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通讯作者:

曾晓宁,E-mail:zeng_xiao_njng@njmu.edu.cn;

姚欣,yaoxin@njmu.edu.cn

中图分类号:R562.25

文献标识码:A

文章编号:1007-4368(2022)06-790-07

DOI:10.7655/NYDXBNS20220604

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参考文献 10
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参考文献 14
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参考文献 15
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参考文献 20
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目录contents

    摘要

    目的:探讨蛋白酶激活受体2(protease⁃activated receptor 2,PAR2)在人支气管上皮细胞间充质转化(epithelial⁃mesen⁃ chymal transition,EMT)中的作用及可能机制。方法:16HBE细胞予以不同浓度类胰蛋白酶(PAR2激动剂)处理,采用划痕实验、Transwell小室、蛋白质免疫印迹等方法观察类胰蛋白酶对细胞EMT过程的影响;通过DCFH⁃DA探针测定细胞活性氧(re⁃ active oxygen species,ROS)的水平;外源性加入ROS清除剂N⁃乙酰半胱氨酸(N⁃acety⁃1⁃cysteine,NAC)后,划痕、Transwell小室观察类胰蛋白酶诱导的细胞修复与迁移功能。结果:类胰蛋白酶呈剂量依赖性地促进16HBE修复与迁移;下调E⁃cadherin、上调N⁃cadherin及α⁃平滑肌肌动蛋白(α⁃smooth muscle actin,α⁃SMA)表达(P < 0.05)。类胰蛋白酶增加16HBE细胞内ROS水平, 而PAR2抑制剂FSLLRY⁃NH2可逆转上述改变(P < 0.05)。NAC可有效抑制类胰蛋白酶诱导的细胞修复与迁移(P < 0.05)。结论:激活PAR2可经由ROS信号调控人支气管上皮细胞EMT过程,是细胞表型转化与重塑调控的潜在靶标。

    Abstract

    Objective:This study aims to investigate the role of protease ⁃ activated receptor 2(PAR2)involved in epithelial ⁃ mesenchymal transition(EMT)of human bronchial epithelial(HBE)cells. Methods:The 16HBE cells were stimulated with various concentrations of tryptase(a nature agonist of PAR2). Cell migration and repair were assessed by transwell and scratch assay. Western blot was used to examine the expressions of EMT associated biomarkers including E⁃cadherin,N⁃cadherin and α⁃smooth muscle actin (α⁃ SMA). DCFH ⁃DA probe was employed to measure the generation of reactive oxygen species(ROS). The effects of N ⁃ acety ⁃ 1⁃ cysteine(NAC)on 16HBE induced by tryptase were also examined by transwell and scratch assay. Results:Tryptase dramatically promoted cell migration and repair with loss of E⁃cadherin and increase of N⁃cadherin and α⁃SMA in a dose⁃dependent manner(P < 0.05). Tryptase enhanced generation of ROS in 16HBE cells,and this effect can be inhibited by PAR2 antagonist FSLLRY⁃NH2(P < 0.05). ROS scavenger NAC significantly inhibited the EMT changes induced by tryptase(P < 0.05). Conclusion:Activation of PAR2 triggered EMT process via ROS signal in HBE cells,which highlights a potential target for the regulation of HBE phenotype shift involved in airway remodeling.

  • 气道重塑是支气管哮喘(简称哮喘)发生发展的重要事件,包括一系列关键环节,如上皮间充质转化(epithelial⁃mesenchymal transition,EMT)、成纤维细胞活化及分化为肌成纤维细胞、细胞外基质(extracellular matrix,ECM)沉积等[1-2]。随着EMT机制的不断探索,其与气道疾病、尤其与哮喘的相关性已倍受关注。新近研究证实,EMT在上皮异常修复、纤维生成等过程中扮演了重要角色[3-4]。人支气管上皮细胞(human bronchial epithelial cell,HBE)作为机体接触外界的第一道防线,应激后发生EMT,同时产生多种促纤维化因子如转化生长因子⁃β (transforming growth factor⁃β,TGF⁃β)等加速EMT进程[5]。EMT时,HBE历经细胞间连接丢失等一系列改变,启动整个气道重塑过程。然而,哪些因素参与调控EMT及其具体机制目前仍不明确。

