心功能衰竭发生率明显高于治疗组(P=0.045);两组主要心血管事件发生率比较,治疗组为6.7%,对照组为33.3%,治疗组明显低于对照组(P=0.024)。
2.实验研究:
(1)组织学观察:球囊损伤后,可见动脉内皮剥脱,个别处内弹力板破裂,内弹力板由正常时的波浪状变平坦,失去弹性。球囊损伤后4周内膜增生明显,8周形成的增生内膜有少量减少,12周时形成的新生内膜减少明显。空白盐水组球囊损伤后4周内膜增生达到高峰。通冠胶囊使损伤后4周、8周、12周形成的IA较盐水对照组显著减少(均P＜0.01):IA/MA较盐水对照组明显降低(均P＜0.01):与4周、8周同时段辛伐他汀组内膜面积及IA/MA无统计学差异。与12周辛伐他汀组内膜面积及IA/MA比较,差异有显著性意义(均P＜0.05)。辛伐他汀组的变化趋势与通冠胶囊组相似,与同时段盐水对照组比较有显著性差异(均P＜0.01)。
(2)VEGF免疫组织化学检测:通冠胶囊组、辛伐他汀组分别与盐水对照组比较,术后4W、8W和12W时差异均有显著性(P＜0.01)。4周、8周通冠胶囊组与辛伐他汀组比较无统计学差异。12周时通冠胶囊组较辛伐他汀组VEGF表达增高明显。
(3)eNOS免疫组织化学检测:通冠胶囊组、辛伐他汀组分别与盐水对照组比较,术后4W、8W和12W时差异均有显著性(P＜0.01)。各时段通冠胶囊组与辛伐他汀组比较无统计学差异。
(4)VEGF酶联免疫吸附法检测:通冠胶囊组、辛伐他汀组分别与盐水对照组比较,术后4W、8W和12W时差异均有显著性(均P＜0.01,表2)。4周、8周通冠胶囊组与辛伐他汀组比较无统计学差异。12周时通冠胶囊组较辛伐他汀组VEGF表达增高明显(P＜0.05)。
(5)SDF-1酶联免疫吸附法检测:通冠胶囊组、辛伐他汀组分别与盐水对照组比较,术后4W、8W和12W时差异均有显著性(均P＜0.01)。4周通冠胶囊组与辛伐他汀组比较,差异无显著性意义,8周通冠胶囊组与辛伐他汀组比较,差异有显著性意义。而12周时通冠胶囊组SDF-1的表达与辛伐他汀组比较,差异无显著性意义,但二者均可促进SDF-1的表达,从数值上看,通冠胶囊组似乎略显突出(P=0.053)。
结论:
1.大鼠颈动脉球囊损伤模型能较好地模拟冠心病内皮损伤及PTCA术后再狭窄的病理变化过程,对临床研究有指导价值。
2.VEGF、SDF-1参与抑制大鼠颈动脉球囊损伤后内膜增生和管腔狭窄。
3.血清中VEGF、SDF-1水平在冠心病介入术后患者中下降与冠状动脉再通有关。而通冠胶囊可以上调PCI术后患者血清VEGF、SDF-1的表达,可能是通过动员EPCs归巢到损伤血管处修复血管内皮,建立侧支循环,在改善左室收缩功能方面起重要作用。
4.通冠胶囊可通过上调VEGF和SDF-1表达以及eNOS的表达参与抑制大鼠颈动脉球囊损伤后内膜增生和管腔狭窄,对PTCA术后再狭窄有防治作用。
Research Purposes:
1.Through applying the randomized,double-blind,placebo-controlled comparison method,to research the possible mechanism on repairing arterial endothelial injury of the Tongguan capsule with percutaneous coronary intervention(PCI) of coronary heart disease(CHD).
2.Based on the rat carotid artery balloon injury models,to obsere the correlation of the Tongguan capsule,VEGF and SDF-1 during endothelial repairing and intimal hyperplasia after carotid artery injury,to investigate the possible mechanism on repairing arterial endothelial injury of the Tongguan capsule.
Methodology:
1.Clinical research:
Taking the randomized,double-blind,placebo-controlled comparison method,sixty patients treated with percutaneous coronary intervention(PCI) were randomized to two groups:group A(n=30,treated with Tongguan Capsule and conventional western medicine) and group B(n=30,treated with placebo and conventional western medicine).
