基于5-HT信號系統(tǒng)探討電針治療便秘型腸易激綜合征的機(jī)制
發(fā)布時間：2018-09-01 14:42
【摘要】：背景:腸易激綜合征是臨床最常見的功能性胃腸病。IBS病因及發(fā)病機(jī)制復(fù)雜,目前認(rèn)為腦腸互動異常引起的內(nèi)臟敏感性增加和腸動力異常構(gòu)成了 IBS的病理生理基礎(chǔ)。IBS臨床較難治愈,消耗大量社會醫(yī)療資源。患者及家庭承受病痛、心理和經(jīng)濟(jì)三重壓力。針灸能顯著緩解IBS患者腹痛、排便功能紊亂等癥狀,提高患者生存質(zhì)量。但迄今為止針灸治療IBS的機(jī)制尚未完全闡明。目的:5-HT是參與腸道動力、感覺調(diào)節(jié)的重要化學(xué)信號和神經(jīng)遞質(zhì)。5-HT_3型和5-HT_4型受體及5-HT轉(zhuǎn)運體是其腸道代謝途徑的主要受體,也是目前研究的熱點。鑒于5-HT信號系統(tǒng)對腸功能的重要調(diào)節(jié)作用及與IBS病機(jī)的密切相關(guān)性,本文立足于驗證"針灸通過調(diào)節(jié)5-HT信號系統(tǒng)治療IBS-C"這一科學(xué)假設(shè),旨在闡述電針治療IBS-C的重要作用機(jī)制。方法:本研究以野生型(Wildtype,WT)C57BL6小鼠和Htr4基因敲除小鼠(Htr4+/-)為研究對象。C57BL6小鼠分為WT空白組、WT模型組和WT模型電針組,采用冰水灌胃法構(gòu)建IBS-C模型并評價;采用Crispr/Cas9法構(gòu)建Htr4+/-小鼠模型并評價,構(gòu)建成功后分為Htr4+/-模型組和Htr4+/-模型電針組。通過檢測首粒黑便排出時間、糞便顆粒數(shù)和含水量、結(jié)腸肌電評價電針對IBS-C模型小鼠胃傳輸功能的影響;通過糖水消耗試驗評價IBSC-模型小鼠心理行為學(xué)改變;通過酶聯(lián)免疫吸附法、免疫組織化學(xué)法、免疫印跡法、RT-PCR等分子生物學(xué)技術(shù)觀察各組小鼠血清及結(jié)腸組織5-HT含量,結(jié)腸5-HT_3受體、5-HT_4受體及SERT蛋白含量及mRNA表達(dá),以闡明電針對IBS-C模型小鼠5-HT信號系統(tǒng)的調(diào)控作用。結(jié)果:(1)模型評價:與WT空白組相比,WT模型組小鼠首粒黑便排出時間延長,糞便顆粒數(shù)減少、含水量下降,糖水偏好度下降,結(jié)腸肌電頻率增加,幅值增大,變異系數(shù)升高,差異均有統(tǒng)計學(xué)意義(P0.05);Htr4+/-模型組首粒黑便排出時間顯著延長,糞便顆粒數(shù)減少,含水量顯著較正常值下降,糖水偏好度,結(jié)腸肌電頻率增加,幅值增大,變異系數(shù)升高,差異有統(tǒng)計學(xué)意義(P0.05)。(2)電針對胃腸道傳輸功能的影響:與WT模型組相比,WT模型電針組小鼠首粒黑便排出時間縮短、糞便顆粒數(shù)增加,含水量升高。結(jié)腸肌電慢波頻率降低,幅值減少,變異系數(shù)降低,差異有統(tǒng)計學(xué)意義(P0.05);與Htr4+/-模型組相比,Htr4+/-模型電針組小鼠首粒黑便排出時間縮短(P0.05)、糞便顆粒數(shù)增加(P0.05),含水量升高(P0.05)。結(jié)腸肌電慢波放電頻率降低,放電頻率變異系數(shù)降低,差異有統(tǒng)計學(xué)意義(P0.05)。幅值、幅值變異系數(shù)有下降趨勢,但差異無統(tǒng)計學(xué)意義(P0.05)。(3)電針對5-HT的影響:與WT空白組比,WT模型組、Htr4+/-模型組血清5-HT含量及結(jié)腸組織5-HT表達(dá)均顯著增加(P0.05);與WT模型組相比,WT模型電針組小鼠血清和結(jié)腸5-HT水平下調(diào),差異有統(tǒng)計學(xué)意義(P0.05);與Htr4+/-模型組相比,Htr4+/-模型電針組小鼠血清和結(jié)腸5-HT水平下調(diào),差異無統(tǒng)計學(xué)意義(P0.05)。(4)電針對結(jié)腸5-HT_4R、5-HT_3R的影響:與WT空白組比,WT模型組、Htr4+/-模型組結(jié)腸5-HT_4R蛋白含量及mRNA表達(dá)均明顯下降,差異有統(tǒng)計學(xué)意義(P0.05)。