基于5-HT信号系统探讨电针治疗便秘型肠易激综合征的机制
发布时间：2018-09-01 14:42
【摘要】：背景:肠易激综合征是临床最常见的功能性胃肠病。IBS病因及发病机制复杂,目前认为脑肠互动异常引起的内脏敏感性增加和肠动力异常构成了 IBS的病理生理基础。IBS临床较难治愈,消耗大量社会医疗资源。患者及家庭承受病痛、心理和经济三重压力。针灸能显著缓解IBS患者腹痛、排便功能紊乱等症状,提高患者生存质量。但迄今为止针灸治疗IBS的机制尚未完全阐明。目的:5-HT是参与肠道动力、感觉调节的重要化学信号和神经递质。5-HT_3型和5-HT_4型受体及5-HT转运体是其肠道代谢途径的主要受体,也是目前研究的热点。鉴于5-HT信号系统对肠功能的重要调节作用及与IBS病机的密切相关性,本文立足于验证"针灸通过调节5-HT信号系统治疗IBS-C"这一科学假设,旨在阐述电针治疗IBS-C的重要作用机制。方法:本研究以野生型(Wildtype,WT)C57BL6小鼠和Htr4基因敲除小鼠(Htr4+/-)为研究对象。C57BL6小鼠分为WT空白组、WT模型组和WT模型电针组,采用冰水灌胃法构建IBS-C模型并评价;采用Crispr/Cas9法构建Htr4+/-小鼠模型并评价,构建成功后分为Htr4+/-模型组和Htr4+/-模型电针组。通过检测首粒黑便排出时间、粪便颗粒数和含水量、结肠肌电评价电针对IBS-C模型小鼠胃传输功能的影响;通过糖水消耗试验评价IBSC-模型小鼠心理行为学改变;通过酶联免疫吸附法、免疫组织化学法、免疫印迹法、RT-PCR等分子生物学技术观察各组小鼠血清及结肠组织5-HT含量,结肠5-HT_3受体、5-HT_4受体及SERT蛋白含量及mRNA表达,以阐明电针对IBS-C模型小鼠5-HT信号系统的调控作用。结果:(1)模型评价:与WT空白组相比,WT模型组小鼠首粒黑便排出时间延长,粪便颗粒数减少、含水量下降,糖水偏好度下降,结肠肌电频率增加,幅值增大,变异系数升高,差异均有统计学意义(P0.05);Htr4+/-模型组首粒黑便排出时间显著延长,粪便颗粒数减少,含水量显著较正常值下降,糖水偏好度,结肠肌电频率增加,幅值增大,变异系数升高,差异有统计学意义(P0.05)。(2)电针对胃肠道传输功能的影响:与WT模型组相比,WT模型电针组小鼠首粒黑便排出时间缩短、粪便颗粒数增加,含水量升高。结肠肌电慢波频率降低,幅值减少,变异系数降低,差异有统计学意义(P0.05);与Htr4+/-模型组相比,Htr4+/-模型电针组小鼠首粒黑便排出时间缩短(P0.05)、粪便颗粒数增加(P0.05),含水量升高(P0.05)。结肠肌电慢波放电频率降低,放电频率变异系数降低,差异有统计学意义(P0.05)。幅值、幅值变异系数有下降趋势,但差异无统计学意义(P0.05)。(3)电针对5-HT的影响:与WT空白组比,WT模型组、Htr4+/-模型组血清5-HT含量及结肠组织5-HT表达均显著增加(P0.05);与WT模型组相比,WT模型电针组小鼠血清和结肠5-HT水平下调,差异有统计学意义(P0.05);与Htr4+/-模型组相比,Htr4+/-模型电针组小鼠血清和结肠5-HT水平下调,差异无统计学意义(P0.05)。(4)电针对结肠5-HT_4R、5-HT_3R的影响:与WT空白组比,WT模型组、Htr4+/-模型组结肠5-HT_4R蛋白含量及mRNA表达均明显下降,差异有统计学意义(P0.05)。与WT模型组相比,电针干预后,WT模型电针组结肠5-HT_4R蛋白含量及mRNA表达均上调(P0.05),差异有统计学意义。与Htr4+/-模型组比,Htr4+/-模型电针组结肠5-HT_4R蛋白含量上调,但差异无统计学意义(P0.05)。5-HT_4RmRNA表达上调,差异有统计学意义(P0.05)与WT空白组比,WT模型组结肠5-HT_3R蛋白含量及mRNA表达均明显下降,差异有统计学意义(P0.05);Htr4+/-模型组5-HT_3R蛋白含量及mRNA表达均显著上升(P0.05)。电针干预后,WT模型电针组结肠5-HT_3R蛋白含量及mRNA表达较WT模型组均上调(P0.05);Htr4+/-模型电针组结肠5-HT_3R蛋白含量及5-HT_4RmRNA较Htr4+/-模型组明显下调,差异有统计学意义(P0.05)(5)电针对结肠SERT的影响:WT空白、WT模型组、WT模型电针组、Htr4+/-模型组、Htr4+/-模型电针组结肠SERT蛋白含量及mRNA表达比较差异无统计学意义(P0.05)。结论:(1)冰水灌胃法可以复制便秘型肠易激综合征动物模型,模型小鼠首粒黑便时间延长、粪便颗粒数减少、含水量降低、糖水偏好度下降,慢性冰水刺激可以促进EC过度释放5-HT,诱导5-HT信号系统紊乱,继而导致肠道动力、感觉异常;Htr4+/-模型组表现为先天5-HT_4R受体表达下调,5-HT信号系统紊乱,出现粪便颗粒数减少、含水量降低、糖水偏好度下降等类IBS-C症状,证实了 5-HT_4受体是胃肠道5-HT信号系统的关键受体。两种IBS-C模型的成功建立,一方面证实5-HT信号系统异常是IBS-C的重要发病机制,另一方面提示在5-HT信号系统中,任何平衡被打破,都会继发整个代谢过程功能紊乱,导致疾病的发生。(2)电针可以良性下调冰水灌胃IBS-C模型及Htr4+/-模型小鼠异常升高的血清5-HT含量及结肠组织5-HT表达;上调冰水灌胃IBS-C模型及Htr4+/-模型小鼠结肠5-HT_4R蛋白及mRNA含量;上调冰水灌胃IBS-C模型小鼠结肠组织中表达减少的5-HT_3R,而下调Htr4+/-模型电针组表达异常增高的5-HT_3R。促使5-HT、5-HT_4R、5-HT_3R恢复正常水平。提示了电针可以通过调节5-HT释放、激活各个环节达到调控5-HT信号系统的作用。体现了针灸双向、良性调节的优势。(3)电针可以通过良性调控5-HT信号系统,改善肠道动力和肠道分泌,达到治疗IBS-C的效果,从而改善冰水灌胃和Htr4+/-基因敲除所致的胃肠动力紊乱,增强胃肠动力、增加粪便颗粒数、含水量、促使结肠肌电恢复正常节律,缓解IBS-C模型小鼠排便困难的症状。
[Abstract]:BACKGROUND: Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder in clinic. The etiology and pathogenesis of IBS are complex. It is considered that visceral hypersensitivity and abnormal bowel motility caused by abnormal brain-intestinal interaction constitute the pathophysiological basis of IBS. Objective: 5-HT is an important chemical signal and neurotransmitter involved in intestinal motility and sensory regulation. 