【作者】 黄武锋；

【导师】 蔡绍曦；

【作者基本信息】 南方医科大学 ， 内科学， 2014， 博士

【摘要】 支气管哮喘(简称哮喘)是多种细胞(如嗜酸性粒细胞、T淋巴细胞、中性粒细胞、气道上皮细胞等)和细胞组份(cellular elements)参与的慢性气道炎症性疾病,伴有气道高反应性、气道阻塞和气道重塑。哮喘患病率在全球范围内有逐年增加的趋势,目前全球至少有3亿以上,我国有近3000万哮喘患者,哮喘已成为仅次于癌症的世界第2大致死和致残疾病,严重危害人类健康。哮喘是一种基因和环境因素相互作用导致的复杂疾病,即使在今天生物-环境-心理的疾病评估模式中,其发病、发展等环节仍不清楚。因此探讨哮喘发病机制、寻找新的治疗乃至预防靶点仍然是哮喘研究的重大课题。随着研究的不断拓展和深入,关于哮喘发病的假说不断增多,其中气道上皮结构、功能受损和免疫作用异常成为研究热点,气道上皮在哮喘发病中的地位被大大提升。目前认为气道上皮细胞是哮喘发病关键环节。气道上皮正常情况处于免疫耐受状态,是机体抵抗气道内外环境有害刺激的第一道屏障。上皮细胞维持正常结构及功能,需要相邻细胞通过不同连接蛋白及信号分子形成紧密连接、粘附连接及桥粒连接,并通过连接蛋白协调表达和相互作用,保持上皮细胞结构的完整及极性,调节物质转运。紧密连接位于气道上皮顶端,由occludin及claudin蛋白家族构成,主要负责调控旁细胞通透性以及维持上皮极性。紧靠紧密连接下方是粘附连接,是由Ca2+依赖的E-cadherin以及与E-cadherin相结合的多种连环蛋白(如a、p连环蛋白)和肌动蛋白细胞骨架相互作用形成的,E-cadherin具有负性免疫调控作用,与上皮免疫耐受有关。般认为,粘附连接是紧密连接形成的前提,它的形成活化了细胞内多种有活性的蛋白质分子,最终导致紧密连接相关蛋白聚集于相邻细胞接触部位,形成一个完整的顶端连接复合体。气道上皮细胞不但参与气道上皮物理屏障的形成,而且通过分泌多种活性物质发挥免疫调节作用。气道上皮细胞表面及胞浆中存在模式识别受体(PRRs),主要为Toll样受体(TLRs)和晚期糖基化终产物受体(RAGE)。模式识别受体(PRRs)被激活后,经过一系列衔接蛋白的信号转导后,激活NF-κB或IRF-3/7等转录调节因子,调节相关效应分子的基因转录,最终表现为抗菌蛋白、干扰素、促炎因子、趋化因子的产生和释放,参与哮喘慢性气道炎症的启动和维持。当哮喘发病时,气道上皮及固有免疫细胞可释放以高迁移率族蛋白1(HMGB1)为代表的DAMP(损伤相关分子模式)分子,进而启动气道固有免疫及获得性免疫,打破气道上皮免疫耐受,释放更多炎症介质,炎症介质相互作用于气道上皮,形成复杂细胞因子调节网络,引发和促进哮喘发生。因此,炎症介质致气道上皮功能失调是促发和维持哮喘发病的重要机制之一。高迁移率族蛋白(HMGB1)是真核细胞大量存在的一种染色体结合蛋白,广泛分布于淋巴组织、脑、肝、肺、心、脾、肾等组织中,其基本功能参与调控DNA重组、修复、复制和基因转录。正常情况下肺组织HMGB1主要位于结构细胞核中,处于低表达水平,但对维持肺及气道的正常生理功能具有重要作用。近年来在动物模型和人类疾病的研究上发现：HMGB1可释放到胞外,发挥促炎因子的功能。HMGB1可以从激活的免疫细胞如单核巨噬细胞中释放出来,也能够从损伤和坏死的细胞被动释放出来。HMGB1可以诱导许多炎症因子如TNF-α,IL-1, IL-6和IL-8等的释放,也能激活人内皮细胞,导致其粘附分子的表达上调。因此,HMGB1作为一种重要上游免疫调节因子,当细胞损伤后其释放出细胞外,会激发免疫系统产生炎症反应。HMGB1是一种重要的炎症介质,与脓毒症、免疫性疾病、恶性肿瘤等疾病相关,近期研究显示HMGB1可能参与多个呼吸系统疾病的病理生理过程,在哮喘等慢性炎症气道疾病的地位也越来越受到人们的重视。当气道上皮细胞受到过敏原、微生物等有害因素攻击而受损时,根据Matzinger的危险模式理论,可产生某些内源性危险信号分子,也就是DAMPs分子,又称警报素(alarmin)。HMGB1是DAMPs家族的重要组成成分之一。气道上皮受到有害因素攻击后,HMGB1可由胞核释放到胞外,与Toll样受体(TLRs)、晚期糖基化终产物受体(RAGE)等膜受体结合,引起前炎症因子IL-1释放,刺激机体固有免疫系统产生防御反应及组织修复。释放到胞外的HMGB1其本身也能够诱导单核／巨噬细胞、中性粒细胞和树突状细胞等获得性免疫细胞合成并分泌TNF-α、INF-γ及IL-1β等炎症介质,这些炎症介质又能够加强HMGB1的分泌效应,从而形成一个复杂的细胞因子分泌调节网络。这说明以HMGB1为代表的DAMPs分子在气道炎症过程致敏阶段固有免疫向获得性免疫转化中起重要作用,是调控TNF-α、INF-y及IL-1β等炎症介质的关键上游分子。本实验室早期,在卵蛋白(OVA)诱导的小鼠哮喘模型观察到肺组织及肺泡灌洗液HMGB1表达显著升高。我们进一步研究也发现,哮喘患者诱导痰及血浆中HMGB1水平显著升高,且诱导痰HMGB1水平与肺功能指标显著负相关,这提示HMGB1参与哮喘气道炎症。为进一步评价HMGB1在哮喘发病中作用,韩国学者在OVA诱导的小鼠哮喘模型阻断HMGB1活性,发现能够显著降低气道高反应性,改善气道局部炎症反应及病理改变。2013年最新研究表明HMGB1受体RAGE缺失的小鼠,显著减少屋尘螨(HDM)致哮喘Th2型细胞因子表达,气道嗜酸性粒细胞浸润以及血浆IgE水平,这一发现使得HMGB1-RAGE受体轴在哮喘发病的重要作用愈发受人关注。据此,我们认为DAMPs分子-HMGB1是参与哮喘发病的重要炎症介质,可能是一种新的哮喘慢性气道炎症调控机制。我们已证实,哮喘患者诱导痰及血清中HMGB1水平显著上调,且与疾病严重程度相关,提示HMGB1是参与哮喘发生、发展过程重要炎症介质。目前,关于哮喘气道上皮功能失调的研究主要集中在TNF-α、IFN-γ及IL-1β等下游炎症介质的致伤作用,而对于调控TNF-α、IFN-γ及IL-1β的关键上游DAMPs分子-HMGB1对气道上皮功能的影响及调节机制研究相对较少,有必要深入的研究。目的：1.探讨HMGB1致气道上皮屏障功能损伤作用及可能参与的信号转导调节机制；2.进一步明确HMGB1协同IL-1β是否对气道上皮屏障功能损伤有促进作用；方法：1.培养气道上皮细胞株16-HBE及A-549。2.MTT比色法检测HMGB1对气道上皮细胞株16-HBE及A-549细胞活力的影响：选取不同浓度(100、200、400和800ng/ml) HMGB1与16-HBE及A-549细胞共培养,MTT比色法检测细胞活力。3.观察HMGB1对气道上皮屏障功能的损伤作用-剂量效应实验。