Roscovitine挽救帕金森病小鼠相关脑区神经元丢失和神经炎症

Roscovitine rescuing neuronal loss and neuroinflammation in brain regions associated with Parkinson’s disease mice

目的探讨细胞周期依赖性激酶(Cdk)5抑制剂Roscovitine对1-甲基4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导的帕金森病(PD)模型小鼠相关脑区病理变化的影响及可能机制。方法经MPP~+处理的细胞,采用Western blotting检测Roscovitine对P25、Cdk5蛋白的相对表达水平的影响;ELISA法检测Roscovitine对多巴胺的释放的影响。将15只雄性C57BL/6 N小鼠随机分为3组,分别为PBS组、MPTP组、MPTP+Roscovitine组,每组5只。MPTP组与MPTP+Roscovitine组从第3天起连续7 d腹腔注射25 mg/(kg·d)MPTP制备PD模型小鼠,MPTP+Roscovitine组连续10 d腹腔注射10 mg/(kg·d)Roscovitine,PBS组给予等容量的PBS。末次给药24 h后,采用步态分析、旷场实验、转棒实验等检测Roscovitine对PD模型小鼠行为学的影响;采用免疫组织化学法检测Roscovitine对PD模型小鼠黑质、纹状体酪氨酸氢化酶(TH)及PD相关脑区的神经元、神经胶质细胞、神经炎症等相关指标表达的影响。结果Western blotting和ELISA结果显示,经MPP~+处理的细胞P25、Cdk5蛋白表达水平升高,多巴胺释放量相对降低(P<0.01),Roscovitine可降低P25、Cdk5蛋白表达水平(P<0.05)、增加多巴胺释放量(P<0.05);与PBS组相比,MPTP组PD模型小鼠存在运动功能障碍,且黑质、纹状体内TH+细胞数下降(P<0.01),PD相关脑区内胶质纤维酸性蛋白(GFAP)、Iba1、核因子κB(NF-κB)抑制物激酶α(IKKα)、p-IKK阳性细胞数升高(P<0.05);而Roscovitine干预显著改善了运动能力(P<0.01),增加TH(P<0.01)、降低GFAP、Iba1、IKKα、p-IKK(P<0.05)的表达。结论Roscovitine可减少MPTP模型小鼠PD相关脑区的多巴胺能神经元丢失和胶质细胞激活,抑制NF-κB信号通路激活,从而发挥神经保护作用。

Objective To investigate the effect and possible mechanism of cell cycle-dependent kinase(Cdk)5 inhibitor Roscovitine on 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced pathological changes in brain regions associated with Parkinson's disease(PD) model mice. Methods The effect of Roscovitine on the relative expression levels of P25 and Cdk5 proteins was detected by Western blotting in MPP~+-treated cells. The ELISA method detected the effect of Roscovitine on the release of dopamine. Fifteen male C57BL/6N mice were randomly divided into 3 groups, PBS group, MPTP group, and MPTP + Roscovitine group, 5 mice in each group. PD model mice were prepared by intraperitoneal injection of 25 mg/(kg·d) MPTP for 7 consecutive days from the 3rd day in the MPTP + Roscovitine group, and 10 mg/(kg·d) Roscovitine was injected intraperitoneally for 10 days in the MPTP + Roscovitine group, and the PBS group was given the same volume of PBS. Twenty-four hours after the last dose, the effect of Roscovitine on the behavior of PD model mice was detected by gait analysis, open field experiment, and rod rotation experiment. The effect of Roscovitineon the expression of neurons, glial cells, neuroinflammation and other related indexes in PD model mice such as nigrostriatal tyrosine hydrogenase(TH) and PD-related brain regions was detected by immunohistochemistry. Results Western blotting and ELISA showed that the expression levels of P25 and Cdk5 proteins and the release of dopamine decreased relatively low in MPP+ treated cells(P<0.01), Roscovitine could reduce the expression level of P25 and Cdk5 protein(P<0.05), increased the release of dopamine(P< 0.05);Compared with the PBS group, the PD model mice in the MPTP group had motor dysfunction and decreased the number of TH~+ cells in the substantia nigra and striatum(P< 0.01). The number of positive cells in PD-related brain regions increased in glial fibrillary acidic protein(GFAP), Iba1, inhibitor of nuclear factor kappa B(NF-κB) kinase subunit α(IKKα), and p-IKK(P<0.05), and Roscovitine intervention significantly improved exercise capacity(P<0.01), increased TH(P<0.01), reduced GFAP, Iba1, IKKα, p-IKK(P< 0.05). Conclusion Roscovitine can reduce the loss of dopaminergic neurons and glial cell activation in PD-related brain regions of MPTP model mice, and inhibit the activation of NF-κB signaling pathway, thereby exerting neuroprotective effects.