5-HT_3 and 5-HT_4 receptors and 5-HT transporters are among them. In view of the important regulation of 5-HT signaling system on intestinal function and its close relationship with the pathogenesis of IBS, this paper is based on verifying the scientific hypothesis that acupuncture can treat IBS-C by regulating 5-HT signaling system, aiming at explaining the important mechanism of electroacupuncture in treating IBS-C. C57BL6 mice were divided into WT blank group, WT model group and WT model electroacupuncture group. IBS-C model was constructed and evaluated by ice water gavage method. Htr4+/-mouse model was constructed by Crispr/Cas9 method and evaluated. After successful construction, C57BL6 mice were divided into Htr4+/-model group and H tr4+/-model group. TR4 + / - model electroacupuncture group. The gastric transport function of IBS-C model mice was evaluated by detecting the first black stool discharge time, fecal granule number and water content, and colon electromyography; the psychological and behavioral changes of IBSC-model mice were evaluated by glucose consumption test; the enzyme-linked immunosorbent assay, immunohistochemistry, immunoblotting, RT-PCR were used. The content of 5-HT, 5-HT_3 receptor, 5-HT_4 receptor, SERT protein and mRNA expression in serum and colon of mice in each group were observed by molecular biology techniques to clarify the regulatory effect of Electroacupuncture on 5-HT signaling system in IBS-C model mice. Fecal granules decreased, water content decreased, sugarwater preference decreased, colon EMG frequency increased, amplitude increased, coefficient of variation increased, the difference was statistically significant (P 0.05); Htr4 +/- model group first black stool discharge time significantly prolonged, fecal granules decreased, water content significantly decreased compared with normal value, sugar water preference, colon EMG frequency increased. (2) The effect of Electroacupuncture on gastrointestinal transport function: Compared with WT model group, the first black stool discharge time of WT model group was shortened, the number of fecal particles was increased, and the water content was increased. Compared with the Htr4 + / - model group, the first black stool discharge time (P 0.05), fecal granule number (P 0.05) and water content (P 0.05) of the Htr4 + / - model group were shortened, and the slow wave discharge frequency of colon EMG was decreased, and the coefficient of variation of discharge frequency was decreased (P 0.05). Trend, but no significant difference (P 0.05). (3) Effect of Electroacupuncture on 5-HT: Compared with WT blank group, serum 5-HT content and colon 5-HT expression in WT model group, Htr4+/- model group were significantly increased (P 0.05); Compared with WT model group, serum and colon 5-HT levels in WT model electroacupuncture group were significantly decreased (P 0.05); Compared with Htr4+/-model group, serum and colon 5-HT levels in WT model group were significantly decreased (P 0.05). Compared with WT blank group, 5-HT_4R protein content and mRNA expression in colon of Htr4+/-model group and Htr4+/-model group were significantly decreased (P 0.05). Compared with the Htr4 + / - model group, the content of 5-HT_4R protein in the colon of the Htr4 + / - model group was up-regulated, but there was no significant difference (P 0.05). The expression of 5-HT_4R mRNA was up-regulated in WT model group (P 0.05) and WT blank group (P 0.05). Compared with WT model group, the