Cao Yang, Zhong Feng, Wen Qian, Fang Chuang, Xia Ye-Wan, Luo Rong, Kuang Hong-Jun, Zhang Wei
Postgraduate School of Hunan University of Chinese Medicine, Changsha 410208, China.
Department of Acupuncture and Tuina Rehabilitation, The First Affiliated Hospital of Hunan University of Chinese Medicine, Changsha 410007.
Zhen Ci Yan Jiu. 2022 Feb 25;47(2):141-7. doi: 10.13702/j.1000-0607.20210253.
To observe the effect of electroacupuncture (EA) of "Tianshu"(ST25) and "Dachangshu"(BL25) on the intestinal transit function， expression level of glial cell-derived neurotrophic factor (GDNF) and methylation level of GDNF gene promoter region in colon tissue of rats with slow transit constipation (STC)， so as to explore its mechanisms underlying improvement of STC.
Male Sprague-Dawley (SD) rats were randomized into control， saline， model and EA groups (=16 in each group). The STC model was replicated by gavage of compound diphenoxylate suspension (10 mL· kg· d) for 28 days. Rats of the saline group received the same dose of normal saline via gavage. EA (2 Hz/15 Hz， 0.1-1 mA) was applied to bila-teral ST25 and BL25 for 15 min， once daily for 14 days. The intestinal transmission function (the intestinal propulsion rate) was assessed by recording the first black grain stool discharge time and the number and weight of the discharged stool grains in 30 min after gavage of the activated carbon suspension (1 mL/100 g， 150 g/L). The score of fecal trait and the weight of stool within 24 h were recorded. The ultrastructural changes of Cajal interstitial cells in the colon tissue were observed by transmission electron microscope. The expression levels of GDNF protein and mRNA in the colon tissue were detected by using Western blot and real-time fluorescent quantitative PCR， separately， and changes of methylation level in the promoter region of GDNF gene detected by using Bisulfite sequencing method.
Compared with the control group， the time of the 1 black stool grain discharging was obviously prolonged， and the number and weight of the discharged black stool grains were significantly decreased in the mo-del group (<0.05)， suggesting a success of STC. The weight and trait score of stool in 24 h， intestinal propulsive rate， and the expression levels of GDNF protein and mRNA were significantly lower in the model group than in the control group (<0.01， <0.05). After EA， the weight and trait score of stool within 24 h， intestinal propulsive rate，and the expression levels of GDNF protein and mRNA were significantly increased in the EA group in contrast to the model group (<0.01，<0.05). The total CpGs methylation level of GDNF gene in colon tissue was considerably higher in the model group than in the control group (<0.05)， and markedly lower in the EA group than in the model group (<0.05). No significant differences were found between the control and saline groups in all the above-mentioned indexes (>0.05).
EA of back-shu and front-mu acupoints can effectively improve symptoms of constipation and intestinal transport function in STC rats， which may be related to its function in up-regulating the expression of GDNF and down-regulating the methylation level in the promoter region of GDNF gene in colon tissue.
观察电针“天枢”（ST25）、“大肠俞”（BL25）对慢传输型便秘（STC）大鼠肠道传输功能、结肠组织中胶质细胞源性神经营养因子（GDNF）表达水平及GDNF基因启动子区甲基化水平的影响，以探讨其改善STC的作用机制。
将雄性Sprague-Dawley（SD）大鼠随机分为对照组、生理盐水组、模型组和电针组（每组16只）。采用复方地芬诺酯混悬液（10 mL·kg·d）灌胃28天复制STC模型。生理盐水组大鼠给予等量生理盐水灌胃。电针组采用2 Hz/15 Hz、0.1 - 1 mA电流强度针刺双侧ST25、BL25，留针15分钟，每日1次，共14天。通过记录活性炭混悬液（1 mL/100 g，150 g/L）灌胃后首次排出黑色粪便的时间及30分钟内排出粪便颗粒的数量和重量，评估肠道传输功能（肠道推进率）。记录24小时内粪便性状评分及粪便重量。采用透射电镜观察结肠组织中Cajal间质细胞的超微结构变化。分别采用蛋白质免疫印迹法和实时荧光定量PCR检测结肠组织中GDNF蛋白和mRNA的表达水平，采用亚硫酸氢盐测序法检测GDNF基因启动子区甲基化水平变化。
与对照组相比，模型组首次排出1粒黑色粪便的时间明显延长，排出黑色粪便颗粒的数量和重量显著减少（P<0.05），提示STC模型复制成功。模型组24小时内粪便重量和性状评分、肠道推进率以及GDNF蛋白和mRNA表达水平均显著低于对照组（P<0.01，P<0.05）。电针后，电针组24小时内粪便重量和性状评分、肠道推进率以及GDNF蛋白和mRNA表达水平与模型组相比均显著升高（P<0.01，P<0.05）。模型组结肠组织中GDNF基因总CpGs甲基化水平显著高于对照组（P<0.05），电针组显著低于模型组（P<0.05）。上述各指标在对照组和生理盐水组之间差异均无统计学意义（P>0.05）。
背俞穴与募穴电针可有效改善STC大鼠便秘症状及肠道传输功能，其机制可能与上调结肠组织中GDNF表达、下调GDNF基因启动子区甲基化水平有关。
