四磨汤通过调节TGR5/TRPA1信号通路对慢传输型便秘大鼠肠道动力的影响

Effect of Simo decoction on intestinal motility in rats with slow transit constipation by regulating the Takeda G protein-coupled receptor 5/transient receptor potential ankyrin 1 signaling pathway

摘要

目的 四磨汤通过调节胆汁酸G蛋白偶联受体5（G-protein coupled-receptor kinase5，TGR5）/瞬时受体电位锚蛋白1（transient receptor potential ankyrin-1，TRPA1）信号通路对慢传输型便秘（slow transit constipation，STC）大鼠肠道动力的影响。 方法 取SD大鼠采用复方地芬诺酯混悬液灌胃法制备STC模型，随机分为模型组、四磨汤低剂量（1.8 g/kg）组、四磨汤高剂量（3.6 g/kg）组、四磨汤高剂量（3.6 g/kg）+SBI-115（TGR5抑制剂，55.4 mg/kg）组，每组10只，另取10只SD大鼠灌胃等剂量生理盐水设为对照组，采用四磨汤与SBI-115分组处理后测量各组大鼠首粒黑便排出时间、6 h内排便粒数及排便重量、小肠炭末推进率、结肠平滑肌收缩力；以苏木精-伊红（hematoxylin eosin，HE）染色检测各组大鼠结肠病理形态；以免疫组织化学染色检测各组大鼠结肠组织受体5-羟色胺受体3（5-hydroxytryptamine receptor 3，5-HT3R）表达；以酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）试剂盒测量各组大鼠结肠组织5-羟色胺（5-hydroxytryptamine，5-HT）与炎症因子白细胞介素（interleukin，IL）-6、IL-1β水平；以免疫印迹法检测各组大鼠结肠组织TGR5/TRPA1通路蛋白表达。 结果 与对照组比较，模型组大鼠结肠组织发生明显病理损伤，首粒黑便排出时间、结肠组织IL-6及IL-1β水平明显升高（P<0.05），6 h内排便粒数、排便重量、小肠炭末推进率、结肠平滑肌收缩力、结肠组织5-HT3R阳性表达、5-HT水平及TGR5、TRPA1蛋白表达明显降低（P<0.05）；与模型组比较，四磨汤低剂量组、四磨汤高剂量组大鼠结肠组织病理损伤均减轻，首粒黑便排出时间、结肠组织IL-6及IL-1β水平均降低（P<0.05），6 h内排便粒数、排便重量、小肠炭末推进率、结肠平滑肌收缩力、结肠组织5-HT3R阳性表达、5-HT水平及TGR5、TRPA1蛋白表达均升高（P<0.05），且高剂量四磨汤作用更强；SBI-115可减弱四磨汤对STC大鼠各指标的作用。 结论 四磨汤可通过促进TGR5/TRPA1信号活化而抑制STC大鼠结肠炎症，升高大鼠结肠组织5-HT水平及其受体5-HT3R表达，减轻大鼠结肠组织病理损伤，进而增强大鼠结肠平滑肌收缩力及肠道动力，减轻其便秘症状。

Abstract

Objective To investigate the effect of Simo decoction on intestinal motility in rats with slow transit constipation（STC） by regulating the Takeda G protein-coupled receptor 5（TGR5）/transient receptor potential ankyrin 1（TRPA1） signaling pathway. Methods Sprague-Dawley rats were given compound diphenoxylate suspension by gavage to establish a model of STC，and then they were randomly divided into model group，low-dose（1.8 g/kg） Simo decoction group，high-dose（3.6 g/kg） Simo decoction group，and high-dose（3.6 g/kg） Simo decoction+SBI-115（TGR5 inhibitor，55.4 mg/kg） group，with 10 rats in each group； another 10 rats were given an equal volume of normal saline by gavage and were selected as control group. After treatment with Simo decoction and SBI-115，the rats in each group were measured in terms of the time to first black stool defecation，the number and weight of feces granules within 6 hours，small intestine charcoal powder propulsion rate，and colonic smooth muscle contractibility； HE staining was used to observe colon pathomorphology； immunohistochemical staining was used to measure the expression level of 5-hydroxytryptamine receptor 3（5-HT3R） in colon tissue； ELISA was used to measure the levels of 5-hydroxytryptamine（5-HT） and the inflammatory factors interleukin-6（IL-6） and interleukin-1β（IL-1β） in colon tissue； Western blotting was used to measure the expression of TGR5/TRPA1 pathway proteins in colon tissue. Results Compared with the control group，the model group had marked pathological damage of colon tissue，significant increases in the time to first black stool defecation and the levels of IL-6 and IL-1β in colon tissue（P<0.05），and significant reductions in the number and weight of feces granules within 6 hours，small intestine charcoal powder propulsion rate，colonic smooth muscle contractibility，positive expression of 5-HT3R in colonic tissue，5-HT level，and the protein expression levels of TGR5 and TRPA1（P<0.05）. Compared with the model group，the low- and high-dose Simo decoction groups had alleviation of the pathological damage of colon tissue，significant reductions in the time to first black stool defecation and the levels of IL-6 and IL-1β in colon tissue（P<0.05），and significant increases in the number and weight of feces granules within 6 hours，small intestine charcoal powder propulsion rate，colonic smooth muscle contractibility，positive expression of 5-HT3R in colonic tissue，5-HT level，and the protein expression levels of TGR5 and TRPA1（P<0.05），and high-dose Simo decoction showed a stronger effect. SBI-115 could weaken the effect of high-dose Simo decoction on various indicators of STC rats. Conclusion Simo decoction can inhibit colitis in STC rats by promoting the activation of TGR5/TRPA1 signaling，increase the levels of 5-HT and its receptor 5-HT3R in rat colon tissue，and alleviate pathological damage of colon tissue，thereby enhancing the contractibility of colon smooth muscle and intestinal motility and alleviating the symptoms of constipation.

关键词

四磨汤 / TGR5/TRPA1 / 慢传输型便秘 / 肠道动力

Key words

Simo decoction / Takeda G protein-coupled receptor 5/transient receptor potential ankyrin 1 / slow transit constipation / intestinal motility

基金资助

国家自然科学基金资助项目(81573990)