content of 5-HT_3R protein and the expression of 5-HT_3R mRNA in colon were significantly decreased (P 0.05), while the content of 5-HT_3R protein and the expression of mRNA in Htr4+/- model group were significantly increased (P 0.05). Intestinal 5-HT_3R protein content and 5-HT_4R mRNA were significantly down-regulated compared with Htr4+/-model group, and the difference was statistically significant (P 0.05). (5) The effect of Electroacupuncture on colon SERT was not significant (P 0.05). WT blank, WT model group, WT model electroacupuncture group, Htr4+/-model group, Htr4+/-model electroacupuncture group, colon SERT protein content and mRNA expression were not significantly different (P 0.05). The animal model of constipation-predominant irritable bowel syndrome (IBS) can be reproduced by gavage. The first black stool prolongs, fecal particles decrease, water content decreases, sugar preference decreases. Chronic ice water stimulation can promote EC to release 5-HT excessively, induce 5-HT signal system disorders, and then lead to intestinal motility and abnormal sensation. Inborn 5-HT_4R receptor expression is down-regulated, 5-HT signaling system is disordered, fecal particles are reduced, water content is reduced, sugar preference is decreased and other IBS-C symptoms, confirmed that 5-HT_4 receptor is the key receptor of 5-HT signaling system in gastrointestinal tract. The successful establishment of two IBS-C models, on the one hand, confirmed that abnormal 5-HT signaling system is an important pathogenesis of IBS-C. The mechanism, on the other hand, suggests that any balance breaking in the 5-HT signaling system will lead to the disorder of the whole metabolic process, leading to the occurrence of disease. (2) EA can benignly down-regulate the abnormally elevated serum 5-HT content and colon 5-HT expression in IBS-C model and Htr4+/- model mice by intragastric ice water infusion; up-regulate the IBS-C model and Htr-C model by intragastric ice water infusion. The contents of 5-HT_4R protein and mRNA in colon of 4+/-model mice were up-regulated, the expression of 5-HT_3R in colon of IBS-C model mice was down-regulated, and the abnormally high expression of 5-HT_3R in Htr4+/-model electroacupuncture group was down-regulated, which promoted the recovery of 5-HT, 5-HT_4R and 5-HT_3R to normal levels. (3) Electroacupuncture can improve intestinal motility and intestinal secretion by benign regulation of 5-HT signal system, so as to improve the effect of IBS-C treatment, thereby improving the gastrointestinal motility disorder caused by ice water gavage and Htr4+/-gene knockout, enhancing gastrointestinal motility and increasing the number of fecal particles. Water content promotes the recovery of normal rhythm of colon electromyography and relieves the symptoms of defecation difficulty in IBS-C mice.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R245
本文编号：2217512