不同浓度(100.200、400ng/ml) HMGB1刺激16-HBE及A-549气道上皮细胞株24h,观察其对气道上皮屏障功能损伤作用,屏障功能观察指标主要有：①单层气道上皮细胞通透性检测：Transwell技术检测单层16-HBE及A-549细胞跨膜电阻值(TER)以及FITC右旋糖苷通透性(Pa/Pc)的改变。②Western bolt方法检测紧密连接蛋白(occludin和claudin-2)及粘附连接蛋白(E-cadherin和β-catenin)表达情况；③免疫荧光化学方法检测紧密连接蛋白(occludin和claudin-2)或粘附连接蛋白(E-cadherin和β-catenin)分布情况；4.观察HMGB1对气道上皮屏障功能的损伤作用-时间效应实验。400ng/ml HMGB1在不同时间点(0、1、3、6、12、24和48h)刺激16-HBE和A-549细胞,观察其对气道上皮屏障功能的损伤作用,屏障功能观察指标参照方法3；5.观察HMGB1协同IL-1β对气道上皮屏障功能损伤的作用。实验分为4组：①对照组；②单独IIMGB1(100ng/ml)刺激组；③单独IL-1β (2.5ng/ml)刺激组；④HMGB1(100ng/ml)/IL-1β (2.5ng/ml)联合刺激组。按上述分组处理16-HBE细胞24h,观察其对气道上皮屏障功能损伤作用,屏障功能观察指标参照方法3；6.评价连接蛋白occludin在HMGB1致气道上皮屏障功能损伤中的作用：构建含野生型全长occludin基因质粒,扩增、提取并鉴定质粒。应用碱裂解法提取目的质粒,采用琼脂凝胶电泳和DNA测序鉴定目的基因,并测定滴度。应用阳离子脂质体法分别将occludin基因转染气道上皮细胞并过表occludin蛋白,通过倒置荧光显微镜观察GFP情况以了解转染效率,再应用QPCR和Western bolt技术确定有效的基因转染；转染16-HBE细胞。①检测过表达occludin蛋白对跨膜电阻(TER)、FITC右旋糖苷通透性(Pa/Pc)以及其他连接蛋白表达的影响；②评价连接蛋白occludin在HMGB1致气道上皮屏障功能损伤中的作用：HMGB1(400ng/ml)分别刺激过表达和正常表达occludin蛋白的16-HBE细胞；实验分为4组：正常表达组(未转染occlduin基因正常细胞),过表达组(转染并过表达occlduin蛋白的16-HBE细胞),正常表达+HMGB1刺激组(HMGB1刺激正常表达occludin蛋白的16-HBE细胞24h)和过表达+HMGB1刺激组(HMGB1刺激过表达occludin蛋白的16-HBE细胞24h)。按照分组给予相应处理后,观察和比较跨膜电阻值(TER)及FITC右旋糖苷通透性(Pa/Pc)的变化。7.探讨参与HMGB1致气道上皮屏障功能损伤作用的信号通路。观察HMGB1致气道上皮屏障功能损伤过程中HMGB1受体(TLR2、TLR4及RAGE)蛋白表达水平变化,以及其下游MAPK信号通路活化情况。在上述实验基础上,选择相应抗-膜受体单克隆抗体和MAPK信号通路阻断剂,检测其对跨膜电阻(TER)、FITC右旋糖苷通透性(Pa/Pc)及连接蛋白的影响,发现参与HMGB1致气道上皮屏障损伤的可能信号通路途径。结果：1、与对照组比较,100ng/ml、200ng/ml、400ng/ml HMGB1对细胞活力无明显影响(P值均>0.05,n=6),而浓度达到800ng/ml的HMGB1刺激细胞明显降低细胞活力(P值均<0.001,n=6)。2, HMGB1对气道上皮细胞株16HBE和A549屏障功能的影响：HMGB1可导致16HBE和A549单层细胞跨膜电阻值(TER)显著下降和FITC右旋糖苷通透性(Pa/Pc)显著增加,跨膜电阻值(TER)和FITC右旋糖苷(FITC-DX)通透性改变呈时间剂量依赖效应。与对照组比较,分别给予100ng/ml、200ng/ml、400ng/ml HMGB1刺激16HBE或A549细胞,均可导致跨膜电阻值(TER)下降和FITC右旋糖苷通透性(Pa/Pc)增加,其中400ng/ml HMGB1引起改变最为显著(P<0.001)。在400ng/ml HMGB1刺激A549细胞6h或者16HBE细胞12h,即可观察到跨膜电阻值(TER)下降,呈时间依赖关系(A549细胞：F=80.46,P<0.001;16HBE细胞：F=49.62,P<0.001)。与对照组比较,400ng/ml HMGB1刺激细胞12h、24h、48h均可导致FITC右旋糖苷通透性(Pa/Pc)增加(P<0.001),且呈时间依赖关系。与16HBE细胞比较,HMGB1刺激A549细胞对跨膜电阻值(TER)和FITC右旋糖苷通透性(Pa/Pc)的改变更加显著。3、HMGB1对气道上皮细胞株16HBE和A549连接蛋白表达及分布的影响：①A549细胞：400ng/ml HMGB1刺激细胞12h后,紧密连接蛋白occludin和claudin-2表达水平显著减少；而刺激细胞48h后粘附连接蛋白E-cadherin和β-catenin表达水平才显著减少；刺激细胞48h对连接蛋白表达的影响最明显。②16HBE细胞:400ng/ml HMGB1刺激细胞24h时,紧密连接蛋白occludin和claudin-2表达水平显著减少,刺激细胞48h时蛋白水平进一步减少；而粘附连接蛋白E-cadherin和β-catenin表达水平无明显变化。但免疫荧光定位显示HMGB1可促进E-cadherin蛋白由胞膜向胞浆移位并破坏β-catenin细胞膜结构稳定性。4、紧密连接蛋白occludin参与HMGB1致气道上皮屏障功能损伤作用：与正常表达occludin蛋白的16HBE细胞比较,过表达occludin蛋白导致16HBE细胞的跨膜电阻值(TER)增加、FITC右旋糖苷通透性(Pa/Pc)减少,有统计学差异(P<0.001),但过表达occludin蛋白对连接蛋白E-cadherin、β-catenin和claudin-2的表达水平无显著影响；16HBE细胞过表达occludin蛋白可以显著抑制HMGB1导致的气道上皮屏障功能损伤[跨膜电阻值(TER)降低和对FITC右旋糖苷通透性(Pa/Pc)增加],与HMB1刺激正常的16HBE细胞引起屏障功能损伤相比较,有统计学差异(P<0.001,n=5)。5、HMGB1协同IL-1β促进气道上皮屏障功能损伤作用的结果：①与对照组比较,单独HMGB1(100ng/ml)刺激16HBE细胞24h,跨膜电阻值(TER)无明显改变(P=0.091),但可增加FITC右旋糖苷通透性(Pa/Pc)(P<0.001)。