Caspase-1通过炎症小体激活MPTP诱导的PD小鼠神经炎症反应的相关研究

Role of NLRP3-dependent activation of caspase-1 in mediating neuroinflammatory response in a mouse model of MPTP-induced Parkinson＇s disease

目的NOD样受体热蛋白结构域3（NLRP3）-含半胱氨酸的天冬氨酸蛋白水解酶1（caspase-1）炎症小体在1-甲基，4-苯基-1，2，3，6-四氢吡啶（MPTP）诱导的帕金森病（PD）小鼠神经损伤中的作用及机制。方法将小鼠随机分为模型组和对照组，模型组腹腔注射MPTP溶液（30 mg·kg－1·d－1），制备PD模型，对照组给予等量生理盐水；爬杆试验和悬挂试验观察小鼠行为学变化；免疫印迹试验（Western blot）检测小鼠中脑组织中炎症小体的含量。将体外培养的人源性神经母细胞SH-SY5Y随机分为对照组、无义组、siRNA NLRP3组、1-甲基-4-苯基吡啶（MPP＋）组、siRNA＋MPP＋组；采用脂质体转染的方法，将siRNA-NLRP3转染至人源性神经母细胞SH-SY5Y中，加入MPP＋孵育细胞48 h，体外观察NLRP3炎症小体对神经细胞的保护作用。噻唑蓝法（MTT）检测细胞活力的变化，流式细胞仪检测细胞的凋亡率，Western blot检测NLRP3、B细胞淋巴瘤/白血病-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、活化的含半胱氨酸的天冬氨酸蛋白水解酶3（cleaved caspase-3）的蛋白质水平。结果与对照组相比，模型组小鼠从木杆顶端爬至底端所耗时间显著增加（P〈0.05），悬挂得分显著降低（P〈0.05），中脑黑质部中NLRP3炎症小体、IL-1β、Pro-IL-1β以及caspase-1的表达量显著增加（P〈0.05）。与MPP＋组相比，体外转染siRNA-NLRP3的可显著降低MPP＋介导的SH-SY5Y细胞中NLRP3蛋白的水平（P〈0.05），提高细胞的活力（P〈0.05），抑制其凋亡（P〈0.05），促进Bcl-2蛋白表达（P〈0.05），显著抑制cleaved caspase-3、Bax的蛋白表达。结论NLRP3-caspase-1炎症小体在PD神经损伤过程中发挥重要作用；体外抑制NLRP3表达可显著增强SH-SY5Y细胞的活力，抑制其凋亡，可能通过调控线粒体凋亡信号通路对神经细胞发挥保护作用，为PD的临床治疗提供新的药物靶点。

ObjectiveTo understand the role of NOD-like receptor pyrin domain containing 3 （NLRP3）-dependent activation of cysteinyl aspartate specific proteinase 1 （caspase-1） in mediating the neuroinflammatory response in mice with MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine）-induced Parkinson′s disease（PD） and to investigate the underlying mechanism.MethodsMale C57BL/6 mice were randomly divided into two groups： experimental and control groups.MPTP solution （30 mg·kg-1·d-1） was given to mice through intraperitoneal injection to prepare the PD model and equal amount of saline was used to set up the control group.Changes in mouse behavior were observed through pole climbing and suspension experiment.Expression of inflammasome in the midbrain of mice was detected by Western blot.SH-SY5Y cells, a human neuroblastoma cell line, were cultured in vitro and randomly divided into five groups including control, nonsense, siRNA-NLRP3, MPP＋ （1-methyl-4-phenylpyridinium） and siRNA＋ MPP＋ groups.siRNA-NLRP3 was transfected into SH-SY5Y cells using Lipofectamine 2000 to silence the expression of NLRP3 gene.Then the transfected cells were incubated with MPP＋ for 48 h to observe the protective effect of NLRP3 on nerve cells.MTT assay and flow cytometry were performed to measure the viability and the apoptosis rate of SH-SY5Y cells, respectively.Western blot was used to detect the levels of NLRP3, B-cell lymphoma 2 （Bcl-2）, Bcl-2-associated X protein （Bax） and cleaved caspase-3.ResultsCompared with the control group, the mice in the experimental group spent more time climbing from the top to the bottom of the pole （P〈0.05）, got a lower score on suspension experiment （P〈0.05） and showed enhanced expression of NLRP3, IL-1β, Pro-IL-1β and caspase-1 in the substantia nigra （P〈0.05）. Compared with the MPP＋ group, the siRNA＋ MPP＋ group showed significantly inhibited expression of NLRP3, cleaved caspase-3 and Bax （P〈0.05）, enhanced cell viability （P〈0.05）, suppressed cell apoptosis （P〈0.05） and promoted expression of Bcl-2 （P〈0.05）.ConclusionNLRP3-dependent activation of caspase-1 plays an important role in the nerve injury in PD.Inhibition of NLRP3 expression in vitro can significantly enhance the vitality and inhibit the apoptosis of SH-SY5Y cells, which may have a protective effect on nerve cells through regulating mitochondrial apoptosis signaling pathway and provide a new drug target for the treatment of PD.