[Abstract]:BACKGROUND: Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder in clinic. The etiology and pathogenesis of IBS are complex. It is considered that visceral hypersensitivity and abnormal bowel motility caused by abnormal brain-intestinal interaction constitute the pathophysiological basis of IBS. Objective: 5-HT is an important chemical signal and neurotransmitter involved in intestinal motility and sensory regulation. 5-HT_3 and 5-HT_4 receptors and 5-HT transporters are among them. In view of the important regulation of 5-HT signaling system on intestinal function and its close relationship with the pathogenesis of IBS, this paper is based on verifying the scientific hypothesis that acupuncture can treat IBS-C by regulating 5-HT signaling system, aiming at explaining the important mechanism of electroacupuncture in treating IBS-C. C57BL6 mice were divided into WT blank group, WT model group and WT model electroacupuncture group. IBS-C model was constructed and evaluated by ice water gavage method. Htr4+/-mouse model was constructed by Crispr/Cas9 method and evaluated. After successful construction, C57BL6 mice were divided into Htr4+/-model group and H tr4+/-model group. TR4 + / - model electroacupuncture group. The gastric transport function of IBS-C model mice was evaluated by detecting the first black stool discharge time, fecal granule number and water content, and colon electromyography; the psychological and behavioral changes of IBSC-model mice were evaluated by glucose consumption test; the enzyme-linked immunosorbent assay, immunohistochemistry, immunoblotting, RT-PCR were used. The content of 5-HT, 5-HT_3 receptor, 5-HT_4 receptor, SERT protein and mRNA expression in serum and colon of mice in each group were observed by molecular biology techniques to clarify the regulatory effect of Electroacupuncture on 5-HT signaling system in IBS-C model mice. Fecal granules decreased, water content decreased, sugarwater preference decreased, colon EMG frequency increased, amplitude increased, coefficient of variation increased, the difference was statistically significant (P 0.05); Htr4 +/- model group first black stool discharge time significantly prolonged, fecal granules decreased, water content significantly decreased compared with normal value, sugar water preference, colon EMG frequency increased. (2) The effect of Electroacupuncture on gastrointestinal transport function: Compared with WT model group, the first black stool discharge time of WT model group was shortened, the number of fecal particles was increased, and the water content was increased. Compared with the Htr4 + / - model group, the first black stool discharge time (P 0.05), fecal granule number (P 0.05) and water content (P 0.05) of the Htr4 + / - model group were shortened, and the slow wave discharge frequency of colon EMG was decreased, and the coefficient of variation of discharge frequency was decreased (P 0.05). Trend, but no significant difference (P 0.05). (3) Effect of Electroacupuncture on 5-HT: Compared with WT blank group, serum 5-HT content and colon 5-HT expression in WT model group, Htr4+/- model group were significantly increased (P 0.05); Compared with WT model group, serum and colon 5-HT levels in WT model electroacupuncture group were significantly decreased (P 0.05); Compared with Htr4+/-model group, serum and colon 5-HT levels in WT model group were significantly decreased (P 0.05). Compared with WT blank group, 5-HT_4R protein content and mRNA expression in colon of Htr4+/-model group and Htr4+/-model group were significantly decreased (P 0.05). Compared