阿折地平對西羅莫司洗脫支架致血管內(nèi)皮損傷的干預(yù)作用及機(jī)制探討

阿折地平對西羅莫司洗脫支架致血管內(nèi)皮損傷的干預(yù)作用及機(jī)制探討

袁玲，聶衛(wèi)，高萍，李昕，劉偉偉，崔曉雪

(天津市醫(yī)藥科學(xué)研究所，天津300020)

摘要：目的觀察阿折地平對西羅莫司洗脫支架致血管內(nèi)皮損傷的干預(yù)作用，并探討其作用機(jī)制。方法培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞(HUVEC)，將HUVEC分為對照組、模型組和實(shí)驗(yàn)組。對照組不加藥物，模型組給予西羅莫司500 nmol/L，實(shí)驗(yàn)組給予阿折地平10 μmol/L+西羅莫司500 nmol/L。各組處理24 h后，HE染色觀察細(xì)胞形態(tài)變化，測定培養(yǎng)液中的NO，F(xiàn)luo-3/AM 熒光探針標(biāo)記細(xì)胞內(nèi)Ca2+，JC-1熒光探針檢測線粒體膜電位，Annexin V FITC/PI染色法測算細(xì)胞凋亡率。結(jié)果各組處理24 h后，對照組細(xì)胞呈多角形、單層鋪路石樣排列；模型組細(xì)胞腫脹，胞質(zhì)出現(xiàn)空泡，部分細(xì)胞核固縮、碎裂；實(shí)驗(yàn)組細(xì)胞基本呈多角形、單層鋪路石樣排列，少見細(xì)胞腫脹及胞質(zhì)空泡。與對照組相比，模型組與實(shí)驗(yàn)組細(xì)胞培養(yǎng)液中NO水平降低、胞質(zhì)Ca2+濃度升高、細(xì)胞凋亡率升高、線粒體膜電位下降(P均<0.05)；與模型組相比，實(shí)驗(yàn)組細(xì)胞培養(yǎng)液中NO水平升高、胞質(zhì)Ca2+濃度及細(xì)胞凋亡率降低、線粒體膜電位升高(P均<0.05)。結(jié)論 阿折地平可在一定程度上抑制西羅莫司洗脫支架導(dǎo)致的血管內(nèi)皮損傷，其機(jī)制可能與提高細(xì)胞內(nèi)NO水平、降低細(xì)胞內(nèi)Ca2+濃度、抑制線粒體膜電位降低、減少細(xì)胞凋亡有關(guān)。

關(guān)鍵詞：鈣通道阻滯劑；阿折地平；藥物洗脫支架；人臍靜脈內(nèi)皮細(xì)胞；血管內(nèi)皮損傷

doi：10.3969/j.issn.1002-266X.2015.39.003

中圖分類號：R543.3；R965.3 文獻(xiàn)標(biāo)志碼：A

基金項(xiàng)目：天津市衛(wèi)生局科技

作者簡介：第一袁玲(1979-)，女，碩士，助理研究員，主要從事藥物毒理、病理及醫(yī)療器械安全性評價(jià)方面的研究。E-mail： 1128sun@163.com

收稿日期：(2015-07-10)

Protective effect of azelnidipine on sirolimus eluting stent-induced vascular

endothelial injury and its mechanism

YUANLing,NIEWei,GAOPing,LIXin,LIUWei-wei,CUIXiao-xue

(TianjinInstituteofMedicalScience,Tianjin300020,China)

