【作者】 朱向东；

【导师】 梅晓云；

【作者基本信息】 南京中医药大学 ， 中医基础理论， 2013， 博士

【摘要】 研究目的与意义溃疡性结肠炎(uleerativeeolitis, UC)是一种病因不明的,主要累及直肠和乙状结肠的黏膜和黏膜下层的浅表性、非特异性炎症病变为主的消化道疾病,可扩展至降结肠、横结肠,少数可累及全结肠。其治疗难度非常大,且反复发作,缠绵难愈,被公认为是结肠癌的癌前病变,已经被世界卫生组织(WHO)列为现代社会最难治疗的疾病之一。现代医学对UC缺乏特效治疗药物,患者常因为并发症、手术、激素耐药或依赖问题而痛苦异常、造成患者严重的经济、家庭负担。UC在国外十分常见,我国UC发病率和患病率也非常高,且呈现逐年增加的态势。由于UC的病因和发病机制十分复杂,因而临床缺乏特异性治疗药物,部分患者因缺乏有效治疗而伴随终身,患者的生活质量已经受到严重影响。目前,针对UC的治疗药物主要有肾上腺皮质激素、水杨酸类、免疫抑制剂等,这些药物因为具有副反应多、停药后易复发,部分顽固性病例疗效不理想等缺点,限制了其在临床的使用。中医药治疗UC具有副反应少、价格低廉、疗效显著、无依赖性、多靶点整体调节、复发率低、治病求本以及远期疗效稳定等优势,具有较好的深入研究与应用开发价值,受到消化内科专家的青睐。中医药防治UC的机理研究亦是近年来研究者追踪的前沿热点。目前,在中医药防治UC的实验研究方面,医家多偏重从“湿热”病机入手,以“清化湿热”立法,对治疗泄泻的经典方,如“白头翁汤”、“芍药汤”、“参苓白术散”等进行了深入研究,取得了诸多成果。但缺乏对UC“肝郁脾虚”病机及“疏肝健脾法”的探讨。根据临床所见及证候分布和治法研究,发现“肝郁脾虚证”是UC的重要证候,“疏肝健脾法”亦是治疗UC的重要方法,尤其是精神情绪因素“肝郁”既可能是UC发病的始动因素,亦可能是UC病情加重或缠绵难愈的重要原因。治则治法是连接病机和方药疗效的核心环节,从“肝郁脾虚”病机视角研究“疏肝健脾法”治疗UC的疗效机制和作用靶点,不仅可以拓展中医药治疗UC的思路,揭示中医治法的科学内涵,亦可为开发防治UC的特效中成药提供理论和实验依据,所以本研究具有较好的应用前景和科学意义、现实意义。痛泻要方出自《丹溪心法》,由炒白术、炒白芍、防风、陈皮四味药组成,功能疏肝健脾,是治疗肝郁脾虚所致腹泻的经典方。回顾并研究古今近10年文献,发现临床运用痛泻要方或者痛泻要方的加减方治疗UC取得良好疗效的报道甚多,有较好的临床基础,但从实验角度研究其治疗UC的作用机理却鲜有报道。基于“肝郁脾虚证”是UC的重要证型,“肝郁脾虚”是UC的重要病机,“疏肝健脾法”是治疗UC的重要治法的认识,结合国内外鲜有研究“疏肝健脾法”治疗UC的作用机制,本研究以“疏肝健脾法”为指导,选用“疏肝健脾法”的代表方痛泻要方,针对UC“肝郁脾虚”基本病机,运用分子生物学实验技术,从基因和蛋白表达水平揭示痛泻要方治疗UC的分子机制,为其临床广泛运用提供实验依据。研究方法：本论文研究分为理论研究、实验研究两个部分。理论研究部分是在系统整理中医学对UC认识古今相关文献的基础上,结合现代医学对UC的研究进展,重点探讨了UC的中医病因病机,UC的免疫学发病机制,溃疡性结肠炎的证候分型、治法的分布规律,疏肝健脾法治疗UC的现状及本论文研究的主要思路。实验研究部分首先采用TNBS/乙醇灌肠法复建UC大鼠模型,在造模的不同时间段采用RT-PCR及ELISA法、免疫组化技术动态观察大鼠结肠组织NF-κB p65、PAR-γ、ICAM-1及血清TNF-α、IL-1β的动态表达、探讨了其在UC发病中的作用。其次是研究采用TNBS/乙醇灌肠+束缚法+饮食失节的复合方法建立大鼠肝郁脾虚型UC模型,并给予痛泻要方干预治疗,采用RT-PCR、免疫印迹及ELISA法检测其对UC大鼠结肠组织中PPARγ、NF-κB p65、ICAM-1表达以及血清中IL-1β、TNF-α水平的影响,一方面为治疗UC寻找新的作用靶点,另外一方面是从分子水平进一步探讨疏肝健脾法治疗UC作用机理。研究结论及意义1. TNBS/乙醇灌肠法可成功建立UC大鼠模型,肉眼观察及光镜镜观察均出现明显的炎症、溃疡。且结肠损伤随着造模时间不同呈现动态变化趋势,其中以3d和7d的病变最为严重,14d和21d天则炎症和溃疡有所修复。2.在UC模型的急性期,结肠组织中PPAR-γ表达显著降低,慢性期PPAR-γ表达略有升高,但仍明显低于空白组。UC模型的急性期NF-κB p65、ICAM-1表达显著升高,慢性期,NF-κB p65、ICAM-1表达略有降低,但仍明显高于空白组。3.促炎因子TNF-α、IL-1β和抗炎因子IL-4的平衡参与了UC的发生、发展。4. PPAR-γ的表达与结肠组织损伤程度、IL-1β、TNF-α、NF-κB p65、ICAM-1的表达之间呈不同程度的负相关。5.UC的发生与发展与PPAR-γ的表达受抑密切相关,上调PPAR-γ的表达有望成为UC治疗新靶向。6.肝郁脾虚是UC的重要病机,肝郁脾虚证是UC的重要证候类型,疏肝健脾法是治疗UC的重要治法。7.痛泻要方对肝郁脾虚型UC模型大鼠结肠组织具有良好的保护和修复作用,其分子机制可能是上调PPAR-y mRNA及蛋白表达,抑制结肠组织ICAM-1、 NF-κB p65mRNA及蛋白表达、抑制血清IL-1β、TNF-α的表达,从而减轻结肠炎症反应,起到治疗UC的作用。痛泻要方可能具有PPAR-γ激动剂的效用。更多还原

【Abstract】 Purpose and SignificanceUlcerative colitis (uleerativeeolitis, UC) is a kind of unknown etiology, mainly involving the rectum and sigmoid colon mucosa and submucosa of the superficial, nonspecific inflammatory disease of digestive tract disease, can be extended to the descending colon, the transverse colon, minority can affects the colon, is difficult to cure, often recurrent, is considered to be precancerous lesions of the colon cancer, World Health Organization has been listed it as one of the modern difficult to treat. Modern medicine to UC is lack of a specific treatment, patients often bearing complications, surgery, hormone resistant or dependent abnormal pain, causing severe economic and family burden. Ulcerative colitis is quite common in Western countries, According to the ulcerative colitis-related documents and statistics the number of hospital cases show that its incidence is increasing year by year. Due to the complexity of the pathogenesis of UC, the