王艷道， 余 晨

(同濟(jì)大學(xué)附屬同濟(jì)醫(yī)院腎內(nèi)科，上海 200065)

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·基礎(chǔ)研究·

Nintedanib對Ang Ⅱ誘導(dǎo)腎臟成纖維細(xì)胞產(chǎn)生細(xì)胞外基質(zhì)的拮抗作用

王艷道， 余 晨

(同濟(jì)大學(xué)附屬同濟(jì)醫(yī)院腎內(nèi)科，上海 200065)

目的 探討尼達(dá)尼布(Nintedanib)對抗血管緊張素Ⅱ(angiotensin Ⅱ, Ang Ⅱ)誘導(dǎo)的腎臟成纖維細(xì)胞產(chǎn)生細(xì)胞外基質(zhì)的作用及其機(jī)制。方法 Ang Ⅱ處理大鼠腎臟成纖維細(xì)胞(NRK- 49F)，Western 印跡法檢測相關(guān)分子的蛋白表達(dá)量，DHE熒光探針法檢測活性氧簇(ROS)的水平。結(jié)果 腎臟成纖維細(xì)胞中Ang Ⅱ通過誘導(dǎo)PI3K/Akt 信號通路的激活，引起纖維連接蛋白和膠原蛋白Ⅰ的積聚。Nintedanib能抑制Ang Ⅱ誘導(dǎo)的PI3K/Akt信號通路的激活。Nintedanib還可以抑制Ang Ⅱ引起的ROS水平升高。結(jié)論 Nintedanib可以抑制Ang Ⅱ誘導(dǎo)的ECM成分升高，其作用機(jī)制與其抑制PI3K/Akt激活并抑制ROS產(chǎn)生有關(guān)。

尼達(dá)尼布； PI3K/Akt； 活性氧簇； 血管緊張素Ⅱ； 腎臟纖維化； 大鼠

腎臟纖維化是各種慢性腎臟疾病持續(xù)進(jìn)展為終末期腎病的共同途徑，主要標(biāo)志為細(xì)胞外基質(zhì)(extracellular matrixc, ECM)過度沉積，導(dǎo)致正常腎臟組織結(jié)構(gòu)破壞及腎臟功能進(jìn)行性下降。腎素-血管緊張素系統(tǒng)(renin-angiotensin system, RAS)的過度激活在腎臟纖維化的病理進(jìn)程中有重要作用。作為RAS的最重要活性肽，血管緊張素Ⅱ(angiotensin Ⅱ， Ang Ⅱ)可引起與腎臟纖維化密切相關(guān)的細(xì)胞因子如TGF-β、TNF-α等的表達(dá)增多，并引起多條相關(guān)信號通路如TGF-β-Smads，MAPK，PI3K/Akt的激活[1]。

尼達(dá)尼布(Nintedanib)是一種三重酪氨酸激酶抑制劑，對血小板源生長因子(platelet derived growth factor, PDGF)、血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)和堿性纖維母細(xì)胞生長因子(basic fibroblast growth factor, bFGF)均有抑制作用[2]。初起主要用于腫瘤治療[3-4]，如腎癌、卵巢癌等，近年來發(fā)現(xiàn)Nintedanib對特發(fā)性肺間質(zhì)纖維化(idiopathic pulmonary fibrosis, IPF)[5]有明顯的療效。本研究旨在探索Nintedanib對Ang Ⅱ引起的大鼠腎成纖維細(xì)胞細(xì)胞外基質(zhì)過度增生的抑制作用，并探討其作用機(jī)制。

1 材料與方法

1.1 試劑

DMEM高糖培養(yǎng)基購自美國Gibco公司；Nintedanib購自美國Selleck公司；Ang Ⅱ、坎地沙坦(candesartan， can)、LY294002、DPI、纖維連接蛋白(fibronectin， FN)一抗、超氧化物陰離子熒光探針二氫乙錠(dihydroethidium， DHE)購自美國Sigma公司；β-Actin一抗購自中國上海碧云天生物技術(shù)有限公司；Akt一抗、p-Akt一抗購自美國Cell Signaling公司；膠原蛋白 Ⅰ一抗購自美國Millipore公司。

