NOXs在肝纖維化中的作用機(jī)制研究

卞勉勵，陳星燃，張晨曦，金歡歡，趙士峰，張 峰，3，鄭仕中, 3

(1. 南京中醫(yī)藥大學(xué)藥學(xué)院，2. 南京中醫(yī)藥大學(xué)中藥學(xué)一級學(xué)科，3. 江蘇省中藥藥效與安全性評價重點(diǎn)實(shí)驗(yàn)室，江蘇 南京 210023)

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NOXs在肝纖維化中的作用機(jī)制研究

卞勉勵1, 2，陳星燃1, 2，張晨曦1, 2，金歡歡1, 2，趙士峰1, 2，張 峰1, 2，3，鄭仕中1, 2, 3

(1. 南京中醫(yī)藥大學(xué)藥學(xué)院，2. 南京中醫(yī)藥大學(xué)中藥學(xué)一級學(xué)科，3. 江蘇省中藥藥效與安全性評價重點(diǎn)實(shí)驗(yàn)室，江蘇 南京 210023)

肝纖維化；NOXs；肝細(xì)胞凋亡；炎癥反應(yīng)；氧化應(yīng)激；內(nèi)質(zhì)網(wǎng)應(yīng)激；IRE1α-XBP1信號通路

在肝纖維化發(fā)生發(fā)展過程中常伴有繼發(fā)性的炎癥反應(yīng)，而作為肝臟中的固有免疫細(xì)胞，肝臟炎癥反應(yīng)過程中，枯否細(xì)胞(Kupfer cell, KC)大量活化并釋放多種促炎因子，同時還可分泌趨化因子，募集其他炎性細(xì)胞聚集于損傷部位，從而進(jìn)一步加重炎癥損傷[1]。其釋放的細(xì)胞因子如活性氧簇(reactive oxygen species, ROS)，能直接刺激靜息態(tài)肝星狀細(xì)胞(hepatic stellate cells, HSC)活化并轉(zhuǎn)化為肌成纖維細(xì)胞(myofibroblast, MFB)，后者大量增殖并合成胞外基質(zhì)(extracellular matrix, ECM)，從而導(dǎo)致肝纖維化的形成[2]。此外，ROS還能使促纖維化因子大量合成，如轉(zhuǎn)化生長因子-β(transforming growth factor-beta, TGF-β)、腫瘤壞死因子-α(tumor necrosis factor -α, TNF-α)等，并刺激HSC發(fā)生表型轉(zhuǎn)化，ECM進(jìn)一步合成增多，炎癥因子表達(dá)增加[3]。

新近研究發(fā)現(xiàn)，還原型煙酰胺腺嘌呤二核苷酸磷酸氧化酶(nicotinamide adenine dinucleotide phosphate oxidase, NOXs)產(chǎn)生的ROS能夠介導(dǎo)細(xì)胞內(nèi)多種信號通路的激活，進(jìn)而調(diào)控肝細(xì)胞的生長、增殖、分化、遷移、衰老及凋亡等生理活動[4-5]。

1 NOXs的分類及功能調(diào)控

NOXs參與調(diào)控HSC的活化和肝細(xì)胞的凋亡，在不同種類的細(xì)胞中特異性表達(dá)NOXs的催化亞基，分別為NOX1、NOX2、NOX3、NOX4、NOX5、DUOX1、DUOX2，后統(tǒng)稱為NOX蛋白家族[5]。NOX蛋白家族廣泛表達(dá)于各種哺乳動物細(xì)胞中，且其活性普遍受到還原型煙酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate, NADPH)膜結(jié)合酶類的調(diào)節(jié)。其中，肝細(xì)胞和HSC表達(dá)NOX1、NOX2、NOX4、DUOX1和DUOX2；血管內(nèi)皮細(xì)胞主要表達(dá)NOX1、NOX2和NOX4；KC主要表達(dá)NOX2[6]。

