瑞巴派特對(duì)氯吡格雷所致人胃黏膜上皮細(xì)胞損傷的保護(hù)作用及其機(jī)制研究

瑞巴派特對(duì)氯吡格雷所致人胃黏膜上皮細(xì)胞損傷的保護(hù)作用及其機(jī)制研究

陸宏梅段兆濤張振玉*

南京醫(yī)科大學(xué)附屬南京醫(yī)院(南京市第一醫(yī)院)消化科(210006)

背景：長(zhǎng)期服用氯吡格雷可引起胃腸道損傷。瑞巴派特是一種新型胃黏膜保護(hù)劑，其用于防治氯吡格雷相關(guān)胃黏膜損傷的療效尚不明確。目的：探討瑞巴派特對(duì)氯吡格雷所致人胃黏膜上皮細(xì)胞損傷的保護(hù)作用及其可能機(jī)制。方法：以氯吡格雷處理的人胃黏膜上皮細(xì)胞株GES-1作為氯吡格雷組，以氯吡格雷聯(lián)合不同濃度(0.2、0.5、1.0 mmol/L)瑞巴派特預(yù)處理的GES-1細(xì)胞作為瑞巴派特組，同時(shí)設(shè)立陰性對(duì)照組。采用MTT法檢測(cè)細(xì)胞增殖抑制情況；采用倒置相差顯微鏡觀察細(xì)胞形態(tài)學(xué)變化；采用蛋白質(zhì)印跡法檢測(cè)三葉因子1(TFF1)、磷酸化細(xì)胞外信號(hào)調(diào)節(jié)激酶1/2(p-ERK1/2)表達(dá)。結(jié)果：MTT法檢測(cè)結(jié)果顯示，瑞巴派特0.2、0.5、1.0 mmol/L組GES-1細(xì)胞增殖抑制率均顯著低于氯吡格雷組(P<0.05)。倒置相差顯微鏡觀察發(fā)現(xiàn)，氯吡格雷可誘導(dǎo)GES-1細(xì)胞損傷，而瑞巴派特可減輕氯吡格雷所致的細(xì)胞損傷。蛋白質(zhì)印跡法檢測(cè)結(jié)果顯示，氯吡格雷組GES-1細(xì)胞TFF1蛋白表達(dá)較陰性對(duì)照組顯著升高(P<0.05)；瑞巴派特0.2、0.5、1.0 mmol/L組TFF1蛋白表達(dá)均較氯吡格雷組進(jìn)一步升高，且作用呈濃度依賴性(P<0.05)。氯吡格雷組GES-1細(xì)胞p-ERK1/2蛋白表達(dá)較陰性對(duì)照組顯著升高(P<0.05)；瑞巴派特0.2、0.5、1.0 mmol/L 組p-ERK1/2蛋白表達(dá)均較氯吡格雷組顯著降低，作用亦呈濃度依賴性(P<0.05)。結(jié)論：瑞巴派特可能通過(guò)抑制ERK信號(hào)通路上調(diào)TFF1蛋白表達(dá)，從而參與調(diào)控氯吡格雷所致GES-1細(xì)胞損傷的修復(fù)，此過(guò)程是瑞巴派特防治氯吡格雷相關(guān)胃黏膜損傷的可能機(jī)制之一。

關(guān)鍵詞瑞巴派特；胃黏膜；上皮細(xì)胞；氯吡格雷；三葉因子1；細(xì)胞外信號(hào)調(diào)節(jié)激酶1/2

Extracellular Signal Regulated Kinase 1/2

氯吡格雷作為噻吩吡啶類抗血小板藥物已廣泛應(yīng)用于心腦血管疾病的防治，但長(zhǎng)期服用引起胃腸道損傷的報(bào)道亦隨之增加[1-2]。近年來(lái)多采用質(zhì)子泵抑制劑(PPIs)預(yù)防氯吡格雷相關(guān)胃腸道損傷，但有研究[3-4]表明PPIs預(yù)防氯吡格雷相關(guān)胃腸道損傷會(huì)導(dǎo)致心血管事件發(fā)生率和死亡率升高。因此，尋找其他能減輕氯吡格雷相關(guān)胃腸道損傷的胃黏膜保護(hù)劑顯得尤為重要。瑞巴派特是一種新型胃黏膜保護(hù)劑，兼有保護(hù)胃黏膜和抑制炎癥的雙重作用，對(duì)胃炎、胃潰瘍具有良好療效[5-6]，但其用于防治氯吡格雷相關(guān)胃黏膜損傷的療效尚不明確。本研究旨在觀察瑞巴派特對(duì)氯吡格雷所致人胃黏膜上皮細(xì)胞損傷的保護(hù)作用，并對(duì)其可能作用機(jī)制進(jìn)行探討。

