大气细颗粒物诱导的内质网整合应激效应对气道黏蛋白加工的影响机制

Airway mucus hypersecretion is the major pathologic feature of chronic airway inflammation induced by PM2.5 exposure. It is known that PM2.5 could induce inflammation and airway epithelia injury, Mucin(MUC) 5AC is the secondary effects induced by inflammation, the regulation of mucin secretion, which is known related to the regulation of MUC5AC transcription, can not well explain the substantial increasing of mucus hypersecretion. We speculate that under the stimulation of PM2.5, post-translational MUC5AC protein modifications are altered by oxidative stress-induced endoplasmic reticulum integrated stress response(ISR). This effect is achieved by affecting the signal recognition particle (SRP) and the endoplasmic reticulum (ER) membrane protein translocator Sec61, which interacted to mediate prototype peptide transferring to endoplasmic reticulum. In this research, we will detect the expression and distribution of ISR-related protein in airway epithelial cell cultured in vitro, the alteration of SRP and Sec61 protein and their interaction will be observed by co-immunoprecipitation. Then we will verify the effects of SRP and Sec61 on MUC5AC protein secretion by negative-gene transfection, postive-gene transfection, and RNA interference, also the effect of ISR on the interaction of SRP and Sec61 will be elucidated. This study will improve our understanding of the mechanism involved in mucus hypersecretion under PM2.5 exposure, also it may provide some effective targets for the prevention and intervention of mucus secretion.

黏液高分泌是PM2.5长期暴露所致气道炎症的重要表现，此高分泌粘蛋白（MUC）的表型主要为MUC5AC。目前PM2.5对气道的致炎效应及上皮损害作用已有较明确认知，但对其导致黏液中高分泌的认识仍停滞于源自炎症因子的继发效应，此只限于转录水平提升的机制无法解释MUC5AC后期加工能力大幅提升的需求。考虑到氧化应激是PM2.5的主要致炎机制，故气道上皮细胞应可藉氧化应激诱导内质网整合应激反应（ISR）提升蛋白加工负荷，该效应系通过影响信号识别颗粒（SRP）和亲水性移位通道复合体Sec61介导雏形肽向内质网移位这两个节点实现的。本研究采用体外培养细胞，观察PM2.5刺激下ISR相关效应蛋白的表达及分布特点，以及SRP和Sec61的变化特征，并观测两组蛋白间的相互作用。并籍缺陷基因转染、RNA干扰等节点抑制的方法，验证此两个门控因子的地位，了解ISR对其介导作用的影响，有望为防治提供有效靶点。

大气环境在呼吸道疾病发生发展中起着重要的作用，PM2.5是近年来研究的热点。内质网应激作为细胞的一种防御性反应，可抵抗机体免受损伤，但过度的有害刺激，则使内质网应激持续激活，导致一系列伤害性反应。本课题采用PM2.5及中性粒细胞弹力蛋白酶（NE）作为诱导因素，探讨内质网应激在诱导气道炎症及黏蛋白生成中的作用。通过比较未吸烟无慢支炎、吸烟慢支炎的患者手术支气管肺组织标本，了解慢性炎症时内质网应激相关蛋白的表达差异；构建COPD大鼠模型，予以NAC、4-PBA干预，比较干预前后气道炎症及黏蛋白生成情况；体外培养气道上皮细胞，分别予以PM2.5及NE刺激，并予以不同通路内质网应激蛋白抑制剂处理，流式细胞术检测细胞凋亡情况，ELISA、Real-time PCR及Western blot检测各组的内质网应激相关蛋白及细胞炎性因子、黏蛋白MUC5AC的表达变化情况；进一步构建了IRE1α siRNA、NOD1siRNA、SRP54 siRNA、SRPRα siRNA及Sec61 siRNA并转染细胞，免疫荧光双染色检测IRE1α及NOD1的共表达情况，检测IRE1α、NOD1、细胞炎性因子、黏蛋白MUC5AC的表达变化。研究结果显示，慢性炎症患者及COPD大鼠模型气道组织中内质网应激蛋白表达增多，予以抗氧化剂及内质网应激抑制剂干预可减轻气道炎症，减少黏蛋白生成；PM2.5及NE均能诱导ROS 生成，引起内质网应激反应，抑制PERK、ATF6、IRE1α通路均能使炎性因子表达及MUC5AC生成降低，但IRE1α组降低明显。IRE1α siRNA及XBP1siRNA能降低NE诱导的气道炎症及黏液分泌，同时在PM2.5诱导的上皮细胞中，IRE1α siRNA、NOD1siRNA导致NOD1表达降低，NF-κBp65核转运减少，IL-6、TNF-α、MUC5AC生成减少。SRP54 siRNA、SRPRα siRNA及Sec61 siRNA均能使MUC5AC表达降低。表明内质网应激通路及转运分子参与了PM2.5及NE诱导的气道炎症及黏液分泌。PM2.5及NE产生氧化应激，诱导内质网应激相关蛋白活化。多种信号通路如IRE1α/XBP1及IRE1α/NOD1/NF-κB参与了气道炎性因子及黏液分泌生成增多的过程。同时，信号识别颗粒（SRP）和亲水性移位通道复合体Sec61也介导了黏蛋白M