NOXs是調(diào)節(jié)機(jī)體產(chǎn)生ROS的重要酶類，主要分布于內(nèi)皮、血管平滑肌等非吞噬細(xì)胞中。在TGF-β、TNF-α、炎癥介質(zhì)、鈣離子、重金屬、部分藥物等刺激時，通過激活NOXs進(jìn)一步誘導(dǎo)ROS的大量產(chǎn)生，參與多種疾病的發(fā)生和發(fā)展[7-8]。此外，NOXs極易受細(xì)胞外環(huán)境變化刺激，Paik等[9]研究表明，高氧、缺氧、高溫、高糖及胞外酸性環(huán)境變化等能夠使胞質(zhì)內(nèi)p47phox磷酸化，與細(xì)胞色素b558結(jié)合，進(jìn)而激活NOXs，導(dǎo)致一系列疾病的發(fā)生。

2 NOXs與肝纖維化

Piskounova等[5]研究發(fā)現(xiàn)，NOXs在肝纖維化的發(fā)生發(fā)展中扮演重要角色。在丙型肝炎病毒和非酒精性脂肪肝病(nonalcoholic fatty liver disease, NAFLD)患者中，NOX4的水平明顯升高，而NOX4 的升高又進(jìn)一步依賴性上調(diào)TGF-β/Smad3下游調(diào)節(jié)因子，促進(jìn)HSC活化及膠原的形成，進(jìn)而加重肝纖維化[10-11]。NOX1主要介導(dǎo)內(nèi)源性肝細(xì)胞的促纖維化作用，通過抑癌基因(phosphatase and tensin homolog deleted on chromosome ten, PTEN)的失活及上調(diào)Akt/FOXO4/p27信號傳導(dǎo)途徑，進(jìn)一步引發(fā)炎癥反應(yīng)，從而促進(jìn)環(huán)氧合酶-2表達(dá)和前列腺素的合成[12]。

各種NOXs激動劑能夠刺激HSC和MFB中促纖維化因子的釋放，從而誘發(fā)肝纖維化反應(yīng)，在細(xì)胞內(nèi)直接對細(xì)胞膜、蛋白質(zhì)等進(jìn)行攻擊，造成氧化損傷或間接激活氧化還原信號途徑，進(jìn)而誘導(dǎo)肝細(xì)胞的凋亡[13]。NOXs作為ROS 的主要供體，介導(dǎo)氧化應(yīng)激和內(nèi)質(zhì)網(wǎng)損傷，分泌細(xì)胞因子、趨化因子和微粒，觸發(fā)KC和HSC的活化[14]。HSC在轉(zhuǎn)變成MFB的同時，激活NOX2，誘發(fā)肝細(xì)胞凋亡小體發(fā)揮吞噬作用，從而加重肝纖維化[15-16]。

基于NOXs在肝纖維化發(fā)生發(fā)展中的重要作用，進(jìn)一步證實(shí)NOXs在肝臟疾病演變過程中存在多種致病機(jī)制。本文接下來將對其進(jìn)行重點(diǎn)闡述。

2.1 NOXs與炎癥反應(yīng) 研究發(fā)現(xiàn)[17]，NOXs介導(dǎo)的炎癥反應(yīng)參與多種疾病的發(fā)生發(fā)展。肝纖維化是肝臟遭遇損傷后，通過許多細(xì)胞因子的釋放誘發(fā)的一種慢性炎癥反應(yīng)，并伴有大量炎癥細(xì)胞的滲入。巨噬細(xì)胞、淋巴細(xì)胞和嗜酸性細(xì)胞等廣泛參與肝纖維化的炎癥反應(yīng)過程。其中，淋巴細(xì)胞可通過釋放淋巴因子激活巨噬細(xì)胞，反過來，巨噬細(xì)胞又能夠分泌TNF-α進(jìn)一步刺激淋巴細(xì)胞、MFB和其他炎癥細(xì)胞，從而加劇炎癥反應(yīng)[18]。有研究表明，NOXs在肝臟缺血/再灌注、過度飲酒、異源物質(zhì)或重金屬中毒、細(xì)菌、病毒及寄生蟲感染誘發(fā)的肝臟炎癥反應(yīng)過程中發(fā)揮了重要作用[13, 19]。