cause is not yet very clear, due to the lack of effective treatment and specific clinical drug, for some patients accompanied by lifelong with abnormal pain, have serious impact on people’s quality of life. At present, Drugs for UC are often salicylic acid, adrenal corticosteroids, immunosuppressants, etc. Most of them are easy to relapse after treatment, long-term medication side effects, for some stubborn cases, the effect is not ideal shortcomings.In recent years, Chinese medicine in the treatment of UC achieved more satisfactory results, and has little side effects, low recurrence rate and long-term efficacy advantage with better research and development value, favored by the Department of Gastroenterology experts, it is the tracked hotspots in recent years that the Mechanism research on Traditional Chinese Medicine to UC. At present, mechanism on Traditional Chinese Medicine to treatment UC, physicians more emphasis to start from the "Shire" pathogenesis and treatment of diarrhea classic recipe of the "Qinghuashire", such as "Pulsatilla soup","Peony soup","Shenlingbaishu powder" made a lot of achievements. But, according to clinical diagnosis, stagnation and spleen deficiency is important pathogenesis of UC,"Shuganjianpi" is an important method for the treatment of UC, especially the mental factor "liver depression" is probably UC incidence initiating factor, or hard to be cured reasons. The therapeutic principle is the core aspects of connected to the pathogenesis and recipe efficacy, clarify its efficacy mechanism and target of the treatment of UC’s efficacy, from perspective of stagnation and spleen deficiency to study curative effect mechanism of "Shuganjianpi" treatment of UC, can not only expand the idea of Chinese medicine treatment of UC, revealing the scientific connotation of TCM treatments, also provide theoretical support for the development of prevention and treatment of UC proprietary Chinese medicines, all in all, this study has good application prospects and scientific significance and practical significance. Tong Xie Yao Fang from the "Danxi Experiences", composed of four herbs of fried Baizhu, fried Baishao, Fangfeng and Chenpi, its function is Shuganjianpi, it is the classic recipe for diarrhea raised by liver depression and spleen. Reviewed and studied nearly10years ancient and modern literature, it is found that the clinical use Tongxieyaofang or it’s extend recipe for treatment of UC achieved good effect, with good clinical basis, but it is rarely reported of treatment UC mechanism from the experimental perspective. Liver depression and spleen deficiency syndrome is important UC pattern of syndrome, stagnation and spleen deficiency is an important pathogenesis of UC,"Shuganjianpi" is the important treatment of UC, combined with domestic and international fresh research on "Shuganjianpi "treatment mechanism of UC, this study as guide of the "Shuganjianpi" treatment, choice its representative recipe of Tongxieyaofang, aim at basic pathogenesis of UC stagnation and spleen deficiency, focusing on the use of the experimental techniques of molecular biology, from geneand the level of protein expression reveal the TongxieYaofang treatment of the molecular mechanisms of UC, provided experiment basis for the clinical extensive