  • 蛋白酶激活受体2(protease ⁃activated receptor 2,PAR2)作为独特的PAR家族成员,在呼吸系统 (如支气管上皮、成纤维、内皮细胞等)广泛表达[6],且参与多种呼吸系统疾病体内稳态的调节。作为细胞表面的“传感器”,PAR2主要介导细胞对胞外丝氨酸蛋白酶的应答。其天然配体——类胰蛋白酶(是机体内PAR2的天然激动剂,主要源于肥大细胞,被认为是哮喘中的一种重要炎症介质),通过裂解PAR2胞外段、激活受体[7],在哮喘、COPD、肺纤维化等慢性呼吸系统疾病中对成纤维细胞的增殖具有促进作用[8]。急性肺损伤中,激活PAR2不仅可引起成纤维细胞增殖[9],还可导致内皮屏障功能障碍、血管通透性增加[10],可见肺组织中PAR2对多种细胞的功能具有调控作用。然而,PAR2对气道重要结构细胞HBE的功能、尤其是其EMT表型转化过程是否存在影响,迄今尚未见报道。

  • 机体多种病理生理过程中,活性氧(reactive ox⁃ ygen species,ROS)是一类重要的信号分子[11]。研究发现,ROS生成与PAR2活化密切相关[12]。本研究通过体外培养HBE、建立TGF⁃β诱导的EMT模型,着重观察PAR2活化对HBE细胞EMT的影响、并阐明ROS信号在该过程中的作用。

  • 1 材料和方法

  • 1.1 材料

  • 类胰蛋白酶(T7063)、ROS检测试剂盒(reactive oxygen species,DCFH⁃DA)(MAK143,Sigma⁃Aldrich公司,美国)。ROS清除剂N⁃乙酰半胱氨酸(N⁃acety ⁃1⁃cysteine,NAC)(616⁃91⁃1,中国国药集团)。PAR2抑制剂FSLLRY⁃NH2(FS)(C7865BL120⁃1,南京金斯瑞公司)。TGF⁃β1(7754⁃BH,R&D system公司,美国)。抗α⁃平滑肌肌动蛋白(α⁃smooth muscle ac⁃tin,α⁃SMA)(ab5694)、N⁃cadherin(ab76011)、E⁃cad⁃ herin(ab40772)特异性抗体(Abcam公司,英国)。抗甘油醛⁃3⁃磷酸脱氢酶(glyceraldehyde⁃3⁃phosphate dehydrogenase,GAPDH)特异性抗体(2118,CST公司,美国)。辣根过氧化物酶标记的山羊抗兔IgG抗体(sc⁃2004,Santa Cruz公司,美国)。

  • 1.2 方法

  • 1.2.1 细胞培养

  • 人支气管上皮细胞系16HBE购自北京癌症研究所,培养于RPMI 1640培养基(Invitrogen Gibco公司,美国)、1%青霉素/链霉素(Invitrogen Gibco公司,美国)及10%胎牛血清(fetal bovine serum, FBS,Invitrogen Gibco公司,美国)配制的完全培养基,置于37℃、5%CO2条件的细胞培养箱孵育,每隔2~3d更换培养基,直至细胞密度达80%后,0.5%胰酶(Invitrogen Gibco公司,美国)消化、传代。

  • 1.2.2 划痕实验

  • 划痕实验检测16HBE修复迁移功能。16HBE接种于6孔板,培养24h后以无血清培养基饥饿6~8h;使用200 μL无菌移液枪头尖端在细胞上进行一次笔直划痕,PBS清洗漂浮的细胞;每孔加入含不同浓度类胰蛋白酶、NAC、TGF⁃β的无血清培养基,立即显微镜拍照;培养箱孵育24h后,再次记录划痕区域。使用Image J软件计算迁移平均距离,重复3次实验进行统计学分析。

