法舒地爾聯(lián)合羅格列酮對(duì)脂多糖誘導(dǎo)人冠狀動(dòng)脈內(nèi)皮細(xì)胞MCP-1和VCAM-1表達(dá)的影響

李世云，巫相宏，黃文，王淳，馬國(guó)添，閉奇

(廣西醫(yī)科大學(xué)附屬第一醫(yī)院，南寧 530021)

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法舒地爾聯(lián)合羅格列酮對(duì)脂多糖誘導(dǎo)人冠狀動(dòng)脈內(nèi)皮細(xì)胞MCP-1和VCAM-1表達(dá)的影響

李世云，巫相宏，黃文，王淳，馬國(guó)添，閉奇

(廣西醫(yī)科大學(xué)附屬第一醫(yī)院，南寧 530021)

目的觀察法舒地爾聯(lián)合羅格列酮對(duì)脂多糖(LPS)誘導(dǎo)的人冠狀動(dòng)脈(冠脈)內(nèi)皮細(xì)胞(HCAEC)血管細(xì)胞黏附因子1(VCAM-1)、單核細(xì)胞趨化因子1(MCP-1)表達(dá)的影響。方法選用體外培養(yǎng)的第3～5代HECAC進(jìn)行實(shí)驗(yàn)，將細(xì)胞分為空白對(duì)照組、LPS組(100 ng/mL LPS作用細(xì)胞)、法舒地爾組(10 nmol/L法舒地爾作用細(xì)胞)、羅格列酮組(10 μg/mL羅格列酮作用細(xì)胞)、LPS+法舒地爾組(先予10 nmol/L的法舒地爾干預(yù)細(xì)胞2 h后再給予100 ng/mL LPS共同干預(yù)22 h)、LPS+法舒地爾+羅格列酮組(先予10 nmol/L的法舒地爾和10 μg/mL羅格列酮干預(yù)細(xì)胞2 h后再給予濃度為100 ng/mL LPS共同干預(yù)22 h)，實(shí)時(shí)熒光定量PCR檢測(cè)各組細(xì)胞VCAM-1、MCP-1 mRNA，ELISA法檢測(cè)細(xì)胞上清液中可溶性VCAM-1、MCP-1蛋白。結(jié)果與空白對(duì)照組相比，LPS組細(xì)胞VCAM-1、MCP-1 mRNA及上清液VCAM-1、MCP-1蛋白表達(dá)高(P均<0.05)。與LPS組相比，LPS+法舒地爾組細(xì)胞VCAM-1、MCP-1 mRNA及上清液VCAM-1、MCP-1蛋白表達(dá)低(P均<0.05)。與LPS+法舒地爾組相比，LPS+法舒地爾+羅格列酮組細(xì)胞VCAM-1、MCP-1 mRNA及上清液VCAM-1、MCP-1蛋白表達(dá)低(P均<0.05)。結(jié)論法舒地爾聯(lián)合羅格列酮可協(xié)同抑制LPS誘導(dǎo)的HCAEC中VCAM-1、MCP-1的表達(dá)，可能與兩者共同抑制p38MAPK、NF-κB信號(hào)通路有關(guān)。

動(dòng)脈粥樣硬化；人冠狀動(dòng)脈內(nèi)皮細(xì)胞;血管細(xì)胞黏附因子1;單核細(xì)胞趨化因子1

動(dòng)脈內(nèi)皮細(xì)胞炎癥反應(yīng)及可能存在的信號(hào)通路、炎癥因子為目前動(dòng)脈粥樣硬化發(fā)病機(jī)制的研究熱點(diǎn)。羅格列酮可通過(guò)p38MAPK信號(hào)通路抑制炎癥反應(yīng)，法舒地爾可通過(guò)下調(diào)p38MAPK和NF-κB的活性從而下調(diào)MCP-1的表達(dá)[1]。本研究通過(guò)觀察法舒地爾和羅格羅酮對(duì)脂多糖(LPS)誘導(dǎo)的人冠狀動(dòng)脈(冠脈)內(nèi)皮細(xì)胞(HCAEC)血管細(xì)胞黏附因子1(VCAM-1)、單核細(xì)胞趨化因子1(MCP-1)表達(dá)的影響，旨在觀察法舒地爾與羅格列酮是否有協(xié)同抑制血管炎癥反應(yīng)的作用。

