基于AMPK/mTOR/ULK1信号通路介导的自噬探讨芪黄益气摄血方治疗免疫性血小板减少症模型小鼠的作用机制  

Mechanism of Qihuang Yiqi Shexue Prescription in Treatment of ITP Model Mice Based on Autophagy Mediated by AMPK/mTOR/ULK1 Signaling Pathway

作　　者：罗雅琴[1] 于新阳 张月明 黄伟[3,4] LUO Yaqin;YU Xinyang;ZHANG Yueming;HUANG Wei(The Affiliated Hospital of Shandong University of Traditional Chinese Medicine(TCM),Jinan 250014,China;School of Traditional Chinese Medicine,Shandong University of TCM,Jinan 250014,China;Shandong Academy of Chinese Medicine,Jinan 250014,China;Shandong Analysis and Test Center,Qilu University of Technology(Shandong Academy of Sciences),Jinan 250014,China)

机构地区：[1]山东中医药大学附属医院 [2]山东中医药大学中医学院 [3]山东省中医药研究院 [4]齐鲁工业大学(山东省科学院)山东省分析测试中心

出　　处：《中国实验方剂学杂志》2022年第17期43-51,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81704029);齐鲁卫生与健康杰出青年人才资助项目(鲁卫人才字[2020]3号);中央本级重大增减支项目“名贵中药资源可持续利用能力建设项目”(2060302);全国名老中医药专家刘持年传承工作室建设项目(国中医药人教发[2014]20号);山东省中医药科技面上项目(2020M027)。

摘　　要：目的:基于腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白/Unc-51样激酶1(AMPK/mTOR/ULK1)信号通路介导的自噬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。检测外周血血小板计数(PLT)和血红蛋白(Hb)浓度变化;分离脾脏、胸腺组织,称质量,计算脏器指数;取胸骨做骨髓涂片,显微镜下进行骨髓巨核细胞分类;酶联免疫吸附测定法(ELISA)检测血清血小板生成素(TPO)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、肿瘤坏死因子-α(TNF-α)、转化生长因子-β1(TGF-β1)、γ干扰素(IFN-γ)水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测脾脏AMPK、mTOR、ULK1、微管相关蛋白1轻链3(LC3)、自噬关键分子酵母Atg6同系物(Beclin1)、p62 mRNA表达水平;蛋白免疫印迹法(Western blot)检测脾脏AMPK、p-AMPK、p-mTOR、p-ULK1、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水平明显下降(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),血清IL-6、TNF-α、IFN-γ水平显著升高(P<0.01),而IL-10、TGF-β1水平显著降低(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显著增加模型小鼠PLT计数、TPO水平(P<0.01),明显降低脾脏和胸腺指数(P<0.05,P<0.01),明显增加骨髓产板巨核细胞数(P<0.05,P<0.01),明显降低血清IL-6、TNF-α、IFN-γ水平(P<0.05,P<0.01),明显提高IL-10、TGF-β1水平(P<0.05,P<0.01);与芪黄益气摄血方低剂量组比较,芪黄益气摄血方高剂量组和强的松组在改善PLT计数、细胞炎性因子水平方面呈现不同程度的�Objective:To explore the mechanism of Qihuang Yiqi Shexue prescription(QHYQSX)in the treatment of immune thrombocytopenia(ITP)model mice based on the autophagy mediated by the adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)/Unc-51-like kinase1(ULK1)signaling pathway.Method:Fifty BALB/c mice were randomly divided into normal group,model group,high-and low-dose QHYQSX groups,and prednisone group,with 10 mice in each group.The ITP model was induced by intraperitoneal injection of anti-platelet serum(APS)of guinea pig.On the 8th day of the APS injection,drugs were administered by gavage for 14 days.Peripheral blood platelet(PLT)count and hemoglobin(Hb)concentration were detected.Spleen and thymus were separated,weighed,and the organ index was calculated.Sternum was sampled for bone marrow smear,and bone marrow megakaryocytes were classified under a microscope.Thrombopoietin(TPO),interleukin-6(IL-6),IL-10,tumor necrosis factor-α(TNF-α),transforming growth factor-β1(TGF-β1),and interferon-γ(IFN-γ)in the serum were detected by enzyme-linked immunosorbent assay(ELISA).AMPK,mTOR,ULK1,microtubule-associated protein light chain 3(LC3),Beclin1,and p62 mRNA expression levels in the spleen were detected by Real-time fluorescence-based quantitative polymerase chain reaction(Real-time PCR).The protein expression of AMPK,p-AMPK,p-mTOR,p-ULK1,LC3 Ⅱ/LC3 Ⅰ,Beclin1,and p62 in the spleen was detected by Western blot.Result:Compared with the normal group,the model group showed reduced peripheral blood PLT count,Hb,and TPO levels(P<0.05,P<0.01),increased spleen and thymus indexes(P<0.01),decreased number of bone marrow megakaryocytes(P<0.01),elevated serum levels of IL-6,TNF-α,and IFN-γ(P<0.01),and reduced IL-10 and TGF-β1levels(P<0.01).Compared with the model group,the groups with drug intervention showed increased PLT counts and TPO levels(P<0.01),decreased spleen and thymus indexes(P<0.05,P<0.01),elevated number of bone marrow megakaryocytes(P<0.05,P<0.01),reduced serum levels o

关 键 词：芪黄益气摄血方 免疫性血小板减少症(ITP) 自噬 腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白/Unc-51样激酶1(AMPK/mTOR/ULK1)信号通路 炎症反应 

分 类 号：R2-0[医药卫生—中医学] R33R259R554+.6