摘要
经皮腔内冠状动脉介入(PCI)治疗是目前治疗冠心病的主要手段之一,但是介入治疗后再狭窄的发生严重影响了其远期疗效。因此,如何有效防治再狭窄已成为基础研究和临床研究的热点和难点课题。许多研究表明,再狭窄的主要机制是血管损伤局部中膜平滑肌细胞增殖,向内膜迁移及细胞外基质形成引起的内膜增生和血管重构。DIM是I3C的衍生物,I3C属于吲哚类化合物,它具有多种药理学活性。国外学者通过细胞培养发现DIM具有抑制肿瘤细胞的增殖,诱导肿瘤细胞凋亡的作用,而再狭窄与肿瘤细胞在细胞的增殖上有相似之处。近年研究显示DIM可抑制平滑肌细胞增殖,但其作用机制尚未阐明。故我们推测DIM是否也具有抑制VSMC增殖和诱导平滑肌细胞凋亡的作用?能否用于预防PCI后再狭窄?DIM对再狭窄方面的研究尚未见报道。本实验就是通过研究DIM对兔颈总动脉球囊损伤后内膜增生的影响,为此做了一系列实验,旨在探讨其防治RS发生的机制。
目的
①建立实验兔颈动脉再狭窄模型。评价DIM对血管成形术(PTA)后再狭窄的影响,分析DIM对血管再狭窄的干预作用,为再狭窄的防治提供新的理论依据。②观察PTA术后相关细胞生长因子mRNA水平表达以及DIM对其表达变化的影响。③观察PTA术后,bcl-2在蛋白水平及mRNA水平表达的变化特点,探讨bcl-2在血管再狭窄过程中的作用和意义。④评价DIM在本实验研究中的安全性。
方法
1动物模型和分组:选用新西兰兔,随机分为假手术组、模型组和大小DIM剂量治疗组共4组。将实验兔右颈外动脉结扎,造成内膜增生、颈动脉再狭窄模型。DIM做成乳剂后予腹腔注射给药。术前3天开始给药,大小DIM剂量组每只兔分别予8mg/kg/d与2mg/kg/d ,腹腔注射给药,假手术组与模型组予等体积的生理盐水。术前3天开始阿司匹林经灌胃给药,50 mg/d,1次/d,持续至动物处死。给药4周后取损伤区域的血管段制备标本,根据HE染色标本情况,进行后续染色及处理。
2病理学检查:所有标本行HE染色。对PDGF、TGF-β、VEGF和bFGF采用免疫组织化学染色观察其表达的变化,而bcl-2进行原位杂交实验观察其mBNA表达的改变。
3病理学资料图像分析:采用病理图像分析系统对标本HE染色片,在光镜下进行形态学和组织学分析,同时分别计数PDGF、TGF-β、VEGF和bFGF免疫组化染色片和bcl-2原位杂交染色片内膜、中膜,外膜阳性细胞表达率。
4通过检测实验兔肝功能及肝肾脏的病理形态变化来评价DIM对动物的安全性。
结果
1. DIM对PTA术后血管内膜增生和重塑的影响
1.1病理改变
肉眼可见假手术组颈动脉柔软,有弹性,与周围组织没有粘连,镜下显示内膜光滑完整,无内膜增生,内弹力板完整,中膜平滑肌细胞排列整齐,形态一致,细胞核呈长梭形;模型组动脉成灰白色,僵硬,与周围组织粘连,动脉明显变细甚至闭塞,镜下新生内膜明显增厚,管腔横切面减少,呈偏心性狭窄,内含大量增殖和迁移的血管平滑肌细胞(VSMC)及纤维组织,内弹力板不连续,中膜可见损伤,外膜增厚。DIM小剂量组与模型组没有明显差异,而DIM大剂量组内皮比较光滑完整,内膜增生减轻,管腔明显增大。
1.2内膜、中膜和外膜面积的变化
HE染色光镜下可见损伤动脉内膜面积和内膜/中膜面积显著增加,提示造模成功。与模型组比较,DIM小剂量组内膜面积、内膜/中膜面积、内膜/中膜厚度都有所降低,但差异不显著(P>0.05),大剂量组则效果明显,具有显著性差异(P <0.01)。中膜与外膜面积没有显著变化。
1.3内膜、中膜与外膜厚度的变化
与模型组比较,DIM小剂量组内膜厚度与内膜/中膜厚度,都有所降低,但差异不显著(P>0.05),大剂量组则效果明显,差异具有显著性。(P<0.01)。中膜与外膜厚度无明显变化。
2细胞生长因子的表达
根据免疫组织化学染色的石蜡切片发现,正常组PDGF与TGF-β仅内膜微量表达或没有表达。模型组与治疗组主要在损伤动脉管壁表面及新生内膜表达,中膜有少许表达。模型组PDGF、VEGF、bFGF及TGF-β的明显升高;DIM小剂量组也升高明显,与模型组比没有显著差异(P>0.05);而DIM大剂量组表达明显减少,与模型组比具有显著差异﹙P<0.01﹚。
3 BCL-2的表达结果
根据原位杂交实验发现,凋亡基因bcl-2在假手术组中只有极少量表达或没有表达,模型组阳性的细胞主要集中在新生内膜,并呈棕黄色,中膜也有表达,其表达明显增加,而DIM小剂量组与模型组没有明显差别﹙P>0.05);大剂量DIM组bcl-2的表达明显减少,较模型组明显改善﹙P<0.01﹚。
4 DIM的安全性评价
通过检测肝功能的ALT与AST及肝肾的病理变化均没有发现明显病变,而肝肾的重量及大小也无异常改变,因此DIM对动物没有肝肾损害。
结论
1 DIM可抑制实验兔颈动脉损伤后内膜增生与血管重构,这与其抑制平滑肌增生作用有关。
2相关细胞生长因子mRNA基因表达的增加是血管重构的重要机制,抑制其基因表达,是DIM抑制血管重构、防治再狭窄的主要机理之一。
3 DIM能够促进实验兔动脉血管成形术后血管局部SMC的凋亡,其作用与下调Bcl一2的表达的表达密切相关。
4内膜增殖与血管重塑均是RS形成的主要病理机制,RS形成由内膜增殖与血管重塑的失衡所决定,两者协同导致管腔狭窄。
5 DIM对实验动物是安全的。
Coronary heart disease(CHD)is a common disease,which severely threatens human health and affects life quality of patients .Main various measures used for the treatment of CHD includes pharmacological agents,percutaneous transluminal coronary angioplasty(PTCA) and coronary artery bypass grafting(CABG). Angioplasty is an effective, minor-wounded method for CHD. The incidence of restenosis(RS) which confirmed with coronary angiongraphy over the first six months