电针通过内源性大麻素系统缓解山羊内脏超敏
发布时间：2018-08-12 15:07
【摘要】：内脏超敏是肠道疾病的临床常见症状,如炎症性肠病(IBDs)和肠易激综合征(IBS)等。大约50%~70%IBDs和20%~90%IBS患者都存在这一症状。内脏超敏指的是内脏组织对多种刺激的敏感性和反应性增强,包括痛觉过敏、触摸痛、肠动力异常以及肠道黏膜分泌增多等。内脏超敏与中枢神经敏感化、外周神经致敏和脑肠轴失调等因素相关,其药物治疗存在严重的副作用,已经成为困扰人们多时的严重医疗问题。近年来,针刺治疗内脏超敏在临床上取得了良好的成效。然而,针刺治疗内脏超敏的机制尚未阐明。研究证实阿片肽系统在电针镇痛方面具有重要作用,然而在伴有炎症反应的慢性疼痛模型中,阻断阿片肽系统并不能完全逆转电针镇痛效果,而阻断内源性大麻素系统能够成功逆转电针效应。这说明了在炎症条件下内源性大麻素系统在电针缓解疼痛方面有独特的作用。内源性大麻素系统主要是由特殊的G蛋白偶联受体(大麻素受体1和大麻素受体2)、内源性大麻素配体以及内源性大麻素合成和降解酶组成,广泛存在于中枢神经系统和外周神经系统,尤其是扣带回皮层、中脑导水管周围灰质、延髓、脊髓背角以及肠道神经系统等。花生四烯乙醇胺(AEA)和2-花生四烯酰甘油(2-AG)作为主要的内源性大麻素,能够特异性地识别、结合并激活大麻素受体1(CB1),从而抑制神经元的过度兴奋。有研究表明电针治疗能够显著上调CB1的表达量,并抑制关节炎大鼠痛觉过敏。脂肪酸酰胺水解酶(FAAH)和单酰基甘油酯酶(MAGL)分别是AEA和2-AG的降解酶,研究发现FAAH和MAGL的拮抗剂显著增加了大鼠体内AEA与2-AG的表达量,同样能够缓解大鼠痛敏。然而,CB1、FAAH和MAGL是否同时参与电针诱导的超敏抑制未见报道。本实验采用山羊回肠炎诱导的内脏超敏模型研究电针的治疗效果及其机制。选择24只雄性健康山羊,将三硝基苯磺酸(TNBS)注射到其中的18只山羊回肠壁中建立内脏超敏模型;等体积的无菌生理盐水代替TNBS注射到另外的6只山羊回肠壁中,作为对照组(Saline)。18只内脏超敏山羊被随机分为三组:内脏超敏组(TNBS)、电针组(EA+TNBS)、假针组(Sham+TNBS),每组6只。TNBS组:不进行电针操作;EA+TNBS组:手术7 d后开始电针,采用双侧“后三里”穴(ST36),60 Hz,30 min,1次/3 d,总计6次;Sham+TNBS组:只扎针,不通电,其余与电针组相同。在手术后第7、10、13、16、19和22进行电针,之后立即采用腹肌电图(EMG)测量实验山羊由不同压力梯度(20、40、60、80和100 mmHg)的结直肠扩张(CRD)刺激引起的内脏运动反应(VMR)。第22 d,VMR测量后立即取山羊脑组织、T11脊髓和回肠样品,采用免疫组织化学法检测内脏感觉相关的大脑核团与区域(前皮质扣带回、中脑导水管周围灰质、中缝大核、巨细胞网状核、孤束核、迷走运动背核、延髓头端腹内侧核群)、脊髓背角以及回肠中CB1、FAAH和MAGL的表达量。结果显示,TNBS组山羊7 d时VMR显著高于Saline组(P0.05),并且维持到第22 d。假针组山羊的VMR与TNBS组相比无显著性差异(P0.05)。与TNBS组相比,电针组山羊在7 d时VMR无显著性变化(P0.05),在10、13、16、19和22 d时VMR均显著升高(P0.05),表明回肠受到炎症损伤后山羊出现内脏超敏反应而电针能够有效的缓解内脏超敏,且具有累积效应。手术后第22 d,TNBS组山羊前皮质扣带回(ACC)、中脑导水管周围灰质(PAG)、中缝大核(NRM)、巨细胞网状核(GI)、孤束核(NTS)、迷走运动背核(DMV)、延髓头端腹内侧核群(r VLM)、脊髓背角(SDH)以及回肠中CB1的蛋白表达量均显著低于对照组(P0.05),而FAAH和MAGL的蛋白表达量显著高于对照组(P0.05)。与TNBS组相比,电针组山羊上述核团和脑区、脊髓及回肠中CB1的含量显著上升(P0.05),FAAH和MAGL的含量显著下降(P0.05),显示电针能够促进回肠炎诱导的内脏超敏模型山羊CB1的上调和FAAH、MAGL的下调。本研究通过向山羊回肠壁中注射TNBS诱导回肠炎建造内脏超敏模型,探讨电针对山羊内脏运动反应以及CB1、FAAH和MAGL表达水平的影响,表明电针能够有效的缓解山羊内脏超敏并增加CB1和降低FAAH、MAGL在ACC、PAG、NRM、GI、NTS、DMV、RVLM、SDH及回肠中的表达量。该研究有助于揭示电针缓解山羊回肠炎诱导的内脏超敏的中枢和外周机制,促进胃肠道疾病治疗的发展。
[Abstract]:Visceral hypersensitivity is a common clinical symptom of intestinal diseases, such as inflammatory bowel disease (IBDs) and irritable bowel syndrome (IBS). It occurs in about 50%-70% of IBDs and 20%-90% of IBS patients. Visceral hypersensitivity refers to the increased sensitivity and responsiveness of visceral tissues to a variety of stimuli, including hyperalgesia, touch pain, abnormal bowel motility and intestinal mucus. Visceral hypersensitivity is associated with central nervous system sensitization, peripheral nervous system sensitization and brain-gut axis disorder. Its drug treatment has serious side effects, which has been a serious medical problem for many years. In recent years, acupuncture treatment of visceral hypersensitivity has achieved good results in clinical practice. However, acupuncture treatment of visceral hypersensitivity has been proved effective. Studies have shown that opioid peptide system plays an important role in EA analgesia. However, blocking the opioid peptide system does not completely reverse EA analgesia in chronic pain models with inflammation, and blocking the endocannabinoid system can successfully reverse EA effect. Endogenous cannabinoid system plays a unique role in pain relief by electroacupuncture. The endogenous cannabinoid system consists of special G protein-coupled receptors (cannabinoid receptor 1 and cannabinoid receptor 2), endogenous cannabinoid ligands, and endogenous cannabinoid synthase and degrading enzymes, which are widely distributed in the central nervous system and peripheral nervous system. Arachidonethanolamine (AEA) and 2-arachidonyl glycerol (2-AG), as the main endogenous cannabinoids, can specifically recognize, bind and activate cannabinoid receptor 1 (CB1), thereby inhibiting neuronal hyperexcitability. Fatty acid amide hydrolase (FAAH) and monoacylglycerol esterase (MAGL) are degrading enzymes of AEA and 2-AG, respectively. It is found that antagonists of FAAH and MAGL can significantly increase the expression of AEA and 2-AG in rats, and also alleviate the pain sensitivity of rats. However, it has not been reported whether CB1, FAAH and MAGL are involved in EA-induced hypersensitivity inhibition. The therapeutic effect and mechanism of EA were studied by using the visceral hypersensitivity model induced by goat ileitis. Twenty-four healthy male goats were injected with TNBS into the ileum wall to establish visceral hypersensitivity models. 