Foo等[20]研究表明，不同細(xì)胞內(nèi)NOXs的激活主要由肝纖維化炎性因子TGF-β所誘導(dǎo)，進(jìn)一步通過ROS的大量產(chǎn)生介導(dǎo)胞內(nèi)不同信號傳導(dǎo)途徑的激活。TGF-β促進(jìn)HSC轉(zhuǎn)化為MFB的過程也依賴于NOXs誘導(dǎo)ROS的產(chǎn)生、基質(zhì)金屬蛋白酶(matrix metalloproteinases, MMPs)和基質(zhì)金屬蛋白酶抑制劑(matrix metalloproteinase inhibitors, TIMPs)的平衡來調(diào)節(jié)ECM大量沉積[21]。在膽管結(jié)扎(bile duct ligation, BDL)和CCl4誘導(dǎo)的肝纖維化模型中，運(yùn)用NOX4/NOX1抑制劑GKT137831處理后，肝纖維化標(biāo)志物表達(dá)明顯降低、肝細(xì)胞炎癥反應(yīng)也明顯減輕[22]。此外，NOX4抑制劑也能夠明顯抑制肝細(xì)胞中TGF-β信號的表達(dá)，降低ROS產(chǎn)生，進(jìn)一步使纖維黏連蛋白和膠原產(chǎn)生降低[23]。因此，抑制NOXs功能的發(fā)揮，能夠有效緩解肝纖維化過程中的炎癥反應(yīng)。

2.2 NOXs與氧化應(yīng)激 Altenh?fer 等[24]研究發(fā)現(xiàn)，ROS與促炎癥介質(zhì)的過度表達(dá)有著密切的聯(lián)系，主要表現(xiàn)在其可以激活多種炎癥轉(zhuǎn)錄因子，進(jìn)一步導(dǎo)致ROS 過量產(chǎn)生和堆積。而細(xì)胞內(nèi)ROS的產(chǎn)生又主要依賴于NOXs的介導(dǎo)[25-26]。這提示，NOXs可通過促進(jìn)ROS的產(chǎn)生誘發(fā)機(jī)體內(nèi)環(huán)境失衡，導(dǎo)致細(xì)胞器功能失調(diào)，如內(nèi)質(zhì)網(wǎng)鈣穩(wěn)態(tài)遭到破壞、蛋白質(zhì)的加工轉(zhuǎn)運(yùn)能力及生理狀態(tài)發(fā)生紊亂，進(jìn)而參與宿主細(xì)胞生長發(fā)育、死亡和衰老等生命過程中的氧化應(yīng)激反應(yīng)[27]。

2.2.1 NOXs調(diào)控ROS產(chǎn)生介導(dǎo)內(nèi)質(zhì)網(wǎng)應(yīng)激的發(fā)生 肝纖維化形成過程中，活化型HSC產(chǎn)生大量的ECM，蛋白質(zhì)的大量合成增加了對內(nèi)質(zhì)網(wǎng)折疊能力的需求，擾亂內(nèi)質(zhì)網(wǎng)功能，ROS可以改變內(nèi)質(zhì)網(wǎng)的穩(wěn)態(tài)激活細(xì)胞未折疊蛋白響應(yīng)(unfolded protein response, UPR)機(jī)制[28]。肝細(xì)胞具有豐富的內(nèi)質(zhì)網(wǎng)結(jié)構(gòu)，是蛋白質(zhì)折疊加工與質(zhì)量監(jiān)控的重要細(xì)胞器，其穩(wěn)態(tài)平衡對于維持肝細(xì)胞功能至關(guān)重要。當(dāng)肝細(xì)胞受到各種化學(xué)、物理等刺激時，極易誘發(fā)蛋白質(zhì)代謝障礙，進(jìn)而引起內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress, ERS)[29]。研究發(fā)現(xiàn)，當(dāng)內(nèi)質(zhì)網(wǎng)不能承擔(dān)蛋白折疊的高負(fù)荷時，HSC中NOXs所介導(dǎo)ROS產(chǎn)生，將引發(fā)ERS，從而啟動細(xì)胞UPR信號，激活抗氧化應(yīng)答基因NOXs的表達(dá)，進(jìn)一步增強(qiáng)內(nèi)質(zhì)網(wǎng)在蛋白質(zhì)折疊、組裝和運(yùn)輸方面的功能[30]。