材料與方法

一、細(xì)胞株和主要試劑

人胃黏膜上皮細(xì)胞株GES-1由江蘇省人民醫(yī)院消化科饋贈(zèng)。硫酸氯吡格雷購(gòu)自北京諾德恒信化工技術(shù)有限公司；瑞巴派特購(gòu)自湖北康寶泰精細(xì)化工有限公司；MTT購(gòu)自美國(guó)Sigma公司；兔抗人磷酸化細(xì)胞外信號(hào)調(diào)節(jié)激酶1/2(p-ERK1/2)單克隆抗體購(gòu)自南京凱基生物科技發(fā)展有限公司；兔抗人三葉因子1(TFF1)單克隆抗體購(gòu)自美國(guó)Cell Signaling Technology公司；β-actin抗體購(gòu)自美國(guó)Santa Cruz公司。

二、實(shí)驗(yàn)方法

1. 細(xì)胞培養(yǎng)：將GES-1細(xì)胞置于DMEM培養(yǎng)基(含15%胎牛血清，pH 7.4)中，于37 ℃、5% CO2溫箱內(nèi)培養(yǎng)，隔天更換培養(yǎng)液，每3 d按1∶3比例傳代。

2. 實(shí)驗(yàn)分組：取對(duì)數(shù)生長(zhǎng)期GES-1細(xì)胞，以每孔1×105個(gè)加入96孔培養(yǎng)板，每孔體積200 μL，培養(yǎng)24 h，吸出培養(yǎng)液，氯吡格雷組加入含50%抑制濃度(IC50)氯吡格雷(0.36 mmol/L)[7]的培養(yǎng)液200 μL，瑞巴派特組分別以不同濃度(0.2、0.5、1.0 mmol/L)瑞巴派特預(yù)處理2 h，再加入含IC50氯吡格雷(0.36 mmol/L)的培養(yǎng)液200 μL。每組設(shè)4個(gè)復(fù)孔，同時(shí)設(shè)不予藥物干預(yù)的陰性對(duì)照組。

3. MTT法檢測(cè)細(xì)胞增殖抑制情況：取陰性對(duì)照組、氯吡格雷組以及0.2、0.5、1.0 mmol/L瑞巴派特組細(xì)胞，培養(yǎng)24 h，每孔加入MTT 50 μL，繼續(xù)培養(yǎng)4 h，棄上清液，每孔加入DMSO 150 μL，室溫避光振蕩10 min，以空白孔調(diào)零，于酶標(biāo)儀490 nm波長(zhǎng)處讀取吸光度(A)值。細(xì)胞增殖抑制率(%)=(1-實(shí)驗(yàn)組A值/陰性對(duì)照組A值)×100%。

4. 倒置相差顯微鏡觀察細(xì)胞形態(tài)：取陰性對(duì)照組、氯吡格雷組以及0.2、0.5、1.0 mmol/L瑞巴派特組細(xì)胞，于倒置相差顯微鏡下觀察細(xì)胞形態(tài)學(xué)變化。

5. 蛋白質(zhì)印跡法檢測(cè)TFF1、p-ERK1/2蛋白表達(dá)：取陰性對(duì)照組、氯吡格雷組以及0.2、0.5、1.0 mmol/L 瑞巴派特組細(xì)胞，加入蛋白裂解液，4 ℃裂解，14 000 r/min離心15 min，取上清液(總蛋白提取物)，以BCA法測(cè)定蛋白濃度。取蛋白樣品上樣，行SDS-PAGE電泳，轉(zhuǎn)膜，封閉后加入TFF1抗體(1∶1 000)、p-ERK1/2抗體(1∶1 000)，4 ℃孵育過(guò)夜，加入HRP標(biāo)記的二抗，孵育后ECL法顯色，以Gel-Pro Analyzer軟件分析電泳條帶灰度值，以目的蛋白條帶灰度值與內(nèi)參照β-actin條帶灰度值的比值作為目的蛋白相對(duì)表達(dá)量。