与对照组及单独HMGB1(100ng/ml)刺激比较,HMGB1(100ng/ml)协同IL-1β(2.5ng/ml)可显著降低跨膜电阻值(TER)(P=0.002和P=0.04),并显著增加FITC右旋糖苷通透性(Pa/Pc)(P<0.001);②对连接蛋白表达及分布的影响：单独HMGB1或IL-1β刺激对连接蛋白表达及分布无影响,HMGB1协同IL-1β显著减少occludin及claudin-2蛋白的表达水平(P=0.004和P=0.001)；免疫荧光定位结果显示HMGB1可促进E-cadherin蛋白由胞膜向胞浆移位,并破坏β-catenin细胞膜结构稳定性。6、RAGE/ERK信号通路参与HMGB1致气道上皮屏障损伤及连接蛋白破坏：①Western bolt结果显示：HMGB1可显著增加16HBE细胞RAGE蛋白表达水平,1h开始明显增加,24减少至正常水平,而对TLR2和TLR4蛋白表达水平无影响；抗RAGE单克隆抗体预处理16HBE细胞1h,可部分抑制HMGB1引起跨膜电阻值(TER)下降及FITC右旋糖苷通透性(Pa/Pc)增加(P=0.001和P=0.002)；②HMGB1可引起16HBE细胞MAPK信号通路活化,分别在HMGB1刺激后6,7,5h可观察到ERK、PI3K/akt及JN磷酸化水平明显增加；使用ERK通路抑制剂(U0126)后,可以部分抑制HMGB1引起跨膜电阻值(TER)下降及FITC右旋糖苷通透性(Pa/Pc)增加,而使用PI3K/akt和JNK信号通路抑制剂(LY294002和SP600125)并未观察到类似效应；③抗RAGE单克隆抗体能抑制HMGB1引起ERK磷酸化；④使用ERK通路抑制剂(U0126)后能减少HMGB1引起16HBE细胞occludin及claudin-2蛋白的破坏,并抑制E-cadherin蛋白由胞膜向胞浆移位及β-catenin细胞膜结构的破坏。结论：1、HMGB1可导致气道上皮细胞屏障功能损伤,主要表现为跨膜电阻值(TER)显著下降和FITC右旋糖苷通透性(Pa/Pc)显著增加,这种损伤作用呈现为时间-剂量依赖效应；HMGB1协同IL-1p可以促进对气道上皮屏障功能损伤作用；2、在HMGB1引起气道上皮细胞屏障功能损伤同时,减少紧密蛋白occludin及claudin-2蛋白表达水平,其中紧密蛋白occludin在HMGB1引起气道上皮细胞屏障功能损伤中发挥重要作用。此外HMGB1还可以促进粘附连接蛋白E-cadherin由胞膜向胞浆移位及破坏β-catenin细胞膜结构稳定性。3、HMGB1可能通过RAGE/ERK信号通路参与气道上皮细胞屏障功能损伤,连接蛋白表达及分布异常；更多还原

【Abstract】 Asthma is a chronic inflammatory airway disorder involved multiple cells (eosnophils, neutrophils, T lymphocytes and airway epithelium) and cellular element, characterized by airway hyper-responsiveness, airflow obstruction and airway remodeling. There has been a sharp increase in the global prevalence, morbidity, mortality, and economic burden associated with asthma over recent years.The bronchial epithelium is central to the pathogenesis of asthma and plays a important role in immune regulation during initiation of allergic responses. The epithelial barrier function is dependent on cellular integrity, as well as coordinate expression and interaction of proteins in cellular junctional complexes, including apical tight junctions and adherens junctions. Tight juctional proteins localize apically and are considered to be the main regulators of paracellular permeability and movement of ions and solutes between epithelial cells. Adherent junction localizes below tight junction and is composed of a calcium-dependent E-cadherin and various kinds of catenin proteins. E-cadherin is thought to provide the architecture that is required to form other junctional complexes, including tight junctions.It is now clear that the pro-inflammatory cytokine release drive airway pathology in asthma. A number of observations suggest that airway epithelial barrier function is compromised in asthma. These observations have revealed structural abnormalities in apical junctional complexes of asthmatics, which comprise the interacting proteins occludin and claudins, and adherens junction proteins E-cadherin and β-catenin, compared with that in healthy subjects. The junction proteins are supposed to be gatekeeper in charge of different aspects of epithelial barrier function. These structural and functional abnormalities of junction proteins might lead to enhanced signaling between the