摘要
目的:探讨糖蛋白非转移性黑色素蛋白B(GPNMB)调节小胶质细胞M2极化减轻缺血性脑卒中(CIS)后神经损伤的机制。方法:采用SD大鼠建立大鼠大脑中动脉闭塞(MCAO)模型,缺氧缺糖(OGD)培养神经元、星形胶质细胞与小胶质细胞,Western blot测定组织与细胞GPNMB的表达情况;分别向大鼠脑组织注射腺病毒相关病毒(AAV)包裹的Gpnmb或sh RNA-Gpnmb过表达或抑制GPNMB,采用改良神经功能缺损评分(mNSS)、转棒疲劳试验(Rotarod)与胶带去除试验测试大鼠神经功能,TTC染色测定脑梗死比例,免疫荧光与RT-PCR检测小胶质细胞M1/M2极化标志物,Western blot测定磷脂酰肌醇3-激酶(PI3K)/丝氨酸/苏氨酸激酶(Akt)通路表达情况;体外OGD培养小胶质细胞,过表达Gpnmb的同时使用LY294002抑制PI3K表达,测定M1/M2极化标志物。结果:与正常大鼠/正常培养细胞比较,MCAO模型/OGD培养的小胶质细胞中GPNMB表达均上调(P<0.05);在MCAO大鼠脑组织内过表达Gpnmb后,mNSS评分下降,Rotarod延迟降落时间延长,胶带去除试验中接触时间与去除时间缩短,且脑梗死比例降低,小胶质细胞M1极化水平降低,M2极化水平升高,PI3K/Akt通路激活,差异均有统计学意义(P<0.05);抑制PI3K表达能逆转过表达Gpnmb促进体外小胶质细胞M2极化的效果(P<0.05)。结论:GPNMB通过激活PI3K/Akt通路促进小胶质细胞M2极化,从而减轻CIS后的神经损伤。
Objective:To investigate the mechanism by which glycoprotein non-transferable melanin B(GPNMB)regulates microglia M2 polarization to reduce nerve damage after cerebral ischemic stroke(CIS).Methods:SD rats were used for establishment of middle cerebral artery occlusion(MCAO)model.Neurons,astrocytes and microglia were cultured under oxygen and glucose depri⁃vation(OGD)conditions,and the expression of GPNMB in tissues and cells were measured by Western blot.Gpnmb wrapped with adenoassociated virus(AAV)or shRNA-Gpnmb were injected into rat brain tissues for overexpression or inhibition of GPNMB,modified neurological deficit score(mNSS),Rotarod fatigue test and tape removal test were used to evaluate rat nerve function,the proportion of cerebral infarction was determined by TTC staining,microglia M1/M2 polarization markers were detected by immunofluorescence and RT-PCR,and the expression of Phosphatidylinositol 3-kinase(PI3K)/Serine/threonine kinase(Akt)pathway was determined by Western blot.Microglia was cultured under OGD conditions in vitro,Gpnmb was overexpressed and PI3K expression were inhibited by LY294002,and M1/M2 polarization markers were measured.Results:Compared with normal rats or normal cultured cells,the expres⁃sion of GPNMB in MCAO model or OGD-intervened microglia was up-regulated(P<0.05);when Gpnmb was overexpressed in the brain tissue of MCAO rats,the mNSS score decreased,the Rotarod time of latency to fall lengthened,the contact time and removal time shortened in the tape removal test,the proportion of cerebral infarction decreased,the M1 polarization level of microglia decreased while the M2 polarization level increased,PI3K/Akt pathway activated,and these difference were statistically significant(P<0.05);inhibition of PI3K reversed the effect of overexpression of Gpnmb on promoting M2 polarization of microglia in vitro(P<0.05).Conclusion:GPNMB promotes M2 polarization of microglia by activating the PI3K/Akt pathway,thereby reducing nerve damage after CIS.
作者
刘梦
朱洋洋
方敬献
LIU Meng;ZHU Yangyang;FANG Jingxian(Nanyang First People's Hospital,Nanyang 473010,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2023年第12期2483-2488,共6页
Chinese Journal of Immunology
基金
河南省科技发展计划项目(202102310219)资助。
