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
发布时间：2018-11-10 19:10
【摘要】：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P0.05)及IL-1β和NF-κB(P0.05或P0.01)。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(P0.05或P0.01),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P0.05);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。
[Abstract]:To observe the intervention effect of Shenlian tablet on atherosclerotic (As) unstable plaque model in apoE~ (- / -) mice and its mechanism. Rat peritoneal mast cells were cultured in vitro. The mast cells were induced to degranulate by the stimulation of substance P for 2 h after pretreatment with Shenlian tablets (100,50,12.5 mg L ~ (-1) and 200 渭 g L ~ (-1). The contents of histamine, trypsin, IL-1 尾 and NF- 魏 B in supernatant were determined. ApoE~ (- / -) mouse common carotid artery cannula combined with high-fat diet was used to induce plaque formation of As. The model of unstable plaque was established by injecting substance P into the adventitia of the vessel and mediating the activation of mast cells in the outer membrane. Carotid cannula sham-operation group (M1), carotid cannula high-fat diet group (M2), M2 cannula group (0.5 渭 g / mouse), Shenlian tablet group (95190 and 380 mg kg~ (-1) D1),) Atto vastatin group (2.6 mg kg~ (-1) d ~ (-1) and normal control group (C). After the experiment, total cholesterol (total cholesterol,TC), high density lipoprotein (high-density lipoprotein,HDL-C), Gao Min C-reactive protein (high-sensitivity C-reactive protein,hs-CRP) and matrix metalloproteinase-9 (matrix metallo proteinases 9 were measured. MMP-9) and histamine (histamin). The pathological changes were observed by hematoxylin-eosin (hematoxylin and eosin staining,HE staining, the degranulation of mast cells was observed by toluidine blue staining, and the expression of CD117 antigen in mast cells was stained by immunofluorescence. Bio-Rad phosphorylation assay kit was used to detect the phosphorylation of eight inflammatory signaling molecules in the diseased vascular tissues. Objective: to evaluate the protective effect of Shenlian tablets on unstable plaques. The results showed that Shenlian tablet could stabilize rat peritoneal mast cell membrane and reduce its activation and release histamine. Trypsin (P0.05) and IL-1 尾 and NF- 魏 B (P0.05 or P0.01). The proliferation and degranulation of mast cells in the adventitia of apoE~ (- / -) mice increased significantly, and the release of active substances increased significantly. It induced the infiltration of a large number of inflammatory cells in the outer membrane of the lesion, the hemorrhage (erythrocyte deposition) under and within the plaque, the thinning of the medial smooth muscle, and the increase of serum plaque inflammatory activation related index (hs-CRP,MMP-9). The results suggest that As plaques are unstable. Shenlian tablet could reduce mast cell proliferation and degranulation, decrease the content of hs-CRP,MMP-9 and histamine (P0.05 or P0.01), reduce the inflammatory reaction in the focus and reduce the phosphorylation of I 魏 B and p38 MAPK in vascular tissue (P0.05). Reduce plaque bleeding and collagen degradation, thus increasing the stability of As plaque. The model of unstable plaque mediated by the activation of adventitial mast cells was successfully established. Shenlian tablet can stabilize mast cells, reduce inflammatory reaction, slow down the development of As and increase the stability of As plaque.
【作者单位】： 中国中医科学院中药研究所;
【基金】：科技部第二十次中泰科技合作联委会长期合作项目(20-602J) 国家自然科学基金面上资助项目(30973901,81573649)
【分类号】：R285.5;R-332
本文编号：2323352
[Abstract]:To observe the intervention effect of Shenlian tablet on atherosclerotic (As) unstable plaque model in apoE~ (- / -) mice and its mechanism. Rat peritoneal mast cells were cultured in vitro. The mast cells were induced to degranulate by the stimulation of substance P for 2 h after pretreatment with Shenlian tablets (100,50,12.5 mg L ~ (-1) and 200 渭 g L ~ (-1). The contents of histamine, trypsin, IL-1 尾 and NF- 魏 B in supernatant were determined. ApoE~ (- / -) mouse common carotid artery cannula combined with high-fat diet was used to induce plaque formation of As. The model of unstable plaque was established by injecting substance P into the adventitia of the vessel and mediating the activation of mast cells in the outer membrane. Carotid cannula sham-operation group (M1), carotid cannula high-fat diet group (M2), M2 cannula group (0.5 渭 g / mouse), Shenlian tablet group (95190 and 380 mg kg~ (-1) D1),) Atto vastatin group (2.6 mg kg~ (-1) d ~ (-1) and normal control group (C). After the experiment, total cholesterol (total cholesterol,TC), high density lipoprotein (high-density lipoprotein,HDL-C), Gao Min C-reactive protein (high-sensitivity C-reactive protein,hs-CRP) and matrix metalloproteinase-9 (matrix metallo proteinases 9 were measured. MMP-9) and histamine (histamin). The pathological changes were observed by hematoxylin-eosin (hematoxylin and eosin staining,HE staining, the degranulation of mast cells was observed by toluidine blue staining, and the expression of CD117 antigen in mast cells was stained by immunofluorescence. Bio-Rad phosphorylation assay kit was used to detect the phosphorylation of eight inflammatory signaling molecules in the diseased vascular tissues. Objective: to evaluate the protective effect of Shenlian tablets on unstable plaques. The results showed that Shenlian tablet could stabilize rat peritoneal mast cell membrane and reduce its activation and release histamine. Trypsin (P0.05) and IL-1 尾 and NF- 魏 B (P0.05 or P0.01). The proliferation and degranulation of mast cells in the adventitia of apoE~ (- / -) mice increased significantly, and the release of active substances increased significantly. It induced the infiltration of a large number of inflammatory cells in the outer membrane of the lesion, the hemorrhage (erythrocyte deposition) under and within the plaque, the thinning of the medial smooth muscle, and the increase of serum plaque inflammatory activation related index (hs-CRP,MMP-9). The results suggest that As plaques are unstable. Shenlian tablet could reduce mast cell proliferation and degranulation, decrease the content of hs-CRP,MMP-9 and histamine (P0.05 or P0.01), reduce the inflammatory reaction in the focus and reduce the phosphorylation of I 魏 B and p38 MAPK in vascular tissue (P0.05). Reduce plaque bleeding and collagen degradation, thus increasing the stability of As plaque. The model of unstable plaque mediated by the activation of adventitial mast cells was successfully established. Shenlian tablet can stabilize mast cells, reduce inflammatory reaction, slow down the development of As and increase the stability of As plaque.
【作者单位】： 中国中医科学院中药研究所;
【基金】：科技部第二十次中泰科技合作联委会长期合作项目(20-602J) 国家自然科学基金面上资助项目(30973901,81573649)
【分类号】：R285.5;R-332
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