epithelium and underlying immune and structural cells.High-mobility group box-1protein (HMGB1) has been known to be a nuclear DNA-binding protein, participating in many pathological processes. HMGB1is not only actively secreted by innate immune cells such as macrophages and monocytes, but also passively released by injured and necrotic cells. It is secreted into the extracellular milieu and acts as a pro-inflammatory cytokine. By binding to cytokines such as IL-1β, IFN-γ and TNF-α, HMGB1protein can enhance pro-inflammatory and immune response. There are accumulating evidences that HMGB1is involved in pathological processes including inflammatory disorders of the respiratory tract. When airway epithelial cells were attacked by allergen and microorganism, it can release endogenous dangerous signaling molecule, named damage associated pattern molecules (DAMPs). HMGB1, a typical molecule of DAMPs, can be released into the extracellular milieu by the damaged epithelial cells and binds pattern-recognition receptors (PRRs) such as RAGE, TLR2and TLR4. They can function in an autocrine manner (secreted and acting on airway epithelial cell).In a previous study, we investigated the expression of HMGB1in plasma and induced sputum of patients with asthma and explored the relationship with lung function. We demonstrate that a potential role of HMGB1in the development of asthma. However, it has remained undefined how HMGB1participates in the pathogenesis of asthma. Recent studies further suggest that both HMGB1and RAGE contributes to pathogenesis of asthma. Blocking HMGB1activity decreased magnitude of the allergic response in either HDM or OVA induced mouse asthma model. RAGE knockout mice exhibit attenuated airway hypersensitivity, eosinophilic inflammation, and airway remodeling. To date, there appears to have no studies about the role of HMGB1/RAGE axle on epithelial barrier function.Therefore, the aims of our study were to determine the effects of HMGB1on epithelial barrier structure and function and to characterize possible mechanisms involved in these modulatory properties. Our findings indicate that HMGB1not only increases barrier permeability, but also selectively disrupts expression of occludin and claudin-2, dislocates E-cadherin and P-catenin via RAGE/ERK1/2signal pathways. In addition, the presence of IL-1β may facilitate HMGB1induced epithelial barrier dysfunction.Objectives:1. To investigate the effect of HMGB1induced barrier function defect in airway cells;2. To investigate the possible signaling pathway involved in HMGB1mediacted epithelial barrier dysfunction.3. To study the effect of HMGB1in synergy with IL-1β on epithelial barrier property.Methods:1. Epithelial Cell lines (16HBE and A549) culture.2. Methyltetrazolium (MTT) assay was used to assess the16HBE and A549 cell viabilities under different concentration (100,200and400ng/ml) of HMGB1stimulation.3. To