1.2 細(xì)胞培養(yǎng)

NRK-49F細(xì)胞株用含10%FBS的DMEM高糖培養(yǎng)基，在37℃、5%CO2條件下培養(yǎng)、傳代。用0.5% FBS的DMEM高糖培養(yǎng)基同步化24h后進(jìn)行藥物處理。給予細(xì)胞Ang Ⅱ(1μmol/L)刺激0、24、48h或者0、5、15、30、60、120min；加藥處理細(xì)胞主要分為3組: 對照組(con)；Ang Ⅱ組(1μmol/L)；Ang Ⅱ(1μmol/L)+預(yù)處理坎地沙坦(10μmol/L)/Nintedanib(100nmol/L)/LY294002(10μmol/L)/DPI(10μmol/L) 1h組。

1.3 Western印跡法檢測

以SDS細(xì)胞裂解液(加入蛋白酶抑制劑)提取細(xì)胞總蛋白，BCA法測定蛋白濃度。40μg蛋白上樣，行SDS-PAGE電泳，轉(zhuǎn)膜至PVDF膜。TBST沖洗，脫脂奶粉封閉2h。然后將膜放入稀釋的一抗(按照說明書),4℃孵育過夜。次日，TBST沖洗后，將膜放入相應(yīng)的辣根過氧化物酶標(biāo)記的二抗(稀釋倍數(shù)均為1∶1000)，室溫孵育2h，TBST沖洗后，用凝膠成像系統(tǒng)拍照，Smart viewer軟件進(jìn)行灰度分析，內(nèi)參照為β-actin。

1.4 ROS水平檢測

利用DHE熒光探針檢測方法反映細(xì)胞內(nèi)超氧陰離子(·O2-)的水平，DHE脫氫后和RNA或DNA結(jié)合產(chǎn)生紅色熒光。Ang Ⅱ處理NRK-49F細(xì)胞24h后，用10μmol/L DHE在37℃下孵育細(xì)胞30min進(jìn)行熒光探針裝載，用PBS洗滌3次后在熒光顯微鏡下觀察細(xì)胞內(nèi)熒光強(qiáng)度變化。

1.5 統(tǒng)計學(xué)處理

2 結(jié) 果

2.1 Nintedanib抑制Ang Ⅱ誘導(dǎo)的細(xì)胞外基質(zhì)合成

NRK-49F細(xì)胞株中，給予Ang Ⅱ(1μmol/L)刺激0、24、48h后，Ang Ⅱ引起膠原蛋白 Ⅰ、纖維連接蛋白水平明顯升高，其作用強(qiáng)度呈時間依賴性，見圖1。

用AT1R阻斷劑坎地沙坦(10μmol/L)預(yù)處理細(xì)胞1h后，再給予Ang Ⅱ刺激48h，Western 印跡法結(jié)果顯示坎地沙坦顯著抑制Ang Ⅱ誘導(dǎo)的膠原蛋白 Ⅰ、纖維連接蛋白水平升高，見圖2。

用Nintedanib(100nmol/L)預(yù)處理細(xì)胞1h后，再加入Ang Ⅱ(1μmol/L) 刺激細(xì)胞48h，Western 印跡法檢測結(jié)果顯示，Nintedanib明顯抑制Ang Ⅱ 引起膠原蛋白 Ⅰ、纖維連接蛋白水平升高，見圖3。

2.2 Nintedanib抑制Ang Ⅱ所誘導(dǎo)的Akt磷酸化

Ang Ⅱ(1μmol/L)刺激48h NRK-49F細(xì)胞后，在0、5、15、30、60、120min，Western 印跡法檢測Akt磷酸化水平，結(jié)果顯示，細(xì)胞Akt磷酸化明顯增強(qiáng)，見圖4。