三、統(tǒng)計(jì)學(xué)分析

結(jié)果

一、細(xì)胞增殖抑制情況

MTT法檢測(cè)結(jié)果顯示，陰性對(duì)照組、氯吡格雷組、瑞巴派特0.2、0.5、1.0 mmol/L組GES-1細(xì)胞增殖抑制率分別為(3.56±0.50)%、(49.15±2.97)%、(38.52±2.84)%、(29.88±3.29)%和(14.20±3.42)%。氯吡格雷組細(xì)胞增殖抑制率顯著高于陰性對(duì)照組(P<0.05)，不同濃度瑞巴派特組細(xì)胞增殖抑制率均顯著低于氯吡格雷組(P<0.05)。

二、細(xì)胞形態(tài)學(xué)變化

倒置相差顯微鏡觀察顯示，陰性對(duì)照組GES-1細(xì)胞呈梭形，均一貼壁生長(zhǎng)；氯吡格雷組貼壁細(xì)胞數(shù)量較陰性對(duì)照組減少，部分細(xì)胞變圓，懸浮，細(xì)胞核濃縮；不同濃度瑞巴派特組貼壁細(xì)胞數(shù)量均較陰性對(duì)照組減少，有不同程度的細(xì)胞損傷，但損傷細(xì)胞 數(shù)量與氯吡格雷組相比有所減少，瑞巴派特1.0 mmol/L 組細(xì)胞損傷最輕(圖1)。

陰性對(duì)照組GES-1細(xì)胞有一定程度的ERK1/2磷酸化，氯吡格雷組p-ERK1/2蛋白相對(duì)表達(dá)量較陰性對(duì)照組顯著升高(P<0.05)，瑞巴派特0.2、0.5、1.0 mmol/L組p-ERK1/2蛋白相對(duì)表達(dá)量均較氯吡格雷組顯著降低(P<0.05)，作用亦呈濃度依賴性(P<0.05)(圖3)。

A：陰性對(duì)照組；B：氯吡格雷組；C：瑞巴派特0.2 mmol/L組；D：瑞巴派特0.5 mmol/L組；E：瑞巴派特1.0 mmol/L組

圖1倒置相差顯微鏡下各組GES-1細(xì)胞形態(tài)學(xué)表現(xiàn)(×200)

三、TFF1和p-ERK1/2蛋白表達(dá)

蛋白質(zhì)印跡法檢測(cè)結(jié)果顯示，氯吡格雷組GES-1細(xì)胞TFF1蛋白相對(duì)表達(dá)量較陰性對(duì)照組顯著升高(P<0.05)；瑞巴派特0.2、0.5、1.0 mmol/L組TFF1蛋白相對(duì)表達(dá)量均較氯吡格雷組進(jìn)一步升高(P<0.05)，且作用呈濃度依賴性(P<0.05)(圖2)。

1 Da=0.992 1 u

A：陰性對(duì)照組；B：氯吡格雷組；C：瑞巴派特0.2 mmol/L組；D：瑞巴派特0.5 mmol/L組；E：瑞巴派特1.0 mmol/L組

圖2各組GES-1細(xì)胞TFF1蛋白相對(duì)表達(dá)量(蛋白質(zhì)印跡法)

A：陰性對(duì)照組；B：氯吡格雷組；C：瑞巴派特0.2 mmol/L組；D：瑞巴派特0.5 mmol/L組；E：瑞巴派特1.0 mmol/L組

圖3各組GES-1細(xì)胞p-ERK1/2蛋白相對(duì)表達(dá)量(蛋白質(zhì)印跡法)