study the dosage effect of HMGB1on airway epithelial barrier function impairment.16HBE or A549cell monolayer was treated with100,200and400ng/ml HMGB1for24h, respectively. And then the indicators as follows were measured:a) Transepithelial electrical resistance (TER) was measured in real-time using MILLICELL-ERS Voltohmmeter. Fluorescein isothiocyanate-dextran flux (FITC) across monolayers of cultured epithelial cells was evaluated using a fluorescent plate reader.b) Western bolt was used to analyze protein expression of junction proteins.c) Immunofluorescence microscopy was measured to evaluate the delocalization of junction proteins (E-cadhrein, β-catenin occludin and claudin-2).4. To study time effct of HMGB1on airway epithelial barrier function impairment.16HBE or A549cell monolayer was treated with400ng/ml HMGB1for0,1,3,6,12,24,48h, respectively. And then the indicators the same as method3were measured.5. To study the effect of HMGB1in synergy with IL-1βon barrier properties in16HBE cells. Groups as follows:the normal control group,100ng/ml HMGB1stimulation group,2.5ng/ml IL-1βstimulation group and HMGB1in synergy with IL-1βstimulation group. After stimulated for24h, ion and macromolecular barrier permeability were measured and Western blot and immunohistochemistry were used to detect the expression and distribution of junctional proteins.6. To investigate the crucial role of tight junction protein occludin in the HMGB1induced barrier function defect in16HBE cells. Plasmid containing wild type of full length occludin gene were constructed, amplified, extracted and identified. Plasmid was extracted by alkaline lysis method.After the agar gel electrophoresis and DNA sequencing were used to identify the target gene. Titer of occludin gene was measured. Wild type of full length occludin gene was transfected to16HBE cells by Lipofectin Reagent. QPCR and western bolt were used to analyze transfection efficiency.a) To investigate the effect of over-expressing occludin protein on barrier function and other junction protein expression (E-cadhrein, β-catenin and claudin-2) in the16HBE cells. Groups as follows:the normal vector group and over-expressing occludin group. Ion and macromolecular barrier permeability were measured and Western blot were used to detect the expression of other junctional proteins (E-cadhrein, β-catenin and claudin-2).b) To investigate the crucial role of tight junction protein occludin in the HMGB1induced barrier function defect in16HBE cells. Over-expressing and normal-expressing occludin protein in16HBE cells were stimulated with400ng/ml HMGB1and then the change of epithelial barrier properties were measured.7. To explore the possible signaling pathway involved in HMGB1induced epithelial barrier function defect. Western bolt was used to detect the expression of main membrane receptors (TLR2, TLR2and RAGE) and activation of