  • 1.2.3 迁移实验

  • Transwell小室迁移实验检测16HBE迁移功能。完全培养基中培养16HBE至密度80%后,无血清培养基饥饿;细胞计数后以100 μL细胞悬液 (2.0×104 个/孔)接种于Transwell(Corning公司,美国)上室,置于24孔板中;加入含不同浓度的类胰蛋白酶、NAC、TGF⁃β的完全培养基于下室,细胞培养箱中孵育24h;弃培养基、PBS清洗,多聚甲醛固定30min后,棉签擦拭小室细胞,结晶紫孵育5min; PBS清洗3次后,显微镜拍照。随机取4个不同视野计数,并行统计学分析。

  • 1.2.4 蛋白质免疫印迹实验

  • 蛋白质免疫印迹实验检测16HBE中α⁃SMA、E⁃ cadherin、N⁃cadherin蛋白表达量。用不同浓度类胰蛋白酶刺激16HBE 48h后收集细胞、PBS洗涤2次,200 μL RIPA冰上裂解15min;收集裂解物, 14 000r/min离心20min,十二烷基硫酸钠(sodium dodecyl sulfate,SDS)蛋白变性;制胶上样,80~120V恒压电泳,300mA恒流PVDF转膜;Tween 20的Tris洗涤缓冲液(TBST)配制5%脱脂奶粉溶液,37℃封闭1h;加入抗α⁃SMA(1∶100)、E⁃cadherin(1∶1 000)、 N⁃cadherin(1∶1 000)、GAPDH(1∶1 000)抗体4℃孵育过夜;TBST洗涤后,与辣根过氧化物酶标记的山羊抗兔IgG抗体孵育;ECL化学发光液(Thermo公司,美国)曝光,并用Bio⁃Rad Gel Doc/Chemi Doc成像系统及Quantity One软件进行分析。

  • 1.2.5 ROS测定

  • DCFH⁃DA探针检测16HBE胞内ROS。细胞内ROS可与DCFH反应生成DCF的荧光产物。本研究中,10mmol/L DCFH⁃DA溶于无血清培养基中,稀释1 000倍得到10 μmol/L工作液;将密度达80%的16HBE接种于6孔板中,实验组试剂处理6h;37℃ DCFH⁃DA探针避光作用30min,PBS洗涤2次,激光扫描共聚焦显微镜(LSM 5LIVE,Zeiss公司,德国) 调节为FL1Log通道,525nm波长,观察细胞并拍照。随机取4个不同视野行统计学分析。

  • 1.3 统计学方法

  • 使用Graphpad 6.0和SPSS 20统计软件进行分析处理,计量资料以均值±标准差(x-±s)表示,单因素方差分析比较多组间差异。P <0.05为差异有统计学意义。

  • 2 结果

  • 2.1 类胰蛋白酶促HBE细胞修复与迁移

  • 上皮细胞发生EMT时,修复、迁移功能增强。本研究结果显示,对照组HBE细胞形态呈典型的鹅卵石样特征,修复、迁移功能较弱(图1);然而给予不同浓度(1、10、50、100pmol/L)类胰蛋白酶作用后,细胞呈现纺锤形,修复、迁移功能明显增强(图1)。当浓度≥50pmol/L时,类胰蛋白酶可显著上调细胞的修复、迁移能力(P< 0.05,图1);浓度达100pmol/L时,细胞修复、迁移能力与TGF⁃β阳性对照组(10ng/mL)相近(图1)。

  • 图1 类胰蛋白酶促16HBE细胞修复与迁移

  • Fig.1 Tryptase promoted the repair and migration of 16HBE cells

  • 2.2 类胰蛋白酶下调HBE细胞E⁃cadherin、上调N⁃ cadherin及α⁃SMA蛋白表达

  • EMT过程中,往往伴随上皮、间充质细胞特征标志物水平的变化。蛋白印迹结果显示,不同浓度类胰蛋白酶处理16HBE后,间充质细胞标志物 (N⁃cadherin及α⁃SMA)表达上调,上皮细胞标志物 (E ⁃cadherin)表达降低(P< 0.05,图2)。故选择100pmol/L浓度进行后续研究。

