基于PI3K/Akt信号通路探究当归补血汤干预大鼠实验性脑缺血再灌注损伤的作用机制  

Mechanism of Danggui Buxue Decoction in intervention of experimental cerebral ischemia reperfusion injury in rats based on PI3K/Akt signaling pathway

作　　者：石娅 刘文 刘兴德[1] 宋信莉 李鑫 舒万芬 张甘纯 秦琴 王洪鑫 SHI Ya;LIU Wen;LIU Xing-de;SONG Xin-li;LI Xin;SHU Wan-fen;ZHANG Gan-chun;QIN Qin;WANG Hong-xin(Guizhou Medical University,Guiyang 550025,China;Guizhou Traditional Chinese Medicine Processing and Preparation Engineering Technology Research Center,National Miao Medicine Engineering Technology Research Center,School of Pharmacy,Guizhou University of Traditional Chinese Medicine,Guiyang 550025,China;Guizhou University of Traditional Chinese Medicine,Guiyang 550025,China)

机构地区：[1]贵州中医药大学,贵州贵阳550025 [2]贵州中医药大学药学院,国家苗药工程技术研究中心,贵州中药炮制与制剂工程技术研究中心,贵州贵阳550025 [3]贵州医科大学,贵州贵阳550025

出　　处：《中草药》2022年第16期5052-5065,共14页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(82060704);贵州省特色功能食品与中药制剂开发攻关平台项目(黔教合KY字[2020]006);贵州中医药大学药用高分子材料研究中心项目(贵中医党办发[2019]70)。

摘　　要：目的基于网络药理学结合体内实验探讨当归补血汤对脑缺血再灌注损伤(cerebral ischemia reperfusion injury,CIRI)大鼠的干预作用及相关作用机制。方法通过数据库收集当归补血汤中的活性成分及其治疗CIRI的靶点,构建蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络,并进行基因本体(gene ontology,GO)功能和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析;通过Auto Dock软件对关键作用靶点与主要活性成分进行分子对接验证。SD大鼠给予尼莫地平或当归补血汤2周后,采用改良Longa线栓法复制大脑中动脉栓塞/再灌注(middle cerebral artery occlusion/reperfusion,MCAO/R)模型,对神经行为学进行评分,并测定脑组织梗死面积;采用TUNEL染色法测定脑组织细胞凋亡情况;采用苏木素-伊红(HE)染色法考察脑组织病理变化;采用ELISA测定血清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)和IL-1β水平;采用qRT-PCR检测脑组织TNF-α、IL-6和IL-1βmRNA表达;采用Western blotting检测脑组织蛋白激酶B(protein kinase B,Akt)、磷酸化Akt(phosphorylated Akt,p-Akt)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)和Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)蛋白表达。结果筛选出当归补血汤30种活性成分,得到当归补血汤治疗CIRI的靶点467个,核心靶点55个。KEGG通路富集分析表明磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)/Akt信号通路是当归补血汤抗CIRI的关键机制之一。分子对接显示,当归补血汤中主要活性成分与核心作用靶点中的Akt1、信号传导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)、转录因子AP-1(transcription factor AP-1,JUN)、表皮生长因子受体(epidermal growth factor receptor,EGFR)、细胞肿瘤抗原p53(cellular tumor antigen p53,TP53)、TNF和IL-6等具有较为稳定的结合活性。动物实验�Objective To explore the interventional effect and related mechanism of Danggui Buxue Decoction(当归补血汤)on rats with cerebral ischemia reperfusion injury(CIRI)based on network pharmacology combined with in vivo experiments.Methods Active ingredients in Danggui Buxue Decoction and targets for the treatment of CIRI were collected from databases,protein-protein interaction(PPI)network was constructed,gene ontology(GO)function and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis were performed;Key targets and main active ingredients were selected for molecular docking by AutoDock software.After SD rats were given nimodipine or Danggui Buxue Decoction for 2 weeks,middle cerebral artery occlusion/reperfusion(MCAO/R)model was established by modified Longa suture method,neurobehavioral scores were evaluated And brain tissue infarct size was determined;TUNEL staining was used to determine the apoptosis of brain tissue;Hematoxylin-eosin(HE)staining was used to investigate the pathological changes of brain tissue;Levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and IL-1βin serum were determined by ELISA;qRT-PCR was used to detect the mRNA expressions of TNF-α,IL-6 and IL-1βin brain tissue;Western blotting was used to detect protein expressions of protein kinase B(Akt),phosphorylated Akt(p-Akt),Bcell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax)in brain tissue.Results Thirty active ingredients of Danggui Buxue Decoction were screened out,467 targets and 55 core targets of Danggui Buxue Decoction in the treatment of CIRI were obtained.KEGG pathway enrichment analysis showed that phosphatidylinositol-3-kinase(PI3K)/Akt signaling pathway was one of key mechanisms of Danggui Buxue Decoction against CIRI.Molecular docking showed that main active components in Danggui Buxue Decoction had relatively stable binding activities with core targets such as Akt1,signal transducer and activator of transcription 3(STAT3),and transcription factor AP-1(JUN),epidermal growth factor recepto

关 键 词：当归补血汤 脑缺血再灌注损伤 网络药理学 磷脂酰肌醇-3-激酶/蛋白激酶B信号通路 炎症 细胞凋亡 毛蕊异黄酮葡萄糖苷 毛蕊异黄酮 阿魏酸 芒柄花素 藁本内酯 黄芪甲苷 

分 类 号：R285.5[医药卫生—中药学]