down-stream MAPK signaling. Based on the above experiments, anti-membrane receptors antibody or inhibitor of MAPK signaling was used to detect the effect on epithelial barrier dysfunction and junction proteins disruption.Results:1. HMGB1induced an increase in ion and macromolecular barrier permeabilityIon Barrier PermeabilityThe ion barrier permeability effect of HMGB1was manifested in a concentration-and time-dependent manner. The transmembrane resistance (TER) levels started to decline at6h, with a significant effect after stimulation with HMGB1for24h(A549cells:F=82.815,P=0.001;16HBE cells:F=57.887,P=0.002). Additions of400ng/ml HMGB1induced a more significant decline in TER than the other two low concentrations HMGB1treated groups and control group (all P value＜0.001, n=3). This indicates high concentration of HMGB1displays potent ability to changes in ion barrier permeability. In addition, A549cells were more susceptible to the stimulation of HMGB1, compared with16HBE cells (maximal mean+SEM decrease,59+7% and42+2%, respectively; n=3).Macromolecular Barrier PermeabilityIn addition to its effects on ion barrier permeability, HMGB1also increased macromolecular transmission. We found that addition of HMGB1induced an increase in macromolecular permeability to FITC-labeled4-kDa dextran. A549and16HBE cells monolayer treated with400ng/ml HMGB1for48h showed an over50%increase in macromolecular permeability (all P value<0.001, n=3),compared with controls. Concentration-and time-dependent changes in FITC dextran permeability were also observed.2. HMGB1mediated disruption in the expression and localization of junction proteins.To investigate the mechanism of HMGB1induced epithelial barrier dysfunction, we studied whether the changes in barrier function were paralleled by changes in intercellular junction proteins. First we treated16HBE cells with100,200, and400ng/ml HMGB1respectively in culture medium for24h. Low expression of tight junction proteins occludin and claudin-2was observed with incubation of400ng/ml HMGB1, other than100and200ng/ml HMGB1(all P value<0.05). Then both two epithelial cell line monolayers were treated with400ng/ml HMGB1for1,6, 12,24and48h, respectively. Western blot analyses showed variable changes in tight junction proteins and adhesion junction proteins in response to HMGB1. HMGB1caused a time dependent down-regulation of occludin and claudin-2, which was already detectable at12h in A549cells and24h in16HBE cells. An even stronger time-dependent effect on tight junction proteins down-regulation was observed when cell monolayers were treated with HMGB1for48h. These modifications were in accordance with changes in ion and macromolecular barrier permeability.Surprisingly, no similar changes in the expression of E-cadherin and β-catenin were seen between the two cell lines. Down-regulated expression of E-cadherin and P-catenin was observed