基于5-HT通道探讨天灸法治疗内脏高敏性肠易激综合征小鼠的机制

Effect mechanism of blistering moxibustion on visceral hypersensitivity of irritable bowel syndrome in mice based on 5-HT signal pathway

目的:观察天灸法对内脏高敏性肠易激综合征(IBS)小鼠结肠组织5-羟色胺(5-HT)及其受体蛋白表达的影响,探讨天灸法治疗IBS的作用机制。方法:将40只SPF级新生昆明小鼠随机分为正常组、模型组、拮抗组和天灸组,每组10只。造模前拮抗组于侧脑室注射0.2m L对氯苯丙氨酸(PCPA),模型组、拮抗组和天灸组小鼠应用直肠内冰乙酸刺激结合夹尾刺激制备内脏高敏性IBS模型。造模后,天灸组小鼠行天灸法干预,穴取“中脘”“天枢”“足三里”,每周各穴贴药1次,每次贴药2 h,共3周。采用腹壁回缩反射(AWR)评分和腹壁肌电波幅评价小鼠内脏高敏状态;HE法观察小鼠结肠组织形态变化;免疫组化法检测小鼠结肠组织5-HT及其受体蛋白表达。结果:与正常组比较,20、40 mm Hg(1 mm Hg=0.133 kPa)压力下模型组腹壁肌电波幅升高(P<0.05),60、80 mm Hg压力下模型组AWR评分及腹壁肌电波幅均升高(P<0.05);与模型组比较,20 mm Hg压力下天灸组腹壁肌电波幅降低(P<0.05),40 mm Hg压力下天灸组与拮抗组AWR评分均升高(P<0.05),60、80 mm Hg压力下天灸组与拮抗组AWR评分及腹壁肌电波幅均降低(P<0.05)。与正常组比较,模型组黏膜上层结构轻度紊乱,黏膜下层可见中度炎性细胞;与模型组比较,天灸组与拮抗组黏膜上层固有腺体形状规则,可见少量炎性细胞。与正常组比较,模型组结肠组织5-HT及5-羟色胺受体3(5-HT3R)蛋白平均阳性面积率增加、5-羟色胺受体4(5-HT4R)蛋白平均阳性面积率降低(P<0.05);与模型组比较,天灸组和拮抗组结肠组织5-HT及5-HT3R蛋白平均阳性面积率降低(P<0.05)。结论:天灸法可改善内脏高敏性IBS小鼠内脏高敏状态,可能与5-HT信号通路介导的肠脑轴的调节有关。

Objective To observe the effect of blistering moxibustion on the expression levels of 5-hydroxytyptamine(5-HT)and its receptors of the colon tissue in the mice with visceral hypersensitivity of irritable bowel syndrome(IBS),so as to explore the effect mechanism of blistering moxibustion in treatment of IBS.Methods Forty SPF-grade newborn Kunming mice were randomly divided into a normal group,a model group,an antagonist group and a blistering moxibustion group,10 mice in each one.Before modeling,the injection with 0.2 mL parachlorophenylalanine(PCPA)was given on the lateral ventricle in the antagonist group.The endorectal glacial acetic acid stimulation combined with tail clipping was used to prepare the model of visceral hypersensitivity of IBS in the model group,the antagonist group and the blistering moxibustion group.After modeling,in the blistering moxibustion group,the intervention with blistering moxibustion was exerted at"Zhongwan"(CV 12),"Tianshu"(ST 25)and"Zusanli"(ST 36),once herbal irritant plaster at each acupoint,for 2 h each time,once a week,consecutively for 3 weeks.Abdominal withdrawal reflex(AWR)score and electromyographic(EMG)amplitude of abdominal muscles were adopted to evaluate the visceral hypersensitivity.HE staining was applied to observe the morphological changes in colon tissue,and immunohistochemistry was to determine the expression levels of 5-HT and its receptors.Results Compared with the normal group,EMG amplitude of abdominal muscles was increased under 20,40 mm Hg(1 mm Hg=0.133 kPa)in the model group(P<0.05),AWR scores and EMG amplitude of abdominal muscles under 60,80 mm Hg were all increased in the model group(P<0.05).In comparison with the model group,EMG amplitude of abdominal muscles was reduced under 20 mm Hg in the blistering moxibustion group(P<0.05),AWR scores were increased under 40 mm Hg in both the blistering moxibustion group and the antagonist group(P<0.05);AWR scores and EMG amplitude of abdominal muscles under 60,80 mm Hg were all reduced in both the blistering moxibustion group and the antagonist group(P<0.05).Compared with the normal group,in the model group,the mucosa was slightly disturbed,while,the moderate inflammatory cells were visible in the submucosa.In comparison with the model group,the inherent glands of mucosa were regular in shape and a small number of inflammatory cells were visible in both the blistering moxibustion group and the antagonist group.In comparison with the normal group,the average positive staining area percentage(APSAP)of 5-HT and 5-HT3 R of the colon tissue was increased,while,APSAP of 5-HT4 R was reduced in the model group(P<0.05).Compared with the model group,APSAP of 5-HT and 5-HT3 R was reduced in both the blistering moxibustion group and the antagonist group(P<0.05).Conclusion Blistering moxibustion can relieve the visceral hypersensitivity of the mice with visceral hypersensitive IBS and the underlying mechanism is related to the regulation of the gut-brain axis mediated by 5-HT signaling pathway.