Abstract:ObjectiveTo investigate the protective effect of calcium-channel blockers azelnidipine on sirolimus eluting stent-induced vascular endothelial injury and its mechanism. MethodsHuman umbilical vein endothelial cells (HUVECs) were cultivated and divided into the control group (treated with culture medium), model group (treated with sirolimus 500 nmol/L) and the experimental group (azelnidipine 10 μmol/L + sirolimus 500 nmol/L), respectively. After 24 h of treatment, changes in cell morphology were observed by HE staining. Effects on the production of nitric oxide (NO) were detected by Nitric Oxide Assay Kit. The intracellular calcium ion (Ca2+) concentration was assayed with Fluo-3/AM staining, the changes of mitochondrial membrane potential was detected by JC-1 fluorescence labeling, and the apoptosis rate of HUVECs was analyzed by Annexin V FITC/PI staining. ResultsAfter 24 h of treatment, in the control group, cells were polygonal and in the single cobblestone arrangement; in the model group, cell swelled, cytoplasm had vacuoles, part of the nucleus had pycnosis, and nuclear fragmentation were observed; in the experimental group, cells were substantially polygonal, and in the single cobblestone arrangement, the cell swelling and cytoplasmic vacuoles were rare. Compared with the control group, the levels of NO in the cell culture fluid were reduced, the levels of intracellular free Ca2+ were increased, apoptosis rate was increased and mitochondrial membrane potential was reduced in the model group and experimental group (all P<0.05). Compared with the model group, the levels of NO in the cell culture fluid were increased, the levels of intracellular free Ca2+ were reduced, the apoptosis rate was reduced and the mitochondrial membrane potential was increased in the experimental group (all P<0.05). ConclusionsAzelnidipine has protective effect on sirolimus eluting stent-induced vascular endothelial injury. The possible mechanism might be related to the decrease of intracellular Ca2+ which could alleviate calcium overload and mitochondrial membrane potential and finally reduce apoptosis.

Key words: calcium-channel blockers; azelnidipine; drug eluting stent; human umbilical vein endothelial cells; vascular endothelial injury

西羅莫司洗脫支架(SES)能明顯減少冠狀動脈介入術(shù)后血管再狹窄的發(fā)生率，但近年研究發(fā)現(xiàn)，SES與支架內(nèi)血栓形成有關(guān)[1~3]。鈣通道阻滯劑是心血管疾病常用的藥物。Kubota等[4]發(fā)現(xiàn)鈣通道阻滯劑阿折地平能在一定程度上恢復(fù)豬冠脈支架植入模型的血管內(nèi)皮功能并上調(diào)內(nèi)皮型一氧化氮合酶(eNOS)表達(dá)。但有關(guān)阿折地平對SES致血管內(nèi)皮損傷的干預(yù)作用和機(jī)制目前研究較少。為此，我們于2013年6月~2015年5月進(jìn)行了如下研究。

1材料與方法

1.1西羅莫司及阿折地平作用濃度篩選將HUVEC培養(yǎng)于完全培養(yǎng)液，置于37 ℃、5% CO2及飽和濕度的細(xì)胞培養(yǎng)箱中培養(yǎng)，細(xì)胞貼壁并生長至融合80%以上時(shí)，用0.25%胰酶-EDTA消化傳代。取對數(shù)生長期細(xì)胞用0.25%胰酶-EDTA消化，制成單細(xì)胞懸液，按5×103/孔接種于96孔板，分為正常對照組(磷酸鹽緩沖液刺激)、西羅莫司組(分別給予西羅莫司62.5、125、250、500 nmol/L處理24 h)、阿折地平組(分別給予阿折地平1、10、20 μmol/L+西羅莫司刺激24 h)。處理后，每孔加入MTT試劑10 μL，繼續(xù)培養(yǎng)4 h，吸去上清液，加入150 μL DMSO，振蕩10 min，酶標(biāo)儀測定570 nm處的吸光度值(A值)。以僅加入150 μL DMSO的孔作為空白對照。按公式計(jì)算細(xì)胞存活率：細(xì)胞存活率(%)=(A加藥-A空白)/(A正常對照-A空白)×100%。每組設(shè)置6個(gè)復(fù)孔，實(shí)驗(yàn)重復(fù)3次。選擇500 nmol/L西羅莫司與10 μmol/L阿折地平進(jìn)行后續(xù)實(shí)驗(yàn)。

1.2細(xì)胞分組與阿折地平干預(yù)方法將HUVEC按5×104/孔接種于6孔板，分為對照組、模型組和實(shí)驗(yàn)組。對照組不加藥物，模型組給予西羅莫司500 nmol/L，實(shí)驗(yàn)組給予阿折地平10 μmol/L+西羅莫司500 nmol/L，各組處理24 h。

