星形胶质细胞外泌体对癫痫大鼠小胶质细胞活化及JAK2/STAT3通路的影响

The effect of astrocyte exosomes on the activation of microglia and JAK2/STAT3 pathway in epileptic rats

目的探讨星形胶质细胞外泌体(Astrocyte exosomes,Ast-exos)对癫痫大鼠小胶质细胞活化及Janus激酶2(Janus kinase,JAK2)/信号转导与转录激活子3(Signal transducer and activator of transcription,STAT3)通路的影响。方法将雄性SD大鼠用随机数字表法分为正常对照组、模型组、Ast-exos组(尾静脉注射Ast-exos,0.5 mg/kg)、Coumermycin A1组(JAK2通路激活剂,4.0 mg/kg)、Ast-exos+Coumermycin A1组,每组各10只;腹腔注射戊四氮(35 mg/kg)建立大鼠癫痫模型,各组给药14 d后观察大鼠行为,对癫痫症状进行评分;处死大鼠,取海马组织,免疫组化法检测小胶质细胞活化数目;酶联免疫吸附测定法(Enzyme-linked immunosorbent assay,ELISA)法检测海马组织炎性因子-白细胞介素(Interleukin,IL)-1β、IL-6及神经递质γ氨基丁酸水平;尼氏及原位末端标记法(TdT-mediated dUTP nick end labeling,TUNEL)染色观察海马组织神经元损伤及凋亡状况;免疫荧光共定位法检测JAK2磷酸化蛋白(Phosphorylated JAK2,p-JAK2)与活化小胶质细胞共表达数目;蛋白免疫印迹法(Western blot)法检测JAK2/STAT3通路磷酸化蛋白p-JAK2、磷酸化STAT3(Phosphorylated STAT3,p-STAT3)、通路下游炎症相关蛋白IL-1β、IL-6、凋亡靶蛋白-半胱氨酸天冬氨酸酶-3(Cysteyl aspartate specific protease,Caspase-3)表达水平。结果与正常对照组比较,模型组大鼠癫痫发作频率及发作等级评分升高,海马神经元损伤及凋亡加重,海马组织中炎性因子表达、小胶质细胞活化数目、p-JAK2与活化小胶质细胞阳性共表达数目均升高,JAK2/STAT3磷酸化活化蛋白及其介导的下游炎症及凋亡途径相关蛋白表达水平升高(P<0.05);Ast-exos可改善癫痫发作症状,缓解癫痫大鼠海马神经元损伤及凋亡,抑制JAK2/STAT3磷酸化活化及其介导的下游炎症及凋亡途径激活,降低p-JAK2在活化小胶质细胞中表达,降低小胶质细胞活化数目及炎症因子表达水平(P<0.05);JAK2通路激活剂-Coumermycin A1可逆转Ast-exos的上述作用(P<0.05)。结论Ast-exos可能通过抑制JAK2/STAT3通路活化来缓解癫痫大鼠小胶质细胞活化介导的神经炎性损伤及凋亡。

Objective To explore the effect of astrocyte exosomes(Ast-exos)on the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)pathway in epileptic rats,and its effect on the activation of microglia.Methods Male SD rats were randomly divided into normal control group,model group,Ast-exos group(tail vein injection of Ast-exos,0.5 mg/kg),Coumermycin A1 group(JAK2 pathway activator,4.0 mg/kg),and Ast-exos+Coumermycin A1 group,with 10 rats in each group.Pentylenetetrazol(35 mg/kg)was injected intraperitoneally to establish a rat epilepsy model,and each group was evaluated after 14 days of intervention.The behaviors of rats were observed and the symptoms of epilepsy were scored.The hippocampus tissue was taken after the rats were killed,and the number of activated microglia was measured with immunohistochemical method.The levels of inflammatory factors in hippocampus including interleukin(IL)-1β,IL-6 and neurotransmitterγ-aminobutyric acid were estimated with ELISA method.The neuron damage and apoptosis in hippocampus were assessed with Nissl and TUNEL stainings.The expression of JAK2 phosphorylated protein(p-JAK2)in activated microglia was detected with immunofluorescence co-localization method.The expression of phosphorylated proteins in JAK2/STAT3 pathway including p-JAK2 and p-STAT2 and inflammation-related proteins such as IL-1β,IL-6 and apoptosis target protein-cysteine aspartase 3(Caspase-3)were detected with Western blot method.Results Compared with the control group,the epileptic seizure frequency and seizure grade scores of the model group increased and the hippocampal neuron damage and apoptosis were aggravated.The expression levels of inflammatory factors and the number of activated microglia with or without expression of p-JAK2 increased in hippocampus.The levels of JAK2/STAT3 phosphorylation and the downstream inflammation and apoptosis pathway-related protein expression were increased(P<0.05).Ast-exos could improve the symptoms of epileptic seizures and relieve the damage and apoptosis of hippocampal neurons in epileptic rats,inhibit JAK2/STAT3 phosphorylation and the activation of downstream inflammation and apoptosis pathways,decrease the expression of p-JAK2 in activated microglia,and reduce the number of activated microglia and the expression of inflammatory factors(P<0.05).The JAK2 pathway activator(Coumermycin A1)could reverse the above effects of Ast-exos(P<0.05).Conclusion Ast-exos may inhibit the activation of JAK2/STAT3 pathway and alleviate the neuroinflammatory injury and apoptosis mediated by the activation of microglia in epileptic rats.