圖1 Ang Ⅱ誘導(dǎo)細(xì)胞外基質(zhì)合成增加Fig.1 Ang Ⅱ induced the synthesis of extracellular matrix與Ang Ⅱ處理細(xì)胞0h相比，*P<0.01,n=3

圖2 坎地沙坦阻斷Ang Ⅱ誘導(dǎo)的細(xì)胞外基質(zhì)合成增加Fig.2 Candesartan blocked the synthesis of extracellular matrix induced by Ang Ⅱ與con組相比，*P<0.01，與Ang Ⅱ組相比，#P<0.01;n=3

圖3 Nintedanib阻斷Ang Ⅱ誘導(dǎo)的細(xì)胞外基質(zhì)合成增加Fig.3 Nintedanib blocked the synthesis of extracellular matrix induced by Ang Ⅱ與con組相比，*P<0.01，與Ang Ⅱ組相比，#P<0.01;n=3

給予PI3K/Akt抑制劑LY294002(10μmol/L)預(yù)處理細(xì)胞1h，再給予Ang Ⅱ(1μmol/L)刺激48h，Western印跡法結(jié)果顯示LY294002可以抑制Ang Ⅱ誘導(dǎo)的膠原蛋白 Ⅰ、 纖維連接蛋白水平明顯升高，見圖5。

Nintedanib(100nmol/L)預(yù)處理細(xì)胞1h后，給予Ang Ⅱ(1μmol/L)刺激0、5、15、30、60、120min，收集細(xì)胞蛋白，Western印跡法結(jié)果顯示，Nintedanib 明顯抑制Ang Ⅱ引起的Akt磷酸化水平升高，見圖6。

圖4 Ang Ⅱ激活A(yù)kt信號通路Fig.4 Ang Ⅱ induced the activation of Akt signaling與Ang Ⅱ作用0min組相比，*P<0.01；n=3

圖5 LY294002阻斷Ang Ⅱ誘導(dǎo)的細(xì)胞外基質(zhì)合成增加Fig.5 LY294002 blocked the synthesis of extracellular matrix induced by Ang Ⅱ與con組相比，*P<0.01，與Ang Ⅱ組相比，#P<0.01；n=3

2.3 Nintedanib抑制Akt信號通路激活引起ROS水平增高

Ang Ⅱ(1μmol/L)處理細(xì)胞24h后，利用DHE熒光探針檢測細(xì)胞內(nèi)ROS的水平。熒光結(jié)果顯示，與對照組相比，Ang Ⅱ處理組的ROS水平明顯升高，見圖7。

圖6 Nintedanib阻斷Ang Ⅱ誘導(dǎo)的Akt激活Fig.6 Nintedanib blocked the activation of Akt induced by Ang Ⅱ與con組相比，*P<0.01,與Ang Ⅱ組相比，#P<0.05,##P<0.01;n=3

圖7 Ang Ⅱ誘導(dǎo)ROS水平增高Fig.7 Ang Ⅱ induced the production of ROS(DHE,×400)A: con組; B: Ang Ⅱ組

給予PI3K/Akt抑制劑LY294002(10μmol/L)預(yù)處理細(xì)胞1h，再給予Ang Ⅱ(1μmol/L)刺激24h，結(jié)果顯示LY294002可以抑制Ang Ⅱ誘導(dǎo)的ROS水平升高。Nintedanib(100nmol/L)預(yù)處理細(xì)胞1h后，給予Ang Ⅱ刺激24h，利用DHE熒光探針檢測細(xì)胞內(nèi)ROS的水平。結(jié)果表明，Nintedanib可顯著抑制Ang Ⅱ所引起的ROS水平升高，見圖8。

