李慧婷 趙杰 李海泉 施萍 張衍民 杜永亮
[摘要] 目的 觀察在不同濃度的莫西沙星作用下,人氣道平滑肌細(xì)胞(HASMC)小凹蛋白-1(Caveolin-1,Cav-1)蛋白及mRNA表達(dá)的變化,探討莫西沙星對(duì)HASMC發(fā)揮抗炎調(diào)節(jié)的可能機(jī)制。 方法 購買HASMC,體外傳代培養(yǎng),按隨機(jī)數(shù)字表法分為空白對(duì)照組(M0)、莫西沙星5 mg/L(M5組)、10 mg/L(M10組)、20 mg/L(M20組),均孵育48 h后,分別應(yīng)用Western blot和qRT-PCR檢測(cè)Cav-1蛋白水平及mRNA表達(dá)。 結(jié)果Western blot檢測(cè)顯示,各莫西沙星濃度組(M5組、M10組、M20組)Cav-1蛋白表達(dá)分別為(3.02±0.43),(4.18±0.07),(4.44±0.14),均明顯高于空白對(duì)照組(M0組)(1.00±0.15)(P<0.05),同時(shí)也存在濃度依賴性(r=0.77)。qRT-PCR結(jié)果顯示各莫西沙星組(M5組、M10組、M20組)Cav-1 mRNA表達(dá)量(2.54±0.35),(3.26±0.22),(5.57±0.51)均較空白對(duì)照組(M0組)表達(dá)量(1.00±0.11)明顯增加(P<0.05),同時(shí)存在濃度依賴性(r=0.99)。 結(jié)論 莫西沙星可濃度依賴性增加HASMC Cav-1蛋白及mRNA表達(dá),這可能與莫西沙星對(duì)ASMC發(fā)揮抗炎作用相關(guān)。
[關(guān)鍵詞] 莫西沙星;肌細(xì)胞,平滑??;小凹蛋白-1
[中圖分類號(hào)] R562.2 [文獻(xiàn)標(biāo)識(shí)碼] A [文章編號(hào)] 1674-0742(2019)03(c)-0011-04
[Abstract] Objective To culture human airway smooth muscle cells (HASMCs) with moxifloxacin at various concentrations, then to observe the effects of moxifloxacin on the expression of the protein and mRNA of Cav-1, and to explore the possible mechanisms of anti-inflammatory effects of moxifloxacin on HASMCs. Methods HASMCs were bought and cultured in vitro. The cells were randomly divided into 4 groups including a control group (M0) and 3 groups to which moxifloxacin was added at different concentrations (5,10,20)mg/L, groups M5, M10 and M20 respectively). Then the cells of different groups were incubated for 48 h. Finally the protein of Cav-1 was measured by Western blot and the mRNA of Cav-1 was measured by qRT-PCT respectively. Results The results of Western blot indicated that the expression of Cav-1 increased with the concentrations of moxifloxacin increasing (3.02±0.43), (4.18±0.07),(4.44±0.14) respectively, and the expression of Cav-1 of these groups were all higher than that of the control group (1.00±0.15)(P<0.05), The expression of Cav-1 showed a positive correlation with the concentration of moxifloxacin (r=0.77). The results of qRT-PCR showed that the expression of mRNA of Cav-1 of different moxifloxacin groups M5,M10 and M20 (2.54±0.35),(3.26±0.22),(5.57±0.51) respectively were all higher than that of the control group (1.00±0.11)(P<0.05). There was also a positive correlation between the mRNA of Cav-1 and the concentration of moxifloxacin (r=0.99). Conclusion Moxifloxacin could increase the expression of Caveolin-1 of HASMCs in concentration-dependent manner, which may be the possible mechanism concerning the anti-inflammatory effects of moxifloxacin.
