大鼠主动脉内膜损伤后新生内膜各种生长因子表达与表没食子儿茶素没食子酸酯的干预

Effects of epigallocatechin-3 gallate on the expression of various growth factors in neoformative intima of rats after aorta injury

目的:观察表没食子儿茶素没食子酸酯(epigallocatechin-3 gallate,EGCG)对大鼠主动脉内膜损伤后再狭窄的影响,探讨其对新生内膜各种生长因子表达的干预效应。方法:实验于2006-03/10在汕头大学医学院细胞衰老实验室完成。实验分组:选用清洁级雄性健康SD大鼠72只,体质量(300±35)g,随机数字表法分为对照组、模型组、EGCG低剂量组和EGCG高剂量组,每组18只。实验方法:以球囊损伤方法建立大鼠主动脉内膜损伤模型。①对照组:结扎左颈总动脉,不插入球囊导管,不给药。②模型组:结扎左颈总动脉并球囊拉伤主动脉内膜,不给药。③EGCG低剂量组:拉伤主动脉内膜,给予20mg/(kg.d)EGCG灌胃。④EGCG高剂量组:拉伤主动脉内膜,给予50mg/(kg.d)EGCG灌胃,药物组术前3d给药。对照组及模型组予生理盐水灌胃,直至实验结束。实验评估:每组于术后14,28d分别随机选取9只大鼠,取其主动脉进行苏木精-伊红染色并作血管形态学检测;同时进行免疫组织化学染色检测增殖细胞核抗原、血小板源性生长因子BB、碱性成纤维细胞生长因子、生长转化因子β1的表达水平。结果:纳入大鼠72只,其中模型组28d时1只鼠死于腹主动脉血栓;14d时1只鼠右后肢出现血栓性跛行;EGCG低剂量组14d时1只死于手术急性呼吸窘迫综合征,1只死于术后21d胸水形成,无血栓形成现象;EGCG高剂量组14,28d各有1只分别因感染、误灌入气管死亡,无血栓形成现象;对照组动物生长状况良好;最后67只大鼠进入结果分析。①模型组新生内膜显著增厚,新生内膜平均厚度、面积自胸主动脉到腹主动脉上段及下段逐渐增加、狭窄逐渐明显。②EGCG低剂量组新生内膜平均厚度及面积于术后14d下降,与模型组比较,无显著差异[(45.89±9.54),(60.75±17.70)μm;(1.10±0.29),(1.55±0.61)mm2;P>0.05],但两者于术后28d则显著降低(P<0.05);EGCG高剂量组术后14,28d的新生内膜平均厚度、面积均显著下降[14d:(20.21±5.55),(60.75±17.70)μm;(0.48±0.22),(1.55±0.61)mm2;28d:(35.08±12.39),(80.75±22.27)μm;(0.82±0.50),(2.35±1.03)mm2;P<0.01]。③模型组动脉增殖细胞核抗原、血小板源性生长因子BB、碱性成纤维细胞生长因子、转化生长因子β1表达显著增加;低剂量EGCG对内膜损伤后增殖细胞核抗原、血小板源性生长因子、碱性成纤维细胞生长因子表达无明显抑制作用;而高剂量在术后14,28d均显著抑制增殖细胞核抗原、血小板源性生长因子、碱性成纤维细胞生长因子表达。EGCG对转化生长因子β1表达无影响。结论:EGCG剂量依赖性地抑制动脉内膜损伤后再狭窄,其作用与抑制血小板源性生长因子、碱性成纤维细胞生长因子表达有关。

AIM： To investigate the effects of epigallocatechin-3 gallate （EGCG） on the vascular restenosis in rats after aorta injury, and explore the intervention of EGCG to the expression of growth factors in neoformative intima. METHODS： The experiment was conducted in the Laboratory of Cell Senescence, Medical College of Shantou University from March to October 2006. The models of aorta injury were established by balloon catheter. Seventy-two male healthy SD rats of clean grade and （300±35） g were selected and randomly divided into ①control group, which only received left common carotid ligation; ②model group, which was subjected to left common carotid llgation and aorta injury by balloon catheter, but not given any medicine; ③low dose EGCG group, which underwent aorta injury and infused with 20 mg/kg daily EGCG intragastricaUy; ④high dose group, which was infused with 50 mg/kg daily EGCG -after the previous operation. All administration was given three days before operation, and matching normal saline was infused to the control and model groups until the end of the experiment. The aorta of 9 randomly selected rats from each group were harvested on postoperative days 14 and 28 for H-E staining and vessel morphological detection; meanwhile, immunohistochemical staining was performed to measure the expressions of proliferating cell nuclear antigen （PCNA）, platelet-derived growth factor-BB （PDGF-BB）, basic fibroblast growth factor （bFGF）, and transforming growth factor-β1 （TGF-β1）, RESULTS： Among the 72 rats, 1 of the model group died of abdominal aortic thrombosis on day 28, and 1 was of thrombotic lameness in right limbs on day 14; 1 of the low dose group died of operative acute respiratory distress syndrome, and 1 died of hydrothorax with no thrombosis on day 21; 1 of the high dose group died of infection and intrabronchial injection with no thrombosis on days 14 and 28, respectively. Finally, 67 rats were involved in the result analysis. ①Neoformative intima in the model group was increased In thickness （NT） and area （NA） markedly from aorta thoracica to the upper and lower vessels of abdominal aorta, and the stenosis became evident gradually. ②NT and NA of the low dose EGCG group were decreased since the postoperative day 14, and not significantly different from those of the model group [（45.89±9.54）, （60.75±17.70）μm; （1.10±0.29）, （1.55±0.61） mm^2; P 〉 0.05]; while they were significantly decreased from the 28th day （P 〈 0.05）, NT and NA of the high dose group were significantly decreased on the 14^th and 28^th days after aorta injury [14^th day： （20.21±5.55）, （60.75±17.70） μm; （0.48±0.22）, （1.55±0.61） mm^2; 28^th day： （35.08±12.39）, （80.75±22.27） μm; （0.82±0.50）, （2.35±1.03） mm^2; P 〈 0.01].③Expressions of PCNA, PDGF-BB, bFGF, and TGF-β1 were increased remarkably in the model group; low dose EGCG had no obvious inhibition to the expressions of PCNA, PDGF-BB and bFGF, while high dose EGCG significantly inhibited the expressions of PCNA, PDGF-BB and bFGF, EGCG had no effect on the expression of TGF-β1. CONCLUSION： EGCG could reduce the artedal restenosis after aorta injury by inhibiting the expressions of PDGF-BB and bFGF.