  • 2.3 类胰蛋白酶上调HBE细胞ROS的水平

  • 既往肺纤维化研究提示,ROS是疾病进展的重要信号,胞内ROS水平与EMT诱导密切相关。本研究采用DCFH⁃DA探针结合荧光显微镜观察ROS水平变化,结果显示与对照组相比,100pmol/L类胰蛋白酶显著上调了HBE细胞ROS的水平,其效应与TGF ⁃β相近(P< 0.05,图3)。200 μmol/L浓度的PAR2阻断剂FS,可有效阻断类胰蛋白酶的促ROS生成效应(P< 0.05,图3)。

  • 图2 类胰蛋白酶对间充质细胞标志物(N⁃cadherin及α⁃SMA)和上皮细胞标志物(E⁃cadherin)的影响

  • Fig.2 Effects of tryptase on mesenchymal markers(N⁃cadherin and α⁃SMA)and epithelial marker(E⁃cadherin)

  • 2.4 ROS介导类胰蛋白酶促HBE细胞修复、迁移效应

  • 为了探讨类胰蛋白酶引起的上皮细胞ROS水平上调是否是诱导EMT过程的重要信号,应用ROS强效清除剂NAC处理细胞,观察类胰蛋白酶的促HBE细胞修复、迁移效应是否被抑制。如图4显示,与类胰蛋白酶单处理组相比,同时给予2mmol/L NAC下调ROS水平,可显著抑制16HBE细胞的修复及迁移(P< 0.05),提示ROS水平增高是类胰蛋白酶上调细胞修复与迁移的重要环节。

  • 图3 类胰蛋白酶通过激活PAR2上调16HBE ROS水平

  • Fig.3 Tryptase increased ROS production in 16HBE by activating PAR2

  • 3 讨论

  • 多种慢性疾病如器官纤维化、上皮源性肿瘤侵袭转移中,EMT是病程进展的关键环节,可引发后续一系列复杂的不良事件,包括细胞骨架重构, ECM沉积,肿瘤细胞扩散、迁移、分化等[13]。呼吸系统疾病中,EMT作为气道重塑的重要特征,与支气管细胞功能及结构损伤密切相关,是肺功能障碍的重要预测因素[14]。哮喘患者肺组织中,往往出现活化的成纤维/肌成纤维细胞,ECM(如胶原蛋白、纤连蛋白、基质金属蛋白酶⁃9)堆积、气道重塑[15]。EMT作为肌成纤维细胞的重要来源途径,其事件特征为上皮细胞逐步转化为α⁃SMA阳性的肌成纤维细胞,失去E⁃cadherin等上皮标记物[16]。本研究通过体外培养16HBE、建立支气管上皮细胞模型,首次证实选择性激活PAR2可通过氧化还原机制、诱导气道上皮发生EMT,细胞表型向间充质转化。PAR2的天然激动剂——类胰蛋白酶,通过上调细胞ROS信号、实现促EMT效应,可能是哮喘气道重塑、尤其是上皮重塑的重要机制之一。

  • 图4 NAC抑制类胰蛋白酶诱导的细胞修复与迁移

  • Fig.4 NAC inhibited cell repair and migration induced by tryptase

  • 肺和气道(包括支气管上皮细胞)存在大量PAR2表达,但其病理生理功能研究甚少。近期研究发现,肺纤维化患者肺组织PAR2表达上调[17];激活PAR2可诱导肺部炎症,引发成纤维细胞活化如增殖、迁移、分化为肌成纤维细胞等[18];同时,多项证据显示PAR2可通过介导气道上皮活化,促进基质金属蛋白酶⁃9的合成与分泌[19]。上述系列研究结果均提示,PAR2的状态与气道结构及功能存在紧密联系。

