基于网络药理学并辅以生物学验证探讨固肠止泻丸治疗炎症后肠易激综合征内脏痛敏的分子机制
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摘要
目的基于网络药理学并辅以生物学验证探讨固肠止泻丸治疗炎症后肠易激综合征(post-inflammation irritable bowel syndrome,PI-IBS)内脏痛觉敏感的分子机制。方法经ADME参数过滤分析从固肠止泻丸中筛选出96个活性成分,利用抗PI-IBS的上市药物上传到PharmMapper中进行抗PI-IBS疾病靶标预测。活性成分与预测疾病靶标的二者进行分子对接验证,用Cytoscape软件构建成分-靶标网络;借助CluoGO插件对固肠止泻丸治疗PI-IBS内脏痛觉敏感的相关靶标进行生物学通路分析,最后以TNBS诱导PI-IBS大鼠模型为研究材料,采用低、中、高不同剂量GCZX干预,然后从内脏痛觉阈值、结肠EC细胞数量,及若干关键细胞因子、关键靶标蛋白表达等多种指标,进行动物、细胞、分子等多个层面的验证性实验。验证前期计算机模拟结果的可靠性。结果计算机模拟实验预测结果表明,89个活性药效分子作用于39个关键靶标,其中核心靶标为TPH1、TNF-α、IL-6、IFN-γ、MAO-A、IL-10,且靶标主要作用于5-HT信号通路上。生物学验证实验结果表明,经GCZX的干预后,显著降低了TNBS诱导的PI-IBS大鼠的内脏痛觉敏感度、结肠嗜铬细胞数目,且有效调控了结肠组织内TPH、5-HT、5-HIAA及细胞炎症因子TNF-α、IFN-γ、IL-6、IL-10的表达,以及SERT蛋白表达量无显著变化。结论固肠止泻丸的药效分子可能主要通过调节5-HT信号通路发挥药效,并主要作用于6个核心靶标。本研究为进一步开展固肠止泻丸治疗PI-IBS作用机制的研究提供了新思路和新方法。
Objective To explore the molecular mechanism of Guchang Zhixie Pills(GCZX) in treating visceral hyperalgesia of post-inflammation irritable bowel syndrome(PI-IBS) based on network pharmacology and the biological verification. Methods This study screened 96 active components from GCZX by ADME parameter filtration analysis,and conventional anti PI-IBS drugs were uploaded to PharmMapper for target prediction of PIIBS resistance. Then active ingredients were used for molecular docking to predict disease targets and to construct component target-network with Cytoscape software. Finally,animal-cell-molecular experiments using TNBS induced PI-IBS rat models were carried out for verification. Results The biological pathways of GCZX in the treatment of PIIBS visceral hyperalgesia were analyzed by CluoGO plug-in. Eighty-nine active molecules acted on 39 key targets,including TPH1,TNF-α,IL-6,IFN-γ,MAO-A,IL-10,and the targets mainly acted on the 5-HT signaling pathway. The results of biological verification experiments show that GCZX significantly decreased the visceral pain sensitivity and the number of colon chromaffin cells in PI-IBS rats induced by TNBS,and effectively regulated the expression of TPH,5-HT,5-HIAA and inflammatory cytokines in colon tissues such as TNF-α,IFN-γ,IL-6,IL-10,and have no effect on the expression of SERT protein. Conclusion GCZX may exert pharmacological effects mainly by regulating 5-HT signaling pathway,and acts on six core targets. The study built a foundation for drug development and innovative research.
Objective To explore the molecular mechanism of Guchang Zhixie Pills(GCZX) in treating visceral hyperalgesia of post-inflammation irritable bowel syndrome(PI-IBS) based on network pharmacology and the biological verification. Methods This study screened 96 active components from GCZX by ADME parameter filtration analysis,and conventional anti PI-IBS drugs were uploaded to PharmMapper for target prediction of PIIBS resistance. Then active ingredients were used for molecular docking to predict disease targets and to construct component target-network with Cytoscape software. Finally,animal-cell-molecular experiments using TNBS induced PI-IBS rat models were carried out for verification. Results The biological pathways of GCZX in the treatment of PIIBS visceral hyperalgesia were analyzed by CluoGO plug-in. Eighty-nine active molecules acted on 39 key targets,including TPH1,TNF-α,IL-6,IFN-γ,MAO-A,IL-10,and the targets mainly acted on the 5-HT signaling pathway. The results of biological verification experiments show that GCZX significantly decreased the visceral pain sensitivity and the number of colon chromaffin cells in PI-IBS rats induced by TNBS,and effectively regulated the expression of TPH,5-HT,5-HIAA and inflammatory cytokines in colon tissues such as TNF-α,IFN-γ,IL-6,IL-10,and have no effect on the expression of SERT protein. Conclusion GCZX may exert pharmacological effects mainly by regulating 5-HT signaling pathway,and acts on six core targets. The study built a foundation for drug development and innovative research.
