血管外膜肥大细胞激活介导的不稳定斑块模型建立及参莲片的干预

Establishment of vulnerable plaque model by mast cell activation in adventitia and evaluation of effectiveness of intervention of Shenlian tablet

观察参莲片对apoE^(-/-)小鼠动脉粥样硬化(As)不稳定斑块模型的干预作用及其机制。大鼠腹腔肥大细胞体外培养,以参莲片各剂量组(100、50、25和12.5 mg·L^(-1))和色甘酸钠(200μg·L^(-1))预处置2 h后加入P物质刺激诱导肥大细胞脱颗粒,检测上清中组胺、类胰蛋白酶、IL-1β和NF-κB含量。采用apoE^(-/-)小鼠颈总动脉套管法合并高脂饮食诱导As斑块形成,在套管部位血管外膜处滴加P物质建立外膜肥大细胞活化介导的不稳定斑块模型,分设颈动脉套管假手术组(M1)、颈动脉套管+高脂饮食组(M2)、M2+套管部位滴加P物质(0.5μg/只)组(M3)、参莲片组(95、190和380 mg·kg^(-1)·d^(-1))、阿托伐他汀组(2.6 mg·kg^(-1)·d^(-1))和正常对照组(C)。实验结束后,取血检测总胆固醇(total cholesterol,TC)、高密度脂蛋白(high-density lipoprotein,HDL-C)、高敏C反应蛋白(high-sensitivity C-reactive protein,hs-CRP)、基质金属蛋白酶-9(matrix metallo proteinases 9,MMP-9)和组胺(histamin)含量。取动脉组织通过苏木素-伊红(hematoxylin and eosin staining,HE)染色观察病理变化,甲苯胺蓝染色观察肥大细胞脱颗粒情况,免疫荧光法染色肥大细胞CD117抗原表达。采用Bio-Rad磷酸化检测试剂盒检测病变血管组织中8个炎症相关信号分子磷酸化。综合评价参莲片对不稳定斑块的保护作用。结果表明,参莲片可以稳定大鼠腹腔肥大细胞细胞膜,减少其活化释放组胺、类胰蛋白酶(均P<0.05)及IL-1β和NF-κB(P<0.05或P<0.01)。apoE^(-/-)小鼠M3模型组血管外膜处肥大细胞增殖和脱颗粒显著增多,释放出的活性物质显著升高,诱发病灶处外膜大量炎性细胞的浸润,内膜下及斑块内出血(红细胞沉积),中膜平滑肌变薄,同时血清斑块炎性活化相关指标hs-CRP、MMP-9等显著增高,提示As斑块呈不稳定状态;参莲片可减少肥大细胞增殖和脱颗粒,降低hs-CRP、MMP-9和组胺含量(P<0.05或P<0.01),减少病灶处炎症反应,减轻血管组织中IκB和p38 MAPK磷酸化程度(均P<0.05);减少斑块内出血及胶原降解,从而增加As斑块的稳定性。血管外膜肥大细胞激活介导的不稳定斑块模型建立成功。参莲片可以通过稳定肥大细胞,减少病灶处炎症反应,减缓As发生发展,增加As斑块的稳定性。

This study was designed to investigate the activity of Shenlian tablet in stabilization of the atherosclerosis（As） plaque in apoE（-/-） mice and explore the mechanisms. Rat peritoneal mast cells were randomly allocated and treated with Shenlian tablet（100, 50, 25, 12.5 mg·L（-1）） or cromoglicate sodium（200 μg·L（-1）） for 2 h before exposure to substance P. Histamine, tryptase, IL-1β and NF-κB were measured in the cell culture supernatant by ELISA assay. The plaque formation was induced by common carotid artery cannula method combined with high-fat diet in apoE（-/-） mice, and the plaque instability was induced by substance P through local mast cell degranulation. Mice were divided into eight groups that included the model 1（M1, sham-operated group）, M2（carotid artery cannula combined with high-fat diet）, M3（M2 combined with substance P〈0.5 μg/mouse）, Shenlian extract（95, 190 and 380 mg·kg（-1）·d（-1））, atorvastatin（2.6 mg·kg（-1）·d（-1）） and normal control group. Total cholesterol（TC）, high-density lipoprotein（HDL-C）, high-sensitivity C-reactive protein（hs CRP）, matrix metalloproteinases 9（MMP-9） and histamine were measured by ELISA. Thickness, plaque area, mast cell degranulation were observed by hematoxylin and eosin staining, toluidine blue staining. CD117 antigen expression were observed by confocal microscopy. Intracellular phosphorylation was detected using the Bio-Plex 6-plex phosphoprotein assay kit. The results show that the mast cell membrane was stabilized by Shenlian tablet. Histamine, tryptase, interleukin l-β and NF-κB exhibited a significantly reduction in the Shenlian tablet-treated group（P〈0.05 or P〈0.01）. Substance P significantly enhanced activation and degranulation of adventitial mast cells. In addition, it increased adventitia inflammatory cells infiltration and promoted intraplaque hemorrhages in apo E-/- mice model group. The proliferation, degranulation and inflammation of mast cell were significantly inhibited by Shenlian tablet. On the other hand, the same treatment decreased hs-CRP, MMP-9 and histamine in serum. IκB, p38 MAPK phosphorylation, intraplaque hemorrhage and collagen degradation were reduced in the presence of Shenlian tablet, which increased the stability of the As plaque. The results show that the vulnerable plaque model induced by mast cell activation in adventitia was established. Shenlian tablet exhibited a protective effect in this model. Shenlian tablet may increase the plaque stability via inhibition of mast cell-mediated inflammatory response.