  • 类胰蛋白酶,作为机体内PAR2的天然激动剂,主要来源于肥大细胞,在炎症及过敏反应中发挥了重要作用[20]。多项研究证实,其可促进成纤维细胞分化[21]、诱导气道平滑肌细胞增殖[22],在肺纤维化的发生发展中十分关键。然而,类胰蛋白酶对气道上皮是否存在效应、与气道上皮发生EMT是否相关,目前所知甚少。本研究首次观测了PAR2活化对气道上皮表型及功能的影响,探究了16HBE细胞PAR2激活能否诱发EMT及其可能机制。结果显示予气道上皮细胞以类胰蛋白酶处理后,细胞逐步丧失上皮特征而向间充质表型转化,且呈浓度依赖性,提示类胰蛋白酶可诱导气道上皮EMT过程。

  • 支气管上皮细胞EMT的潜在机制目前仍未完全阐明。研究表明,PAR2活化与线粒体ROS生成相关[12]。事实上,细胞ROS水平是影响EMT过程的重要因素[23]。而激活PAR2诱发气道上皮EMT是否经由氧化应激通路,至今仍未可知。本研究结果揭示,类胰蛋白酶通过激活气道上皮中的PAR2、促ROS生成,从而介导细胞EMT的发生。上皮细胞接受不同应激如理化因素、细胞因子等,可显著上调ROS水平、调控下游重要事件。上皮细胞ROS主要源自线粒体,是上皮实现诸多功能的公认信号分子,对细胞存活、细胞增殖分化、迁移、血管生成等至关重要[11]。然而,过量产生的ROS可致上皮功能障碍、引起细胞氧化损伤,因此成为多种呼吸系统疾病如哮喘、肺纤维化、急性肺损伤的重要致病因子[24-26]

  • 越来越多的证据显示,PAR2活化可促进不同类型细胞ROS产生。本研究围绕气道上皮表型与功能,发现PAR2经ROS信号、调控上皮EMT过程,可能为治疗多种慢性气道疾病如哮喘等提供新的治疗策略。本研究结果表明:①类胰蛋白酶促进16HBE细胞表型向间充质转化,即诱导气道上皮发生EMT;②气道上皮PAR2活化可引起细胞ROS大量产生;③ROS信号参与PAR2介导的EMT过程。研究首次证实激活PAR2可诱发和加重上皮细胞EMT,揭示ROS在PAR2介导EMT中的重要作用,为临床多种慢性气道疾病的防治提供了潜在靶点。然而作为胞内重要的信号分子,ROS来源较广。本研究揭示了ROS在类胰蛋白酶调控EMT过程中的重要作用,但未进一步探讨类胰蛋白酶上调ROS的具体分子机制;同时16HBE作为转化的永生系气道上皮细胞,与气液交界培养的原代气道上皮细胞有所差异,因此有待进一步深入探索。

  • 参考文献

    • [1] HOUGH K P,CURTISS M L,BLAIN T J,et al.Airway re⁃ modeling in asthma[J].Front Med(Lausanne),2020,7:191

    • [2] LIU G,PHILP A M,CORTE T,et al.Therapeutic targets in lung tissue remodelling and fibrosis[J].Pharmacol Ther,2021,225:107839

    • [3] JOLLY M K,WARD C,EAPEN M S,et al.Epithelial ⁃ mesenchymal transition,a spectrum of states:role in lung development,homeostasis,and disease[J].Dev Dyn,2018,247(3):346-358

    • [4] HADDAD A,GAUDET M,PLESA M,et al.Neutrophils from severe asthmatic patients induce epithelial to mesen⁃ chymal transition in healthy bronchial epithelial cells[J].Respir Res,2019,20(1):234

    • [5] KHALIL N,O'CONNOR R N,UNRUH H W,et al.In⁃ creased production and immunohistochemical localization of transforming growth factor ⁃ beta in idiopathic pulmo⁃ nary fibrosis[J].Am J Respir Cell Mol Biol,1991,5(2):155-162

