斑蝥素对大鼠颈动脉球囊损伤后新生内膜增生的影响

目的:通过细胞培养及建立大鼠颈动脉球囊损伤模型,探讨斑蝥素对血管内皮损伤后新生内膜增生的作用及机制。方法:采用组织块贴壁法体外培养SD大鼠胸主动脉血管平滑肌细胞(VSMC),以脂多糖(LPS)作为刺激因素,选择吡咯烷二硫代氨甲基甲酸盐(PDTC)作为核转录因子(NF-κB)信号通路抑制剂,将VSMC分为对照组、1μg/ml LPS刺激组(LPS组)、LPS刺激后加用5μmol/L斑蝥素组(斑蝥素组)、LPS刺激后加用80μmol/L PDTC处理组(PDTC组)、LPS刺激后加用5μmol/L斑蝥素和80μmol/L PDTC处理组(斑蝥素+PDTC组)共5组;应用伤口愈合实验和Transwell实验检测斑蝥素对VSMC迁移的影响,蛋白免疫印迹法(Western blot)检测斑蝥素对NF-κB p65、磷酸化NF-κB p65(p-p65)和基质金属蛋白酶9(MMP-9)表达的影响。采用Fogarty(2 F)球囊导管建立大鼠颈动脉球囊损伤模型,SD大鼠随机分为假手术组、损伤组、斑蝥素(2 mg/kg)组,各组于造模前1周开始腹腔注射给药,连续给药3周,术后14 d取血检测血清肿瘤坏死因子α(TNF-α)及白细胞介素-6(IL-6)的水平,同时取损伤颈动脉进行苏木精-伊红染色、免疫组织化学法检测MMP-9、NF-κB p65、TNF-α及IL-6的表达。结果:伤口愈合实验和Transwell实验结果显示,LPS组细胞的迁移能力显著增强(P<0.01);与LPS组比较,斑蝥素组、PDTC组、斑蝥素+PDTC组细胞迁移能力均受到抑制(P<0.01),另外与斑蝥素组比较,斑蝥素+PDTC组细胞迁移未见差异(P>0.05)。Western blot结果显示,与对照组比较,LPS组细胞p-p65和MMP-9表达增加,斑蝥素组或PDTC组表达显著降低(P均<0.05);与斑蝥素组比较,斑蝥素+PDTC组中p-p65和MMP-9表达未见差异(P均>0.05)。在大鼠颈动脉球囊损伤模型中,损伤组大鼠血清内TNF-α和IL-6浓度明显升高,斑蝥素组大鼠血清中TNF-α和IL-6浓度较损伤组显著降低(P均<0.05);损伤组大鼠颈动脉内膜增生明显,斑蝥素组较损伤组内膜面积、内膜面积/中膜面积减小(P均<0.05);与损伤组比较,斑蝥素组血管内膜中MMP-9、NF-κB p65、TNF-α和IL-6的表达明显降低(P均<0.05)。结论:斑蝥素对LPS诱导的VSMC的迁移以及对受损大鼠颈总动脉内膜的增生具有显著的抑制作用;其机制主要与抑制NF-κB信号通路介导的炎症反应密切相关。

Effect of Cantharidin on Neointimal Hyperplasia After Carotid Balloon Injury in Rats

Objectives:To investigate the effect of cantharidin on neointimal hyperplasia in a rat carotid balloon injury model.Methods:Vascular smooth muscle cells(VSMCs)were isolated from thoracic aorta of male Sprague-Dawley rats by tissue adherent method,lipopolysaccharide(LPS)was used as a stimulator and Ammonium pyrrolidinedithiocarbamate(PDTC)was selected as NF-κB signaling pathway inhibitor.The cells were divided into 5 groups:control group,1μg/ml LPS stimulation group(LPS group),LPS stimulation plus 5μmol/L cantharidin group(cantharidin group),LPS stimulation plus 80μmol/L PDTC treatment group(PDTC group),5μmol/L cantharidin and 80μmol/L PDTC treatment group(cantharidin+PDTC group).Wound healing assay and Transwell assay were applied to detect the effect of cantharidin on VSMC migration.The effects of cantharidin on the expression of nuclear factor p65(NF-κB p65),phosphorylated NF-κB p65(p-p65)and matrix metalloproteinase-9(MMP-9)were detected by Western blot.Rat carotid balloon injury model was established by Fogarty(2 F)balloon catheter.SD rats were randomly divided into sham operation group(equal volume saline/day),injury group equal volume saline/day and cantharidin(2 mg/[kg·d])group.Rats received intraperitoneal injection of assigned agent 1 week before modeling and the treatment was continued for 3 weeks.14 days after operation,the serum levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were detected,and the injured carotid artery was taken for HE staining and immunohistochemistry examinations.Results:The wound healing assay and Transwell assay showed that the migration ability of cells stimulated by LPS was significantly increased(P<0.01).Compared with LPS group,the cell migration ability were significantly inhibited in cantharidin group,PDTC group and cantharidin+PDTC group(P all<0.01).There was no difference in cell migration between cantharidin+PDTC group,cantharidin group(P>0.05).Western blot showed that the expression of p-p65 and MMP-9 was significantly upregulated in the LPS group compared with the control group(both P<0.05),and which could be significantly downregulated by cotreatment with cantharidin or PDTC(both P<0.05).In addition,there was no difference in the expression of p-p65 and MMP-9 between the cantharidin+PDTC group and cantharidin group(P>0.05).In the rat carotid balloon injury model,the serum concentration of TNF-αand IL-6 was significantly increased in the injured group,which was significantly reduced by treatment with cantharidin(P<0.05).Carotid intima hyperplasia as assessed by intima area and intimal area/medial area were significantly decreased in the cantharidin group compared with the injured group(P<0.05).Compared with the injured group,the expression of MMP-9,NF-κB p65,TNF-αand IL-6 in the intima in cantharidin group was also significantly decreased(Pall<0.05).Conclusions:Cantharidin could inhibite the migration of VSMCs induced by LPS and the neointimal hyperplasia of the injured rat common carotid artery through inhibiting the inflammatory response mediated by NF-κB signaling pathway.