基于生物钟调控探讨肠安菌泰对腹泻型肠易激综合征的作用机制研究  

Study on the Mechanism of Chang’an Juntai on Diarrhea-Predominant Irritable Bowel Syndrome Based on Circadian Rhythm Modulation

作　　者：沈丹婷 祝旺 张佳河 侯秋科[2] 刘凤斌[2,3] SHEN Danting;ZHU Wang;ZHANG Jiahe;HOU Qiuke;LIU Fengbin(The First Clinical Medical School,Guangzhou University of Chinese Medicine,Guangzhou 510405 Guangdong,China;Department of Spleen and Stomach Diseases,The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405 Guangdong,China;Baiyun Hospital,The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510470 Guangdong,China)

机构地区：[1]广州中医药大学第一临床医学院,广东广州510405 [2]广州中医药大学第一附属医院脾胃病科,广东广州510405 [3]广州中医药大学第一附属医院白云医院,广东广州510470

出　　处：《中药新药与临床药理》2023年第10期1327-1335,共9页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(82174303)。

摘　　要：目的基于生物钟调控探讨肠安菌泰对腹泻型肠易激综合征(IBS-D)的疗效及其作用机制。方法通过采用母婴分离、乙酸灌肠联合饥饱失常的方法构建IBS-D小鼠疾病模型。模型评价后,IBS-D小鼠称质量,按照随机数字将IBS-D小鼠分成模型组、肠安菌泰组和匹维溴铵组。给药两周后,评价各组小鼠的IBS-D发病情况,包括糖水偏嗜实验(SPT)、大便含水率测定(FMC)、排便频率(FF)和避水应激实验(WAS)。实时荧光定量PCR(RT-qPCR)法检测紧密连接蛋白ZO-1、Occludin、E-cadherin,生物钟基因CRY1,炎症因子肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6),褪黑素合成酶Aanat、Asmt,褪黑素受体MT1、MT2的mRNA表达情况;免疫组化法检测Aanat、褪黑素Melatonin、肥大细胞类胰蛋白酶Tryptase的蛋白表达情况;免疫荧光法检测CRY1、p-IκBα、p65、p-p65的蛋白表达情况;蛋白免疫印迹(Western Blot,WB)法检测IκBα、p-IκBα、p65、p-p65的蛋白表达情况。结果与正常组比较,模型组小鼠糖水偏嗜率SP降低(P<0.01),FMC、FF和WAS均明显升高(P<0.01);而肠安菌泰可不同程度改善上述发病情况(P<0.05,P<0.01)。RT-qPCR结果显示,与正常组比较,模型组小鼠ZO-1、Occludin、E-cadherin、CRY1、Aanat、Asmt、MT1和MT2的mRNA表达水平降低(P<0.01),TNF-α和IL-6的mRNA表达水平升高(P<0.01);而肠安菌泰可不同程度升高ZO-1、Occludin、E-cadherin、CRY1、Aanat、Asmt、MT1和MT2的mRNA表达水平(P<0.05,P<0.01),降低TNF-α和IL-6的mRNA表达水平(P<0.01)。免疫组化结果显示,与正常组比较,模型组小鼠Aanat和Melatonin的蛋白表达平均光密度值降低(P<0.01),Tryptase蛋白表达平均光密度值升高(P<0.01);而肠安菌泰可上调Aanat和Melatonin的蛋白表达(P<0.01),降低Tryptase蛋白表达(P<0.01)。免疫荧光和WB结果显示,与正常组比较,模型组小鼠CRY1蛋白的相对荧光强度降低(P<0.01),p-IκBα和p-p65的蛋白表达水平升高(P<0.01);而肠安菌泰可提Objective To investigate the efficacy and mechanism of Chang’an Juntai(CAJT)on diarrheapredominant irritable bowel syndrome(IBS-D)based on circadian rhythm modulation.Methods The IBS-D mouse disease model was constructed by using mother-infant separation,acetic acid enema combined with hunger and satiety disorders.After the model was evaluated,IBS-D mice were weighed and divided into model group,CAJT group and Pivetonium Bromide group according to random numbers.Two weeks after administration,the IBS-D progression was evaluated in each group of mice,including sugar-water preference test(SPT),measurement of fecal moisture content(FMC),fecal frequency(FF)and water avoidance stress test(WAS).The real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the mRNA expressions of tight junction protein(ZO-1,Occludin and E-cadherin),clock gene CRY1,inflammatory factors(TNF-αand IL-6),key enzymes for melatonin synthesis(Aanat and Asmt)and melatonin receptors(MT1 and MT2).Immunohistochemistry was performed to detect the protein expressions of Aanat,Melatonin and Tryptase.The protein expressions of CRY1,p-IκBα,p65 and p-p65 were detected by immunofluorescence.The protein expressions of IκBα,p-IκBα,p65 and p-p65 were detected by Western Blot.Results Compared with the control group,mice in the model group had lower SP(P<0.01)and significantly higher FMC,FF and WAS(P<0.01),while CAJT improved the above morbidity to different degrees(P<0.05,P<0.01).RT-qPCR showed that,compared with the control group,the mRNA expressions of ZO-1,Occludin,E-cadherin,CRY1,Aanat,Asmt,MT1 and MT2 were decreased(P<0.01)and the mRNA expressions of TNF-αand IL-6 were increased(P<0.01)in the model group mice.CAJT could differentially increase the mRNA expressions of ZO-1,Occludin,E-cadherin,CRY1,Aanat,Asmt,MT1 and MT2(P<0.05,P<0.01),as well as decrease the mRNA expressions of TNF-αand IL-6(P<0.01).Immunohistochemical showed that compared with the control group,the mean optical density values of protein expressions of Aanat and Melato

关 键 词：肠安菌泰 腹泻型肠易激综合征(IBS-D) 生物钟 CRY1 NF-ΚB 小鼠 机制 

分 类 号：R285.5[医药卫生—中药学]