基于VIP/PACAP介导的肠道免疫功能失调探讨强的松治疗免疫性血小板减少症的效应机制  

Investigation of the mechanisms of high-dose methylprednisolone treatment for immune thrombocytopenia based on VIP/PACAP mediated intestinal immune dysfunction

作　　者：车明璐 严香 王冬雨 孙叙敏 郎海燕[1] 黄子明 何昊[3] 赵倩[1] 张雅月[1] Che Ming-lu;Yan Xiang;Wang Dong-yu;Sun Xu-min;Lang Hai-yan;Huang Zi-ming;He Hao;Zhao Qian;Zhang Ya-yue(Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China;Emergency General Hospital,Beijing 100028,China;Xi'an Medical University,Xi'an,Shaanxi 710021,China)

机构地区：[1]北京中医药大学东直门医院,北京100700 [2]应急总医院,北京100028 [3]陕西省西安医学院,陕西西安710021

出　　处：《中国现代医学杂志》2023年第21期33-40,共8页China Journal of Modern Medicine

基　　金：国家自然科学基金青年基金(No:81703903)。

摘　　要：目的 通过检测脑、结肠、血清中血管活性肠肽(VIP)、垂体腺苷酸环化酶激活多肽(PACAP)水平,探讨强的松治疗免疫性血小板减少症(ITP)的潜在机制。方法 BALB/c小鼠18只,SPF级,体重18~22 g,雌雄各半,采用全自动血细胞计数仪检测血小板计数,依据血小板计数采用区间分组法随机分为正常组、模型组及强的松组,每组6只。正常组注射生理盐水,模型组和强的松组注射抗小鼠血小板血清(APS)复制ITP动物模型。强的松组在模型复制第8天起开始强的松干预,正常组和模型组则予以生理盐水;注射APS前、注射后第4天、实验第8天(给药第1天)、实验第12天(给药第5天)、实验第15天(给药第8天)动态检测血小板计数;实验结束后采用酶联免疫吸附试验(ELISA)分别检测小鼠脑、结肠、血清中VIP、PACAP含量;用免疫组织化学法观察小鼠肠系膜淋巴结淋巴细胞P53蛋白表达的变化;原位末端转移酶标记法(TUNEL)检测小鼠肠系膜淋巴结淋巴细胞凋亡率的变化。采用ELISA检测小鼠脾脏中γ干扰素(IFN-γ)、白细胞介素-4(IL-4)、白细胞介素-10(IL-10)、白细胞介素-17A(IL-17A)的水平。结果 与正常组比较,模型组及强的松组注射APS后,小鼠生命活力明显减弱,并于实验第8天出现皮肤渗血等现象;强的松组经药物干预后,小鼠饮食量和体重均增加,毛色恢复光亮,渗血点减少。实验各组小鼠注射APS前、注射后第4天、实验第8天(给药第1天)、实验第12天(给药第5天)、实验第15天(给药第8天)不同时间点的外周血小板计数比较:(1)不同时间点的外周血小板计数有差异(F=20.618,P=0.000);(2)3组的外周血小板计数有差异(F=59.621,P=0.000);(3)3组的外周血小板计数随时间变化趋势有差异(F=7.072,P=0.000)。与正常组比较,模型组小鼠肠系膜淋巴结淋巴细胞P53蛋白表达升高(P <0.05),淋巴细胞凋亡率升高(P <0.05)。与模型组比较,强的松组小鼠肠系膜Objective To investigate the potential mechanisms of high-dose methylprednisolone treatment for immune thrombocytopenia(ITP)by assessing the levels of vasoactive intestinal peptide(VIP)and pituitary adenylate cyclase-activating polypeptide(PACAP)in the brain,colon,and serum.Methods A total of 18 BALB/c mice,SPF grade,weighing 18-22 g,with an equal distribution of males and females,were utilized.Blood platelet counts were measured using an automatic hematology analyzer.Based on platelet counts,the mice were randomly divided into three groups:a normal group,a model group,and a high-dose methylprednisolone group,each consisting of 6 mice.The normal group received injections of physiological saline,while the model and high-dose methylprednisolone groups were injected with anti-mouse platelet serum(APS)to replicate an ITP animal model.High-dose methylprednisolone treatment began in the high-dose methylprednisolone group on the 8th day after model induction,while the normal and model groups received physiological saline.Peripheral blood platelet counts were monitored at different time points:before APS injection,4 days after injection,the 8th day of the experiment(day 1 of treatment),the 12th day of the experiment(day 5 of treatment),and the 15th day of the experiment(day 8 of treatment).Following the experiment,enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of VIP and PACAP in the brain,colon,and serum.Immunohistochemistry was employed to observe changes in P53 protein expression in lymphocytes of the mesenteric lymph nodes of mice,and the terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)method was used to assess the apoptosis rate of lymphocytes in mesenteric lymph nodes.ELISA was also conducted to evaluate the levels of IFN-γ,IL-4,IL-10,and IL-17A in the spleen of mice.Results Compared to the normal group,both the model group and the high-dose methylprednisolone group exhibited reduced vitality and developed symptoms such as skin hemorrhage after APS injection.High-dose met

关 键 词：免疫性血小板减少症 血管活性肠肽 肠道免疫 强的松 

分 类 号：R558.2[医药卫生—血液循环系统疾病]