討論

瑞巴派特具有促進(jìn)動(dòng)物和人胃黏膜黏液分泌，提高胃黏膜表層黏液總量和可溶性黏液量，增加黏膜內(nèi)前列腺素E2(PGE2)含量和胃黏膜血流量，促進(jìn)胃黏膜上皮細(xì)胞增生和成熟，從而增強(qiáng)胃黏膜防御功能的作用[8-9]。研究表明，瑞巴派特對(duì)乙醇[10]、非甾體消炎藥(NSAIDs)[11]、幽門螺桿菌(Hp)感染[12]等損傷因子引起的胃腸道損傷均具有一定保護(hù)作用，可促進(jìn)胃黏膜修復(fù)再生，提高胃黏膜屏障功能。然而，目前關(guān)于瑞巴派特防治氯吡格雷相關(guān)胃腸道損傷的研究報(bào)道尚少。本研究采用MTT法結(jié)合倒置相差顯微鏡觀察分析氯吡格雷和瑞巴派特對(duì)人胃黏膜上皮細(xì)胞株GES-1增殖的影響，結(jié)果顯示氯吡格雷可抑制GES-1細(xì)胞增殖，導(dǎo)致細(xì)胞損傷，而給予瑞巴派特預(yù)處理能顯著減輕氯吡格雷引起的GES-1細(xì)胞增殖抑制和損傷。

TFF1是三葉肽家族成員之一，又名乳癌相關(guān)肽(pS2)，是一類小分子蛋白肽，于1982年由Masia-kowski等[13]在雌激素誘導(dǎo)的人乳腺癌細(xì)胞株MCF-7中獲得，主要表達(dá)于胃黏膜小凹和上皮。研究表明TFF1是一種保護(hù)因子，在胃黏膜保護(hù)中發(fā)揮重要作用，參與黏液屏障組成，促進(jìn)細(xì)胞遷移和損傷修復(fù)[14-15]。其機(jī)制可能為：與黏液中的糖蛋白結(jié)合形成穩(wěn)定的凝膠復(fù)合物，加強(qiáng)黏液凝膠層，從而加固碳酸氫鹽屏障，減少胃腔表面有害物質(zhì)以及機(jī)械應(yīng)力等因素對(duì)黏膜的損傷[16-17]。研究[18-19]證實(shí)，發(fā)生急性胃黏膜損傷時(shí)，TFF1表達(dá)增強(qiáng)以促進(jìn)損傷修復(fù)。提前或同時(shí)口服TFF1能防治阿司匹林引起的大鼠胃潰瘍，使胃潰瘍指數(shù)不同程度地下降[20]。莫喜晶等[21]在研究七方胃痛顆粒對(duì)Hp感染人胃腺癌細(xì)胞中TFF1表達(dá)的影響時(shí)發(fā)現(xiàn)，七方胃痛顆粒濃度越高，保護(hù)作用越強(qiáng)，TFF1蛋白表達(dá)量越高。上述研究表明，急性胃黏膜損傷時(shí)，機(jī)體可通過(guò)上調(diào)TFF1蛋白表達(dá)修復(fù)胃黏膜，而給予胃黏膜保護(hù)劑后，TFF1蛋白表達(dá)可進(jìn)一步上調(diào)，對(duì)胃黏膜損傷的修復(fù)作用亦顯著增強(qiáng)。

本研究同時(shí)觀察了瑞巴派特預(yù)處理對(duì)氯吡格雷作用下GES-1細(xì)胞TFF1表達(dá)的影響。研究采用蛋白質(zhì)印跡法檢測(cè)GES-1細(xì)胞TFF1蛋白表達(dá)的變化，結(jié)果顯示與陰性對(duì)照組相比，氯吡格雷組TFF1蛋白表達(dá)顯著上調(diào)，而給予瑞巴派特預(yù)處理的GES-1細(xì)胞隨著瑞巴派特濃度的增加，TFF1蛋白表達(dá)較氯吡格雷組進(jìn)一步升高且作用呈濃度依賴性，推測(cè)氯吡格雷誘導(dǎo)GES-1細(xì)胞損傷，使TFF1表達(dá)代償性升高，而瑞巴派特在減輕氯吡格雷引起的細(xì)胞損傷的同時(shí)，使TFF1表達(dá)進(jìn)一步升高，以促進(jìn)損傷黏膜修復(fù)，這可能是瑞巴派特防治氯吡格雷相關(guān)胃黏膜損傷的機(jī)制之一。目前瑞巴派特上調(diào)TFF1表達(dá)的具體機(jī)制尚不明確，有學(xué)者[22]指出TFF1的激活依賴于RAS和ERK1/2的激活，由此推測(cè)TFF1表達(dá)上調(diào)可能與ERK信號(hào)通路有關(guān)，本研究對(duì)此推測(cè)進(jìn)行了驗(yàn)證。