following angioplasty remains 30%-50%.But restenosis is still the most important long-term limitation.Therefore how to reduce the ratio of restenosis is challenges both in basic and clinic research. Pathological studies have suggested that restenosis is correlation to vessel chronic elastic recoil, thrombus formation in situs, vascular smooth muscle cells(SMCS) migration and proliferation, and excessive extracellular matrix (ECM)synthesis. Cells in the restenostic plaques were mainly composed of VSMCs. Neointimal hyperplasia was formed by VSMCs migration from vessel media to subendothelium and proliferation. So, it is a key step to inhibit VSMCs proliferation for preventing restenosis.The process of cells proliferation is bound to follow by cells apoptosis in cytology. Growth factors-related and bcl-2 play an important role in the VSMC proliferation. If apoptosis of VSMCs is induced in the duration of restenosis,certainly neointimal formation will be decreased.In the end restenosisis may be prevented.
The search for an appropriate agent which inhibits VSMC proliferation and promote VSMC proliferation will contribute to better treatment of restenosis.
DIM is one of the active components of I3C, and it has various pharmacological activities that may affect many tumor cells. In addition, it's reported recently that DIM can inhibit proliferation of tumor cells.It is a much better agent used for induction of cancers,via suppressing some cell growth factors and cancer cells expression to inhibit cancers proliferation and induce cancer cells apoptosis. And cell apoptosis is found in human atherosclerosis plaque and in the process of restenosis. However, little is known about its antiproliferative mechanisms, and there is no directive evidence about its effect of preventing restenosis.We wondered whether DIM may be inhibit restenosis after angioplasty.So we performed the initial experiment of DIM on prevention of restenosis.
Objective
①To create the common carotid artery injury model of rabbit and to evaluate the effccts of DIM on neointimal proliferation and vascular remodeling after percutaneous transluminal angioplasty( PTA),and confirm DIM may prevent and treat rabbit' s experimental restenosis after PTA.
②To investigate the role for some growth factors-related in restenosis experiment. Here ,we confirm the expression and significance of GF-related during restenosis.
③To demonstrate DIM may induce VSMC apoptosis to prevent RS after artery injury,via downregulation of gene bcl-2 expression. We discuss the effect and significance of cell apoptosis gene during restenosis. .
④To assess the safety of DIM on neointimal proliferation of rabbit artery after balloon angioplasty.
