基于AQP和TLR/NF-κB信号通路研究风药在溃疡性结肠炎治疗中的增效作用机制

Ulcerative colitis（UC） is characterized by chronic relapsing intestinal inflammation. It has been a worldwide health-care problem with a continually increasing incidence. Although the etiology of UC remains largely unknown, it involves a complex interaction between the genetic, environmental or microbial factors and the immune responses. In recent years, most study focus on immunologic aspect and more and more evidence revealed that aquaporins and TLR/NF-κB signaling pathway play an important role in its pathogenesis. In traditional Chinese medicine , it is believed that the dampness evil is the main factor in its pathomechanism. The use of wind medicine can improve the therapeutic effect. In the present study, rats of dextran sulphate sodium (DSS)-induced colitis been used for animal model. The rats was divided into wind medicine group, clear heat and drain dampness medicine group, clear heat and drain dampness plus wind medicine group and 5-ASA group. Disease activity index(DAI), colonic mucosal damage index(CMDI) and histopathological features been measured for treatment effect. The expression of AQP4、AQP8、TLR4、TLR2、NF-κBp65、TNF-α、IL-1、IL-6、IL-8 were detected by fluorescent PCR and immunohistochemical method. The aims of the study are to investigate the molecular mechanism of the synergy effect of wind medicine in UC treatment, reveal the scientific connotation of wind can overcome dampness, create a new route and provide scientific basis for UC treatment.

溃疡性结肠炎是慢性肠道炎症性疾病，由于发病率不断增加引起广泛关注。其病因和发病机制目前尚未完全明确，一般认为与免疫、遗传、环境等因素的相互作用有关。近年来对其发病机理研究多集中在免疫学方面，越来越多的证据显示黏膜免疫系统中水通道蛋白和TLR/NF-κB信号通路在其中发挥着重要作用。中医认为湿邪为患是本病主要病机，临床发现加用风药可明显提高疗效。本研究采用葡聚糖硫酸钠制作溃疡性结肠炎大鼠模型，观察比较风药组、清热利湿药组、风药加清热利湿药组以及西药美沙拉秦组的疾病活动指数、黏膜损伤指数、黏膜组织病理改变，并通过荧光实时定量PCR法和免疫组化法检测结肠黏膜AQP4、AQP8、TLR4、TLR2、NF-κBp65、TNF-α、IL-1、IL-6、IL-8的表达，探索风药在UC治疗中的增效作用的分子机制，阐释"风能胜湿"理论的科学内涵，为中医药治疗UC提供新的思路和理论依据。

溃疡性结肠炎是病因尚未明确的慢性肠道炎症性疾病，发病率不断升高。近年来越来越多的证据显示黏膜免疫系统中水通道蛋白和TLR/NF-κB信号通路在其中发挥着重要作用。依据中医“风能胜湿”理论及临床观察，本研究旨在研究风药治疗溃疡性结肠炎过程中对水通道蛋白和TLR/NF-κB信号通路的作用。对溃疡性结肠炎DSS造模法进行研究，对比不同浓度、相对分子量及暴露时间，证实以5%DSS相对分子量36000-50000自由饮用10天进行造模，成功率高，均衡性较强，结肠大体观察与溃疡性结肠炎相类似。在预实验中，观察到 DSS大鼠模型，造模成功并停用DSS后大鼠表现出较强的自愈能力，掩盖了药物效果，修正实验方案为药物干预与造模同步进行。采用DSS大鼠模型，观察比较风药组低中高剂量、白头翁汤组低中高剂量、风药合白头翁汤组低中高比例、美沙拉嗪组及模型组的DAI积分、结肠损伤情况及组织病理学改变，以ELISA法检测血清TNF-α、IL1、IL6、CINC1等炎症因子浓度，分别以PCR及免疫组化方法检测大鼠结肠组织中AQP4、AQP8、TLR4、TLR2、NF-κBp65、TNF-α、IL1、IL6、CINC1的mRNA含量及蛋白表达情况，结果显示，风药、白头翁汤单独应用均可缓解DSS模型结肠炎相关表现，二者均有一定的抗炎作用，风药可降低血清IL-1浓度并抑制结肠黏膜IL-6、TLR2转录，白头翁汤可降低血清TNF-α及CINC-1浓度。风药与白头翁汤合用对DSS大鼠治疗效果较风药及白头翁汤单用明显，机制在于风药在合用时促进结肠黏膜局部的AQP8转录与表达。证实了风药在溃疡性结肠炎治疗中发挥增效作用，一方面是其单独应用时可下调IL-1、IL-6、TLR2而发挥抗炎作用，另一方面是其与白头翁汤合用可上调结肠黏膜AQP8以改善肠腔水液代谢能力。本研究首次将风药理论引入溃疡性结肠炎的研究，以风药增效机制中对AQP8的调节作用从微观角度阐释了中医“风能胜湿”的科学内涵，为溃疡性结肠炎治疗提供了新的思路。