1　材料與方法

1.1材料HECAC、ECM1001培養(yǎng)基、青霉素/鏈霉素(P/S)、胎牛血清(FBS)、內(nèi)皮細(xì)胞因子(ECGs)均購(gòu)自美國(guó)sciencecell公司，0.25%胰酶購(gòu)自Gibco公司，LPS購(gòu)自Sigma公司，法舒地爾購(gòu)自天津紅日藥業(yè)股份有限公司，羅格列酮購(gòu)自Alexis biochemicals公司，ELISA試劑盒購(gòu)自eBioscience公司。

1.2法舒地爾、羅格列酮、LPS處理細(xì)胞方法將HCAEC從液氮罐中取出后置于37 ℃水浴箱中，溶化后立即轉(zhuǎn)移到含4 mL完全培養(yǎng)基(完全培養(yǎng)基的配制是500 mL ECM+25 mL FBS+5 mL ECGs+5 mL P/S)的培養(yǎng)瓶中，置于37 ℃、5%CO2培養(yǎng)箱中培養(yǎng)，第2天換液，之后每隔3 d換1次液，換液時(shí)倒凈培養(yǎng)瓶中液體，PBS沖洗2遍，加入新完全培養(yǎng)液4 mL，待細(xì)胞長(zhǎng)滿80%左右時(shí)傳代。實(shí)驗(yàn)時(shí)取第3～5代細(xì)胞，用完全培養(yǎng)基稀釋成2×105/孔種植于6孔板中，當(dāng)細(xì)胞長(zhǎng)到約80%時(shí)，將細(xì)胞分成6組：空白對(duì)照組(ECM培養(yǎng)基)、LPS組(培養(yǎng)基中加入終濃度為100 ng/mL LPS[2])、法舒地爾組(培養(yǎng)基中加入終濃度為10 nmol/L法舒地爾[3])、羅格列酮組(培養(yǎng)基中加入終濃度為10 μg/mL羅格列酮[4])、LPS+法舒地爾組(先予終濃度為10 nmol/L的法舒地爾干預(yù)2 h后再給予終濃度為100 ng/mL LPS共同干預(yù)22 h)、LPS+法舒地爾+羅格列酮組(先予終濃度為10 nmol/L的法舒地爾和濃度為10 μg/mL羅格列酮干預(yù)2 h后再給予終濃度為100 ng/mL LPS共同干預(yù)22 h)。上述細(xì)胞均于培養(yǎng)箱中培養(yǎng)24 h，所有細(xì)胞的RNA、上清液均被收集保存于-80 ℃冰箱中備用。

1.3細(xì)胞VCAM-1、MCP-1 mRNA檢測(cè)方法RNA提取、逆轉(zhuǎn)錄分別使用TaKaRa公司的TRIzol試劑盒和逆轉(zhuǎn)錄試劑盒，按照說(shuō)明書(shū)進(jìn)行操作，提取的總RNA用BECKMAN COULTER核酸蛋白分析儀測(cè)其在260 nm和280 nm的吸光度(A)值，所有樣品的A260與A280的比值為1.8～2.1。并根據(jù)A260值對(duì)樣品的總RNA進(jìn)行初步定量，逆轉(zhuǎn)錄成cDNA后保存于-80 ℃冰箱備用。實(shí)時(shí)熒光定量PCR測(cè)定在ABI StePone Plus上完成，mRNA表達(dá)水平用SYBR Premix EX Taq(Perfect Real time染料法實(shí)時(shí)熒光定量試劑盒)檢測(cè)。所有樣品95 ℃熱變性60 s，40個(gè)循環(huán)，每個(gè)循環(huán)包括95 ℃ 5 s、60 ℃ 30 s。引物序列：VCAM-1上游為5′-CAGCCTCTTTCTGAGAATGCAAC-3′，下游為5′-TGACAGTGTCTCCTTCTTTGA-CACT-3′；MCP-1上游為5′-CAGCCTCTTTCTGAGAA-TGCAAC-3′，下游為5′-TGACAGTGTCTCCTTCTTTGACACT-3′；GAPDH上游為5′-ACGGATTTGGTCGTATTGGG-3′，下游為5′-TGATTTTGGAGGGATCTCGC-3′。HECAC中VCAM-1、MCP-1 mRNA表達(dá)水平以GAPDH為內(nèi)參測(cè)算，VCAM-1的cDNA需稀息10倍再行PCR反應(yīng)。以2-ΔΔCt計(jì)算分析mRNA的表達(dá)量，將空白對(duì)照組標(biāo)準(zhǔn)化為1。