at48h in A549cells, while HMGB1had no effect on the expression of the two adhesion junction proteins in16HBE cells. However, Immunofluorescent staining showed delocalization of E-cadherin from cell membrane to cell plasma and disruption of β-catenin in HMGB1treated16HBE, suggesting the function of E-cadherin and β-catenin may also be affected by HMGB1and the redistribution of these proteins was the main effect3. Tight junction protein occludin play a crucial role in HMGB1induced epithelial barrier function defect.Compared with normal16HBE cells, the over-expression occludin16HBE cells showed an increase in transmembrane resistance (TER) levels and an decrease in macromolecular permeability to FITC-labeled4-kDa dextran (all P values<0.001, n=6).Howerver, over-expression of occludin had no effect on the expression of E-cadherin,p-catenine and claudin-2. Overexpression of occludin protein can partially inhibited HMGB1induced the increase in transmembrane resistance (TER) levels and the increase in macromolecular permeability to FITC-labeled4-kDa dextran in16HBE, the differences of transmembrane resistance (TER) levels and macromolecular permeability between two group had statistical significance(all P values<0.001, n=5).4. Involvement of RAGE/ERK cascade in HMGB1induced barrier dysfunction and junction proteins modification.To unravel the mechanisms underlying HMGB1induced epithelial barrier dysfunction, we considered that Pattern Recognition Receptors (PRRs) as well as downstream MAPK signalling may contribute to this type of epithelia barrier function defect. Western blot analysis was performed to evaluate the activation of PRRs [Tolls like receptor (TLR)2, TLR4and RAGE], downstream Mitogen-activated protein kinase (MAPK) signaling [p38MAPK (p38), c-Jun N-terminal Kinase (JNK) and Extracellular Signal-Regulated Kinase (ERK)] and PI3kinase/Akt signalling in16HBE cells treated with400ng/ml HMGB1for different time periods. Present study showed HMGB1increased the protein expression of RAGE at1h and reached a maximum level at12h but decreased at24h, no changes in the protein levels of TLR2and TLR4were observed. In line with this, HMGB1increased threonine/tyrosine phosphorylation of ERK, JNK and Akt, which were noticeable at6h,5h and7h, respectively.Previous studies reported HMGB1and RAGE contributes to immune and inflammatory and responses and we supposed RAGE signaling pathways may involve in HMGB1induced defect in epithelial barrier function. As expected, compared with HMGB1stimulaiton group, pretreatment with RAGE antibody (5ug/ml) significantly attenuated permeability-increasing effect (TER:P=0.001; FITC:P=0.002, n=3). We next investigated whether MAPK and PI3kinase/Akt signalling inhibition could improve HMGB1induced epithelial barrier dysfunction. Treatment of16HBE cells with the ERK1/2tyrosine kinase inhibitor U0126significantly rescued HMGB1induced