LPS介导的TLR4/NF-κB信号通路参与艾灸对IBS-D模型大鼠的免疫修复

Diarrhea-predominant irritable bowel syndrome (IBS-D) is a clinically common chronic functional gastrointestinal disease (FGID). Its pathogenesis is regarded as being closely related to immune system dysregulation, and the imbalance of proinflammatory factors and anti-inflammatory factors caused by immune responses may be the key of the occurrence and progression of IBS.Previous research finds that moxibustion can obviously improve the clinical symptoms of the patients with IBS-D. Moxibustion on Shangjuxu and Tianshu can significantly reduce the level of serum inflammatory factors in IBS-D rats, increase the activity of SOD in colon tissue of rats, and decrease the level of MDA. The project intend to observe the effect of moxibustion on the general condition, intestinal electrical test, intestinal barrier function and mucosa permeability of IBS-D rats from the point of using LPS mediated TLR4/NF- κB signaling pathway. It also intend to observe the effect of moxibustion on IKKβ, IκBɑ, TLR4, Myd88, NF-κBp65 and HBD-2mRNA expression and downstream damage-inducing factors in IBS-D rats hippocampus and colon,so as to investigate the mechanism of action of moxibustion on the immune repair of inflammatory intestinal mucosa injuries and gut-brain regulation.The purpose of the project is to reveal the mechanism of treatment of IBS-D rats by moxibustion from a perspective of molecular biology, to provide an experimental basis for an improvement in clinical effect.

腹泻型肠易激综合征(IBS-D)是一种临床多见的慢性功能性胃肠道疾病，其发病机制被认为与免疫系统调控失调有着密切关系，免疫反应产生的促炎因子与抗炎因子失衡可能是IBS发生发展的关键。前期研究发现，艾灸对IBS-D患者的临床症状有明显改善作用；艾灸上巨虚、天枢能显著改善IBS-D大鼠血清炎症因子的水平，提高大鼠结肠组织SOD活性，降低MDA水平。本项目拟以LPS介导的TLR4/NF-κB信号通路为切入点，观察艾灸对IBS-D大鼠一般情况、肠电检测、肠粘膜屏障功能、粘膜通透性的影响；观察艾灸对IBS-D大鼠海马和结肠组织中的IKKβ、IκBɑ、TLR4、Myd88、NF-κBp65、HBD-2mRNA表达和下游致损伤因子的影响，探讨艾灸对IBS-D肠粘膜炎性损伤的免疫修复及肠-脑双向调节作用机制。旨在从分子生物学水平揭示艾灸对IBS-D大鼠的治疗机制，为提高临床疗效提供实验依据。

临床和动物实验均证明针灸天枢和上巨虚治疗腹泻型肠易激综合征可以改善肠道紊乱的状态,有着独特的疗效,但其作用机制仍不清楚｡本研究以TLR4/NF-κB､SCF/c-kit信号通路和肠-脑互动为切入点,观察针灸天枢､上巨虚治疗IBS-D对炎性因子､免疫球蛋白、神经递质等的调控｡.主要研究内容与结果如下:.①艾灸可改善IBS-D大鼠腹泻症状和内脏高敏感性,其机制与艾灸抑制TLR4/MyD88/NF-κB和IKKβ/IKBα/NF-κB信号通路,降低下游炎性因子的表达水平有关;.②艾灸维持IBS-D大鼠免疫功能稳态作用机制是通过激活SCF/c-kit信号通路而降低大鼠血清中T细胞亚群（CD4、CD8、CD45）、免疫球蛋白（IgA、IgG、IgM）和炎性因子（TNF-α、IL-8）表达水平;.③艾灸治疗IBS-D大鼠的抗炎作用机制可能与艾灸提高miR-345-3p､miR-216a-5p表达水平与降低miR-155､miR-125b､miR-29b､miR-31､miR-18a表达水平进而抑制NF-κB信号通路,降低炎症因子表达有关;.④艾灸干预IBS-D大鼠的作用机制与调控结肠和下丘脑神经肽Orexin及其受体Ox1R高表达和神经肽SP､VIP低表达,从而延缓痛觉传递､减轻炎症反应､调节胃肠功能紊乱有关;.⑤艾灸干预IBS-D大鼠作用机制可能与与降低肠道中神经递质5-HT3R与NO表达水平和提高SERT表达进而改善胃肠道功能的异常有关;.⑥艾灸改善IBS-D大鼠腹泻症状和内脏高敏感性,可能与上调miRNA-133b靶向抑制中脑Pitx3、TH和血浆、结肠及中脑组织中5-HT、DA、NE的表达有关;.⑦电针治疗IBS-D大鼠作用机制可能与上调lncRNA TUG1而抑制miRNA-127/NF-κB信号通路的表达有关｡.本项目实施意在揭示针灸治疗IBS-D的作用机理,为临床针灸治疗IBS-D提供新的科学依据｡