大蒜素抑制硬膜外纤维化中血管内皮细胞增殖、迁移及血管生成

Basic Research Effects of allicin on the biological behavior of vascular endothelial cells and epidural scar

目的:观察大蒜素(allicin)对大鼠血管内皮细胞(rat vascular endothelial cells,RVECs)增殖、凋亡、迁移、血管生成能力的影响,探究大蒜素抑制硬膜外纤维化的作用机制。方法:根据预实验结果将生长状态良好的RVECs分为对照组(0 mg/L)、25 mg/L组、50 mg/L组和100 mg/L组。根据组别加入含相应大蒜素浓度的细胞培养基培养24 h,使用倒置相差显微镜观察细胞形态,AnnexinV-FITC双染法检测细胞凋亡,PI/RNase法检测细胞周期分布百分比,划痕实验和Transwell实验检测细胞迁移能力,体外管腔形成法察看细胞管腔形成能力,Western blot法检测各组细胞JAK2、STAT3、p-STAT3、PCNA、Bax、BcL-2蛋白表达水平。使用随机数字法将36只成年雄性的SD大鼠均分为3组,即假手术组、生理盐水组和大蒜素组,术后第28天察看各组大鼠硬膜外瘢痕产生状况,并使用HE染色、Masson染色及免疫组化染色分析各组大鼠瘢痕组织中成纤维细胞数目、胶原密度、血管数量、p-STAT3蛋白表达情况。结果:随着大蒜素浓度的升高,细胞活力、细胞迁移能力及管腔形成能力显著低于对照组(P<0.05)。大蒜素各组中G1期和S期细胞百分比随大蒜素浓度逐步下降(P<0.05),G2期细胞百分比和细胞凋亡率则随大蒜素浓度逐步上升(P<0.05),细胞被阻滞在G2/M期。随着大蒜素浓度的升高,细胞内JAK2、STAT3、p-STAT3、PCNA、BcL-2蛋白表达水平逐步下调(P<0.05),Bax蛋白表达水平逐步上调(P<0.05),p-STAT3/STAT3比例下降(P<0.05),Bax/Bcl-2比例上升(P<0.05)。所有实验动物无死亡、感染或步态异常。假手术组椎板外区域和生理盐水组硬膜外区域可观察到致密的瘢痕组织,而大蒜素组中硬膜外瘢痕与硬脑膜之间有明显的间隙,胶原密度、血管数量、p-STAT3蛋白密度以及显着低于生理盐水组(P<0.05)。结论:大蒜素抑制椎板切除术后血管生成及硬膜外瘢痕严重程度,其作用机制可能是通过抑制血管内皮细胞JAK2/STAT3信号通路实现。

Objective To investigate the effects of allicin on the proliferation,migration and angiogenesis of rat vascular endothelial cells(RVES),and to explore the influencing mechanism of allicin on epidural fibrosis.Methods According to the results of preliminary experiments,RVECs were divided into control group(0 mg/L),low concentration group(25 mg/L),medium concentration group(50 mg/L)and high concentration group(100 mg/L).The morphology,viability,migration rate,cell cycle,apoptosis rate and cell lumen formation ability were measured using fluorescence microscope,AnnexinV-FITC double staining,PI/RN-asestaining,scratch assay and Transwell experiments test.Western Blot was used to measure the protein expression level of JAK2,STAT3,p-STAT3,PCNA,Bax and Bcl-2 protein.Using random number method,36 adult male SD rats were divided into sham operation group,saline group and allicin group,with 12 rats in each group.Hematoxylin-eosin(HE)staining,Masson staining and immunohistochemical staining were used to analysis the epidural fibrosis in each group.Results With the increase of concentration of allicin,cell viability,cell migration and lumen formation ability significantly lower than that of control group(P<0.05).With the increase of allicin concentration,the percentage of cells in the G1 and S phases gradually decreased(P<0.05),the percentage of cells in the G2 phase and the apoptosis rate gradually increased(P<0.05),and the cells were blocked in the G2/M phase.With the increase of allicin concentration,the protein expression levels of JAK2,STAT3,p-STAT3,PCNA and Bcl-2 were gradually down-regulated(P<0.05),while the protein expression level of Bax was gradually up-regulated(P<0.05),the ratio of p-STAT3/STAT3 was decreased(P<0.05),and the ratio of Bax/Bcl-2 was increased(P<0.05).There was no death,infection or abnormal gait in all the experimental animals.Dense scar tissue could be observed in the extradural area of the sham operation group and the epidural area of the control group,but there was obvious space between the epidural scar and the dura mater in the allicin group,and the density of collagen,the number of blood vessels,and the protein density of p-STAT3 were significantly lower than those in the control group(P<0.05).Conclusion Allicin inhibits angiogenesis and the severity of epidural scar after laminectomy,and the mechanism may be through inhibiting the JAK2/STAT3 signaling pathway of vascular endothelial cells.