with the Htr4 + / - model group, the content of 5-HT_4R protein in the colon of the Htr4 + / - model group was up-regulated, but there was no significant difference (P 0.05). The expression of 5-HT_4R mRNA was up-regulated in WT model group (P 0.05) and WT blank group (P 0.05). Compared with WT model group, the content of 5-HT_3R protein and the expression of 5-HT_3R mRNA in colon were significantly decreased (P 0.05), while the content of 5-HT_3R protein and the expression of mRNA in Htr4+/- model group were significantly increased (P 0.05). Intestinal 5-HT_3R protein content and 5-HT_4R mRNA were significantly down-regulated compared with Htr4+/-model group, and the difference was statistically significant (P 0.05). (5) The effect of Electroacupuncture on colon SERT was not significant (P 0.05). WT blank, WT model group, WT model electroacupuncture group, Htr4+/-model group, Htr4+/-model electroacupuncture group, colon SERT protein content and mRNA expression were not significantly different (P 0.05). The animal model of constipation-predominant irritable bowel syndrome (IBS) can be reproduced by gavage. The first black stool prolongs, fecal particles decrease, water content decreases, sugar preference decreases. Chronic ice water stimulation can promote EC to release 5-HT excessively, induce 5-HT signal system disorders, and then lead to intestinal motility and abnormal sensation. Inborn 5-HT_4R receptor expression is down-regulated, 5-HT signaling system is disordered, fecal particles are reduced, water content is reduced, sugar preference is decreased and other IBS-C symptoms, confirmed that 5-HT_4 receptor is the key receptor of 5-HT signaling system in gastrointestinal tract. The successful establishment of two IBS-C models, on the one hand, confirmed that abnormal 5-HT signaling system is an important pathogenesis of IBS-C. The mechanism, on the other hand, suggests that any balance breaking in the 5-HT signaling system will lead to the disorder of the whole metabolic process, leading to the occurrence of disease. (2) EA can benignly down-regulate the abnormally elevated serum 5-HT content and colon 5-HT expression in IBS-C model and Htr4+/- model mice by intragastric ice water infusion; up-regulate the IBS-C model and Htr-C model by intragastric ice water infusion. The contents of 5-HT_4R protein and mRNA in colon of 4+/-model mice were up-regulated, the expression of 5-HT_3R in colon of IBS-C model mice was down-regulated, and the abnormally high expression of 5-HT_3R in Htr4+/-model electroacupuncture group was down-regulated, which promoted the recovery of 5-HT, 5-HT_4R and 5-HT_3R to normal levels. (3) Electroacupuncture can improve intestinal motility and intestinal secretion by benign regulation of 5-HT signal system, so as to improve the effect of IBS-C treatment, thereby improving the gastrointestinal motility disorder caused by ice water gavage and Htr4+/-gene knockout, enhancing gastrointestinal motility and increasing the number of fecal particles. Water content promotes the recovery of normal rhythm of colon electromyography and relieves the symptoms of defecation difficulty in IBS-C mice.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R245
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