1.4細(xì)胞上清液中可溶性VCAM-1、MCP-1蛋白檢測(cè)實(shí)驗(yàn)前，將上清液予分析緩沖液(ASsay buffer)分別稀釋成1倍、5倍、10倍、20倍、100倍，按照說(shuō)明書(shū)步驟進(jìn)行操作以確定最佳的上清液作用濃度，MCP-1的上清液稀釋100倍后進(jìn)行蛋白檢測(cè)，VCAM-1的上清液無(wú)需稀釋即可進(jìn)行蛋白檢測(cè)。使用試劑盒提供的MCP-1、VCAM-1標(biāo)準(zhǔn)品按操作說(shuō)明建立標(biāo)準(zhǔn)曲線，標(biāo)準(zhǔn)曲線方程式分別為Y=0.001 5X+0.056 9(R2=0.998 5)，Y=0.018 9X+0.092 6(R2=0.999 3)，根據(jù)方程式求出各組細(xì)胞上清液中MCP-1、VCAM-1蛋白的表達(dá)量。

2　結(jié)果

各組細(xì)胞VCAM-1、MCP-1 mRNA比較見(jiàn)表1。各組細(xì)胞上清液VCAM-1、MCP-1蛋白比較見(jiàn)表2。

3　討論

VCAM-1、MCP-1在動(dòng)脈粥樣硬化早期病變中扮演著非常重要的角色[5,6]，抑制血管內(nèi)皮細(xì)胞VCAM-1、MCP-1的表達(dá)對(duì)動(dòng)脈硬化的發(fā)展能起到抑制的作用。LPS在革蘭陰性細(xì)菌感染及疾病演化中有重要作用，被認(rèn)為是造成全身性炎癥反應(yīng)綜合征的主要原因，與細(xì)胞膜上相應(yīng)受體作用后，啟動(dòng)胞內(nèi)信號(hào)傳遞鏈，引起NF-κB等活化，啟動(dòng)基因轉(zhuǎn)錄，表達(dá)和釋放多種細(xì)胞因子，發(fā)揮其毒性作用。本研究以LPS作為促進(jìn)冠脈內(nèi)皮細(xì)胞炎癥反應(yīng)的誘導(dǎo)劑，結(jié)果顯示，與空白對(duì)照組相比，LPS可明顯增加冠脈內(nèi)皮細(xì)胞VCAM-1、MCP-1的表達(dá)。

表1　各組細(xì)胞VCAM-1、MCP-1 mRNA比較

注：與空白對(duì)照組相比，*P<0.05；與LPS組相比，△P<0.05；與LPS+法舒地爾組相比，#P<0.05。

表2　　　　各組細(xì)胞上清液VCAM-1、MCP-1蛋白比較

注：與空白對(duì)照組相比，*P<0.05；與LPS組相比，△P<0.05；與LPS+法舒地爾組相比，#P<0.05。

Rho/ROCK信號(hào)通路在動(dòng)脈粥樣硬化的發(fā)生發(fā)展中發(fā)揮了重要的作用，其通過(guò)不同途徑活化后促進(jìn)動(dòng)脈粥樣硬化的形成。Rho激酶的活化可能通過(guò)調(diào)節(jié)NF-κB的活性和T淋巴細(xì)胞的增殖，參與早期動(dòng)脈粥樣硬化的形成。Rho激酶及其下游p38MAPK/NF-κB信號(hào)通路的激活可誘導(dǎo)血管內(nèi)皮細(xì)胞MCP-1的表達(dá)。法舒地爾是Rho激酶抑制劑，其可通過(guò)下調(diào)p38MAPK和NF-κB的活性從而下調(diào)MCP-1、VCAM-1的表達(dá)[1,7]，本研究也證明了法舒地爾可以下調(diào)LPS誘導(dǎo)的HCAEC中VCAM-1、MCP-1的表達(dá)。

