羟基红花黄色素A通过调节JAK2/STAT3信号通路对星形胶质细胞OGD/R损伤的神经保护作用机制研究  

Neuroprotective Effect of Hydroxysafflower Yellow Pigment A on OGD/R Injury in Astrocytes Through Modulating JAK2/STAT3 Signaling Pathway

作　　者：马东[1,2] 刘可心 韩光远 殷丽君 夏天晴 温春丽 殷金珠 张嘉旭 黄建军 宋丽娟[2,5] MA Dong;LIU Kexin;HAN Guangyuan;YIN Lijun;XIA Tianqing;WEN Chunli;YIN Jinzhu;ZHANG Jiaxu;HUANG Jianjun;SONG Lijuan(Department of Neurosurgery,General Hospital of Sinopharm Tongmei Group,Datong Shanxi China 037003;Key Laboratory of Multiple Sclerosis,China Research Center for Neurobiology,Shanxi University of Chinese Medicine/Key Research Laboratory of Tonifying Qi and Blood Activation in Treating Multiple Sclerosis,State Administration of Traditional Chinese Medicine,Jinzhong Shanxi China 030619;Oriental University of Hebei,Langfang Hebei China 065001;Liming Vocational College of Science and Technology of Shandong,Tai′an Shandong China 271000;Cell Physiology Key Laboratory of Shanxi Medical University of Ministry of Education Jointly Established by Province and Ministry,Taiyuan Shanxi China 0300015)

机构地区：[1]国药同煤集团总医院神经外科,山西大同037003 [2]山西中医药大学神经生物学研究中心/国家中医药管理局多发性硬化益气活血重点研究室,山西晋中030619 [3]河北东方学院,河北廊坊065001 [4]山东力明科技职业学院,山东泰安271000 [5]山西医科大学细胞生理学省部共建教育部重点实验室,山西太原0300015

出　　处：《中医学报》2024年第9期1954-1964,共11页Acta Chinese Medicine

基　　金：国家自然科学基金项目(82004028);中国博士后科学基金面上资助项目(2020M680912);国家中医药管理局“张仲景传承与创新专项”项目(GZY-KJS-2022-048-1);山西省科技创新人才青年团队项目(202204051001028);山西中医药大学2022年度科技创新团队项目(2022TD2010);山西省卫生健康委员会医学科技领军团队项目(2020TD05);山西中医药大学附属医院国家区域中医医疗中心心血管专项基金项目(XGZX202115);山西中医药大学青年科学家培育项目(2021PY-QN-09);山西中医药大学学科建设经费项目(2023XKJS-02)。

摘　　要：目的:探讨羟基红花黄色素A(hydroxysafflor yellow A,HSYA)在星形胶质细胞(astrocyte,Ast)氧糖剥夺/复氧(oxygen glucose deprivation/reoxygenation,OGD/R)损伤中的作用及潜在分子机制。方法:制备原代Ast,采用第二代Ast建立OGD/R模型,并分为对照组、模型组和HSYA组。常规培养PC12细胞,并分为空白组、LCN2组、LCN2+HSYA组、空白+HSYA组。常规培养Ast,并分为Normal组、Colivelin组、Colivelin+HSYA组、Normal+HSYA组。采用ELISA法检测细胞上清液乳酸脱氢酶(lactate dehydrogenase,LDH)含量;Western blot和免疫荧光(immunofluorescence,IF)法检测细胞胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、神经钙结合蛋白β(S100 calcium binding protein beta,S100β)、脂质运载蛋白2(lipcalin-2,LCN2)、溶质转运蛋白家族22成员17(solute carrier family 22 member 17,SLC22A17)表达量;RT-PCR法检测细胞LCN2 mRNA表达水平。采用Swiss Target Prediction数据库、Similarity ensemble approach(SEA)数据库及GeneCards数据库检索HSYA的靶点。借助GeneCards数据库、OMIM数据库及NCBI数据库检索脑缺血的靶点,并获取其与HSYA的交集靶点。通过STRING数据库构建蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络,使用拓扑分析筛选核心靶点。结果:IF显示,原代Ast中GFAP蛋白阳性比例>95%。RT-PCR显示,与常规培养Ast比较,氧糖剥夺6 h、8 h的LCN2 mRNA水平升高(P<0.05)。与对照组比较,模型组Ast的LCN2 mRNA水平升高,HSYA可降低Ast的LCN2 mRNA水平(P<0.01)。Western blot显示,与对照组比较,模型组Ast中GFAP、S100β、LCN2、SLC22A17表达量升高,HSYA可逆转这一现象(P<0.05)。ELISA显示,与空白组比较,LCN2组细胞上清液中LDH含量升高,HSYA可降低LDH含量(P<0.01)。Western blot显示,与空白组比较,LCN2组细胞LCN2、SLC22A17表达量升高;与LCN2组比较,LCN2+HSYA组细胞LCN2、SLC22A17表达量降低(P<0.05)。HSYA靶点88个,脑缺血靶点5721个,两者的交集靶点60个,信号�Objective:To investigate the role and potential molecular mechanism of hydroxysafflor yellow A(HSYA)in oxygen glucose deprivation/reoxygenation(OGD/R)injury of astrocyte(Ast).Methods:Primary astrocytes(Ast)were prepared,and the OGD/R model was established by the second-generation Ast,and they were divided into control group,model group and hydroxysafflor yellow A(HSYA)group.PC12 cells were routinely cultured and divided into blank group,LCN2 group,LCN2+HSYA group,and blank+HSYA group.Ast was routinely cultured and divided into Normal group,Colivelin group,Colivelin+HSYA group,and Normal+HSYA group.ELISA was used to detect the content of lactate dehydrogenase(LDH)in cell supernatants.Western blot and immunofluorescence(IF)were used to detect glial fibrillary acidic protein(GFAP),S100 calcium binding protein beta(S100ββ),lipcalin-2(LCN2),Expression of solute carrier family 22 member 17(SLC22A17);The expression level of LCN2 mRNA in cells was detected by RT-PCR.The Swiss Target Prediction database,the Similarity ensemble approach(SEA)database and the GeneCards database were used to retrieve the targets of HSYA.The targets of cerebral ischemia were retrieved by GeneCards,OMIM,and NCBI,and the intersection targets with HSYA were obtained.The protein-protein interaction(PPI)network was constructed through the STRING database,and the core targets were screened by MCODE cluster analysis.Results:IF showed that the proportion of GFAP protein positive in primary Ast was>95%.RT-PCR showed that compared with the conventional culture Ast,the level of LCN2 mRNA decreased after 4 h of OGD/R modeling.After 6 h and 8 h of OGD/R modeling,the mRNA level of LCN2 increased(P<0.05).Compared with the control group,the LCN2 mRNA level of Ath in the model group increased,and HSYA decreased the LCN2 mRNA level of Ast(P<0.05).Western blot showed that compared with the control group,the expressions of GFAP,S100β,LCN2 and SLC22A17 in the model group were increased,and HSYA could reverse this phenomenon(P<0.05).ELISA showed that compared wi

关 键 词：羟基红花黄色素A 星形胶质细胞 缺血性脑卒中 JAK2/STAT3信号通路 LCN2 SLC22A17 

分 类 号：R285.5[医药卫生—中药学]