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  • Title: [Effect of electroacupuncture on gastrointestinal motility in rats with slow transit constipation based on GDNF methylation modification].
    Author: Cao Y, Zhong F, Wen Q, Fang C, Xia YW, Luo R, Kuang HJ, Zhang W.
    Journal: Zhen Ci Yan Jiu; 2022 Feb 25; 47(2):141-7. PubMed ID: 35218624.
    Abstract:
    OBJECTIVE: 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. METHODS: Male Sprague-Dawley (SD) rats were randomized into control， saline， model and EA groups (n=16 in each group). The STC model was replicated by gavage of compound diphenoxylate suspension (10 mL· kg-1· d-1) 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. RESULTS: Compared with the control group， the time of the 1st 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 (P<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 (P<0.01， P<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 (P<0.01，P<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 (P<0.05)， and markedly lower in the EA group than in the model group (P<0.05). No significant differences were found between the control and saline groups in all the above-mentioned indexes (P>0.05). CONCLUSION: 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. 目的：观察电针大肠俞募穴“天枢”“大肠俞”对慢传输型便秘(STC)大鼠肠道传输功能的改善情况，通过检测大鼠结肠组织胶质细胞源性神经营养因子(GDNF)的表达以及GDNF基因启动子区CpG岛甲基化状态，探讨电针治疗STC的可能机制。方法：雄性SD大鼠随机分为正常组、盐水组、模型组和电针组，每组16只。采用15 mg/mL复方地芬诺酯混悬液灌胃(10 mL·kg-1·d-1)复制STC大鼠模型。盐水组予同等剂量的0.9%氯化钠溶液灌胃。电针组电针双侧“天枢”“大肠俞”，每次15 min，1次/d，连续14 d。观察大鼠排便情况并检测大鼠肠道传输功能；采用透射电镜观测结肠组织Cajal间质细胞（ICC）超微结构；Western blot法、实时荧光定量PCR法检测结肠组织中GDNF蛋白、mRNA的表达；重亚硫酸盐测序法检测结肠组织GDNF基因启动子区CpG岛甲基化状态。结果：与正常组、盐水组比较，模型组大鼠24 h排便量下降(P<0.01)，粪便性状评分降低(P<0.05)，小肠推进率下降(P<0.01)，ICC形态结构遭到破坏，结肠组织GDNF蛋白及mRNA表达降低(P<0.01)，GDNF基因启动子区CpG岛甲基化阳性率升高(P<0.05)。与模型组比较，电针组大鼠24 h排便量增多(P<0.01)，粪便性状评分升高(P<0.05)，小肠推进率明显升高(P<0.01)，损伤的ICC超微结构得到部分修复，结肠组织GDNF蛋白及mRNA表达升高(P<0.05)，GDNF基因启动子区CpG岛甲基化阳性率降低(P<0.05)。结论：电针大肠俞募配穴可促进结肠组织GDNF的表达，有效改善STC大鼠的便秘症状，修复结肠组织超微结构，从而调整肠道传输功能，其中结肠组织GDNF基因启动子区甲基化状态的改变可能是电针大肠俞募配穴治疗STC的重要机制之一。.
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