KCa3.1诱导内质网应激在JNK-MAPK信号通路介导百草枯肺纤维化中的机制研究

Paraquat poisoning could result in extensive pulmonary fibrosis, thus affecting the prognosis of patients. Fibroblasts have been showed to be majority source of collagen and contribute to pulmonary fibrosis in both physiologic and pathologic settings. We found that, by blocking the Ca2+-activated K+ channel (KCa3.1), both the number of fibroblasts in the lungs and the extent of pulmonary fibrosis were attenuated in the rat model of paraquat (PQ) poisoning. And TRAM34, the blocker of KCa3.1, inhibited the TGF-β1-mediated increase in p-JNK expression. Previous studies have demonstrated that KCa3.1 regulates K+ efflux, increasing the driving force for Ca2+ entry through hyperpolarization of the plasma membrane. Intracellular Ca2+ imbalance can impact and disturb proper functioning of the endoplasmic reticulum, resulting in endoplasmic reticulum stress (ERS). It was reported that ERS contribute to the mediation of JNK pathway. We postulated that the fibroblasts was activated by KCa3.1 channels which contributed to the TGF-β1-mediated JNK pathway via ERS after Ca2+ influx in PQ induced pulmonary fibrosis. We will investigate the role of KCa3.1 in the activation and proliferation of fibroblasts by means of administrating KCa3.1 channel blockers and openers, as well as RNA interference both in vitro and in vivo. The goal of this study is to investigate the role of ion channel in the development of PQ-induced pulmonary fibrosis, which may be a potential therapeutic target.

成纤维细胞活化增殖在百草枯(PQ)中毒后肺纤维化形成中起重要作用。前期研究应用KCa3.1 阻断剂后肺组织中活化成纤维细胞数减少、成纤维细胞中TGF-β1诱导JNK活化减少。国外研究报道KCa3.1活化引起细胞内钙平衡失调，诱发内质网应激(ERS)，而ERS又参与JNK信号通路的调节。因此分析在PQ中毒后，KCa3.1活化可能通过Ca2+内流，诱导ERS，参与TGF-β1激活JNK信号通路，导致成纤维细胞活化增殖。本项目拟通过体内体外中毒模型：①用阻断剂、激动剂、siRNA和基因转染技术，调节KCa3.1表达和状态，检测成纤维细胞表型和细胞增殖变化、肺活化成纤维细胞数和细胞外基质蛋白表达、肺纤维化程度；②应用ERS抑制剂，观察肺成纤维细胞内Ca2+浓度、ERS标志蛋白、JNK信号通路基因和蛋白。探讨PQ中毒后 KCa3.1介导肺成纤维细胞活化增殖的作用机制，为临床治疗提供新思路和干预靶点。

背景和目的：百草枯（PQ）引起不可逆肺纤维化导致的呼吸衰竭是其中毒死亡的主要原因，局部的肺成纤维细胞增殖活化在其中发挥着关键性的作用，新近的研究表明中电导钙激活钾离子通道KCa3.1可能参与激活成纤维细胞并促进器官纤维化的发生发展，同时我们前期研究发现内质网应激（ERS）参与了百草枯致肺纤维化的过程，但其具体机制尚未阐明。本研究着重探讨KCa3.1在PQ所致肺纤维化中的作用以及是否通过调控ERS来促进肺成纤维细胞活化增殖。.方法：建立百草枯中毒大鼠动物模型并提取大鼠原代肺成纤维细胞建立百草枯中毒肺成纤维细胞模型。在应用KCa3.1阻断剂TRAM-34和内质网应激的抑制剂Salubrinal后，采用肺组织H&E染色、Masson染色检测大鼠、肺组织羟脯氨酸含量、肺组织湿/干重比值、ELISA测定肺泡灌洗液I型胶原含量来检测中毒大鼠肺组织炎症和纤维化程度，免疫组化检测KCa3.1、α-SMA、ERS标志物GRP78的表达，Western blot方法检测KCa3.1、α-SMA、I型胶原、GRP78等指标的表达，同时采用免疫荧光法检测KCa3.1和GRP78的表达，采用MTT和BrdU法检测不同干预方法前后肺成纤维细胞的增殖水平。.结果：.1.百草枯中毒后在肺组织以及肺成纤维细胞中KCa3.1表达升高；KCa3.1阻断剂TRAM-34能减轻百草枯导致大鼠肺损伤与肺纤维化；TRAM-34能抑制百草枯中毒后肺成纤维细胞的增殖；TRAM-34不影响百草枯中毒后肺组织TGF-β1的表达；这些都表明KCa3.1可能参与调控PQ所致肺成纤维细胞活化进而影响肺纤维化过程；.2.应用TRAM-34阻断KCa3.1能减轻百草枯中毒后肺组织与肺成纤维细胞的内质网应激；应用Salubrinal抑制内质网应激能减轻百草枯中毒后KCa3.1诱导肺成纤维细胞活化增殖；这表明PQ中毒后KCa3.1促进纤维化的作用可能是通过内质网应激调控肺成纤维细胞活化与增殖实现的。.结论：本研究的结果提示：在百草枯中毒后，KCa3.1参与调控肺成纤维细胞的增殖与活化，进而促进肺纤维化的发生；KCa3.1可能是通过诱发肺成纤维细胞内质网应激来调控其增殖与胶原分泌活性。本研究进一步阐明了百草枯中毒致肺纤维化的机制，并可为其他原因导致的肺纤维化提供借鉴作用。