【摘要】：觀察參蓮片對apoE~(-/-)小鼠動脈粥樣硬化(As)不穩(wěn)定斑塊模型的干預(yù)作用及其機制。大鼠腹腔肥大細胞體外培養(yǎng),以參蓮片各劑量組(100、50、25和12.5 mg·L~(-1))和色甘酸鈉(200μg·L~(-1))預(yù)處置2 h后加入P物質(zhì)刺激誘導(dǎo)肥大細胞脫顆粒,檢測上清中組胺、類胰蛋白酶、IL-1β和NF-κB含量。采用apoE~(-/-)小鼠頸總動脈套管法合并高脂飲食誘導(dǎo)As斑塊形成,在套管部位血管外膜處滴加P物質(zhì)建立外膜肥大細胞活化介導(dǎo)的不穩(wěn)定斑塊模型,分設(shè)頸動脈套管假手術(shù)組(M1)、頸動脈套管+高脂飲食組(M2)、M2+套管部位滴加P物質(zhì)(0.5μg/只)組(M3)、參蓮片組(95、190和380 mg·kg~(-1)·d~(-1))、阿托伐他汀組(2.6 mg·kg~(-1)·d~(-1))和正常對照組(C)。實驗結(jié)束后,取血檢測總膽固醇(total cholesterol,TC)、高密度脂蛋白(high-density lipoprotein,HDL-C)、高敏C反應(yīng)蛋白(high-sensitivity C-reactive protein,hs-CRP)、基質(zhì)金屬蛋白酶-9(matrix metallo proteinases 9,MMP-9)和組胺(histamin)含量。取動脈組織通過蘇木素-伊紅(hematoxylin and eosin staining,HE)染色觀察病理變化,甲苯胺藍染色觀察肥大細胞脫顆粒情況,免疫熒光法染色肥大細胞CD117抗原表達。采用Bio-Rad磷酸化檢測試劑盒檢測病變血管組織中8個炎癥相關(guān)信號分子磷酸化。綜合評價參蓮片對不穩(wěn)定斑塊的保護作用。結(jié)果表明,參蓮片可以穩(wěn)定大鼠腹腔肥大細胞細胞膜,減少其活化釋放組胺、類胰蛋白酶(均P0.05)及IL-1β和NF-κB(P0.05或P0.01)。apoE~(-/-)小鼠M3模型組血管外膜處肥大細胞增殖和脫顆粒顯著增多,釋放出的活性物質(zhì)顯著升高,誘發(fā)病灶處外膜大量炎性細胞的浸潤,內(nèi)膜下及斑塊內(nèi)出血(紅細胞沉積),中膜平滑肌變薄,同時血清斑塊炎性活化相關(guān)指標hs-CRP、MMP-9等顯著增高,提示As斑塊呈不穩(wěn)定狀態(tài);參蓮片可減少肥大細胞增殖和脫顆粒,降低hs-CRP、MMP-9和組胺含量(P0.05或P0.01),減少病灶處炎癥反應(yīng),減輕血管組織中IκB和p38 MAPK磷酸化程度(均P0.05);減少斑塊內(nèi)出血及膠原降解,從而增加As斑塊的穩(wěn)定性。血管外膜肥大細胞激活介導(dǎo)的不穩(wěn)定斑塊模型建立成功。參蓮片可以通過穩(wěn)定肥大細胞,減少病灶處炎癥反應(yīng),減緩As發(fā)生發(fā)展,增加As斑塊的穩(wěn)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.
【作者單位】： 中國中醫(yī)科學(xué)院中藥研究所;
【基金】：科技部第二十次中泰科技合作聯(lián)委會長期合作項目(20-602J) 國家自然科學(xué)基金面上資助項目(30973901,81573649)
【分類號】：R285.5;R-332

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