當(dāng)肝細(xì)胞受到外源性刺激時，細(xì)胞作為一個整體對外界各種刺激發(fā)生反應(yīng)，在細(xì)胞內(nèi)部，細(xì)胞器也會發(fā)生相應(yīng)的應(yīng)激反應(yīng)，進(jìn)而影響蛋白的正常表達(dá)和修飾，導(dǎo)致蛋白的錯誤折疊以及未折疊蛋白在內(nèi)質(zhì)網(wǎng)腔內(nèi)的異常聚集[31]。肝纖維化過程中伴有ROS的大量生成，影響核氧化還原狀態(tài)并誘導(dǎo)核蛋白修飾[32]。其中，UPR相關(guān)激酶c-Jun氨基端激酶(c-Jun-NH2-terminal kinase, JNK)的活化，JNK 的活化通過參與ERS的激活，進(jìn)而促進(jìn)MFB的活化[33]。

新近研究強(qiáng)調(diào)內(nèi)質(zhì)網(wǎng)谷胱甘肽超氧化物清除劑在ERS中發(fā)揮關(guān)鍵作用，并表明在其與谷胱甘肽共同作用下可以在脂肪性肝炎和肝臟纖維化中抑制氯離子通道中超氧陰離子自由基對HSC活化的刺激[34]。此外，天然抗氧化劑，如千層紙素，可通過調(diào)控ROS產(chǎn)生，影響細(xì)胞內(nèi)Ca2+穩(wěn)態(tài)，調(diào)節(jié)蛋白質(zhì)磷酸化以及影響信號通路的級聯(lián)傳遞及轉(zhuǎn)錄因子的表達(dá)，從而有效逆轉(zhuǎn)肝纖維化[35-36]。

2.2.2 NOXs 調(diào)控 ROS 產(chǎn)生介導(dǎo) IRE1α-XBP1信號通路的激活 在肝纖維化發(fā)展過程中，HSC大量活化與增殖的同時，NOXs的表達(dá)也明顯增加，抗氧化防御系統(tǒng)逐漸被破壞，使ROS產(chǎn)生增加，進(jìn)而激活特定的信號傳導(dǎo)通路，如IRE1α-XBP1(inositol-requiring enzyme 1 alpha, IRE1α; X-box binding protein 1, XBP1)信號通路[37]?？v觀UPR的3條通路中，IRE1α-XBP1通路發(fā)揮作用的時間最短、啟動時間最晚，這充分表明了機(jī)體對其調(diào)節(jié)最為敏感[30]。此外，IRE1α以單體的形式表達(dá)于內(nèi)質(zhì)網(wǎng)膜上，同時具備蛋白激酶和RNA內(nèi)切酶雙重活性。當(dāng)內(nèi)質(zhì)網(wǎng)受到壓力刺激時，其單體發(fā)生二聚化，通過自體磷酸化方式被激活，繼而激活其核酸酶活性，對其下游的底物進(jìn)行剪切，翻譯成有活性的轉(zhuǎn)錄因子入核，并激活下游轉(zhuǎn)錄因子，行使其緩解內(nèi)質(zhì)網(wǎng)壓力的功能[38-39]。目前，最為明確的IRE1α核酸酶底物是XBP1 mRNA；另有研究表明，IRE1α的核酸酶活性能夠介導(dǎo)某些特定mRNA的降解[37]。IRE1α自體磷酸化誘導(dǎo)RNA酶的活性增加，是HSC/ MFB以及間質(zhì)干細(xì)胞(mesenchymal stem cells, MSC)定向遷移的一個重要因素[40]。

