巨噬细胞移动抑制因子介导MPP+/MPTP诱导的小胶质细胞NLRP3炎症小体的激活  

Macrophage migration inhibitory factor meditates MPP+/MPTP-induced NLRP3 inflammasome activation in microglia cells

作　　者：黄河灵 高玉元[2] 聂坤[2] 王丽娟[2,1] HUANG Heling;GAO Yuyuan;NIE Kun;WANG Lijuan(School of Medicine,South China University of Technology,Guangzhou 510006,China;Department of Neurology,Guangdong Provincial People's Hospital//Guangdong Neuroscience Institute,Guangzhou 510080,China)

机构地区：[1]华南理工大学医学院,广东广州510006 [2]广东省人民医院神经科//广东省医学科学院,广东广州510080

出　　处：《南方医科大学学报》2021年第7期972-979,共8页Journal of Southern Medical University

基　　金：国家自然科学基金(81671275,81974195);国家重点研发计划(2017YFC1310200);广东省重点领域研发计划项目(2018B030337001);广东省科技计划项目(2020A0505140006);广东省基础与应用基础研究基金项目(2019A1515110061)。

摘　　要：目的探讨巨噬细胞移动抑制因子(MIF)/κb(NF-κB)对1-甲基-4-苯基吡啶离子(MPP+)/1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)激活小胶质细胞NLRP3炎症小体的影响和机制,进而对神经元的影响。方法慢病毒MIF-sh RNA感染Bv-2细胞,敲低MIF表达。Western blot检测MPP+干预的Bv-2 NLRP3和MIF表达水平、转染病毒后细胞NLRP3、p65、caspase-1表达水平及细胞核、浆蛋白p65表达水平。ELISA检测细胞培养上清液IL-1β、IL-18表达水平。将细胞培养上清液作为条件培养基培养MN9D细胞,Western blot检测TH蛋白表达水平。C57BL/6小鼠腹腔注射MPTP构建PD小鼠模型,向中脑黑质致密部立体定位注射腺相关病毒MIF-sh RNA(AAV-MIF-sh RNA)敲低MIF表达。对小鼠进行旷场实验、爬杆实验、悬挂实验行为学评估,组织免疫组化检测小鼠多巴胺能神经元细胞数目和小胶质细胞活化情况,Western blot检测小鼠黑质MIF、NLRP3及TH表达情况。结果感染病毒的细胞MIF m RNA(P<0.001)和MIF蛋白(P=0.014)明显降低。Western blot显示,0.2 mmol MPP+使Bv-2细胞NLRP3(P=0.012)和MIF(P=0.019)表达增高。与MPP组比较,MIF-sh RNA组NLRP3(P=0.042)和caspase-1(P=0.003)表达减少,细胞总蛋白中p65表达没有差异(P=0.978)。ELISA检测细胞上清液发现MIF-sh RNA组较MPP组IL-1β(P<0.001)、IL-18(P=0.002)水平降低。与MPP组比较,MIF-sh RNA组核蛋白p65表达降低(P=0.016),浆蛋白p65表达升高(P<0.001)。相较于MPP+的条件培养基,MN9D细胞在MIF-sh RNA条件培养基中TH(P=0.01)表达增加。与MPTP组比较,注射MIF-sh RNA的小鼠爬杆实验(P=0.024)和旷场实验(P=0.026)评分显著降低,悬挂实验评分显著升高(P=0.001)。组织免疫组化结果显示,相较于MPTP组,AAV-MIF-sh RNA组小鼠TH阳性神经元细胞数量增多(P=0.004),小胶质细胞数量减少(P=0.049)。MIF(P=0.033)、NLRP3表达减少(P=0.045),TH蛋白明显增多(P=0.043)。结论抑制MIF表达可以减少MPP+/MPTP干预所引起的小胶质细胞NLRP3炎Objective To explore the mechanisms of macrophage migration inhibitory factor(MIF)/nucleus factor-κB(NF-κB)in mediating 1-methyl-4-phenylpyridinium iodide(MPP+)/1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced activation of Nod-like receptor protein 3(NLRP3)inflammasome in microglials and the its effects on neurons.Methods Murine microglial cell line Bv-2 was infected with a lentivirus carrying MIF sh RNA for MIF knockdown and then treated with MPP+.The total protein levels of NLRP3,caspase-1,p65 and p65 in the cell nuclei and cytoplasm were detected.ELISA was used to detect the levels of IL-1βand IL-18 in the culture supernatant,which served as the conditioned culture medium for MN9 D cells,whose TH expression level was detected using Western blotting.The effect of stereotactic injection of an adeno-associated virus(AAV)carrying MIF sh RNA on behaviors was assessed in a C57 BL/6 mouse model of Parkinson disease(PD)induced by intraperitoneal MPTP injection.TH and Iba-1 immunohistochemistry was used to evaluate the number of substantia nigra neurons and the activation of microglia cells,and the protein expressions of MIF,NLRP3 and TH in the substantia nigra were detected with Western blotting.Results MPP+significantly increased NLRP3 and MIF expressions in Bv-2 cells(P<0.05).MIF knockdown in Bv-2 cells significantly lowered NLRP3 and caspase-1 protein expressions and IL-1βand IL-18 levels in the culture supernatant(P<0.05)without affecting total protein level of p65.Bv-2 cells with MIF knockdown showed significantlylowered p65 protein expression in the nuclei but increased p65 expression in the cytoplasm(P<0.05).The conditioned medium derived from Bv-2 cells with MIF knockdown,as compared with that from than MPP+-treated Bv-2 cells,significantly increased TH expression in MN9 D cells(P=0.01).Compared with those in MPTP group,the mice receiving injections of AAV-MIF-sh RNA had higher scores in pole test and open field test with lower scores in traction test,and showed increased TH-positive neurons,dec

关 键 词：帕金森病 神经炎症 巨噬细胞移动抑制因子 NLRP3炎症小体 小胶质细胞 

分 类 号：R742.5[医药卫生—神经病学与精神病学]