双特异性磷酸酶-1对小胶质细胞极化及颞叶癫痫发作的影响

Effect of dual-specificity phosphatase-1 on microglial polarization and seizures in temporal lobe epilepsy

目的探讨双特异性磷酸酶-1(DUSP1)对小胶质细胞极化及颞叶癫痫(EP)发作的影响。方法采用慢病毒转染技术构建DUSP1正常表达组(DUSP1^(normal)组)、DUSP1高表达组(DUSP1^(over)组)、DUSP1低表达组(DUSP1^(low)组)小鼠,每组12只,将这些小鼠又分为DUSP1^(normal)+对照(Con)组、DUSP1^(normal)+EP组、DUSP1^(over)+Con组、DUSP1^(over)+EP组、DUSP1^(low)+Con组和DUSP1^(low)+EP组,每组6只。取DUSP1^(normal)+EP组、DUSP1^(over)+EP组及DUSP1^(low)+EP组小鼠分别腹腔注射氯化锂溶液(125 mg/kg)、硫酸阿托品溶液(1 mg/mL)及盐酸匹罗卡品溶液(50 mg/kg),构建颞叶EP模型。评估EP小鼠注射后2 h内EP发作等级,记录达到Ⅳ级的时间为潜伏期。将EP小鼠处死,取完整脑组织用于免疫荧光检测,取颞叶组织用于炎症细胞因子、DUSP1及小胶质细胞调控蛋白检测。结果与DUSP1^(normal)组比较,DUSP1^(over)组DUSP1/β-actin及DUSP1 mRNA表达水平均升高,DUSP1^(low)组DUSP1/β-actin及DUSP1 mRNA表达水平均降低,差异均有统计学意义(P<0.05)。与DUSP1^(normal)+EP组比较,DUSP1^(over)+EP组小鼠EP发作达Ⅳ级潜伏期明显延长,DUSP1^(low)+EP组小鼠EP发作达Ⅳ级潜伏期明显缩短,差异均有统计学意义(P<0.05)。与DUSP1^(normal)+Con组比较,DUSP1^(normal)+EP组小鼠颞叶组织中M1型小胶质细胞极化标志物CD16、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β及诱导型一氧化氮合酶(iNOS)mRNA水平均升高,炎症细胞因子TNF-α、IL-6水平均升高,IL-10水平降低,DUSP1诱导小胶质细胞极化相关蛋白p-胞外调节蛋白激酶(ERK)1/2、p-c-Jun氨基末端激酶(JNK)和p-p38蛋白表达水平均升高,差异均有统计学意义(P<0.05)。与DUSP1^(normal)+EP组比较,DUSP1^(over)+EP组小鼠颞叶组织中M1型小胶质细胞极化标志物CD16、TNF-α、IL-1β和iNOS mRNA水平均降低,M2型小胶质细胞极化标志物CD206、转化生长因子-β、精氨酸酶1与几丁质酶样蛋白mRNA水平均升高,炎症细胞因子TNF-α、IL-6水平均降低,IL-10水平升高,DUSP1诱导小胶质细胞极化相关蛋白p-ERK1/2、p-JNK和p-p38蛋白表达水平均降低,差异均有统计学意义(P<0.05);与DUSP1^(normal)+EP组比较,DUSP1^(low)+EP组小鼠颞叶组织中M1型小胶质细胞极化标志物TNF-α和iNOS mRNA水平均升高,炎症细胞因子TNF-α、IL-6水平均升高,IL-10水平降低,DUSP1诱导小胶质细胞极化相关蛋白p-ERK1/2、p-JNK和p-p38蛋白表达水平均升高,差异均有统计学意义(P<0.05)。结论氯化锂溶液-盐酸匹罗卡品溶液点燃EP小鼠颞叶组织中DUSP1蛋白表达水平降低,高表达DUSP1基因能够延长小鼠EP发作的潜伏期,其机制可能涉及抑制p-ERK1/2、p-JNK和p-p38蛋白表达来调节小胶质细胞向M2型极化。

