芪黄益气摄血方调控PI3K/AKT/mTOR信号通路和自噬治疗免疫性血小板减少症的作用机制  

Mechanism of Qihuang Yiqi Shexue Formula in the treatment of immune thrombocytopenia by regulating PI3K/AKT/mTOR signaling pathway and autophagy

作　　者：罗雅琴[1] 黄伟[2,3] LUO Ya-qin;HUANG Wei(The Afiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan 250014,China;Shandong Academy of Chinese Medicine,Jinan 250014,China;Qilu University of Technology(Shandong Academy of Sciences),Shandong Analysis and Test Center,Jinan 250014,China)

机构地区：[1]山东中医药大学附属医院,济南250014 [2]山东省中医药研究院,济南250014 [3]齐鲁工业大学(山东省科学院)山东省分析测试中心,济南250014

出　　处：《中华中医药杂志》2022年第10期5627-5633,共7页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金项目(No.81704029);齐鲁卫生与健康杰出青年人才资助项目(No.鲁卫人才字[2020]3号);中央本级重大增减支项目(No.2060302);全国名老中医药专家刘持年传承工作室建设项目(No.国中医药人教发[2014] 20号);山东省中医药科技面上项目(No.2020M027)。

摘　　要：目的:基于磷酸肌醇-3-激酶(PI3K)/蛋白激酶B(AKT)/雷帕霉素靶蛋白(mTOR)自噬信号通路探讨芪黄益气摄血方治疗免疫性血小板减少症(ITP)模型小鼠的作用机制。方法:50只BALB/c小鼠随机分为5组,分别为正常组,模型组,芪黄益气摄血方高、低剂量组和强的松组,每组10只。采用豚鼠抗小鼠血小板血清(APS)腹腔注射方法,建立ITP小鼠模型;注射APS后的第8天,各组分别灌胃给药,连续14 d。检测外周血血小板(PLT)计数和血红蛋白(Hb)浓度变化,Elisa法检测血清血小板生成素(TPO)水平;分离脾脏、胸腺组织,称重,计算脾脏、胸腺指数;取胸骨做骨髓涂片,显微镜下进行骨髓巨核细胞分类;qRT-PCR法检测脾脏PI3K、AKT、mTOR、微管相关蛋白轻链3(LC3)、自噬关键分子酵母Atg6同素物(Beclin1)、P62 mRNA表达水平;Western Blot法检测脾脏p-PI3K、p-AKT、p-mTOR、LC3Ⅱ/LC3Ⅰ、Beclin1、P62蛋白表达水平。结果:与正常组比较,模型组小鼠外周血PLT计数、Hb及TPO水平显著下降(P<0.05,P<0.01),脾脏和胸腺指数显著升高(P<0.01),骨髓产板巨核细胞数显著减少(P<0.01);与模型组比较,芪黄益气摄血方高、低剂量组及强的松组PLT计数、TPO水平显著升高(P<0.01),脾脏指数和胸腺指数显著降低(P<0.05,P<0.01),骨髓产板巨核细胞数显著增加(P<0.01),仅芪黄益气摄血方高剂量组Hb水平显著增加(P<0.05);与低剂量组比较,高剂量组PLT计数显著升高(P<0.05)。qRT-PCR和Western Blot结果显示,与正常组比较,模型组小鼠脾脏PI3K、AKT、mTOR、P62 mRNA表达水平显著下调(P<0.05,P<0.01),p-PI3K、p-AKT、p-mTOR、P62蛋白表达水平亦显著下调(P<0.01),而LC3、Beclin1 mRNA和LC3Ⅱ/LC3Ⅰ、Beclin1蛋白表达水平均显著上调(P<0.01);与模型组比较,芪黄益气摄血方高、低剂量组及强的松组可显著上调脾脏PI3K、AKT、mTOR、P62 mRNA表达水平(P<0.05,P<0.01),显著上调p-PI3K、p-AKT、p-mTOR、P62蛋白表达水平(P<0.05,P<0.01)Objective:Based on the PI3K/AKT/mTOR autophagy signal pathway,the mechanism of Qihuang Yiqi Shexue Formula in treating ITP model mice was explored.Methods:Fifty BALB/c mice were randomly divided into 5 groups:normal group,model group,Qihuang Yiqi Shexue Formula high and low dose group and prednisone group,with 10 mice in each group.The intraperitoneal injection of guinea pig anti-mouse platelet serum(APS)was used to establish an ITP mouse model;on the 8th day after the injection of APS,each group was given intragastric administration for 14 consecutive days.To detect the changes in peripheral blood platelet count(PLT)and hemoglobin(Hb)concentration,and use Elisa method to detect serum thrombopoietin(TPO)level;separate spleen and thymus tissue,weigh,calculate organ index;take sternum for bone marrow smear,classification of bone marrow megakaryocytes under a microscope;qRT-PCR method to detect spleen PI3K,AKT,mTOR,LC3,Beclin1,P62 mRNA expression levels;Western Blot method to detect spleen p-PI3K,p-AKT,p-mTOR,LC3Ⅱ/LC3Ⅰ,Beclin1,P62 protein expression level.Results:Compared with the normal group,the peripheral blood PLT count,Hb and TPO levels of the model group were significantly decreased(P<0.05,P<0.01),the spleen and thymus index were significantly increased(P<0.01),and the number of bone marrow-producing megakaryocytes were significantly decreased(P<0.01);compared with the model group,the PLT counts and TPO levels of the Qihuang Yiqi Shexue Formula high and low dose groups and the prednisone group were significantly increased(P<0.01),spleen index and thymus index were significantly decreased(P<0.05,P<0.01),the number of megakaryocytes in the bone marrow plate were significantly increased(P<0.01),only the high-dose group had a significant increase in Hb level(P<0.05);compared with the low-dose group,the PLT counts in the high-dose group were significantly increased(P<0.05).The results of qRT-PCR and Western Blot showed that compared with the normal group,the expression levels of PI3K,AKT,mTOR,and P62 mRNA in th

关 键 词：免疫性血小板减少症 芪黄益气摄血方 PI3K/AKT/mTOR信号通路 自噬 

分 类 号：R285.5[医药卫生—中药学]