Effect of electroacupuncture on enteric neuronal autophagy in functional constipation mice.

To explore the effect mechanism of electroacupuncture (EA) on functional constipation (FC) at the combined lower he-sea and front-mu points of large intestine based on enteric neuronal autophagy.A total of 40 SPF Kunming mice were randomly divided into 5 groups (n = 8), i.e. a control group, a model group, an acupuncture group, a 3-methyl adenine (3-MA) group, and a 3-MA + acupuncture group. Except the control group, the FC model was established by gavage with compound diphenoxylate suspension for 14 days in the other 4 groups. After successful modeling, the mice of the acupuncture group and the 3-MA + acupuncture group received EA at bilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37), stimulated for 30 min with disperse-dense wave, 2 Hz/15 Hz of frequency, 1 mA of intensity. EA was delivered once daily. One course of treatment was composed of 5 days and 2 courses were needed, with an interval of 2 days. An intraperitoneal injection of 3-MA (15 mg/kg) was administered 30 min before EA in the mice of the 3-MA group and the 3-MA + acupuncture group, once daily. Before and after intervention, the time of the first black stool defecation and defecation behaviors in 6 h were observed in each group. After intervention, in every group, the small intestine propulsion rate was calculated, the colon tissue morphology was observed using HE staining, the ultrastructure of enteric neuronal autophagy was observed under transmission electron microscope, and the expressions of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1 and neuronal nuclear antigen protein (NeuN) in neurons of colonic muscularis were determined by immunohistochemistry.Before intervention, when compared with those in the control group, the time of the first black stool defecation was prolonged (P<0.01, P<0.05), and numbers (P<0.01), wet weight (P<0.01, P<0.05) and water content (P<0.05, P<0.01) of stool in 6 h were reduced in the model, acupuncture, 3-MA and 3-MA + acupuncture groups. After intervention, compared with those in the control group, the time of the first black stool defecation was longer (P<0.05), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were decreased in the model group. The time of the first black stool defecation was shortened (P<0.01), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were increased in the acupuncture group when compared with those in the model group. The time of the first black stool defecation was extended (P<0.01), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were declined in the 3-MA + acupuncture group in comparison with those in the acupuncture group. All layers of colon tissue were normal and intact in each group. When compared with the control group, the small intestine propulsion rate and the average optical density (OD) values of LC3, Beclin-1 and NeuN in neurons of colonic muscularis were decreased (P<0.01), and autophagosomes were dropped in the model group. In the acupuncture group, the small intestine propulsion rate and the average OD values of NeuN, LC3 and Beclin-1 in neurons of colonic muscularis increased (P<0.01,P<0.05), and autophagosomes were elevated when compared with those in the model group. The small intestine propulsion rate and the average OD values of NeuN, LC3 and Beclin-1 in neurons of colonic muscularis were dropped (P<0.05,P<0.01) in the 3-MA + acupuncture group in comparison with those in the acupuncture group.Electroacupuncture may promote enteric neuronal autophagy and increase the number of neurons so that the intestinal motility can be improved and constipation symptoms can be relieved in FC mice.目的: 基于肠道神经元自噬探讨电针大肠合募配穴治疗功能性便秘（FC）的作用机制。方法: 将40只SPF级昆明小鼠随机分为对照组、模型组、针刺组、3-甲基腺嘌呤（3-MA）组以及3-MA+针刺组5组，每组8只。除对照组外，其余4组小鼠采用复方地芬诺酯混悬液灌胃14 d制备FC模型。造模成功后，针刺组、3-MA+针刺组电针双侧 “天枢”“上巨虚”，予疏密波，频率3 Hz/15 Hz，电流强度1 mA，每次30 min，每日 1次，5 d为一疗程，疗程之间休息2 d，共治疗2个疗程。3-MA组及3-MA+针刺组于电针前30 min予3-MA溶液腹腔注射（15 mg/kg），每日1次。分别于干预前后观察各组小鼠首粒黑便排出时间和6 h排便情况。干预后计算各组小鼠小肠推进率；HE染色法观察小鼠结肠组织形态；透射电镜观察小鼠肠道神经元自噬情况；免疫组化法检测小鼠结肠肌层神经元微管相关蛋白1轻链3（LC3）、Beclin-1、神经元核抗原蛋白（NeuN）表达。结果: 干预前，与对照组比较，模型组、针刺组、3-MA组及3-MA+针刺组小鼠首粒黑便排出时间延长（P<0.01，P<0.05），6 h排便粒数减少（P<0.01），6 h粪便湿重降低（P<0.01，P<0.05），6 h粪便含水率下降（P<0.05，P<0.01）。干预后，与对照组比较，模型组小鼠首粒黑便排出时间延长（P<0.05），6 h排便粒数减少（P<0.01），6 h粪便湿重降低（P<0.01），6 h粪便含水率下降（P<0.01）；与模型组比较，针刺组首粒黑便排出时间缩短（P<0.01），6 h排便粒数增多（P<0.01），6 h粪便湿重增加（P<0.01），6 h粪便含水率升高（P<0.01）；与针刺组比较，3-MA+针刺组小鼠首粒黑便排出时间延长（P<0.01），6 h排便粒数减少（P<0.01），6 h粪便湿重降低（P<0.01），6 h粪便含水率下降（P<0.01）。各组小鼠结肠组织各层结构均正常、完整。与对照组比较，模型组小鼠小肠推进率及结肠肌层神经元LC3、Beclin-1、NeuN平均吸光度值降低（P<0.01），自噬小体减少；与模型组比较，针刺组小肠推进率及结肠肌层神经元LC3、Beclin-1、NeuN平均吸光度值升高（P<0.01，P<0.05），自噬小体增多；与针刺组比较，3-MA+针刺组小肠推进率及结肠肌层神经元LC3、Beclin-1、NeuN平均吸光度值降低（P<0.05，P<0.01）。结论: 电针可促进肠道神经元自噬，增加神经元数量，从而增加FC模型小鼠肠道运动，改善便秘症状。.