changes in TER and FITC dextran permeability, while there were no such effects with treatment of the PI3K tyrosine kinase inhibitor LY294002and JNK kinase inhibitor SP600125. We also found that ERK1/2downstream signalling was dependent on RAGE, as HMGB1induced phosphorylated-ERKl/2response was suppressed by anti-RAGE antibody. In line with the involvement of tyrosine phosphorylation of ERK1/2in HMGB1induced barrier permeability dysfunction; we found that U0126inhibited HMGB1mediated down-regulation of occludin and claudin-2. As prior experiment showed, redistribution of proteins was the main effect of HMGB1induced disruption of E-cadherin and P-catenin. We assessed adhesion junction proteins distribution in response to HMGB1with U0126. In control cells, immunostaining for E-cadherin and β-catenin showed a similar plasma membrane distribution. Although HMGB1treatment promoted loss of immunostaining for E-cadherin, this was prevented by treatment with U0126. These suggested that RAGE/ERK1/2signalling contributed to HMGB1induced epithelial barrier dysfunction.5. HMGB1in synergy with IL-1β had more significant effect on barrier dysfunction than HMGB1aloneSince previous studies reported HMGB1forming specific complexes with Damage associated molecular pattern (PAMPs) and cytokines has greater pro-inflammatory activity than HMGB1alone, we next investigated whether there was a similar effect on barrier function. Compared with control group and HMB1stimulation group, treatment with of HMGB1(100ng/ml) in synergy with IL-1β (2.5ng/ml) induced a lower TER and higher FITC dextran permeability changes (all P values<0.05). Consistent with this effect, Western blot analyses showed lower expression of occludin, claudin-2and E-cadherin (all P values<0.01). Immunofluorescent staining of16HBE for E-cadherin showed a redistribution of staining from membrane to cytoplasm. HMGB1and in synergy with IL-1β caused loss and interruption in β-catenin staining at plasma membrane. This indicates HMGB1treatment induced epithelial barrier dysfunction was enhanced by IL-1β.Conclusions:1. HMGB1induce barrier function defect in a time and dosage response manner in human bronchial epithelial16HBE and A549cells,largely due to disruption of cell-cell contacts.2. HMGB1induced epithelial barrier function impairment were paralleled by disruption of tight junction proteins occludin and claudin-2.Occludin protein played a crucial role in HMGB1induced epithelial barrier dysfunction. In a addition, HMGB1can cause delocalization and disruption of adhesion junction protein E-cadherin and β-catenin.3. Application of the specific ERK inhibitor U0125and anti-RAGE antibody showed that RAGE/ERK activation contributes to the HMGB1-induced defects in16HBE cells.RAGE/ERK signaling pathway was involved in HMGB1induced barrier function defect and abnormal expression and distribution of junctional proteins.更多还原