基于生物信息挖掘与TLR4/MyD88/NF-κB通路调控效应研究川芎嗪纳米制剂防治术后腹腔粘连机制

Postoperative peritoneal adhesion is closely associated with immune dysfunction. Mining gene chip data of postoperative peritoneal adhesion suggested that TLR4 was highly differentially expressed. Additionally, the TLR4/MyD88/NF-κB regulatory pathway was found after we searched the TLR4 pathway from KEGG pathway Database. Our previous studies have found that tetramethylpyrazine (TMP) nanoparticles can regulate the expression of inflammatory factors in prevention of postoperative peritoneal adhesion. And considering the facts that tetramethylpyrazin participates in the activation of NF-κB, we put forward the regulative logic of “TMP/TLR4/MyD88/NF-κB/ inflammatory factor/ peritoneal adhesion”. Based on the idea, the rat model of peritoneal adhesion and cell model peritoneal mesothelial will be established and be treated with tetramethylpyrazine nanoparticles, then the expression of TLR4/MyD88/NF-κB pathway could be observed. Moreover, we plan to quantificat and analyze the NF-κB nuclear translocation level by multispectral imaging flow cytometry, ananlyze the expression of infammatory factors associated with peritoneal adhesion by using xMAP technology, as well as explore the blinding sites between tetramethylpyrazine nanoparticles and peritoneal mesothelium cell membranes by protein separation and identification technology. These results from this study may verify the mechanism of tetramethylpyrazine nanoparticles preventing peritoneal adhesion by regulating TLR4/MyD88/NF-κB pathway, which will further provide the basis for clinical prevention of peritoneal adhesion.

术后腹腔粘连与局部免疫功能失衡关联密切。腹腔粘连基因芯片数据挖掘后提示TLR4表达高度差异，通过KEGG pathway Database检索TLR4所在通路并分析研究，发现TLR4/MyD88/NF-κB调控通路，结合前期研究发现川芎嗪纳米制剂能够调控炎性因子表达防治术后腹腔粘连，以及川芎嗪调控NF-κB活化事实，提示“川芎嗪/TLR4/MyD88/NF-κB/炎性因子/腹腔粘连”这一调控逻辑。基于此，通过腹腔粘连动物模型及腹膜间皮细胞，观察川芎嗪纳米制剂干预TLR4/MyD88/NF-κB通路表达变化；运用高速成像流式技术量化分析NF-κB核转位水平；运用细胞因子液相芯片分析腹腔粘连局部相关炎性因子表达；运用亲和结合蛋白质分离与鉴定技术探寻川芎嗪纳米制剂与腹膜间皮细胞膜结合位点，验证川芎嗪纳米制剂调控TLR4/MyD88/NF-κB通路防治腹腔粘连效应机制，为中医药有效防治提供依据。

项目背景：1. TLR4/MyD88/NF-κB通路是否是术后腹腔粘连病理机制的关键环节尚不清楚。2. 川芎嗪纳米制剂是否通过调控TLR4/MyD88/NF-κB通路达到防治术后腹腔粘连尚待研究。3. TLR4是否是川芎嗪细胞膜结合位点值得探讨。.研究内容：项目主要涉及了三个方面的实验研究。1. 基于生物信息学挖掘术后腹腔粘连微阵列数据，探寻TLR4/MyD88/NF-κB通路在术后腹腔粘连病理机制中的关键作用。2. 通过现代分子技术，研究川芎嗪纳米制剂调控TLR4/MyD88/NF-κB通路发挥防治术后腹腔粘连效应机制。3. 利用分子对接技术探寻川芎嗪与TLR4膜蛋白潜在结合位点。.重要结果：1. 通过生物信息学，挖掘GEO数据库中术后腹腔粘连基因芯片数据集，发现TLR4/MyD88/NF-κB通路在术后腹腔粘连关键阀门调控机制。2. 通过脂多糖诱导人腹膜间皮细胞（HPMCs）模拟炎症损伤，发现Cox-2对LPS诱导的HPMCs损伤的作用是通过NF-κB途径负调控miR-21和靶向TLR4实现的，揭示了TLR4/MyD88/NF-κB通路在术后腹腔粘连免疫病理机制中起着关键作用。3. 基于整体动物实验，发现川芎嗪纳米制剂具有良好的包封率、载药量和控释缓释特性，可减少胶原纤维的积聚，激活TLR4/MyD88/NF-κB通路，维持Th1（IFN-γ、IL-12）和Th2（IL-4、IL-6）平衡，为川芎嗪纳米制剂防治术后腹腔粘连免疫机制提供有效依据。4. 运用分子对接技术，发现川芎嗪与TLR4膜蛋白的2个氨基酸残基（LYS153和ANS129）产生了重要的氢键相互作用，提示潜在结合位点和调控可能。.科学意义：TLR4/MyD88/NF-κB通路在术后腹腔粘连免疫病理机制中发挥重要作用。川芎嗪纳米制剂能调控TLR4/MyD88/NF-κB通路发挥防治术后腹腔粘连效应机制。川芎嗪与TLR4潜在结合靶点，需进一步研究。通过本项目的实施，加深了术后腹腔粘连复杂病理机制的认识，进一步明晰了川芎嗪纳米制剂防治术后腹腔粘连效应机制，初步探讨了川芎嗪与膜蛋白潜在靶点，为进一步实践中医药防治术后腹腔粘连提供依据和参考。