1.3觀察方法

1.3.1HUVEC形態(tài)觀察吸棄上清液，PBS洗滌細(xì)胞2次，乙醇固定10 min，HE染色，倒置顯微鏡觀察細(xì)胞形態(tài)并采集圖像。

1.3.2HUVEC培養(yǎng)液中NO檢測收集各組細(xì)胞培養(yǎng)液，按NO檢測試劑盒說明操作。

1.3.3HUVEC胞質(zhì)Ca2+濃度檢測消化細(xì)胞制成單細(xì)胞懸液，于細(xì)胞懸液中加入Fluo-3/AM及Pluronic127，37 ℃孵育30 min，以含鈣Hank′s平衡鹽溶液洗滌3次后，在FACS Calibur流式細(xì)胞儀上檢測異硫氰酸熒光素綠色熒光，重復(fù)測量3次，采用FCS Express4.0軟件分析熒光強(qiáng)度，以各組與正常對照的熒光比值代表Ca2+濃度。

1.3.4HUVEC凋亡檢測胰酶消化細(xì)胞，離心，去上清液，緩沖液重懸，于100 μL細(xì)胞懸液中加入5 μL annexin V及5 μL PI室溫孵育15 min，加入400 μL 緩沖液，采用流式細(xì)胞儀檢測細(xì)胞凋亡情況。

1.3.5HUVEC線粒體膜電位檢測0.25%胰酶-EDTA消化細(xì)胞，離心，PBS洗滌，加入0.5 mL JC-1染色工作液。與細(xì)胞培養(yǎng)箱中37 ℃孵育20 min。用JC-1染色緩沖液洗滌2次，重懸，采用流式細(xì)胞儀檢測線粒體膜電位，檢測結(jié)果以FL2與FL1通道紅綠熒光強(qiáng)度比值表示。

1.4統(tǒng)計(jì)學(xué)方法采用SPSS17.0統(tǒng)計(jì)軟件。計(jì)量資料以±s表示，兩組比較用t檢驗(yàn)，多組比較用單因素方差分析。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2結(jié)果

2.1各組細(xì)胞形態(tài)比較對照組細(xì)胞呈多角形、單層鋪路石樣排列；模型組細(xì)胞腫脹，胞質(zhì)出現(xiàn)空泡，部分細(xì)胞核固縮、核碎裂；實(shí)驗(yàn)組細(xì)胞基本呈多角形、單層鋪路石樣排列，少見細(xì)胞腫脹及胞質(zhì)空泡。

2.2細(xì)胞培養(yǎng)液中NO水平、胞質(zhì)Ca2+濃度、細(xì)胞凋亡率、線粒體膜電位比較與對照組相比，模型組與實(shí)驗(yàn)組細(xì)胞培養(yǎng)液中NO水平降低、胞質(zhì)Ca2+濃度升高、細(xì)胞凋亡率升高、線粒體膜電位下降(P均<0.05)；與模型組相比，實(shí)驗(yàn)組細(xì)胞培養(yǎng)液中NO水平升高、胞質(zhì)Ca2+濃度及細(xì)胞凋亡率降低、線粒體膜電位升高(P均<0.05)。見表1。

表1　 各組HUVEC培養(yǎng)液中NO水平、胞質(zhì)Ca 2+濃度、

細(xì)胞凋亡率、線粒體膜電位比較( ± s)

表1　 各組HUVEC培養(yǎng)液中NO水平、胞質(zhì)Ca 2+濃度、

組別NO (μmol/L) Ca2+濃度 (MF1) 細(xì)胞凋亡 率(%) 線粒體膜 電位 實(shí)驗(yàn)組45.7±4.6*#4434±692*#35.1±2.3*#0.49±0.04*#模型組28.3±4.2*5115±352*43.3±2.0*0.24±0.03*對照組60.2±3.93716±463 27.5±1.80.68±0.06

