艾灸干预腹泻型肠易激综合征大鼠miR 24/SERT/5 HT通路改善内脏高敏感状态实验观察

目的 观察艾灸干预对腹泻型肠易激综合征（diarrhea predominant irritable bowel syndrome,IBS-D）大鼠微小RNA-24（micro RNA-24,miR-24）/5-羟色胺转运体（serotonin transporter,SERT）/5-羟色胺（5-hydroxytryptamine,5-HT）通路的影响,探究艾灸改善IBS-D大鼠内脏敏感性的作用机制。方法 将SD大鼠随机分为正常组、模型组、艾灸组共3组,每组12只。采用母子分离＋醋酸刺激＋慢性束缚法制备IBS-D大鼠模型。艾灸组给予艾灸双侧“天枢”“上巨虚”20 min,连续7 d。分别于34、45、53 d检测各组大鼠体质量、稀便率和腹部回撤反射（abdominal withdrawal reflex,AWR）的最小容量阈值;苏木精-伊红染色检测结肠组织病理学变化;ELISA法测定大鼠血清、中脑、结肠组织中5-HT、SERT、5-羟色胺受体4（serotonin receptor 4,5-HT4R）、降钙素基因相关肽（calcitonin gene related peptide,CGRP）、P物质（substance P,SP）水平的变化;Western blot、RT-PCR检测大鼠中脑、结肠组织中SERT、5-HT4R、CGRP、SP的蛋白及mRNA表达水平;RT-PCR检测中脑、结肠组织中miR-24表达水平。结果 与正常组比较,模型组大鼠稀便率升高（P＜0.05）、AWR最小容量阈值下降（P＜0.05）;血清、中脑、结肠组织中5-HT、5-HT4R、CGRP、SP水平均显著升高（P＜0.05）,SERT水平均显著降低（P＜0.05）;miR-24表达水平显著升高（P＜0.05）,SERT蛋白及mRNA表达水平均明显降低（P＜0.05）,5-HT4R、CGRP、SP的蛋白及mRNA表达水平均明显升高（P＜0.05）;与模型组比较,艾灸组大鼠稀便率下降（P＜0.05）,AWR最小容量阈值上升（P＜0.05）;血清、中脑和结肠组织中5-HT、5-HT4R、CGRP、SP水平均显著降低（P＜0.05）,SERT水平显著升高（P＜0.05）;miR-24表达水平显著降低（P＜005）,SERT的蛋白及mRNA表达水平显著升高（P＜0.05）,5-HT4R、CGRP、SP的蛋白及mRNA表达水平显著降低（P＜0.05）。结论 艾灸干预改善IBS-D大鼠内脏高敏感性可能与下调miR-24表达,促进SERT对5-HT的重摄取,降低5-HT水平有关。

Moxibustion Improves Visceral Hypersensitivity in Rats with Diarrhea-predominant Irritable Bowel Syndrome by Exerting an Intervention Effect on the microRNA-24/Serotonin Transporter/5-Hydroxytryptamine Pathway

Objective To investigate the mechanism of action of moxibustion in improving visceral hypersensitivity in rats with diarrhea-predominant irritable bowel syndrome (IBS-D) by observing the intervention effect of moxibustion on the microRNA-24 (miR-24)/serotonin transporter (SERT)/5-hydroxytryptamine (5-HT) pathway.Methods Sprague-Dawley rats were randomly divided into normal group, model group, and moxibustion group, with 12 rats in each group. A rat model of IBS-D was established by mother-child separation combined with acetic acid stimulation and chronic restraint stress. The rats in the moxibustion group were given moxibustion at "Tianshu" and "Shangjuxu" at both sides for 20 minutes, once a day for 7 consecutive days. Body weight, loose stool rate, and minimum volume threshold of abdominal withdrawal reflex (AWR) were measured on days 34, 45, and 53; HE staining was used to observe the histopathological changes of the colon; ELISA was used to measure the changes in the levels of 5-HT, SERT, serotonin receptor 4 (5-HT4R), calcitonin gene-related peptide (CGRP), and substance P (SP) in serum, midbrain, and colon tissue; Western blot and RT-PCR were used to measure the protein and mRNA expression levels of SERT, 5-HT4R, CGRP, and SP in the midbrain and colon tissue, and RT-PCR was used to measure the expression level of miR-24 in the midbrain and colon tissue. Results Compared with the normal group, the model group had a significant increase in loose stool rate (P<0.05), a significant reduction in the minimum volume threshold of AWR (P<0.05), significant increases in the levels of 5-HT, 5-HT4R, CGRP, and SP (P<0.05) and a significant reduction in the level of SERT in serum, midbrain, and colon tissue (P<0.05), a significant increase in the expression level of miR-24 (P<0.05), significant reductions in the protein and mRNA expression levels of SERT (P<0.05), and significant increases in the protein and mRNA expression levels of 5-HT4R, CGRP, and SP (P<0.05). Compared with the model group, the moxibustion group had a significant reduction in loose stool rate (P<0.05), a significant increase in the minimum volume threshold of AWR (P<0.05), significant reductions in the levels of 5-HT, 5-HT4R, CGRP, and SP (P<0.05) and a significant increase in the level of SERT in serum, midbrain, and colon tissue (P<0.05), a significant reduction in the expression level of miR-24 (P<0.05), significant increases in the protein and mRNA expression levels of SERT (P<0.05), and significant reductions in the protein and mRNA expression levels of 5-HT4R, CGRP, and SP (P<0.05). Conclusion Moxibustion improves visceral hypersensitivity in IBS-D rats possibly by downregulating the expression of miR-24, promoting the reuptake of 5-HT by SERT, and reducing the level of 5-HT.