[Key words] Moxifloxacin; Myocytes, Smooth muscle; Caveolin-1
哮喘患者ASMC呈現(xiàn)病理狀態(tài),其可分泌大量炎癥因子(如IL-6、IL-8及Eotaxin)[1],這些炎癥介質(zhì)可導(dǎo)致氣道炎癥持續(xù)級(jí)聯(lián)放大,反過來影響ASMC,致其增殖增加及收縮增強(qiáng),參與哮喘發(fā)病。我們前期研究發(fā)現(xiàn)莫西沙星可抑制大鼠ASMCs炎癥因子IL-8及Eotaxin的分泌[2],但具體機(jī)制尚不清楚。近年來研究發(fā)現(xiàn)Cav-1在氣道炎癥中擔(dān)當(dāng)著重要角色,且哮喘患者Cav-1水平明顯下調(diào)[3],提示Cav-1對(duì)哮喘性氣道炎癥具有保護(hù)作用,但其具體作用機(jī)制尚不完全清楚。該研究旨在探討莫西沙星對(duì)HASMC的Cav-1蛋白及mRNA表達(dá)的影響,探索莫西沙星對(duì)ASMC抗炎作用發(fā)揮的可能機(jī)制。報(bào)道如下。
1 資料與方法
1.1 一般資料
莫西沙星(德國拜耳醫(yī)藥保健股份公司),Cav-1人抗山羊單克隆抗體(16447-1-AP,proteintech公司),二抗山羊抗兔IgG H&L (HRP) (ab6721,abcam公司),HSMC(3410-c,sciencell,上海武光生物科技有限公司),平滑肌細(xì)胞培養(yǎng)液(1101,sciencell,上海武光生物科技有限公司),熒光定量檢測(cè)試劑盒(RR420A,上海馳勇生物科技有限公司)。
1.2 方法
①HASMC的培養(yǎng)及傳代:購買得到HASMC原代細(xì)胞,貼壁法進(jìn)行細(xì)胞培養(yǎng),以胰酶消化法進(jìn)行HASMC傳代并自然純化細(xì)胞,實(shí)驗(yàn)取5-6代細(xì)胞。
②Western blot檢測(cè)Cav-1蛋白表達(dá):用不同濃度(5,10,20 mg/L)莫西沙星處理HASMC 48 h,空白對(duì)照組應(yīng)用生理鹽水處理48 h,提取細(xì)胞總蛋白,經(jīng)電泳、轉(zhuǎn)膜后雜交,一抗為Cav-1人抗山羊單克隆抗體,二抗為山羊抗兔IgG,以NBT/BCIP顯色。條帶灰度用Image J軟件分析。實(shí)驗(yàn)重復(fù)操作3次。
qRT-PCR 定量檢測(cè)Cav-1 mRNA表達(dá)。
①細(xì)胞RNA提取:用生理鹽水及不同濃度莫西沙星干預(yù)HASMC 48 h后,在細(xì)胞液中加入TRIzol試劑,提取細(xì)胞RNA。檢測(cè)RNA純度,結(jié)果顯示OD260/280比值在1.8~2.0之間,表明無RNA降解,無蛋白污染。
②cDNA的合成:取總RNA提取物加入隨機(jī)引物、緩沖液、逆轉(zhuǎn)錄酶等,混勻后在42℃溫浴2 h,94℃,5~10 min,待逆轉(zhuǎn)錄反應(yīng)完成之后,將cDNA保持在-20℃冰箱備用。
③qRT-PCR反應(yīng):引物序列如下:Caveolin-1:上游5-GCGACCCTAAACACCTCAAC-3,下游5-ATGCCG- TCAAAACTGTGTGTC-3;β-actin:上游5-TTGTTA- CAGGAAGTCCCTTGCC-3,下游5-ATGCTATCACCT- CCCCTGTGTG-3;Caveolin-1擴(kuò)增片段為138 bp,β-actin擴(kuò)增101bp。將PCR反應(yīng)體系置于離心機(jī)以4℃、5 500 rpm,離心5 min,上機(jī),循環(huán)程序:95℃,2 min;95℃, 15 s;60℃,50 s;40個(gè)循環(huán)。
④數(shù)據(jù)的分析:首先將實(shí)驗(yàn)所有的基因Ct值整理好,之后每一組樣本自身的目的基因Ct值減去自身內(nèi)參基因Ct值,得到的數(shù)就是△Ct。換成公式就是:△Ct=Ct(目的基因)-Ct(內(nèi)參基因)。用該次實(shí)驗(yàn)研究樣本的△Ct減去對(duì)照組樣本的△Ct并同時(shí)對(duì)結(jié)果取相反數(shù),結(jié)果就是-△△Ct。最后,對(duì)-△△Ct進(jìn)行2的冪運(yùn)算,即2-△△Ct就得出實(shí)驗(yàn)組和對(duì)照組靶基因的轉(zhuǎn)錄倍數(shù)。
1.3 統(tǒng)計(jì)方法
采用SPSS 20.0統(tǒng)計(jì)學(xué)軟件包統(tǒng)計(jì)分析數(shù)據(jù)。所有實(shí)驗(yàn)數(shù)據(jù)使用平均數(shù)±標(biāo)準(zhǔn)誤。兩組數(shù)據(jù)之間的比較使用Student t-檢驗(yàn),多組間采用單因素方差分析,藥物濃度相關(guān)性分析使用pesrson相關(guān)分析檢驗(yàn)。