Observation:
(1) The clinical characteristics were evaluated;
(2) The level of the serum VEGF and SDF-1 with enzyme-linked immunosorbent assay(ELISA) 30 days before and after the operation;
(3) The returning of ECG ST-segment at 2h time point after the operation;
(4) Inspect TEE,LVEF(left ventricular ejection fraction) and ventricular wall motion parameters at 30 days before and after the operation,and observe the cardiac function classification of NYHA.
(5)Major adverse cardiac events(MACE) including death,nonfatal myocardial infarction(MI),target vessel revascularization(TVR) and new heart failure were followed in 1 month after PCI.
2.Experimental research:
A total of 60 male Sprague-Dawley rats which the left common carotid arteries injured were randomly divided into3 groups(n=18):Group 1(10mg/kg/d of PS was given after injury),group 2(2mg/kg/d of Simvastatin was given after injury)and group 3(600mg/kg/d of Tongguan capsule was given after injury). 18 rats from each group were euthanized by exsanguination at week 4,week 8 and week 12 after injury.The injuried left common carotid arteries were carefully removed for HE and elastic fibers staining,intima area,media area and IA / MA were performed in IPP6.0.which is a automatic computerized imaging analysis system.To observe the reparation after injury of blood vessel endothelium through detection of VEGF and eNOS with immunohistochemistry.
Research Results:
1.Clinical research:
(1)There had no difference between the treatment group and the control group before the operation.The level of VEGF and SDF-1 all decreased significantly at 30 days in the the treatment group and the control group,at the same time, The level of VEGF and SDF-1 at 30 days after the operation in the treatment group and which had significant differences compared with the control group(P＜0.01,P＜0.01)。
(2) ST-segment resolution all decreased significantly at 2 hours in the treatment group and the control group,but there had no differences between the treatment group and the control group at 2 hours after the operation(P＞0.05).
(3)Ate the 1 month time point after the operation,the LVEF and ventricular wall motion parameters of the both groups are improved(average P＜0.05), indicating that the PCI treatment can significantly improve the cardiac functions of the CHD patients.Meanwhile,compared with the control group, the LVEF ventricular wall motion scores of the Tongguan capsule treatment group are further improved.
(4)Main Adverse Cardiac Events:At 30 days follow-up,there didn't exist statistics difference in the incidence of death,non-fatal MI and TVR between two groups.The incidence of new heart failure of the treatment group was statistically lower than the control group(P=0.045),thus,the incidence of MACE in the treatment group was statistically lower than the control group (P=0.024).
2.Experimental research on animals:
(1) Histological Observation:Artery endothelium after balloon denudation, internal elastic lamina become flat from wavy and lost its elasticity.4 weeks after the operation,morphological analysis demonstrated that there were markedly differences in the area of neointima proliferation among all the groups.In comparison with Group 1,intima area(IA) of tunica intima in rats after balloon injury in Group 2 and group 3 decreased obviously(average P＜0.01) at 4 weeks,8 weeks and 12 weeks.The trends of ratio of IA/MA in rats were similar to which of IA.
(2) VEGF detected by immunohistochemical assay:the expression of VEGF of Group 3 increased obviously at week 12(P＜0.05).
(3) eNOS detected by immunohistochemical assay:In comparison with Group 1, the expression of eNOS in tunica intima in rats after balloon injury in Group 3 increased obviously(P＜0.01),The changes of the expression of eNOS of Croup 2 were similar to Group 3.There had no difference between Group 2 and Group 3.
(4) detection of VEGF and SDF-1 with enzyme-linked immunosorbent assay(ELISA):In comparison with Group 1,the expression of VEGF and SDF-1 in blood serum in rats after balloon injury in Group 3 increased obviously(P＜0.01),The changes of the expression of VEGF and SDF-1 of Group 2 were similar to Group 3.In comparison with Group 2,the expression of SDF-1 of Group 3 increased obviously at week 8(P＜0.05),however the expression of VEGF of Group 3 increased obviously at week 12(P＜0.05).
Conclusion:
1.Rat carotid balloon-injured model can simulates the process of pathological changes of endothelial injury and restenosis after percutaneous coronary intervention(PCI).
2.After rat carotid artery balloon injury,VEGF and SDF-1 could restrain proliferation and restenosis.
3.The level of VEGF and SDF-1 decreased after the PCI operation is related to coronary artery reoanalization.At the same time the level of VEGF and SDF-1 increased in the Tongguan treatment group,Tongguan capsule played an important role in the improvement of left ventricular systolic function by promoting angiogenesis and reendothelialization after PCI through mobilization of EPCs.
4.Tongguan capsule inhibited intimal proliferation and restenosis by upregulating the VEGF,eNOS and SDF-1 expression.
引文
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