引文
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[3]CARD T,ENCK P,BARBARA G,et al.Post-infectious IBS:Defining its clinical features and prognosis using an internet-based survey.United European Gastroenterol J.2018,6(8):1245-1253.
[4]FORD A C,LACY B E,TALLEY N J.Irritable bowel syndrome[J].N Engl J Med,2017,376(26):2566-2578.
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[8]ASSENOV Y,RAMIREZ F,SCHELHORN S E,et al.Computing topological parameters of biological networks[J].Bioinformatics(Oxford,England),2008,24(2):282-284.
[9]BINDEA G,MLECNIK B,HACKL H,et al.ClueGO:a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks[J].Bioinformatics,2009,25(8):1091-1093.
[10]LIEBREGTS T,ADAM B,BERTEL A,et al.Effect of E coli Nissle 1917 on post-inflammatory visceral sensory function in a rat model[J].Neurogastroenterology&motility,2010,17(3):410-414.
[11]ALCHAER E D,KAWASAKI M,PASRICHA P J.A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development[J].Gastroenterology,2000,119(5):1276-1285.
[12]ZHU X,LIU Z,NIU W,et al.Effects of electroacupuncture at ST25 and BL25 in a Sennae-induced rat model of diarrhoeapredominant irritable bowel syndrome[J].Acupuncture in medicine,2017,35(3):216-223
[13]LI B,XU X,WANG X,et al.A systems biology approach to understanding the mechanisms of action of Chinese herbs for treatment of cardiovascular disease[J].International journal of molecular sciences,2012,13(10):13501-13520.
[14]刘忠政,梁洁萍,聂怡初,等.复方血栓通胶囊基于血液循环和凝血过程相关靶点的网络药理学研究[J].中山大学学报(自然科学版),2013,52(2):97-100.
[15]袁文泽.固肠止泻丸治疗腹泻型肠道易激综合症临床研究[J].中医临床研究,2013,5(17):62-63.
[16]古远明.固肠止泻丸治疗肠易激综合征的临床观察[J].胃肠病学,2003(S1):23.
[17]郭学亮,王子宽,郭艳,等.中药固肠止泻丸治疗溃疡性结肠炎896例临床观察[J].陕西中医学院学报,2000(05):19-23.
[18]ENGELMAN K,LOVENBERG W,SJOERDSMA A.Inhibition of serotonin synthesis by para-chlorophenylalanine in patients with the carcinoid syndrome[J].N Engl J Med,1967,277(21):1103-1108.
[19]KOE B K,WEISSMAN A.P-chlorophenylalanine:a specific depletor of brain serotonin[J].J Pharmacol Exp Ther,1966,154:499-516.
[20]Sanders-Bush E,Sulser F.P-chloroamphetamine:in vivo investigations on the mechanism of action of the selective depletion of cerebral serotonin[J].J Pharmacol Exp Ther,1970,175:419-426.
[21]张玉春,邵云云,左晓,等.冬凌草片对炎症后肠易激综合征大鼠的治疗作用[J].中国现代应用药学,2016,33(10):1235-1239.
[22]BROWN P M,DROSSMAN D A,WOOD A J,et al.The tryptophan hydroxylase inhibitor LX1031 shows clinical benefit in patients with nonconstipating irritable bowel syndrome[J].Gastroenterology,2011,141(2):507-516.
[23]CAO Y N,FENG L J,LIU Y Y,et al.Effect of Lactobacillus rhamnosus GG supernatant on serotonin transporter expression in rats with post-infectious irritable bowel syndrome[J].World JGastroenterol,2018,24(3):338-350.
[24]GRZESIAK M,BESZEJ J A,WASZCZUK E,et al.Serotoninrelated gene variants in patients with irritable bowel syndrome and depressive or anxiety disorders[J].Gastroenterol Res Pract,2017,2017:429-430.
[25]KESZTHELYI D,TROOST F J,JONKERS D M,et al.Visceral hypersensitivity in irritable bowel syndrome:evidence for involvement of serotonin metabolism--a preliminary study[J].Neurogastroenterol Motil,2015,27(8):1127-37.
[26]OLIVEIRA M C G,PELEGRINI-DA-SILVA A,PARADA C A,et al.5-HT acts on nociceptive primary afferents through an indirect mechanism to nduce hyperalgesia in the subcutaneous tissue[J].Neuroscience,2007,145(2):708-714.
[27]MASAND P S,GUPTA S,SCHWARTZ T L,et al.Paroxetine in patients with irritable bowel syndrome:A pilot open-label study[J].Primary Care Companion to The Journal of Clinical Psychiatry,2002,4(1):12-16.
[2]MEARIN F,LACY B E,CHANG L,et al.Bowel disorders[J].Gastroenterology,2016(150):1393-1407.
[3]CARD T,ENCK P,BARBARA G,et al.Post-infectious IBS:Defining its clinical features and prognosis using an internet-based survey.United European Gastroenterol J.2018,6(8):1245-1253.
