大黄灵仙方调控胆管炎症微环境中LPS-TLR4/NF-κB/MAPK通路诱导EMT的机制研究

The diversity of pathogenesis, in which inflammation is one of the hotspots, is the difficulty in treatment of hepatolithiasis and its recurrence. Previous studies showed that lipopolysaccharide (LPS) is closely related to inflammation of bile duct. By down-regulating IL-6 and TGF-β, Da Huang Ling Xian Prescription could attenuate LPS-induced inflammation of bile duct cells (BDECs) in rat model, and inhibit the synthesis of proteins relevant to liver fibrosis. Analyses of miR chip indicated that Da Huang Ling Xian Prescription might target at NF-κB/MAPK signaling. Therefore, we proposed that LPS triggers the crosstalk of TLR4/NF-κB/MAPK pathway and then enhances inflammatory process, thus deteriorating inflammation of bile duct by inducing epithelial-mesenchymal transition (EMT) of BDECs. This project will imitate LPS-induced inflammatory status of bile duct in rat model and explore the alterations in TLR4/NF-κB/MAPK signaling, downstream inflammatory factors, and epithelial and mesenchymal hallmarks. Our results will provide better understanding of the pivotal mechanism through which Da Huang Ling Xian Prescription regulates EMT by LPS-TLR4/NF-κB/MAPK signaling.

治疗肝胆管结石及预防结石复发的难点在于成石机制的多样性，其中炎症相关通路是研究热点。脂多糖（LPS）与胆管炎症密切相关，LPS诱导模型大鼠出现胆管上皮细胞（BDECs）炎症，大黄灵仙方能通过下调炎症因子IL-6、TGF-β，抑制肝纤维化相关蛋白合成，有效缓解胆管炎症。miR芯片功能分析提示大黄灵仙方作用于NF-κB/MAPK通路等预实验结果，推测LPS触发胆管细胞TLR4/NF-κB/MAPK信号通路交互加重炎性进程，导致BDECs发生上皮间质转化（EMT）加重胆管炎症。本项目将以LPS构建大鼠胆道炎症状态，采用Co-IP、WB、qPCR、IHC、PCR及Westbotting技术，探明胆管细胞炎性状态下TLR4/NF-κB/MAPK信号通路转导特点和下游炎症因子表达情况，检测上皮标志物及间叶标志物的表达，阐释大黄灵仙方调控LPS-TLR4/NF-κB/MAPK通路诱导EMT的关键机制。

肝内胆管结石是我国的常见病与多发病，胆管炎症是肝内胆管结石形成的主要原因之一，本研究基于胆道炎症微环境探究TLR4/NF-kB/MAPK信号通路与胆管炎症的关系，以及大黄灵仙方缓解胆管炎症的分子机制。本研究以LPS构建大鼠胆管炎症状态，采用HE染色、Co-IP、WB、qPCR、IHC技术进行验证。HE染色可见，LPS注射后胆管内出现明显炎性浸渍，并且存在胆管上皮细胞的脱落凋亡现象，大黄灵仙方及通路阻断剂干预后炎症反应减轻；同时，PCR及WB结果显示，LPS诱发的胆管炎症激活TLR4/NF-kB/MAPK信号通路，而大黄灵仙方可抑制通路关键因子的表达；免疫共沉淀显示，大黄灵仙方能够促进TAK1与TRAF6 、TAK1与ASK1分子间相互作用，同时胆管炎症反应减轻；免疫组化结果显示，胆管炎症状态下，上皮间质转化标志物E-cad表达量减少而S-100A4、a-SMA表达升高，大黄灵仙方及通路阻断剂干预后，上皮间质转化标志物E-cad表达量升高而S-100A4、a-SMA表达减少。由此得出结论：①大黄灵仙方通过下调TLR4/NF-κB/MAPK信号通路缓解胆管细胞炎性反应；②大黄灵仙方促进ASK1与TAK1、TRAF6与TAK1交互作用和细胞共定位从而减轻炎症反应；③大黄灵仙方通过调控TLR4/NF-κB/MAPK信号通路干预 LPS诱导的胆管上皮细胞发生上皮间质转化。