    • [6] LIN C,BORENSZTAJN K,SPEK C A.Targeting coagula⁃ tion factor receptors ⁃ protease⁃activated receptors in idio⁃ pathic pulmonary fibrosis[J].J Thromb Haemost,2017,15(4):597-607

    • [7] HEUBERGER D M,SCHUEPBACH R A.Protease ⁃acti⁃ vated receptors(PARs):mechanisms of action and poten⁃ tial therapeutic modulators in PAR ⁃ driven inflammatory diseases[J].Thromb J,2019,17:4

    • [8] CARROLL E L,BAILO M,REIHILL J A,et al.Trypsin⁃ like proteases and their role in muco⁃obstructive lung dis⁃ eases[J].Int J Mol Sci,2021,22(11):5817

    • [9] BARDOU O,MENOU A,FRANÇOIS C,et al.Membrane⁃ anchored serine protease matriptase is a trigger of pulmo⁃ nary fibrogenesis[J].Am J Respir Crit Care Med,2016,193(8):847-860

    • [10] ITOH Y,SENDO T,OISHI R.Physiology and pathophysi⁃ ology of proteinase ⁃ activated receptors(PARs):role of tryptase/PAR ⁃ 2 in vascular endothelial barrier function [J].J Pharmacol Sci,2005,97(1):14-19

    • [11] SIES H,JONES D P.Reactive oxygen species(ROS)as pleiotropic physiological signalling agents[J].Nat Rev Mol Cell Biol,2020,21(7):363-383

    • [12] PARK Y,LEE B,KIM D,et al.PAR2 deficiency induces mitochondrial ROS generation and dysfunctions,leading to the inhibition of adipocyte differentiation[J].Oxid Med Cell Longev,2021,2021:6683033

    • [13] DONGRE A,WEINBERG R A.New insights into the mechanisms of epithelial⁃mesenchymal transition and im⁃ plications for cancer[J].Nat Rev Mol Cell Biol,2019,20(2):69-84

    • [14] WALTERS E H,SHUKLA S D,MAHMOOD M Q,et al.Fully integrating pathophysiological insights in COPD:an updated working disease model to broaden therapeutic vi⁃ sion[J].Eur Respir Rev,2021,30(160):200364

    • [15] BURGSTALLER G,OEHRLE B,GERCKENS M,et al.The instructive extracellular matrix of the lung:basic com⁃ position and alterations in chronic lung disease[J].Eur Respir J,2017,50(1);1601805

    • [16] ZHOU J,CHENG H,WANG Z,et al.Bortezomib attenu⁃ ates renal interstitial fibrosis in kidney transplantation via regulating the EMT induced by TNF ⁃ α ⁃ Smurf1 ⁃ Akt ⁃ mTOR ⁃ P70S6K pathway[J].J Cell Mol Med,2019,23(8):5390-5402

    • [17] WYGRECKA M,KWAPISZEWSKA G,JABLONSKA E,et al.Role of protease ⁃activated receptor ⁃ 2 in idiopathic pulmonary fibrosis[J].Am J Respir Crit Care Med,2011,183(12):1703-1714

    • [18] BAGHER M,LARSSON⁃CALLERFELT A K,ROSMARK O,et al.Mast cells and mast cell tryptase enhance migra⁃ tion of human lung fibroblasts through protease ⁃activated receptor 2[J].Cell Commun Signal,2018,16(1):59

    • [19] VLIAGOFTIS H,SCHWINGSHACKL A,MILNE C D,et al.Proteinase⁃activated receptor⁃2⁃mediatedmatrixmetallo⁃ proteinase⁃9 release from airway epithelial cells[J].J Al⁃ lergy Clin Immunol,2000,106(3):537-545

    • [20] MATEJA A,WANG Q,CHOVANEC J,et al.Defining baseline variability of serum tryptase levels improves accuracy in identifying anaphylaxis[J].J Allergy Clin Im⁃ munol,2021,149(3):1010-1017