application.Research MethodsThis thesis is divided into two parts, theoretical and experimental studies. The theoretical part finishing medical UC understanding of the basis of the ancient and modern literature, combined with the progress of modern medicine on UC’s research, focuses on TCM etiology and pathogenesis of UC, immunology for UC, UC syndrome type, conquer the logistic regression, the main idea of the treatment of the UC status quo and the thesis Shuganjianpi treatment. First, study part apply the TNBS/ethanol enema rehabilitation UC rat model of experimental, in the modeling of different time periods by RT-PCR, Western blot and ELISA, immunohistochemistry technology dynamic observation of rat colon tissue of PPAR-γ, NF-κB p65, ICAM-1and serum IL-1β, TNF-α, dynamic expression, and to explore its role in the pathogenesis of UC. Followed by research to establish the rat liver depression and spleen UC model TNBS/ethanol+binding method+diet disloyal composite method and treatment to give TongxueYaofang intervention, observation of UC colon tissue of PPARγ and NF-κB p65, ICAM-1content and the level of serum IL-1β, TNF-α, on the one hand, looking for a new role in the treatment of UC target, other hand, from the genetic level to further explore the mechanism of action of the Shuganjianpi treatment of UC, to provide a theoretical basis for its clinical application and development of new compound Chinese medicine.Conclusions and Significancea. TNBS/ethanol enema can be successfully established rat model of UC, observation from naked eye and microscopic, all showed inflammation and ulcers, and colon injury are dynamic modeling time trend, which is most severe lesions3d and7d,14d and21d days, some repair of the inflammation and ulceration would be appear.b. In the acute phase of the UC model, PPARγ expression in colonic tissue was significantly reduced, the chronic phase PPARγ expression slightly elevated, but still lower than the blank group. UC model of the acute phase of NF-κB p65and ICAM-1expression was significantly increased in the chronic phase of NF-κB p65, ICAM-1expression slightly reduced, but still significantly higher than the blank group.c. Proinflammatory cytokines TNF-α, IL-1β and anti-inflammatory cytokines IL-4balance involved in the occurrence of UC development.d. PPAR-γ expression and colon tissue injury, IL-1β, TNF-α, NF-κB of p65, ICAM-1expression was negative correlation of different.e. The occurrence and development of UC inhibited the expression of PPARγ is closely related to raised expression of PPAR-γ is expected to become the new targeted treatment of UC.f. Liver stagnation and spleen deficiency is an important pathogenesis of ulcerative colitis; stagnation and spleen deficiency is important pathogenesis of UC,"Shuganjianpi" is an important method for the treatment of UCg. Tong Xie Yao Fang has protection and repair function for the model of stagnation and spleen deficiency UC colon tissue, the molecular mechanism is up regulated PPAR-y mRNA and protein expression, inhibition of the colonic tissues ICAM-1, NF-κB p65mRNA and protein expression, suppression of serum IL-1β and TNF-α expression, so as to reduce the inflammatory reaction, and play a role in the treatment of UC. The TongXieYaofang may have PPAR agonist’s utility.更多还原