vlPAG脑区P2X3受体参与电针缓解IBS小鼠内脏高敏感的机制研究

Mechanistic study on P2X3 receptors in the ventrolateral periaqueductal gray underlying electroacupuncture-induced relief of visceral hypersensitivity in irritable bowel syndrome mice

关键词:  肠易激综合征  电针  中腹外侧区脑导水管周围灰质  P2X3受体  内脏高敏感

Key words:  irritable bowel syndrome  electroacupuncture  ventrolateral periaqueductal gray  P2X3 receptor  visceral hypersensitivity

摘要:

[目的] 通过在体钙信号光纤记录技术探讨中腹外侧区脑导水管周围灰质（vlPAG）在肠易激综合征（IBS）小鼠内脏高敏感机制中的作用，采用膜片钳电生理技术探讨vlPAG脑区P2X3受体参与电针缓解IBS小鼠内脏高敏感的作用机制。[方法] 雄性C57和Vgat-Cre小鼠，通过2，4，6-三硝基苯磺酸（TNBS）灌肠液灌肠制备IBS内脏高敏感模型。模型制备4周后运用电针、套管内给药等方法干预。采用苏木素-伊红（HE）染色、腹部撤退反射（AWR）评分判定IBS内脏高敏感模型是否制备成功；运用AWR评分检测IBS小鼠内脏高敏感的变化，在体钙信号光纤记录方法观察IBS小鼠vlPAG脑区神经元的活动变化，全细胞膜片钳技术检测vlPAG脑区神经元的膜电学特性变化。[结果] 1）与模型组比较，电针组小鼠在4个压力等级直结肠扩张（CRD）刺激下的AWR评分均明显下降（P＜0.01）。2）在45 mmHg（1 mmHg≈0.133 kPa） CRD刺激下，与模型组比较，电针组小鼠vlPAG脑区谷氨酸（Glu）能神经元的钙离子内流减少，γ-氨基丁酸（GABA）能神经元的钙离子内流增加。3）与模型组比较，P2X3受体激动剂组小鼠在4个压力等级CRD刺激下的AWR评分均下降（P＜0.05）。4）与模型组比较，电针组和P2X3受体激动剂组小鼠vlPAG脑区神经元的动作电位个数均增加（P＜0.05）。[结论] 电针通过调节IBS内脏高敏感小鼠vlPAG脑区神经元上P2X3受体的表达改变神经元的电生理特性，可能是电针缓解IBS小鼠内脏痛中枢敏化的机制之一。

Abstract:

[Objective] To investigate the role of ventrolateral periaqueductal gray(vlPAG) in irritable bowel syndrome(IBS) mice with visceral hypersensitivity by in-vivo calcium fiber optic recording. To investigate the mechanism of P2X3 receptor in vlPAG participating in relieving visceral hypersensitivity in IBS mice with electroacupuncture by patch-clamp recording.[Methods] Male C57 and Vgat-Cre mice were subjected to enema with 2, 4, 6-trinitrobrnzen sulfonic acid(TNBS) to prepare IBS model with visceral hypersensitivity. Four weeks after model establishment, interventions including electroacupuncture(EA) and intracerebral cannula-guided drug administration were implemented. Successful establishment of the IBS visceral hypersensitivity model was confirmed by Hematoxylin-eosin(HE) staining and abdominal withdrawal reflex(AWR) score. The changes of visceral hypersensitivity in IBS mice was assessed using AWR score, the activity changes of vlPAG neurons in IBS mice were observed by in-vivo calcium fiber optic recording, and the changes of membrane electrical characteristics of vlPAG neurons in IBS mice were detected by patch-clamp recording.[Results] 1) Compared with the model group, the AWR scores of mice in EA group were obviously decreased at all four levels of colorectal distention(CRD) stimulation(P<0.01). 2) Under 45 mmHg CRD stimulation, calcium flow in glutamergic(Glu) neurons in vlPAG of mice in EA group decreased and calcium flow in GABAergic(GABA) neurons increased compared with model group. 3) Compared with model group, the AWR scores of mice in P2X3 receptor agonist group were decreased in all four levels of CRD stimulation(P<0.05). 4) Compared with model group, the number of action potentials of vlPAG neurons in EA group and P2X3 receptor agonist group were increased(P<0.05).[Conclusion] EA can change the electrophysiological characteristics of neurons by regulating the expression of P2X3 receptor in vlPAG of IBS mice, which may be one of the central sensitization mechanisms of EA to relieve the visceral pain in IBS mice.