注：與對照組相比，*P<0.05；與模型組相比，#P<0.05。

3討論

SES將具有抗再狹窄的藥物如西羅莫司等用特殊工藝包被于支架表面的藥物載體上，SES置入血管后，藥物在病變局部緩慢釋放，集中作用于病變部位，發(fā)揮抗再狹窄的作用。目前對于西羅莫司是否損害血管內(nèi)皮仍有爭議，其發(fā)生機(jī)制也尚未明確。有文獻(xiàn)[5，6]報(bào)道，西羅莫司可阻止細(xì)胞周期G1期向S期轉(zhuǎn)化，抑制血管平滑肌細(xì)胞增殖、遷移，防止支架置入后再狹窄，但其同時(shí)也抑制了內(nèi)皮細(xì)胞增殖，造成支架置入部位內(nèi)皮化延遲，導(dǎo)致血小板激活和血栓形成，影響內(nèi)皮細(xì)胞功能。也有學(xué)者[7]發(fā)現(xiàn)，移植血管中西羅莫司給藥組一氧化氮合成酶表達(dá)增高，局部高濃度NO可能是防止動脈硬化的機(jī)制。

我們以不同濃度西羅莫司作用于HUVEC，發(fā)現(xiàn)西羅莫司可影響內(nèi)皮細(xì)胞增殖，降低內(nèi)皮細(xì)胞存活率，同時(shí)細(xì)胞培養(yǎng)基上清液NO水平也減少，提示內(nèi)皮細(xì)胞功能受到了影響，與文獻(xiàn)[8，9]結(jié)果一致。本實(shí)驗(yàn)采用Fluo-3/AM熒光探針特異性標(biāo)記細(xì)胞內(nèi)Ca2+，觀察西羅莫司作用后細(xì)胞內(nèi)游離Ca2+變化，發(fā)現(xiàn)西羅莫司可增加內(nèi)皮細(xì)胞內(nèi)Ca2+水平，同時(shí)降低線粒體膜電位，增加細(xì)胞凋亡，提示西羅莫司造成的內(nèi)皮細(xì)胞損傷與這些因素有關(guān)。

鈣通道阻滯劑是心血管疾病常用藥物。Ma等[10]報(bào)道, 鈣通道阻滯劑阿折地平可提高HUVEC基礎(chǔ)NO水平并減少M(fèi)CP-1表達(dá)，后者與獼猴支架模型(高膽固醇喂養(yǎng)建模)血管內(nèi)膜增生不良有關(guān)。本研究結(jié)果顯示，10 μmol/L阿折地平處理HUVEC后細(xì)胞存活率升高，細(xì)胞凋亡率減少，細(xì)胞形態(tài)得到改善，表明阿折地平對西羅莫司誘導(dǎo)的HUVEC損傷具有保護(hù)作用。我們同時(shí)發(fā)現(xiàn)，實(shí)驗(yàn)組細(xì)胞NO水平升高，提示阿折地平不僅能增加內(nèi)皮細(xì)胞存活率，還可改善內(nèi)皮細(xì)胞功能。另外，實(shí)驗(yàn)組細(xì)胞內(nèi)Ca2+濃度降低，線粒體膜電位升高，提示胞外Ca2+內(nèi)流參與了西羅莫司誘導(dǎo)的內(nèi)皮細(xì)胞損傷，而阿折地平可能通過減少細(xì)胞外Ca2+內(nèi)流、減輕鈣超載、抑制線粒體膜電位降低、減少細(xì)胞凋亡等途徑發(fā)揮保護(hù)內(nèi)皮細(xì)胞的作用，這與相關(guān)研究[11，12]結(jié)果一致。

綜上所述，SES能引起血管內(nèi)皮細(xì)胞損傷，而阿折地平對其誘導(dǎo)的細(xì)胞損傷有一定抑制作用，可提高HUVEC存活率，維持正常細(xì)胞形態(tài)，增加細(xì)胞中NO水平，改善細(xì)胞功能。

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山東醫(yī)藥第55卷第35期刊發(fā)的“血清NGAL、尿KIM-1對擬行CRRT的急性腎損傷患者腎功能轉(zhuǎn)歸及預(yù)后的預(yù)測作用”一文，基金項(xiàng)目為“十二五”國家科技支撐計(jì)劃(2011BAI10B08)、“十二五”全軍重大項(xiàng)目(AWS11J013)。