2 結(jié)果
2.1 HASMC鑒定
通過在倒置顯微鏡下觀察細(xì)胞形態(tài)及免疫細(xì)胞化學(xué)檢測(cè)法鑒定培養(yǎng)細(xì)胞是ASMC。
2.2 莫西沙星對(duì)HASMC Cav-1蛋白表達(dá)的影響
5、10及20 mg/L莫西沙星干預(yù)HASMC48 h后,5、10及20 mg/L莫西沙星干預(yù)HASMC48 h后,各莫西沙星濃度組(M05組、M10組、M20組)Cav-1蛋白表達(dá)分別為(3.02±0.43),(4.18±0.07),(4.44±0.14),均明顯高于空白對(duì)照組(M0組)(1.00±0.15)(P<0.05,n=3),同時(shí)也存在濃度依賴性(r=0.77)。見圖1。
2.3 莫西沙星對(duì)HASMC Cav-1 mRNA表達(dá)的影響
qRT-PCR結(jié)果顯示5、10及20 mg/L莫西沙星干預(yù)HASMC 48 h后,各莫西沙星組(M5組、M10組、M20組)Cav-1 mRNA表達(dá)量(2.54±0.35),(3.26±0.22),(5.57±0.51)均較空白對(duì)照組(M0組)表達(dá)量(1.00±0.11)明顯增加(P<0.05,n=3),同時(shí)存在濃度依賴性(r=0.99)。見圖2。
3 討論
哮喘患者ASMC在哮喘發(fā)病中充當(dāng)著促炎細(xì)胞及主要的炎癥效應(yīng)細(xì)胞雙重角色[4],其病理狀態(tài)是哮喘患者氣道進(jìn)行性狹窄的主要原因[5]。因此,尋找針對(duì)ASMC發(fā)揮抗炎調(diào)節(jié)作用的藥物及作用機(jī)制可能將為重癥及難治性哮喘的控制及治療提供新思路。
我們先期研究發(fā)現(xiàn)莫西沙星可直接作用于大鼠ASMC,干擾ASMC增殖[6]、誘導(dǎo)凋亡[7]及抑制炎癥因子分泌[2]。但莫西沙星是否可直接影響HASMC并改變其生物學(xué)特性,相關(guān)研究較少。該研究中我們應(yīng)用莫西沙星干預(yù)HASMC 48 h后,結(jié)果顯示HASMC形態(tài)及數(shù)量發(fā)生改變,這與我們既往結(jié)論一致,即莫西沙星可直接作用于HASMC并可能改變其生物學(xué)特性。既往研究也證實(shí)莫西沙星可對(duì)多種細(xì)胞發(fā)揮抗炎作用[2,8],但關(guān)于抗炎調(diào)節(jié)的具體機(jī)制尚不清楚。
Cav-1可通過抑制一氧化氮生成[9]及影響ASMC內(nèi)鈣離子濃度在氣道炎癥中發(fā)揮重要作用[10],但莫西沙星對(duì)哮喘氣道炎癥的保護(hù)作用是否與Cav-1相關(guān)少有研究。
該試驗(yàn)研究發(fā)現(xiàn)HASMC中存在Cav-1蛋白(1.00±0.15)及mRNA(1.00±0.11)表達(dá)。應(yīng)用不同濃度莫西沙星(5、10及20 mg/L)干預(yù)HASMC后,結(jié)果顯示HASMC Cav-1蛋白及mRNA表達(dá)均有明顯增加,且均呈現(xiàn)藥物濃度依賴性(r=0.77,r=0.99)。由以上結(jié)論我們推測(cè),當(dāng)莫西沙星干預(yù)HASMC后,影響了Cav-1表達(dá)變化,而Cav-1表達(dá)變化可能將進(jìn)一步影響氣道炎癥反應(yīng)。但莫西沙星抑制哮喘大鼠ASMC炎癥因子IL-8及Eotaxin分泌是否與Cav-1表達(dá)表達(dá)變化相關(guān)仍需更多的研究。
另外,該研究仍有不足之處,我們雖然研究發(fā)現(xiàn)莫西沙星可濃度依賴性影響HASMC Cav-1表達(dá),但關(guān)于莫西沙星通過何種機(jī)制或通路發(fā)揮作用,目前尚無定論。近年來研究發(fā)現(xiàn),霧化吸入莫西沙星后氣道上皮細(xì)胞內(nèi)濃度并未明顯升高,主要因?yàn)槠淇烧T導(dǎo)細(xì)胞膜P糖蛋白表達(dá)增高并啟動(dòng)藥物外排[11]。而Cav-1作為支架蛋白構(gòu)成細(xì)胞表面穴樣內(nèi)陷(Caveolae)介導(dǎo)胞吞,其可參與P糖蛋白的轉(zhuǎn)運(yùn)功能[12]。且有研究發(fā)現(xiàn)過表達(dá)Cav-1可明顯提高食管鱗狀細(xì)胞癌P糖蛋白mRNA及蛋白水平,敲減Cav-1將致P糖蛋白表達(dá)明顯下降,提示Cav-1對(duì)P糖蛋白表達(dá)具有調(diào)控作用[13]。以上結(jié)論為該研究奠定了堅(jiān)實(shí)的理論基礎(chǔ),但需更進(jìn)一步的研究探索莫西沙星影響ASMC Cav-1表達(dá)的具體機(jī)制。
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(收稿日期:2019-01-10)