抑制IRE1α-XBP1信號通路及p38蛋白激酶活性能夠降低HSC活化及自噬的產(chǎn)生，從而有效逆轉(zhuǎn)肝纖維化[41]。IRE1α-XBP1信號通路的活化可誘導(dǎo)ERS，進(jìn)而導(dǎo)致細(xì)胞凋亡[42]。另有文獻(xiàn)報道，IRE1α-XBP1抑制劑MKC-3946通過作用于核糖核酸內(nèi)切酶結(jié)構(gòu)域，有效抑制ERS的發(fā)生[43-45]。

3 結(jié)語

在疾病的發(fā)生和發(fā)展過程中，關(guān)于肝纖維化病理機(jī)制的研究多與炎癥反應(yīng)及氧化應(yīng)激相關(guān)。不同藥物誘發(fā)的慢性肝損傷中，ROS的產(chǎn)生機(jī)制并不相同，研究發(fā)現(xiàn)，NOXs廣泛參與肝纖維化的多種過程，如MFB形成、肝細(xì)胞死亡的調(diào)控等[46]。此外，預(yù)防ROS產(chǎn)生有助于更好地理解ERS在細(xì)胞內(nèi)轉(zhuǎn)導(dǎo)途徑中的作用。未來有望通過使用安全有效的NOXs抑制劑來抑制HSC活化，進(jìn)而發(fā)揮肝細(xì)胞保護(hù)作用。基于肝纖維化發(fā)生過程中嚴(yán)重的炎癥反應(yīng)，通過調(diào)控NOXs在炎癥反應(yīng)中的生物學(xué)作用，可為未來發(fā)展預(yù)防或治療肝纖維化提供新的途徑。

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Study on mechanism of NOXs in liver fibrosis

BIAN Mian-li1, 2, CHEN Xing-ran1, 2, ZHANG Chen-xi1, 2, JIN Huan-huan1, 2,ZHAO Shi-feng1, 2, ZHANG Feng1, 2,3, ZHENG Shi-zhong1, 2,3

(1.CollegeofPharmacy,NanjingUniversityofChineseMedicine,Nanjing210023,China; 2.NationalFirst-ClassKeyDisciplineforTraditionalChineseMedicine,NanjingUniversityofChineseMedicine,Nanjing210023,China;3.JiangsuKeyLaboratoryforPharmacologyandSafetyEvaluationofChineseMateriaMedica,NanjingUniversityofChineseMedicine,Nanjing210023,China)

liver fibrosis; NOXs; hepatocyte apoptosis; inflammation; oxidative stress; endoplasmic reticulum stress; IRE1α-XBP1 signaling pathway

2016-06-23，

2016-07-29

國家自然科學(xué)基金資助項(xiàng)目(No 81270514，31401210，31571455)；江蘇省自然科學(xué)基金青年基金資助項(xiàng)目(No BK20140955)；江蘇省高校自然科學(xué)研究面上項(xiàng)目(No 14KJB310011)；2013年江蘇高校優(yōu)秀科技創(chuàng)新團(tuán)隊計劃；江蘇高校優(yōu)勢學(xué)科建設(shè)工程資助項(xiàng)目(No ysxk-2010)；南京市醫(yī)學(xué)科技發(fā)展項(xiàng)目(No YKK14143)

卞勉勵(1992- )，女，碩士生,研究方向: 天然藥物預(yù)防和治療腫瘤與肝纖維化，Tel:025-85811246，E-mail:1300506693@qq.com;

鄭仕中(1962- )，男，博士，教授，博士生導(dǎo)師,研究方向:天然藥物預(yù)防和治療腫瘤與肝纖維化，通訊作者，Tel/Fax:025-85811246，E-mail:nytws@163.com

時間：2016-10-20 10:29

http://www.cnki.net/kcms/detail/34.1086.R.20161020.1029.006.html

10.3969/j.issn.1001-1978.2016.11.003

A

1001-1978(2016)11-1490-04

R-05；R329.24；R329.25；R345.4；R575.2；R977.3