沉默信息调节因子1抑制核因子κB通路调控癫痫大鼠小胶质细胞活化的机制研究

目的探讨沉默信息调节因子1(SIRT1)在癫痫中对小胶质细胞及炎症因子的影响及作用机制.方法建立氯化锂-匹罗卡品致痫大鼠模型,免疫组化观察各组(对照组和癫痫组)大鼠脑组织内小胶质细胞活化;采用脂多糖(LPS)建立小胶质细胞活化模型,构建pcDNA-SIRT1和si-SIRT1载体,转染至大鼠小胶质细胞中.定量即时聚合酶链反应(qRT-PCR)测定大鼠海马组织及小胶质细胞中SIRT1表达,Western blot检测小胶质细胞的活化标志物Iba1和核因子-κB(NF-κB)通路相关蛋白(p65和IκBα)的蛋白表达水平,酶联免疫吸附测定(ELISA)检测促炎因子白细胞介素1β(IL-1β)、白细胞介素6(IL-6)和肿瘤坏死因子(TNF-α)]的表达水平.结果与Control组比较,癫痫大鼠海马组织中SIRT1 mRNA表达量显著降低(P<0.05),Iba1蛋白表达量显著增加(P<0.05),且对照组CA1、CA2区Iba1阳性细胞数分别为(180±21)、(190±18)/mm2,癫痫组CA1、CA2区Iba1阳性细胞数分别为(412±35)、(470±37)/mm2,两组比较差异均有统计学意义(均P<0.05).同样,与Mock组比较,LPS活化小胶质细胞中SIRT1表达水平显著降低,Iba1蛋白表达水平显著增加,差异均有统计学意义(均P<0.05).转染pcDNA-SIRT1及si-SIRT1至LPS活化的小胶质细胞中,LPS+pcDNA-SIRT1组IL-1β(50.0±3.3)ng/L]、IL-6(55.0±3.2)ng/L]和TNF-α(56.1±3.0)ng/L]表达水平显著低于LPS组和LPS+pcDNA-NC组(均P<0.05);LPS+si-SIRT1组中IL-1β(98.2±4.3)ng/L]、IL-6(108.1±4.5)ng/L]和TNF-α(124.5±4.1)ng/L]表达水平显著高于LPS组和LPS+si-NC组(均P<0.05);同时LPS+pcDNA-SIRT1组NF-κB p65的表达水平显著低于LPS组和LPS+pcDNA-NC组(均P<0.05),IκBα的表达水平显著高于LPS组和LPS+pcDNA-NC组(均P<0.05);而LPS+si-SIRT1组NF-κB p65的蛋白表达水平显著高于LPS组和LPS+si-NC组(均P<0.05),IκBα的表达水平显著低于LPS组和LPS+si-NC组(均P<0.05).加入NF-κB通路激活剂Aconine后,与LPS+pcDNA-SIRT1组比较,LPS+pcDNA-SIRT1+Aconine组大鼠中Iba1表达水平显著升高(P<0.05);LPS+pcDNA-SIRT1+Aconine组大鼠中IL-1β(72.2±4.3)ng/L]、IL-6(80.1±4.0)ng/L]和TNF-α(87.2±4.5)ng/L]表达水平显著高于LPS+pcDNA-SIRT1组的(50.1±2.3)]ng/L、(55.0±3.4)]ng/L和(56.3±4.9)ng/L](均P<0.05).结论SIRT1可能通过抑制NF-κB通路活性来抑制癫痫大鼠小胶质细胞的作用.

Mechanism of SIRT1 regulating the activation of microglia in status epilepticus via inhibiting the NF-κB pathway

Objective To investigate the effect of silent information regulator 1(SIRT1)on microglia in Status epilepticus(SE)and its mechanism.Methods The rat SE model was induced by lithiumpilocarpine injection.The activation of microglia in brain tissues of control and SE rats was observed by immunohistochemistry.Microglial activation model was established by lipopolysaccharide(LPS).PcDNA-SIRT1 and si-SIRT1 vectors were constructed and transfected into rat microglia cells.The expression of SIRT1 in the rat hippocampus and microglia was determined by qRT-PCR.The expression levels of the activation markers of microglia(Iba1)and NF-κB p65 and IκBαwere detected by Western blot,and levels of the inflammatory factors(IL-1β,IL-6 and TNF-α)were detected by ELISA.Results Comparing with the control group,the mRNA expression of SIRT1 in the hippocampus of SE rats was significantly decreased,while the protein expression of Iba1 was significantly increased.The number of Iba1 positive cells in CA1 and CA2 regions in the control group was(180±21)/mm2 and(190±18)/mm2,as(412±35)/mm2 and(470±37)/mm2 in the SE group.The difference in Iba1 positive cells in CA1 and CA2 regions between the two groups was statistically significant(P<0.05).Similarly,the expression level of SIRT1 in LPS activated microglia was significantly decreased,while the expression level of Iba1 protein was significantly increased,with statistical significance(P<0.05).pcDNA-SIRT1 and si-SIRT1 were transfected into the post-microglia cells activated by LPS.The levels of IL-1β(50.0±3.3)ng/L,IL-6(55.0±3.2)ng/L and TNF-α(56.1±3.0)ng/L in LPS+pcDNA-SIRT1 were significantly lower than those in LPS group and LPS+pcDNA-NC group(P<0.05).The levels of IL-1β(98.2±4.3)ng/L,IL-6(108.1±4.5)ng/L and TNF-α(124.5±4.1)ng/L in LPS+si-SIRT1 group were significantly higher than those in LPS group and LPS+si-NC group(P<0.05).Besides,the NF-κB p65 expression in LPS+pcDNASIRT1 group was significantly lower than that in LPS group and LPS+pcDNA-NC group,while the IκBαexpression was higher than those two groups(P<0.05).However,the NF-κB p65 expression in LPS+siSIRT1 group was significantly higher than that in LPS group and LPS+si-NC group,while the IκBαexpression was lower than those two groups(P<0.05).After giving the NF-κB pathway activator(Aconine),compared with the LPS+pcDNA-SIRT1 group,the expression of Iba1 in the LPS+pcDNA-SIRT1+Aconine group was significantly increased(P<0.05).The expression levels of IL-1βwere significantly higher in the LPS+pcDNASIRT1+Aconine group(72.2±4.3)ng/L,IL-6(80.1±4.0)ng/L,and TNF-α(87.2±4.5)ng/L]than that in the LPS+pcDNA-SIRT1 groupIL-1β(50.1±2.3)ng/L,IL-6(55.0±3.4)ng/L,and TNF-α(56.3±4.9)ng/L](P<0.05).Conclusions SIRT1 could inhibit the activation of microglia in SE rats by inhibiting the NF-κB pathway。