Objectives: To investigate the effect of electroacupuncture (EA) on intestinal motility and visceral sensitivity in functional constipation (FC) mice and its regulatory mechanism on Piezo1/2 channels in enterochromaffin (EC) cell.
Methods: Twenty-four male C57BL/6J mice were randomly divided into normal control, model, and EA groups (n=8 in each group). The FC model was established by gavage of loperamide hydrochloride. EA (1 mA, 3 Hz/15 Hz) was applied to unilateral "Tianshu" (ST25) and "Shangjuxu" (ST37) for 20 min per session, once daily for 5 consecutive days per week, two weeks altogether. Fecal parameters (number of stool particles in 6 h, characters, and fecal water content), intestinal motility (the time of the first blue stool excretion after gavage of Evans blue solution and small intestinal transit rate), and visceral sensitivity (assessed by abdominal withdrawal reflex [AWR] in responding to colorectal dilation) were observed. A transmission electron microscopy (TEM) was used to observe EC cells' ultrastructure. Triple immunofluorescence, Western blot and real-time qPCR were used to detect the expressions of Piezo1/2 in the EC cells and colon, and ELISA was employed to detect the contents of serum and colonic 5-HT and colonic Ca2+.
Results: Compared with the normal group, the model group showed a significant reduction in the number of stool particles, fecal character score, and fecal water content, small intestinal transit rate, AWR scores at 0.25 mL, 0.35 mL and 0.65 mL, contents of serum and colonic 5-HT and colonic Ca2+, expression levels of colonic Piezol1 and Piezol 2 proteins and mRNAs, and the positive areas of Piezol1 and Piezol2, EC, Piezol1/EC and Piezol2/EC (P<0.01, P<0.001, P<0.05), and an evident increase in the time of the first blue stool particle excretion (decrease of intestinal propulsion) and AWR 3-points threshold (P<0.01). Following EA treatment, both the decrease and increase of the indexes mentioned above were reversed completely (P<0.05, P<0.001, P<0.01). TEM showed mitochondrial swelling and a reduction in the secretory granules of EC cells in the model group, which was partially restored following EA treatment.
Conclusions: EA of ST25 and ST37 can alleviate FC-related intestinal dysmotility and visceral hypersensitivity in FC mice, which may be associated with its functions in up-regulating Piezo1/2 channels of EC cell, promoting Ca2+ influx and 5-HT secretion.
目的: 探讨电针对功能性便秘（FC）小鼠肠道动力及敏感性障碍的改善作用，及其对肠嗜铬（EC）细胞Piezo通道的调控机制。方法: C57BL/6J雄性小鼠随机分为正常组、模型组和电针组，每组8只。采用盐酸洛哌丁胺灌胃构建FC模型。电针组针刺“天枢”和“上巨虚”，20 min/次，1次/d，干预5 d后休息2 d，共10 d。检测各组小鼠的排便情况、肠道动力和结肠敏感性;透射电镜观察结肠EC细胞超微结构;ELISA法检测血清和结肠5-羟色胺（5-HT）、结肠Ca2+含量;免疫荧光染色法、Western blot法和实时荧光定量PCR法检测结肠和EC细胞中Piezo1/2表达。结果: 与正常组相比，模型组粪便颗粒数、性状评分和含水率均降低（P<0.01，P<0.001，P<0.05），首粒蓝便排出时间和疼痛阈值增加（P<0.01），小肠推进率和腹壁撤退反射（AWR）评分降低（P<0.01，P<0.05）;EC细胞线粒体肿胀，分泌颗粒数量减少;血清和结肠中5-HT及结肠中Ca2+含量均减少（P<0.01，P<0.001）;结肠中Piezo1、Piezo2蛋白及其mRNA表达水平均下调（P<0.001），Piezo1、Piezo2蛋白和EC细胞的阳性面积占比，以及Piezo1/EC、Piezo2/EC的共定位系数均降低（P<0.01，P<0.05，P<0.001）。与模型组相比，电针组粪便颗粒数、性状评分和含水率均增加（P<0.05，P<0.001，P<0.01），首粒蓝便排出时间和疼痛阈值下降（P<0.01，P<0.05），小肠推进率和AWR评分升高（P<0.05，P<0.01）;EC细胞线粒体结构部分恢复，分泌颗粒数量增加;血清和结肠中5-HT及结肠中Ca2+含量增加（P<0.05）;结肠中Piezo1、Piezo2蛋白及其mRNA表达水平均上调（P<0.001），Piezo1、Piezo2蛋白和EC细胞的阳性面积占比，以及Piezo1/EC、Piezo2/EC的共定位系数均上升（P<0.01，P<0.05）。结论: 电针可能通过上调EC细胞Piezo1/2通道表达，促进Ca2+内流及5-HT分泌，改善FC小鼠的肠道动力及敏感性障碍，从而改善FC。.
Keywords: Electroacupuncture; Enterochromaffin cell; Functional constipation; Intestinal motility; Piezo.