ERK是MAPK家族成員之一，主要分為ERK1和ERK2，統(tǒng)稱為ERK1/2，相對(duì)分子質(zhì)量分別為44 kDa 和42 kDa[23]。多種細(xì)胞外信號(hào)均可促使ERK信號(hào)通路激活，活化的ERK進(jìn)一步激活下游核轉(zhuǎn)錄因子，引起特定蛋白表達(dá)和活性改變[24]。本研究結(jié)果顯示，氯吡格雷組GES-1細(xì)胞TFF1和p-ERK1/2蛋白表達(dá)均較陰對(duì)照組顯著升高，如給予瑞巴派特預(yù)處理，TFF1和p-ERK1/2蛋白表達(dá)分別以濃度依賴性的方式上調(diào)和下調(diào)，提示瑞巴派特可能通過(guò)抑制ERK信號(hào)通路上調(diào)TFF1蛋白表達(dá)，從而有效減輕氯吡格雷引起的GES-1細(xì)胞損傷。

綜上所述，瑞巴派特可能通過(guò)抑制ERK信號(hào)通路上調(diào)TFF1蛋白表達(dá)，從而參與調(diào)控氯吡格雷所致GES-1細(xì)胞損傷的修復(fù)，此過(guò)程是瑞巴派特防治氯吡格雷相關(guān)胃黏膜損傷的可能機(jī)制之一。

參考文獻(xiàn)

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(2014-08-15收稿；2014-09-02修回)

·論著·

Protective Effect and Mechanism of Rebamipide on Human Gastric Mucosal Epithelial Cell Injury Induced by Clopidogrel

LUHongmei,DUANZhaotao,ZHANGZhenyu.DepartmentofGastroenterology,NanjingFirstHospitalAffiliatedtoNanjingMedicalUniversity,Nanjing(210006)

Correspondence to: ZHANG Zhenyu, Email: zzy6565@sina.com

Background: Long-term use of clopidogrel may cause gastrointestinal injury. Rebamipide is a novel mucoprotective drug, however, its protective effect on gastric mucosal injury induced by clopidogrel has not been clarified. Aims: To investigate the protective effect and potential mechanism of rebamipide on human gastric mucosal epithelial cell injury induced by clopidogrel. Methods: In clopidogrel group, human gastric mucosal epithelial cell line GES-1 was treated with clopidogrel; GES-1 cells in rebamipide group were pretreated with different concentrations of rebamipide (0.2, 0.5 and 1.0 mmol/L) before clopidogrel treatment, and a negative control group was established simultaneously. Inhibition of cell proliferation was measured by MTT assay. Morphological change of cell was observed by inverted phase contrast microscope. Expressions of trefoil factor family 1 (TFF1) and phosphorylated extracellular signal regulated kinase 1/2 (p-ERK1/2) were determined by Western blotting. Results: MTT assay showed that cell proliferation inhibition rates of GES-1 cells in rebamipide 0.2, 0.5 and 1.0 mmol/L groups were significantly lower than that in clopidogrel group (P<0.05). Injury of GES-1 cells was seen in clopidogrel group by inverted phase contrast microscope and rebamipide could reduce the cell injury. Expression of TFF1 in GES-1 cells in clopidogrel group was significantly higher than that in negative control group (P<0.05), while those in rebamipide 0.2, 0.5 and 1.0 mmol/L groups were further increased in a dose-dependent manner as compared with that in clopidogrel group (P<0.05). Expression of p-ERK1/2 in GES-1 cells in clopidogrel group was significantly higher than that in negative control group (P<0.05), while those in rebamipide 0.2, 0.5 and 1.0 mmol/L groups were significantly lower than that in clopidogrel group in a dose-dependent manner (P<0.05). Conclusions: Rebamipide has a protective effect on GES-1 cells injury induced by clopidogrel, probably through inhibiting ERK signaling pathway, up-regulating TFF1 expression and promoting mucosal healing.

Key wordsRebamipide;Gastric Mucosa;Epithelial Cells;Clopidogrel;Trefoil Factor Family 1;

通信作者*本文，Email: zzy6565@sina.com

DOI:10.3969/j.issn.1008-7125.2015.01.003