過(guò)氧化物酶增殖物激活受體-γ激動(dòng)劑吡格列酮可通過(guò)抑制p38MAPK蛋白活性從而抑制炎癥反應(yīng)過(guò)程，羅格列酮可以通過(guò)抑制NF-κB蛋白活性下調(diào)高糖誘導(dǎo)的VCAM-1的表達(dá)。本研究結(jié)果表明，羅格列酮和法舒地爾聯(lián)用可協(xié)同抑制LPS誘導(dǎo)的HCAEC中VCAM-1、MCP-1的表達(dá)，其作用機(jī)理可能與法舒地爾、羅格列酮抑制某些共同的信號(hào)通路有關(guān)。MAPK及NF-κB是動(dòng)脈內(nèi)皮細(xì)胞內(nèi)兩條非常重要信號(hào)通路，在炎癥信號(hào)轉(zhuǎn)導(dǎo)調(diào)控中具有重要意義。p38MAPK是MAPK家族中的重要成員，是介導(dǎo)細(xì)胞反應(yīng)的重要信號(hào)系統(tǒng)，與血管內(nèi)皮細(xì)胞損傷有關(guān)[8]。NF-κB是調(diào)控轉(zhuǎn)錄多種炎癥因子的中心環(huán)節(jié)和共同通路，是血管內(nèi)皮細(xì)胞損傷進(jìn)展過(guò)程中炎癥反應(yīng)的關(guān)鍵環(huán)節(jié)。研究[9]發(fā)現(xiàn)，p38MAPK被激活后可通過(guò)磷酸化或促炎細(xì)胞因子(如TNF-α、ICAM-1)而活化NF-κB。NF-κB被激活后，也可通過(guò)其促炎細(xì)胞因子產(chǎn)物反過(guò)來(lái)激活p38MAPK。p38MAPK與NF-κB形成的相互激活網(wǎng)絡(luò)可調(diào)控多種炎癥介質(zhì)的基因表達(dá)，促進(jìn)動(dòng)脈粥樣硬化的發(fā)展。

綜上可見(jiàn)，法舒地爾聯(lián)合羅格列酮可協(xié)同抑制LPS誘導(dǎo)的HCAEC中VCAM-1、MCP-1的表達(dá)，可能與兩者共同抑制p38MAPK、NF-κB信號(hào)通路有關(guān)。

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Effects of fasudil combined with rosiglitazone on expression of MCP-1 and VCAM-1 in lipopolysaccharide-induced human coronary arterial endothelial cells

LIShiyun,WUXianghong,HUANGWen,WANGChun,MAGuotian,BIQi

(TheFirstAffiliatedHospitalofGuangxiMedicalUniversity,Nanning530021,China)

Objective To observe the effects of fasudil combined with rosiglitazone on the expression of vascular cell adhesion molecule-1 (VCAM-1)and monocyte chemotactic protein 1 (MCP-1) in lipopolysaccharide (LPS)-induced human coronary arterial endothelial cells (HCAECs). MethodsWe conducted the experiment using the 3rd to the 5th generation of HCAECs in vitro. Then, the cells were divided into the control group (non-intervention), LPS group (HCAECs were intervened with 100 ng/mL LPS), fasudil group (HCAECs were intervened with 10 nmol/L fasudil), rosiglitazone group (HCAECs were intervened with 10 μg/mL rosiglitazone), LPS + fasudil group (HCAECs were intervened with 10 nmol/L fasudil for 2 h followed by 100 ng/mL LPS for 22 h), LPS + fasudil + rosiglitazone group (HCAECs were intervened with 10 nmol /L fasudil and 10 μg/mL rosiglitazone for 2 h followed with 100 ng/mL LPS together for 22 h). The protein expression of VCAM-1 and MCP-1 in supernatant was detected by ELISA and the mRNA was detected by real-time fluorescent quantification PCR. ResultsCompared with the control group, the mRNA expression of VCAM-1 and MCP-1, and the protein expression of VCAM-1 and MCP-1 was increased in the LPS group (allP<0.05). Compared with the LPS group, the mRNA expression of VCAM-1 and MCP-1, and the protein expression of VCAM-1 and MCP-1 was decreased in the LPS + fasudil group (allP<0.05). Compared with LPS+fasudil group, the mRNA expression of VCAM-1 and MCP-1, and the protein expression of VCAM-1 and MCP-1 was decreased in the LPS+fasudil+ rosiglitazone group (allP<0.05).Conclusion Fasudil combined with rosiglitazone collaboratively inhibits the expression of VCAM-1 and MCP-1 in HCAECs induced by LPS, which may be related to their co-suppression of p38MAPK and NF-κB signaling pathway.

atherosclerosis; human coronary artery endothelial cells; vascular cell adhesion molecule 1; monocyte chemotactic protein 1

國(guó)家自然科學(xué)基金資助項(xiàng)目(81360057)。

李世云(1979-)，男，碩士研究生，主要從事冠心病的發(fā)病機(jī)制及介入治療。E-mail: 411955433@qq.com

簡(jiǎn)介：巫相宏(1967-)，男，教授，碩士生導(dǎo)師，主要從事冠心病防治的基礎(chǔ)和臨床研究、冠心病的介入性診斷治療。E-mail: whw780@126.com

10.3969/j.issn.1002-266X.2016.23.004

R543

A

1002-266X(2016)23-0013-03

2016-01-11)