Materials and Methods
Restenosis models of carotid artery after balloon injury was established in rabbits.30 rabbits were randomly divided into 4 groups including sham-operated group,model group,low-dose DIM group and high-dose DIM group. DIM was given to the rabbits in low-dose treatment group[2mg/㎏﹒d ] and high-dose treatment group[8mg/㎏﹒d ]once a day from 3 days before operation to 4 weeks after operation,the two treatment groups were administered with intraperitoneal injection of emulsified DIM,while the other two groups with saline at the same volume. all rabbits were killed after 28 days and carotid arteries were removed. With HE staining、automatic image analysis and immunohistochemistry,We observed artery morphology and measured area and thickness of arterial intima and media,examined platelet derived growth factor﹙ PDGF﹚、transfer growth factorβ1﹙TGF-β1﹚、vascular endothelial growth factor﹙VEGF﹚ and basic fibroblast growth factor﹙bFGF﹚.the expression of bcl-2 gene was observed by in situ hybridization(ISH)technology and computer-assisted picture analysis system .
To evaluate the safety of DIM in experiment by assaying serum alanine aminotransferase and aspartic aminotransferase by examinating serum in all rabbits.
Results
After one week of diet,30 rabbits were balloon injured on carotid artery, then they were randomly separated into sham-operated groups,low-dose groups( 2mg·kg一·d一)and high-dose groups (8mg·kg一·d一)and model groups. At the end of 4 weeks after carotid injury,the rabbits were killed in groups. The arteries were examined with histomorphometrical method, examined PDGF , VEGF , TGF-βand bFGF With immunohistochemical method and bcl-2 with ISH.
1 The efects of captopril on neointima hyperplasia and remodeling during restenosis angiography.
1.1 Pathological changes
The status of the models’arteries after the operation is thrombi-generated in the vessel, as well intimal hyperplasia and lumen stenosis;hyperplastic , shed and partly internal-plastic-membrane-broken in tunica intima .The status is obviously hyperplastic in tunica intima ,antrum-narrow,intimal and medial elastic tissue broken,especially wounded model groups after PTA,all the arteries are narrow and there are SMCs in tunica all intima mainly; there was no obvious difference in area and thickness of intima between model groups and low-dose groups; slightly thickened in tunica adventitia Lumen area,EEL perimeter,IEL perimeter of model group were decreased significantly in high-dose groups than those of model groups.
1.2 Changes of the intimal, medial and adventitial thickenness
The neointimai thickness increased 28 days after injury.There was no significantly change for both medial and adventtial thickness in model groups. There were also no significantly differences among the intimal,medial and Adventitial thickness between the low-dose group and model group; however,Compared with that of model groups ,the thickness、ECM and the size of lumen were markedly declinded in high-dose group (P <0.01).
1.3 Changes of the intimal, medial and adventtial area
The neointmtal area was increased the 28 days following injury in model groups.Compared with that of model group , the intimal area,media area and intima-to-media ratio increased obviously in high-dose group (P <0.01),but there was no statistical significant difference between model group and low-dose groups,high-dose group also showed less extent of restenosis of injured arteries in comparision with the low-dose groups and model group significantly,suggesting the model is successful.
2 The expression and significance of growth factors during restenosis
Immunohistochemical assay in transfected tissues showed a small quantity of positive cells with VEGF、PDGF、TGF-βand bFGF mRNA can be found in the advemitia and media in four groups. The number of positve markedly increased in the neointima at 28 days after balloon injury in model groups. There were no obvious differences for the expression of VEGF、TGF-β、PDGF and bFGF mRNA in the vessel wall between low-dose groups and model groups ,but light density of VEGF、PDGF、TGF-βand bFGF mRNA in intima in high-dose groups less than That of model groups (P <0.01). so DIM may decrease the positive index of VEGF、TGF-β、bFGF and PDGF (P<0.01).
3 The effect of DIM on the expression of apoptosis gene
The results of in situ hybridization(ISH)showed that The increased production of bcl-2 was found in proliferation intima and media of all groups, intensive density of bcl-2 in model groups more than That of high-dose groups (P <0.01):there was not significantly difference between model groups and low-dose groups ( p>0.05).
Conclusion
1 DIM can effectively inhibits intima hyperplasia,which is mainly characterized with the proliferation and migration of smooth muscle cells .DIM effectively prevents intimal thickening and area. This result suggestes that DIM may play a positive role in the prevention of restenosis after PCI.
2 The therapy of DIM can reduced the rabbit carotid artery neointimal hyperplasia induced by ballon injury by decreasing The expression level of growth factors,also DIM can improve constrictive vascular remodeling.
3.bcl-2 may participate in regulating of VSMC apoptosis .So DIM can promotes SMC apoptosis and decreases The expression of Bel一2 in intima and media.
4 The models of vascular restenosis established by balloon intimal injury of rabbit carotid arteries were successfully replicated, and proved to be rational and scientific.lntimal proliferation and vascular remodeling are important pathologic pathogenesis of vascular restenosis. The formation of vascular restenosis was determined by the unbalance of intimal proliferation and vascular remodeling.The both together resulted in lumen narrowing.
5 DIM was little toxicity during performing experiment,so DIM was basically safe for animals.
引文
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