黄芩苷干预PI3K/AKT/NF-κB信号通路对溃疡性结肠炎免疫调节、肠黏膜屏障修复的作用机制研究

Ulcerative colitis is an important pre- cancer disease,PI3K/AKT signaling pathway while participating in the UC inflammation and carcinogenesis.PI3K/AKT signaling pathway,leading to over-expression of NF-κB,starting downstream signaling pathways,the release of inflammatory cytokines and pro-apoptotic proteins,thereby maintaining intestinal inflammation,promote intestinal epithelial cell apoptosis,leading to protracted illness,cancer rates increased, plays an important role in the occurrence and development of ulcerative colitis.Early studies showed that:clearing the intestine and resolving dampness prescription can significantly improve the clinical symptoms of ulcerative colitis patients,inhibit inflammation, promote ulcer healing,the mechanism is connected with the regulation of NF-κB expression in intestinal mucosa,whether with PI3K/AKT pathway remains to be elucidated. In this study,both In vitro and in vivo,SASP and PI3K/AKT inhibitor Wortmannin as a control,observing baicalin PI3K/AKT/NF-κB mediated signaling pathway to inhibit the expression of NF-κB,reduce inflammation, inhibition of intestinal epithelial cell apoptosis,immune mechanisms to repair the intestinal mucosal barrier,to clarify the clearing the intestine and resolving dampness prescription and its main active ingredient therapeutics UC immunological mechanisms to provide a scientific basis for in-depth study of traditional Chinese medicine treatment of UC,in order to further improve ulcerative colitis inflammatory efficacy of treatment,improve existing treatment options for UC to provide ideas .

溃疡性结肠炎是重要的结肠癌前病变，而PI3K/AKT信号通路同时参与了UC的炎症及癌变过程。PI3K/AKT信号通路活化，导致NF-κB过表达，启动下游信号通路，从而维持肠道炎症、促进肠上皮细胞凋亡，导致疾病迁延难愈，癌变率增高，在溃疡性结肠炎的发生、发展过程中起着重要作用。前期研究表明：临床效方清肠化湿方能够显著改善溃疡性结肠炎患者的临床症状，抑制炎症反应，促进溃疡愈合，其作用机制与调控肠黏膜组织NF-κB表达有关，但与PI3K/AKT通路的关系有待进一步阐明。本研究采用从体内外两个方面展开研究，以SASP、PI3K/AKT抑制剂Wortmannin为对照，观察黄芩苷介导PI3K/AKT/NF-κB信号通路从而减轻炎症反应，抑制肠上皮细胞凋亡、修复肠粘膜屏障的免疫机制，阐明黄芩苷治疗UC的免疫学机制，为治疗UC中药的深入研究提供科学依据，为完善UC的现有治疗方案提供思路。

目的：观察黄芩苷对HT-29炎症模型及TNBS诱导的大鼠溃疡性结肠炎模型免疫炎症及黏膜修复的影响，并探讨其与PI3K/AKT/NF-κB信号通路的相关性。方法：体外实验：以TNF-α及LPS共刺激诱导HT-29细胞，制备细胞炎症模型，分别予以黄芩苷、柳氮磺胺吡啶(SASP)、PI3K/AKT抑制剂LY294002和PI3K激动剂IGF-1孵育，采用MTT法检测细胞生长，Elisa法细胞上清中TNF-α，IL-6等炎症因子水平，Western blot法检测p-PI3K/PI3K、p-Akt/Akt、NF-κb、p-IκBα/IκBα、Nucleus NF-κb及凋亡相关蛋白的表达，流式细胞术检测细胞凋亡情况。体内实验：建立TNBS诱导的大鼠溃疡性结肠炎模型，分别予以黄芩苷、SASP、LY294002和IGF-1干预， Elisa和Western-blot检测TNF-α和IL-6等炎症因子表达，TUNEL法检测肠组织凋亡情况，免疫组化和Western blot法检测ZO-1和β-catenin的表达，Western blot检测p-PI3K/PI3K、p-Akt/Akt、NF-κb、p-IκBα/IκBα、NF-κb及FasL、Caspase9等凋亡相关蛋白的表达。结果：体外实验：与模型组相比，黄芩苷组、SASP组的细胞上清中TNF-α、IL-6、IL-8、IL-1水平减少（P＜0.05），PI3K、Akt蛋白的磷酸化水平减少，NF-κB的活化入核水平减少（P＜0.05），Cox-2、β-catenin、Caspase-9、Fasl蛋白表达减少。体内实验：与模型组相比，黄芩苷组、SASP组大鼠结肠组织中TNF-α、IL-6、IL-8、IL-1分泌量减少，且两药联合应用对TNF-α、IL-6、IL-8、IL-1的分泌量的减弱效应更强。黄芩苷组、SASP组中的PI3K蛋白磷酸化水平减少，磷酸化Akt水平减少。黄芩苷组和SASP组中的Cox-2、β-catenin、Caspase-9、Fasl的蛋白表达减少（P＜0.05）。结论：黄芩苷能减轻HT-29炎症细胞模型及大鼠UC模型炎症反应，抑制PI3K磷酸化，下调Akt的活化，抑制NF-κB的活化入核，从而抑制TNF-α、IL-6等炎症因子的分泌，发挥其抗炎效应，这可能是其发挥治疗作用的途径和有效机制。