Objective To investigate the effect of dual-specificity phosphatase-1(DUSP1)on microglia polarization and seizures in temporal lobe epilepsy(EP).Methods Lentivirus transfection technology was used to construct DUSP1^(normal) expression group(DUSP1^(normal) group),DUSP1 high expression group(DUSP1^(over) group),and DUSP1^(low) expression group(DUSP1^(low) group),with 12 mice in each group.These mice were divided into DUSP1^(normal)+control(Con)group,DUSP1^(normal)+EP group,DUSP1^(over)+Con group,DUSP1^(over)+EP group,DUSP1^(low)+Con group and DUSP1^(low)+EP group,with 6 mice in each group.The mice in the DUSP1^(normal)+EP,DUSP1^(over)+EP,and DUSP1^(low)+EP groups were intraperitoneally injected with lithium chloride solution(125 mg/kg),atropine sulfate solution(1 mg/mL),and pilocarpine hydrochloride solution(50 mg/kg)respectively,to establish a model of EP in the temporal lobe.The EP seizure grade of EP mice within 2 h after injection was evaluated,and the time to reach gradeⅣwas recorded as the latency period.The EP mice were sacrificed,and the intact brain tissues were collected for immunofluorescence detection,and the temporal lobe tissues were collected for the detection of inflammatory cytokines,DUSP1,and microglia regulatory proteins.Results Compared with the DUSP1^(normal) group,the expression levels of DUSP/β-actin and DUSP1 mRNA in the DUSP1^(over) group were significantly increased,while the expression levels of DUSP/β-actin and DUSP1 mRNA in the DUSP1^(low) group were significantly decreased(P<0.05).Compared with the DUSP1^(normal)+EP group,the DUSP1^(over)+EP group had a significantly longer latency to gradeⅣEP attack,and the DUSP1^(low)+EP group had a significantly shorter latency to gradeⅣEP attack(P<0.05).Compared with the DUSP1^(normal)+Con group,the mRNA levels of M1 microglial polarization markers CD16,tumor necrosis factor-α(TNF-α),interleukin(IL)-1βand inducible nitric oxide synthase(iNOS)in the temporal lobe of the DUSP1^(normal)+EP group were increased,the levels of inflammatory cytokines(TNF-αand IL-6)were increased,the level of IL-10 was decreased,and the expression levels of microglia induced polarization protein p-extracellular regulated protein kinase(ERK)1/2,p-c-Jun N-terminal kinase(JNK)and p-p38 were increased,and the differences were statistically significant(P<0.05).Compared with the DUSP1^(normal)+EP group,the mRNA levels of M1 microglial polarization markers CD16,TNF-α,IL-1βand iNOS in the temporal lobe of the DUSP1^(over)+EP group were decreased,the mRNA levels of M2 microglial polarization markers CD206,transforming growth factor-β,arginase 1 and Ym1 were increased,and the levels of TNF-αand IL-6 were decreased,the level of IL-10 was increased,and the expression levels of microglia induced polarization protein p-ERK1/2,p-JNK and p-p38 proteins were decreased,and the differences were statistically significant(P<0.05).Compared with the DUSP1^(normal)+EP group,the mRNA levels of M1 microglia polarization markers TNF-αand iNOS in the temporal lobe tissues of the DUSP1^(low)+EP group were increased,the levels of inflammatory cytokines TNF-αand IL-6 were increased,and the level of IL-10 was decreased.DUSP1 microglia induced polarization protein p-ERK1/2,p-p38 lightning and the JNK protein expression levels were elevated,the differences were statistically significant(P<0.05).Conclusion The expression of DUSP1 protein is decreased in the temporal lobe of lithium-pilocarpine induced EP mice.High expression of DUSP1 gene can prolong the latency of EP in mice,and the mechanism may be related to the inhibition of p-ERK1/2,p-JNK and p-p38 protein expression to regulate the polarization of microglia to M2 type.