18 visceral hypersensitivity goats were randomly divided into three groups: visceral hypersensitivity group (TNBS), electroacupuncture group (EA + TNBS), sham acupuncture group (Sham + TNBS), 6 goats in each group. In the Sham + TNBS group, only needling was done, and the rest was the same as the electroacupuncture group. Electroacupuncture was performed at 7, 10, 13, 16, 19 and 22 postoperatively, and then abdominal electromyography (EMG) was used to measure the colorectal dilatation (CRD) induced by different pressure gradients (20, 40, 60, 80 and 100 mmHg). Visceral motor response (VMR). Immediately after the measurement of VMR on the 22nd day, brain tissue, T11 spinal cord and ileum were taken from goats. Immunohistochemical method was used to detect the visceral sensory-related brain nuclei and regions (anterior cingulate gyrus, periaqueductal gray matter, raphe magnus nucleus, giant cell reticular nucleus, solitary tract nucleus, dorsal vagal motor nucleus, ventral end of medulla oblongata). The results showed that the expression of CB1, FAAH and MAGL in the dorsal horn of spinal cord and ileum of the goats in TNBS group was significantly higher than that in Saline group at 7 d (P 0.05) and maintained until 22 D. There was no significant difference between the goats in sham acupuncture group and TNBS group (P 0.05). Compared with TNBS group, there was no significant change in VMR at 7 d in EA group (P 0.05), and at 10, 13, 1 D in TNBS group. VMR increased significantly at 6, 19 and 22 days (P 0.05), indicating that EA could effectively alleviate visceral hypersensitivity in goats with ileal inflammation injury, and had cumulative effect. The expression levels of CB1 in NTS, DMV, R VLM, SDH and ileum were significantly lower than those in control group (P 0.05), while the expression levels of FAAH and MAGL were significantly higher in EA group than those in TNBS group (P 0.05). The amount of FAAH and MAGL increased significantly (P 0.05), and the content of FAAH and MAGL decreased significantly (P 0.05), indicating that EA could promote the up-regulation of CB1 and the down-regulation of FAAH and MAGL in visceral hypersensitivity model goats induced by ileitis. The effects of AAH and MAGL on the expression of AAH and MAGL showed that EA could effectively alleviate visceral hypersensitivity and increase CB1 and decrease FAAH in goats. The expression of MAGL in ACC, PAG, NRM, GI, NTS, DMV, RVLM, SDH and ileum was increased. This study will help to reveal the central and peripheral mechanisms of EA in alleviating visceral hypersensitivity induced by ileitis in goats and promote the treatment of gastrointestinal diseases. Development.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R-332
本文编号：2179431
[Abstract]:Visceral hypersensitivity is a common clinical symptom of intestinal diseases, such as inflammatory bowel disease (IBDs) and irritable bowel syndrome (IBS). It occurs in about 50%-70% of IBDs and 20%-90% of IBS patients. Visceral hypersensitivity refers to the increased sensitivity and responsiveness of visceral tissues to a variety of stimuli, including hyperalgesia, touch pain, abnormal bowel motility and intestinal mucus. Visceral hypersensitivity is associated with central nervous system sensitization, peripheral nervous system sensitization and brain-gut axis disorder. Its drug treatment has serious side effects, which has been a serious medical problem for many years. In recent years, acupuncture treatment of visceral hypersensitivity has achieved good results in clinical practice. However, acupuncture treatment of visceral hypersensitivity has been proved effective. Studies have shown that opioid peptide system plays an important role in EA analgesia. However, blocking the opioid peptide system does not completely reverse EA analgesia in chronic pain models with inflammation, and