[4]FORD A C,LACY B E,TALLEY N J.Irritable bowel syndrome[J].N Engl J Med,2017,376(26):2566-2578.
[5]张声生,许文君,陈贞,等.疏肝健脾法与健脾化湿法治疗腹泻型肠易激综合征对比疗效观察[J].中华中医药杂志,2010,25(1):127~130.
[6]唐娟.固肠止泻丸治疗腹泻型肠道易激综合征疗效探讨[J].内蒙古中医药,2013,32(15):37.
[7]孙光裕,陈壁亮.匹维溴铵联合固肠止泻丸治疗腹泻为主型肠易激综合征[J].广东医学,2003(12):1365-1366.
[8]ASSENOV Y,RAMIREZ F,SCHELHORN S E,et al.Computing topological parameters of biological networks[J].Bioinformatics(Oxford,England),2008,24(2):282-284.
[9]BINDEA G,MLECNIK B,HACKL H,et al.ClueGO:a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks[J].Bioinformatics,2009,25(8):1091-1093.
[10]LIEBREGTS T,ADAM B,BERTEL A,et al.Effect of E coli Nissle 1917 on post-inflammatory visceral sensory function in a rat model[J].Neurogastroenterology&motility,2010,17(3):410-414.
[11]ALCHAER E D,KAWASAKI M,PASRICHA P J.A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development[J].Gastroenterology,2000,119(5):1276-1285.
[12]ZHU X,LIU Z,NIU W,et al.Effects of electroacupuncture at ST25 and BL25 in a Sennae-induced rat model of diarrhoeapredominant irritable bowel syndrome[J].Acupuncture in medicine,2017,35(3):216-223
[13]LI B,XU X,WANG X,et al.A systems biology approach to understanding the mechanisms of action of Chinese herbs for treatment of cardiovascular disease[J].International journal of molecular sciences,2012,13(10):13501-13520.
[14]刘忠政,梁洁萍,聂怡初,等.复方血栓通胶囊基于血液循环和凝血过程相关靶点的网络药理学研究[J].中山大学学报(自然科学版),2013,52(2):97-100.
[15]袁文泽.固肠止泻丸治疗腹泻型肠道易激综合症临床研究[J].中医临床研究,2013,5(17):62-63.
[16]古远明.固肠止泻丸治疗肠易激综合征的临床观察[J].胃肠病学,2003(S1):23.
[17]郭学亮,王子宽,郭艳,等.中药固肠止泻丸治疗溃疡性结肠炎896例临床观察[J].陕西中医学院学报,2000(05):19-23.
[18]ENGELMAN K,LOVENBERG W,SJOERDSMA A.Inhibition of serotonin synthesis by para-chlorophenylalanine in patients with the carcinoid syndrome[J].N Engl J Med,1967,277(21):1103-1108.
[19]KOE B K,WEISSMAN A.P-chlorophenylalanine:a specific depletor of brain serotonin[J].J Pharmacol Exp Ther,1966,154:499-516.
[20]Sanders-Bush E,Sulser F.P-chloroamphetamine:in vivo investigations on the mechanism of action of the selective depletion of cerebral serotonin[J].J Pharmacol Exp Ther,1970,175:419-426.
[21]张玉春,邵云云,左晓,等.冬凌草片对炎症后肠易激综合征大鼠的治疗作用[J].中国现代应用药学,2016,33(10):1235-1239.
[22]BROWN P M,DROSSMAN D A,WOOD A J,et al.The tryptophan hydroxylase inhibitor LX1031 shows clinical benefit in patients with nonconstipating irritable bowel syndrome[J].Gastroenterology,2011,141(2):507-516.
[23]CAO Y N,FENG L J,LIU Y Y,et al.Effect of Lactobacillus rhamnosus GG supernatant on serotonin transporter expression in rats with post-infectious irritable bowel syndrome[J].World JGastroenterol,2018,24(3):338-350.
[24]GRZESIAK M,BESZEJ J A,WASZCZUK E,et al.Serotoninrelated gene variants in patients with irritable bowel syndrome and depressive or anxiety disorders[J].Gastroenterol Res Pract,2017,2017:429-430.
[25]KESZTHELYI D,TROOST F J,JONKERS D M,et al.Visceral hypersensitivity in irritable bowel syndrome:evidence for involvement of serotonin metabolism--a preliminary study[J].Neurogastroenterol Motil,2015,27(8):1127-37.
[26]OLIVEIRA M C G,PELEGRINI-DA-SILVA A,PARADA C A,et al.5-HT acts on nociceptive primary afferents through an indirect mechanism to nduce hyperalgesia in the subcutaneous tissue[J].Neuroscience,2007,145(2):708-714.
[27]MASAND P S,GUPTA S,SCHWARTZ T L,et al.Paroxetine in patients with irritable bowel syndrome:A pilot open-label study[J].Primary Care Companion to The Journal of Clinical Psychiatry,2002,4(1):12-16.