腺苷A2AR拮抗剂对中枢炎症性小胶质细胞形态和功能的影响

Effects of adenosine A2A receptor antagonist on lipopolysaccharide induced inflammatory BV-2 microglial cells’ morphology and function

目的:探讨腺苷A2A受体拮抗剂对实验性自身免疫性脑脊髓炎(EAE)的治疗作用及其对中枢炎症性小胶质细胞形态和功能的影响。方法:MOG35-55免疫诱导建立EAE模型,EAE小鼠出现神经功能缺损症状后开始腹腔注射腺苷A2AR拮抗剂至发病后第10天。ELISA法检测中枢神经系统内IFN-γ、IL-17、TGF-β、IL-10的表达情况,免疫荧光双重染色法检测小胶质细胞内M1型细胞标志物诱导型一氧化氮合酶(iNOS)和M2型细胞标志物精氨酸酶I(ArgI)的合成情况。离体培养BV-2小胶质细胞,LPS诱导的小胶质细胞炎症反应,并予A2AR拮抗剂干预,Real-time PCR和ELISA法检测M1型和M2型细胞相关的细胞因子RNA表达水平和分泌水平。Western Blot法检测各组小胶质细胞内M1型和M2型细胞标志物的表达量。结果:腺苷A2AR拮抗剂能有效缓解发病后EAE小鼠的神经功能缺损症状,降低IFN-γ的分泌水平,使M1型细胞相关的iNOS合成减少,M2型细胞相关的ArgI合成增多。腺苷A2AR拮抗剂能减少LPS刺激后BV-2小胶质细胞M1型细胞相关的细胞因子IL-1β的mRNA表达量和分泌量,对M2型细胞相关的细胞因子及蛋白无显著影响。结论:腺苷A2AR拮抗剂对发病后的EAE小鼠有肯定的治疗作用,其机制可能与改变中枢炎症过程中小胶质细胞的表型(M1/M2转换)改变(形态和功能)有关。

AIM:To investigate the therapeutic effects of adenosine A2A receptor antagonist on experimental autoimmune encephalomyelitis(EAE)and to explore the regulating effects on microglia morphology and function.METHODS:EAE model was induced by MOG35-55 in C57BL/6 mice.Adenosine A2AR antagonists were given by intraperitoneal injection as EAE mice showed neurobehavioral deficits.The treatment lasted ten days until EAE mice reached highest clinical score.ELISA method was used to detect the expression of pro-inflammatory cytokine in central nervous system;immunofluorescence double staining was used to detect M1 cell related markers and M2 cell related markers in microglia.In vitro,activated BV-2 microglia cell line was induced by lipopolysaccharide(LPS)and then was intervened by the A2AR antagonists.Real-time PCR and ELISA method were used to detect the M1 and M2 cells related cytokines.Western Blot method was used to detect the synthesis of M1 and M2 cell related markers.RESULTS:Adenosine A2AR antagonists can effectively ameliorate the EAE-induced neurobehavioral deficits when treated after onset of EAE,and can reduce the secretion of pro-inflammatory cytokine IFN-γ,and M1 cells related iNOS synthesis while increased M2 type cells related ArgI synthesis.Adenosine A2AR antagonists can reduce LPS-induced BV-2 M1 microglial cells related cytokines IL-1β,and had no influence on M2 cells related cytokines and protein.CONCLUSION:Adenosine A2AR antagonists have definite therapeutic effect on EAE mice when treated after the onset of EAE;its underlying mechanism may be involved with shift of phenotype(M1/M2)and changes in morphology and function of microglia during neuro-inflammatory process.