LPS/NF-kappaB/miR-30b参与早期胆管细胞炎性进程特征分析及大黄灵仙胶囊的调控研究

Biliary tract inflammation induces the pathological bile duct rebuilt and accelerate the bile duct stone formation,but the mechanism and the approach isn’t clear.Prophase our research group used lipopolysaccharide (LPS) to triggering the bile duct cells’ continuous inflammatory reaction, micrornas chip to detect bile duct cells endogenous miR - 30b has a high expression, we found that Da Huang Lingxian capsule can alleviate inflammatory injury of bile duct, reduced miR - 30b expression, the analysis found that there are several transcription factor binding sites in NF-kappaB in miR - 30b promoter region.Our research group speculated that Da Huang Lingxian capsule can regulate miR - 30b transcription and impact the downstream signaling cascade to relieve bile duct cell inflammatory reaction.Our project will use LPS to built rats’ biliary tract inflammatory state, Da Huang Lingxian capsule to make intervene, and transfection miR - 30b interfere plasmid in the liver, take a variety of research techniques to understanding the regulatory mechanism of Da Huang Lingxian capsule on NF-kappaB/miR - 30b’s transcription, and through the regulation of miR - 30b play a signal pathway of therapeutic effect.The project will explain Da Huang Lingxian capsule’s mechanism and effect way to relieve the inflammatory injury of bile duct on the miRNA transcription level, and building a stronger foundation to spread this application further.

胆道炎症诱发胆管病理性重塑促进胆管内结石形成，但相关机制和途径不清。前期课题组以脂多糖（LPS）触发胆管细胞持续炎性反应，miRNA芯片检测发现胆管细胞内源性miR-30b呈高表达，大黄灵仙胶囊可缓解胆管炎性损伤，下调miR-30b表达，分析发现在miR-30b启动子区存在NF-kappaB多个转录结合位点，课题组推测大黄灵仙胶囊可通过调控miR-30b转录影响下游信号级联以缓解胆管细胞炎性反应。本项目将以LPS构建大鼠胆道炎症状态，大黄灵仙胶囊进行干预，并在肝内转染miR-30b干扰质粒，采用多种研究技术，明确大黄灵仙胶囊对NF-kappaB/miR-30b起始转录的调控机制及其通过调控miR-30b发挥治疗效应的信号途径。项目的完成将从调控胆管细胞miRNA转录层面洞悉大黄灵仙胶囊缓解胆管炎性损伤的机制和效应途径，为进一步推广应用夯实基础。

原发性肝内胆管结石是我国常见的良性胆道疾病，虽然手术技术不断革新，但术后结石仍有较高的复发率，仍需再次手术或多次手术。长期反复发作的胆道炎症是本病的主要病理特征。胆道炎症诱发胆管病理性重塑促进胆管内结石形成，但相关机制和途径不清。大黄灵仙胶囊临床防治胆石症疗效显著，但相关机制和途径尚未阐明。项目研究以胆道炎症应激、miR-30b及NF-κB为研究切入点，分析miR-30b、NF-κB在早期胆管炎症中的表达特点和大黄灵仙胶囊的调控作用，并构建以miR-30b为核心的胆管细胞炎症应激信号网络，探查关键信号通路在胆管炎性进程中的作用及大黄灵仙胶囊的影响效应。研究发现大黄灵仙胶囊可缓解胆管细胞炎症应激，抑制炎症因子IL-1β、IL-18、TNF-α表达，抑制NF-κB核转位并调控miR-30b表达，反向干扰miR-30b表达后，大黄灵仙胶囊抑制炎症反应的作用被反转。进一步围绕miR-30b 进行RNA-seq 测序与筛选，优选大黄灵仙胶囊影响miR-30b表达进而调控的相关分子功能及关键信号通路，测序得到264个差异基因，其中差异上调基因161个，差异下调基因103个。差异基因主要与RIG-I样受体信号通路、肿瘤坏死因子信号通路、NOD样受体信号通路及TGFβ信号通路相关。研究中初步筛选出与大黄灵仙胶囊影响miR-30b表达进而调控的相关分子：NOD1、SMAD4、BMP2、NLPR3、SMAD2、TGFβ1、CXCL2，提示大黄灵仙胶囊调控NOD1、TGFβ1等信号因子的表达，反向干扰miR-30b表达，并结合使用TGF-b1、NLRP3信号通路干扰剂，证实大黄灵仙胶囊可通过miR-30b调控下游TGF-b1、NLRP3信号通路反应，发挥缓解胆管细胞炎症应激的疗效作用，研究结果丰富了大黄灵仙胶囊临床治疗胆石症的科学依据。