摘要
目的 探讨氟西汀与核因子-E2相关因子2(Nrf2)激活剂联合应用对蛛网膜下腔出血(SAH)早期大鼠小胶质细胞炎症反应的影响及其可能作用机制.方法 选取30只雄性SD大鼠采用血管内穿刺法制备SAH模型,随机分为假手术组、SAH组、氟西汀组、Nrf2激活剂(CDDO-EA)组、氟西汀+CDDO-EA组,每组n=6只.处理1 d后检测各组大鼠脑积水含量、Garcia评分.qRT-PCR法检测大脑皮质中iNOS、COX2 mRNA表达;ELISA法检测脑脊液中TNF-α、IL-6、IL-18水平;TUNEL法检测神经元凋亡情况;Western blot法检测Nrf2蛋白和小胶质细胞标记物CD11b蛋白表达、凋亡蛋白及Toll样受体4(TLR4)/P38丝裂原活化蛋白激酶(P38 MAPK)信号通路相关蛋白表达.结果 与假手术组比较,SAH组Garcia评分降低,脑组织含水量、凋亡指数升高,CD11b、Bax、Cleaved-caspase-3、iNOS、COX2、TLR4、p-P38 MAPK表达量及TNF-α、IL-6、IL-18水平升高,Nrf2、Bcl-2表达量降低(均P<0.05);与SAH组比较,氟西汀组、CDDO-EA组、氟西汀+CDDO-EA组的Garcia评分升高,脑组织含水量、凋亡指数降低,CD11b、iNOS、COX2、Bax、Cleaved-caspase-3、TLR4、p-P38 MAPK表达量及TNF-α、IL-6、IL-18水平降低,Nrf2、Bcl-2表达量升高,且氟西汀+CDDO-EA组变化幅度优于氟西汀组和CDDO-EA组(均P<0.05).结论 氟西汀与Nrf2激活剂联合治疗可抑制SAH后小胶质细胞活化、炎症反应,抑制神经元凋亡,减轻神经功能损伤,其机制可能与抑制TLR4/P38 MAPK信号通路活化有关.
Abstract
Aim To explore the effects of fluoxetine combined with nuclear factor E2 related factor 2(Nrf2)activators on the inflammatory response of microglia in early subarachnoid hemorrhage(SAH)rats and its possible mechanisms of action.Methods Thirty male SD rats were selected to prepare SAH models using intravascular puncture.They were randomly divided into a sham surgery group,a SAH group,a fluoxetine group,a Nrf2 activator(CDDO-EA)group,and a fluoxetine+CDDO-EA group,with 6 rats in each group.After one day of treatment,the hydrocephalus content and Garcia score of each group were measured.qRT-PCR method was used to detect the expression levels of iNOS and COX2 mRNA in the cerebral cortex.The levels of tumor necrosis factor(TNF-α),interleukin-6(IL-6)and interleukin-18(IL-18)in cerebrospinal fluid were detected by ELISA.Neuron apoptosis was detected by TUNEL method.The expression of Nrf2 protein,CD11b protein,apoptotic protein,and Toll like receptor 4(TLR4)/p38 mitogen activated protein kinase(p38 MAPK)signaling pathway related proteins was detected by Western blot.Results Compared with the sham surgery group,in the SAH group,the Garcia score decreased,brain tissue water content and apoptosis index were increased,CD11b,Bax,Cleared-caspase-3,iNOS,COX2,TLR4,p-p38 MAPK expression were increased,and the levels of TNF-α,IL-6,IL-18 were increased,while the expression levels of Nrf2 and Bcl-2 was decreased(P<0.05).Compared with the SAH group,the Garcia score in the fluoxetine group,CDDO-EA group,and fluoxetine+CDDO-EA group were increased,brain tissue water content and apoptosis index were decreased,the expression of CD11b,iNOS,COX2,Bax,Cleared-caspase-3,TLR4,p-p38 MAPK and the levels of TNF-α,IL-6,IL-18 were decreased,while the expression levels of Nrf2 and Bcl-2 were increased.The changes in the fluoxetine+CDDO-EA group were more significant than those in the fluoxetine and CDDO-EA groups(P<0.05).Conclusion Fluoxetine combined with Nrf2 activator could inhibit SAH microglial activation,inflammatory response,alleviate neurological damage,and inhibit neuronal apoptosis.Its mechanism of action may be related to the inhibition of TLR4/p38 MAPK signaling pathway activation.
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