    • [21] LIU B,YANG M Q,YU T Y,et al.Mast cell tryptase pro⁃ motes inflammatory bowel disease ⁃ induced intestinal fibrosis[J].Inflamm Bowel Dis,2021,27(2):242-255

    • [22] BERGER P,PERNG D,THABREW H,et al.Tryptase and agonists of PAR ⁃2 induce the proliferation of human airway smooth muscle cells[J].J Appl Physiol,2001,91(3):1372-1379

    • [23] JIANG J,WANG K,CHEN Y,et al.Redox regulation in tumor cell epithelial ⁃ mesenchymal transition:molecular basis and therapeutic strategy[J].Signal Transduct Tar⁃ get Ther,2017,2:17036

    • [24] VASCONCELOS L,FERREIRA S,SILVA M,et al.Un⁃ covering the role of oxidative imbalance in the develop⁃ ment and progression of bronchial asthma[J].Oxid Med Cell Longev,2021,2021:6692110

    • [25] FU L,ZHAO H,XIANG Y,et al.Reactive oxygen species ⁃evoked endoplasmic reticulum stress mediates 1⁃nitropy⁃ rene ⁃induced epithelial ⁃mesenchymal transition and pul⁃ monary fibrosis[J].Environ Pollut,2021,283:117134

    • [26] CEN M,OUYANG W,ZHANG W,et al.MitoQ protects against hyperpermeability of endothelium barrier in acute lung injury via a Nrf2⁃ dependent mechanism[J].Redox Biol,2021,41:101936

  • 参考文献

    • [1] HOUGH K P,CURTISS M L,BLAIN T J,et al.Airway re⁃ modeling in asthma[J].Front Med(Lausanne),2020,7:191

    • [2] LIU G,PHILP A M,CORTE T,et al.Therapeutic targets in lung tissue remodelling and fibrosis[J].Pharmacol Ther,2021,225:107839

    • [3] JOLLY M K,WARD C,EAPEN M S,et al.Epithelial ⁃ mesenchymal transition,a spectrum of states:role in lung development,homeostasis,and disease[J].Dev Dyn,2018,247(3):346-358

    • [4] HADDAD A,GAUDET M,PLESA M,et al.Neutrophils from severe asthmatic patients induce epithelial to mesen⁃ chymal transition in healthy bronchial epithelial cells[J].Respir Res,2019,20(1):234

    • [5] KHALIL N,O'CONNOR R N,UNRUH H W,et al.In⁃ creased production and immunohistochemical localization of transforming growth factor ⁃ beta in idiopathic pulmo⁃ nary fibrosis[J].Am J Respir Cell Mol Biol,1991,5(2):155-162

    • [6] LIN C,BORENSZTAJN K,SPEK C A.Targeting coagula⁃ tion factor receptors ⁃ protease⁃activated receptors in idio⁃ pathic pulmonary fibrosis[J].J Thromb Haemost,2017,15(4):597-607

    • [7] HEUBERGER D M,SCHUEPBACH R A.Protease ⁃acti⁃ vated receptors(PARs):mechanisms of action and poten⁃ tial therapeutic modulators in PAR ⁃ driven inflammatory diseases[J].Thromb J,2019,17:4

    • [8] CARROLL E L,BAILO M,REIHILL J A,et al.Trypsin⁃ like proteases and their role in muco⁃obstructive lung dis⁃ eases[J].Int J Mol Sci,2021,22(11):5817

    • [9] BARDOU O,MENOU A,FRANÇOIS C,et al.Membrane⁃ anchored serine protease matriptase is a trigger of pulmo⁃ nary fibrogenesis[J].Am J Respir Crit Care Med,2016,193(8):847-860

    • [10] ITOH Y,SENDO T,OISHI R.Physiology and pathophysi⁃ ology of proteinase ⁃ activated receptors(PARs):role of tryptase/PAR ⁃ 2 in vascular endothelial barrier function [J].J Pharmacol Sci,2005,97(1):14-19

    • [11] SIES H,JONES D P.Reactive oxygen species(ROS)as pleiotropic physiological signalling agents[J].Nat Rev Mol Cell Biol,2020,21(7):363-383