給予NADPH氧化酶抑制劑DPI(10μmol/L)預(yù)處理細(xì)胞1h，再給予Ang Ⅱ(1μmol/L)刺激48h，Western印跡法顯示DPI可以顯著抑制Ang Ⅱ誘導(dǎo)的膠原蛋白 Ⅰ、纖維連接蛋白水平升高，見圖9。

圖8 LY294002及Nintedanib均可抑制Ang Ⅱ引起的ROS水平升高Fig.8 LY294002 and Nintedanib inhibited ROS production induced by Ang Ⅱ (DHE,×400)A： 對照組；B： Ang Ⅱ組；C： LY+Ang Ⅱ組；D： Nin+Ang Ⅱ組

圖9 DPI阻斷Ang Ⅱ誘導(dǎo)的細(xì)胞外基質(zhì)合成增加Fig.9 DPI blocked the synthesis of extracellular matrix induced by Ang Ⅱ與con組相比，*P<0.01，與Ang Ⅱ組相比，#P<0.01；n=3

3 討 論

目前，已有的關(guān)于Nintedanib抗纖維化機(jī)制研究均是針對其對肺臟纖維化的拮抗機(jī)制。體外實驗結(jié)果表明，Nintedanib可抑制PDGFR-α，PDGFR-β的激活和正常人肺成纖維細(xì)胞的增殖，也可抑制IPF患者來源的肺成纖維細(xì)胞的增殖[6]。Nintedanib可抑制IPF患者肺成纖維細(xì)胞中PDGF或FGF-2引起的細(xì)胞遷移，同時可抑制TGF-β誘導(dǎo)的成纖維細(xì)胞-肌纖維母細(xì)胞轉(zhuǎn)化。另外，Nintedanib可增加MMP-2的活性，抑制TIMP-2及TGF-β引起的ERK通路激活，從而抑制肺成纖維細(xì)胞中ECM成分的表達(dá)。體內(nèi)實驗表明，Nintedanib可抑制小鼠肺組織中血小板衍生生長因子受體(platelet-derived growth factor receptor, PDGFR)的激活及其下游信號通路。在兩種不同的小鼠IPF模型中，Nintedanib可導(dǎo)致肺組織中膠原蛋白沉積明顯減少并有抗炎作用[7]。

本研究顯示，Nintedanib能抑制Ang Ⅱ刺激所引起PI3K/Akt通路的激活，使用Nintedanib或PI3K/Akt抑制劑LY294002均明顯降低了Ang Ⅱ誘導(dǎo)的ECM合成增多。

PI3K/Akt為細(xì)胞內(nèi)重要的信號通路，在多種細(xì)胞進(jìn)程如增殖、分化和凋亡中發(fā)揮重要作用[8]。PI3K/Akt信號通路與包括腎臟在內(nèi)的多種器官纖維化的形成密切相關(guān)。在腎小球系膜細(xì)胞中，給予PI3K/Akt抑制劑LY294002，可明顯抑制TGF-β引起的Ⅰ型膠原表達(dá)[9]。本研究中，Ang Ⅱ刺激可引起PI3K/Akt通路的激活并導(dǎo)致細(xì)胞外基質(zhì)增多，給予PI3K/Akt抑制劑LY294002后明顯降低了Ang Ⅱ誘導(dǎo)的ECM增多，再次證實了PI3K/Akt通路參與腎臟成纖維細(xì)胞ECM增多。

