健脾理气方通过修复十二指肠屏障治疗功能性消化不良作用机制研究

Functional dyspepsia (FD) is one of the most common functional gastrointestinal disorders with an increasing incidence. Because of the lack of effective treatment options, FD is associated with significantly impaired quality of life and considerable healthcare costs. A lot of studies demonstrated that Traditional Chinese Medicines treatment for functional dyspepsia has the unique advantages. The disorder is thought to be heterogeneous, with different pathophysiological mechanisms underlying varied symptom patterns. Recently, a line of evidence implicates impaired duodenal mucosa barrier function is a major cause of FD. Epithelial barrier dysfunction results in low-grade inflammation and also causes increased sensitivity to duodenal acid and lipids. Tight junction and muctin2 secreted by goblet cells plays an important role in sustaining duodenal barrier function. Chinese medicine, Jianpiliqi formula, has demonstrated remarkable therapeutic effects in the treatment of FD. The major goal of the current proposal is to delineate the molecular mechanisms by which Jianpiliqi formula improves symptoms of FD through restoration of epithelial barrier function in the duodenum. In the guidance of theories of “liver correlate with spleen”and “spleen is the defense of body”, We will test the hypothesis that Jianpiliqi formula could promote the secretion of 5-HT and PGE2 in the duodenum eliciting tight junction activation from both transcriptional and post- transcriptional levels and increase the expression of MUC2 by regulating P53 Signaling pathways. We anticipate that completion of the current work will provide novel therapeutic strategies against FD treatment, and form the molecular basis for the advantages of using Chinese medicine in treating FD.

功能性消化不良（FD）是临床最常见的胃肠病之一，其发病率呈逐年上升，严重影响患者的生活质量，浪费大量医疗资源，中医治疗FD具有独特优势。FD目前发病机制尚不完全清楚，近年来研究发现，十二指肠结构和功能异常是FD重要发病机制， 包括十二指肠对酸、脂类的敏感，十二指肠低程度炎症等，均与十二指肠黏膜屏障功能受损相关。而上皮细胞间的紧密连接和杯状细胞分泌的黏蛋白2（MUC2）在维持十二指肠屏障功能中起重要作用。前期研究证实中药“健脾理气方”治疗FD临床疗效确切，本课题在“肝脾相关”和“脾为之卫”理论的指导下，提出并通过动物和细胞实验验证如下假说：健脾理气方通过提高5-HT和PGE2分泌水平，促进紧密连接蛋白磷酸化，并且通过P53通路，调节上皮杯状细胞分化和MUC2表达，进而修复十二指肠黏膜屏障功能。以期明确健脾理气方可能的作用靶点和信号介导途径，并从分子生物学水平为中药在FD治疗中的优势提供依据。

功能性消化不良（FD）是临床最常见的胃肠病之一，其发病率呈逐年上升，严重影响患者的生活质量，浪费大量医疗资源；中医治疗FD具有独特优势。FD目前发病机制尚不完全清楚，近年来研究发现，十二指肠结构和功能异常是FD重要发病机制，包括十二指肠对酸、脂类的敏感及十二指肠低程度炎症等，均与十二指肠黏膜屏障功能受损相关。而上皮细胞间的紧密连接和杯状细胞分泌的黏蛋白2（MUC2）在维持十二指肠屏障功能中起重要作用。前期研究证实中药“健脾理气方”治疗FD临床疗效确切，为了进一步探索健脾理气方不同的治疗靶点，本课题在“肝脾相关”和“脾为之卫”理论的指导下，从健脾理气方激活不同信号通路，促进紧密连接蛋白磷酸化，调节上皮杯状细胞分化和MUC2表达，进而修复十二指肠黏膜屏障功能探索可能的作用机制。动物实验采用碘乙酰胺灌胃+夹尾刺激法诱导FD大鼠模型，通过HE染色观察十二指肠组织结构的变化，AB-PA染色法检测杯状细胞数量，通过Western blot测定紧密连接蛋白各种成分的蛋白表达水平和磷酸化水平，用免疫荧光法测定紧密连接蛋白定位，通过免疫酶联法测定十二指肠5-HT和PGE2的含量变化，定量PCR、Western blot和免疫组化法检测MUC2蛋白表达变化，通过Western blot测定十二指肠P53含量变化。细胞实验选用 Hutu十二指肠细胞作为模型，检测健脾理气方含药血清、5-HT和PGE2对紧密连接蛋白的表达和定位作用，同时，明确Ca2+-PKC信号途径在 5-HT诱导的紧密连接蛋白活化中的作用和PGE2－cAMP－PKA信号转导途径。研究结果提示健脾理气方通过提高5-HT和PGE2分泌水平，促进紧密连接蛋白磷酸化，并且通过P53通路，调节上皮杯状细胞分化和MUC2表达，进而修复十二指肠黏膜屏障功能。本课题明确了健脾理气方可能的作用靶点和信号介导途径，并从分子生物学水平为中药在FD治疗中的优势提供依据。