    • [12] PARK Y,LEE B,KIM D,et al.PAR2 deficiency induces mitochondrial ROS generation and dysfunctions,leading to the inhibition of adipocyte differentiation[J].Oxid Med Cell Longev,2021,2021:6683033

    • [13] DONGRE A,WEINBERG R A.New insights into the mechanisms of epithelial⁃mesenchymal transition and im⁃ plications for cancer[J].Nat Rev Mol Cell Biol,2019,20(2):69-84

    • [14] WALTERS E H,SHUKLA S D,MAHMOOD M Q,et al.Fully integrating pathophysiological insights in COPD:an updated working disease model to broaden therapeutic vi⁃ sion[J].Eur Respir Rev,2021,30(160):200364

    • [15] BURGSTALLER G,OEHRLE B,GERCKENS M,et al.The instructive extracellular matrix of the lung:basic com⁃ position and alterations in chronic lung disease[J].Eur Respir J,2017,50(1);1601805

    • [16] ZHOU J,CHENG H,WANG Z,et al.Bortezomib attenu⁃ ates renal interstitial fibrosis in kidney transplantation via regulating the EMT induced by TNF ⁃ α ⁃ Smurf1 ⁃ Akt ⁃ mTOR ⁃ P70S6K pathway[J].J Cell Mol Med,2019,23(8):5390-5402

    • [17] WYGRECKA M,KWAPISZEWSKA G,JABLONSKA E,et al.Role of protease ⁃activated receptor ⁃ 2 in idiopathic pulmonary fibrosis[J].Am J Respir Crit Care Med,2011,183(12):1703-1714

    • [18] BAGHER M,LARSSON⁃CALLERFELT A K,ROSMARK O,et al.Mast cells and mast cell tryptase enhance migra⁃ tion of human lung fibroblasts through protease ⁃activated receptor 2[J].Cell Commun Signal,2018,16(1):59

    • [19] VLIAGOFTIS H,SCHWINGSHACKL A,MILNE C D,et al.Proteinase⁃activated receptor⁃2⁃mediatedmatrixmetallo⁃ proteinase⁃9 release from airway epithelial cells[J].J Al⁃ lergy Clin Immunol,2000,106(3):537-545

    • [20] MATEJA A,WANG Q,CHOVANEC J,et al.Defining baseline variability of serum tryptase levels improves accuracy in identifying anaphylaxis[J].J Allergy Clin Im⁃ munol,2021,149(3):1010-1017

    • [21] LIU B,YANG M Q,YU T Y,et al.Mast cell tryptase pro⁃ motes inflammatory bowel disease ⁃ induced intestinal fibrosis[J].Inflamm Bowel Dis,2021,27(2):242-255

    • [22] BERGER P,PERNG D,THABREW H,et al.Tryptase and agonists of PAR ⁃2 induce the proliferation of human airway smooth muscle cells[J].J Appl Physiol,2001,91(3):1372-1379

    • [23] JIANG J,WANG K,CHEN Y,et al.Redox regulation in tumor cell epithelial ⁃ mesenchymal transition:molecular basis and therapeutic strategy[J].Signal Transduct Tar⁃ get Ther,2017,2:17036

    • [24] VASCONCELOS L,FERREIRA S,SILVA M,et al.Un⁃ covering the role of oxidative imbalance in the develop⁃ ment and progression of bronchial asthma[J].Oxid Med Cell Longev,2021,2021:6692110

    • [25] FU L,ZHAO H,XIANG Y,et al.Reactive oxygen species ⁃evoked endoplasmic reticulum stress mediates 1⁃nitropy⁃ rene ⁃induced epithelial ⁃mesenchymal transition and pul⁃ monary fibrosis[J].Environ Pollut,2021,283:117134

    • [26] CEN M,OUYANG W,ZHANG W,et al.MitoQ protects against hyperpermeability of endothelium barrier in acute lung injury via a Nrf2⁃ dependent mechanism[J].Redox Biol,2021,41:101936