與WT模型組相比,電針干預(yù)后,WT模型電針組結(jié)腸5-HT_4R蛋白含量及mRNA表達(dá)均上調(diào)(P0.05),差異有統(tǒng)計學(xué)意義。與Htr4+/-模型組比,Htr4+/-模型電針組結(jié)腸5-HT_4R蛋白含量上調(diào),但差異無統(tǒng)計學(xué)意義(P0.05)。5-HT_4RmRNA表達(dá)上調(diào),差異有統(tǒng)計學(xué)意義(P0.05)與WT空白組比,WT模型組結(jié)腸5-HT_3R蛋白含量及mRNA表達(dá)均明顯下降,差異有統(tǒng)計學(xué)意義(P0.05);Htr4+/-模型組5-HT_3R蛋白含量及mRNA表達(dá)均顯著上升(P0.05)。電針干預(yù)后,WT模型電針組結(jié)腸5-HT_3R蛋白含量及mRNA表達(dá)較WT模型組均上調(diào)(P0.05);Htr4+/-模型電針組結(jié)腸5-HT_3R蛋白含量及5-HT_4RmRNA較Htr4+/-模型組明顯下調(diào),差異有統(tǒng)計學(xué)意義(P0.05)(5)電針對結(jié)腸SERT的影響:WT空白、WT模型組、WT模型電針組、Htr4+/-模型組、Htr4+/-模型電針組結(jié)腸SERT蛋白含量及mRNA表達(dá)比較差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)論:(1)冰水灌胃法可以復(fù)制便秘型腸易激綜合征動物模型,模型小鼠首粒黑便時間延長、糞便顆粒數(shù)減少、含水量降低、糖水偏好度下降,慢性冰水刺激可以促進(jìn)EC過度釋放5-HT,誘導(dǎo)5-HT信號系統(tǒng)紊亂,繼而導(dǎo)致腸道動力、感覺異常;Htr4+/-模型組表現(xiàn)為先天5-HT_4R受體表達(dá)下調(diào),5-HT信號系統(tǒng)紊亂,出現(xiàn)糞便顆粒數(shù)減少、含水量降低、糖水偏好度下降等類IBS-C癥狀,證實了 5-HT_4受體是胃腸道5-HT信號系統(tǒng)的關(guān)鍵受體。兩種IBS-C模型的成功建立,一方面證實5-HT信號系統(tǒng)異常是IBS-C的重要發(fā)病機(jī)制,另一方面提示在5-HT信號系統(tǒng)中,任何平衡被打破,都會繼發(fā)整個代謝過程功能紊亂,導(dǎo)致疾病的發(fā)生。(2)電針可以良性下調(diào)冰水灌胃IBS-C模型及Htr4+/-模型小鼠異常升高的血清5-HT含量及結(jié)腸組織5-HT表達(dá);上調(diào)冰水灌胃IBS-C模型及Htr4+/-模型小鼠結(jié)腸5-HT_4R蛋白及mRNA含量;上調(diào)冰水灌胃IBS-C模型小鼠結(jié)腸組織中表達(dá)減少的5-HT_3R,而下調(diào)Htr4+/-模型電針組表達(dá)異常增高的5-HT_3R。促使5-HT、5-HT_4R、5-HT_3R恢復(fù)正常水平。提示了電針可以通過調(diào)節(jié)5-HT釋放、激活各個環(huán)節(jié)達(dá)到調(diào)控5-HT信號系統(tǒng)的作用。體現(xiàn)了針灸雙向、良性調(diào)節(jié)的優(yōu)勢。(3)電針可以通過良性調(diào)控5-HT信號系統(tǒng),改善腸道動力和腸道分泌,達(dá)到治療IBS-C的效果,從而改善冰水灌胃和Htr4+/-基因敲除所致的胃腸動力紊亂,增強(qiáng)胃腸動力、增加糞便顆粒數(shù)、含水量、促使結(jié)腸肌電恢復(fù)正常節(jié)律,緩解IBS-C模型小鼠排便困難的癥狀。
[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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R245
本文編號：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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R245
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