PI3K/Akt信號通路的激活需要一系列受體酪氨酸激酶的參與，如IGF-1R、HER2/Neu、VEGFR、PDGFR等[10]。活性氧簇(ROS)在維持細(xì)胞的正常生理功能中起重要作用,ROS可激活多條與腎臟纖維化相關(guān)的信號通路。NADPH氧化酶被認(rèn)為是ROS的主要來源之一，研究證實Ang Ⅱ是激活NADPH氧化酶的主要刺激因子。這也是Ang Ⅱ?qū)е履I臟纖維化的重要機(jī)制之一[11]。在病變腎臟中ROS的產(chǎn)生增多很大程度上是由于腎臟中RAS系統(tǒng)的過度激活[12]。Ang Ⅱ通過與其Ⅰ型受體(AT1R)結(jié)合，激活NADPH氧化酶，產(chǎn)生ROS，從而參與機(jī)體功能的調(diào)節(jié)。目前的研究發(fā)現(xiàn)Ang Ⅱ激活NADPH氧化酶主要有以下三條途徑: 一是PKC途徑，二是c-Src激酶途徑；三是受體酪氨酸蛋白激酶途徑: 即當(dāng)Ang Ⅱ與AT1R結(jié)合后，激活PDGFR、EGFR、IGFR，進(jìn)而激活酪氨酸蛋白激酶，并與PI3K的SH2結(jié)構(gòu)結(jié)合，使Rac活化并移位到細(xì)胞膜，從而促進(jìn)NADPH氧化還原反應(yīng)平臺的組裝，產(chǎn)生大量ROS。

在本研究中，Ang Ⅱ 可誘導(dǎo)腎臟成纖維細(xì)胞的ROS水平明顯升高，給予NADPH氧化酶抑制劑DPI則可顯著抑制ECM蛋白的產(chǎn)生，同樣給予Nintedanib也可以明顯抑制Ang Ⅱ 誘導(dǎo)的ROS產(chǎn)生并減少ECM產(chǎn)生。由此認(rèn)為，Nintedanib作為酪氨酸激酶抑制劑，抑制了Ang Ⅱ 所誘導(dǎo)的酪氨酸蛋白激酶的激活，進(jìn)而抑制后者與PI3K的Sh2結(jié)構(gòu)結(jié)合，阻止Rac移位到胞膜，使NADPH氧化酶受到抑制，ROS產(chǎn)生明顯減少，最后減輕腎臟成纖維細(xì)胞ECM沉積。

本研究觀察了Nintedanib對腎臟細(xì)胞過度表達(dá)ECM的拮抗作用，為開展Nintedanib作為治療腎臟纖維化藥物的研究提供了理論依據(jù)，但還需要在后續(xù)研究中進(jìn)行體內(nèi)研究并對其作用機(jī)制做更深入的研究。

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Nintedanib suppresses overexpression of extracellular matrix induced by angiotensin Ⅱ in renal fibroblasts

WANGYan-dao，YUChen

(Dept. of Nephrology, Tongji Hospital, Tongji University, Shanghai 200065, China)

Objective To investigate the effect of nintedanib on extracellular matrix production induced by angiotensin Ⅱ(Ang Ⅱ) in renal fibroblasts and related mechanism. Methods Renal fibroblast cells were treated with Ang Ⅱ. Expression of fibronectin, collagen Ⅰ levels was determined by Western blotting, and the ROS level was detected by DHE fluorescent probe. Results Ang Ⅱ induced the phosphorylation of PI3K/Akt signalling, with the accumulation of fibronectin and collagen Ⅰ in renal fibroblast cells. Nintedanib blocked the phosphorylation of Akt and attenuated the increase of fibronectin and collagen Ⅰ induced by Ang Ⅱ. Nintedanib also inhibited Ang Ⅱ induced ROS generation. Conclusion Nintedanib can attenuate the accumulation of ECM proteins induced by Ang Ⅱ, which may be associated with the suppression effect on PI3K/Akt signalling activation and subsequent ROS overproduction.

Nintedanib; PI3K/Akt; reactive oxygen species; angiotensin Ⅱ; renal fibrosis; rat

10.16118/j.1008-0392.2015.04.003

2015-02-26

國家自然科學(xué)基金(81370790)

王艷道 (1989—)，女，碩士研究生.E-mail: wydao2012@126.com

余 晨.E-mail: yuchen@#edu.cn

R 34

A

1008-0392(2015)04-0013-06