健脾养肝丸调控肠嗜铬细胞及其TPH1-5-HT信号通路治疗IBS-D大鼠的机制研究

Mechanism study on Jianpi Yanggan Pill in regulating enterochromaffin cells and TPH1-5-HT signaling pathway in treating IBS-D rats

关键词:  腹泻型肠易激综合征  内脏高敏感  健脾养肝丸  嗜铬细胞  5-羟色胺  嗜铬粒蛋白A

Key words:  diarrheal irritable bowel syndrome  visceral hypersensitivity  Jianpi Yanggan Pill  enteroendocrine cell  5-hydroxytryptamine  Chromogranin A

摘要:

[目的] 通过研究健脾养肝丸对腹泻型肠易激综合征（IBS-D）模型大鼠嗜铬细胞/5-羟色胺（EC/5-HT）信号通路表达的影响，进一步探索健脾养肝丸是否通过调节嗜铬细胞（EC）中5-羟色胺（5-HT）的分泌来缓解IBS-D的内脏高敏感（VH），从而揭示其治疗IBS-D的内脏敏感机制。[方法] 采用母婴分离结合束缚应激法制备IBS-D大鼠模型，通过粪便评分、粪便含水率测定、腹泻指数检测及内脏敏感性测定法评价IBS-D模型及健脾养肝丸的疗效；采用酶联免疫吸附（ELISA）实验、实时荧光定量聚合酶链反应（qPCR）实验、免疫组化染色、免疫荧光染色及电镜检测EC中5-HT的合成、分泌情况。[结果] 与模型组相比，健脾养肝丸高、中、低剂量组降低了IBS-D大鼠Bristol粪便性状评分、粪便含水量、内脏敏感性、结肠及血清5-HT水平、TPH1的mRNA相对表达量（P<0.05或<0.01），减轻了结肠组织病理损伤，减少了嗜铬粒蛋白A（CgA）阳性表达，且通过健脾养肝丸的治疗，EC呈现椭圆形、分泌颗粒减少。[结论] 健脾养肝丸可能通过降低TPH1介导EC重摄取色氨酸分解成为5-羟色胺酸（5-HTP），进而减少5-HT的生成从而降低内脏高敏感。

Abstract:

[Objective] To explore Jianpi Yanggan Pills alleviate visceral hypersensitivity（VH） in IBS-D by regulating the secretion of 5-hydroxytryptamine（5-HT） in enteroendocrine cells（EC），thereby revealing the visceral hypersensitivity mechanism of its treatment for IBS-DBy studying the effect of Jianpi Yanggan Pills on the expression of the enteroendocrine cells/5-hydroxytryptamine（EC/5-HT） signaling pathway in diarrhea-type irritable bowel syndrome（IBS-D） model rats. [Methods] An IBS-D rat model was established by using the mother-infant separation combined with restraint stress method. The efficacy of the IBS-D model and Jianpi Yanggan Pills was evaluated through fecal scoring，fecal water content determination，diarrhea index detection，and visceral sensitivity measurement. ELISA experiments，qPCR experiments，immunohistochemical staining，immunofluorescence staining，and electron microscopy were used to detect the synthesis and secretion of 5-HT in EC. [Results] Compared with the model group，the high，medium and low-dose Jianpi Yanggan Pills groups decreased the Bristol stool form score，fecal water content，visceral sensitivity，colonic and serum 5-HT levels，and the relative mRNA expression of TPH1 in IBS-D rats（P<0.05，P<0.01，P<0.001），alleviated colonic tissue pathological damage，reduced Chromogranin A positive expression，and through the treatment of Jianpi Yanggan Pills，ECs presented an oval shape and the number of secretory granules decreased. [Conclusion] Jianpi Yanggan Pills may reduce visceral hypersensitivity by decreasing TPH1-mediated EC reuptake of tryptophan to form 5-hydroxytryptophan（5-HTP），thereby reducing 5-HT production.