blocking the endocannabinoid system can successfully reverse EA effect. Endogenous cannabinoid system plays a unique role in pain relief by electroacupuncture. The endogenous cannabinoid system consists of special G protein-coupled receptors (cannabinoid receptor 1 and cannabinoid receptor 2), endogenous cannabinoid ligands, and endogenous cannabinoid synthase and degrading enzymes, which are widely distributed in the central nervous system and peripheral nervous system. Arachidonethanolamine (AEA) and 2-arachidonyl glycerol (2-AG), as the main endogenous cannabinoids, can specifically recognize, bind and activate cannabinoid receptor 1 (CB1), thereby inhibiting neuronal hyperexcitability. Fatty acid amide hydrolase (FAAH) and monoacylglycerol esterase (MAGL) are degrading enzymes of AEA and 2-AG, respectively. It is found that antagonists of FAAH and MAGL can significantly increase the expression of AEA and 2-AG in rats, and also alleviate the pain sensitivity of rats. However, it has not been reported whether CB1, FAAH and MAGL are involved in EA-induced hypersensitivity inhibition. The therapeutic effect and mechanism of EA were studied by using the visceral hypersensitivity model induced by goat ileitis. Twenty-four healthy male goats were injected with TNBS into the ileum wall to establish visceral hypersensitivity models. 18 visceral hypersensitivity goats were randomly divided into three groups: visceral hypersensitivity group (TNBS), electroacupuncture group (EA + TNBS), sham acupuncture group (Sham + TNBS), 6 goats in each group. In the Sham + TNBS group, only needling was done, and the rest was the same as the electroacupuncture group. Electroacupuncture was performed at 7, 10, 13, 16, 19 and 22 postoperatively, and then abdominal electromyography (EMG) was used to measure the colorectal dilatation (CRD) induced by different pressure gradients (20, 40, 60, 80 and 100 mmHg). Visceral motor response (VMR). Immediately after the measurement of VMR on the 22nd day, brain tissue, T11 spinal cord and ileum were taken from goats. Immunohistochemical method was used to detect the visceral sensory-related brain nuclei and regions (anterior cingulate gyrus, periaqueductal gray matter, raphe magnus nucleus, giant cell reticular nucleus, solitary tract nucleus, dorsal vagal motor nucleus, ventral end of medulla oblongata). The results showed that the expression of CB1, FAAH and MAGL in the dorsal horn of spinal cord and ileum of the goats in TNBS group was significantly higher than that in Saline group at 7 d (P 0.05) and maintained until 22 D. There was no significant difference between the goats in sham acupuncture group and TNBS group (P 0.05). Compared with TNBS group, there was no significant change in VMR at 7 d in EA group (P 0.05), and at 10, 13, 1 D in TNBS group. VMR increased significantly at 6, 19 and 22 days (P 0.05), indicating that EA could effectively alleviate visceral hypersensitivity in goats with ileal inflammation injury, and had cumulative effect. The expression levels of CB1 in NTS, DMV, R VLM, SDH and ileum were significantly lower than those in control group (P 0.05), while the expression levels of FAAH and MAGL were significantly higher in EA group than those in TNBS group (P 0.05). The amount of FAAH and MAGL increased significantly (P 0.05), and the content of FAAH and MAGL decreased significantly (P 0.05), indicating that EA could promote the up-regulation of CB1 and the down-regulation of FAAH and MAGL in visceral hypersensitivity model goats induced by ileitis. The effects of AAH and MAGL on the expression of AAH and MAGL showed that EA could effectively alleviate visceral hypersensitivity and increase CB1 and decrease FAAH in goats. The expression of MAGL in ACC, PAG, NRM, GI, NTS, DMV, RVLM, SDH and ileum was increased. This study will help to reveal the central and peripheral mechanisms of EA in alleviating visceral hypersensitivity induced by ileitis in goats and promote the treatment of gastrointestinal diseases. Development.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R-332
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