补肾活血方通过NOX2/NLRP3/CASP1通路改善PD神经炎症的机制研究

Mechanistic Study on the Improvement of Parkinson’s Disease Neuroinflammation by Kidney-tonifying and Blood-activating Recipe Via the NOX2/NLRP3/CASP1 Pathway

关键词:  补肾活血方  帕金森病  黑质纹状体  多巴胺能神经元（DA）  运动障碍

Key words:  Kidney-tonifying and blood-activating Recipe  Parkinson’s disease  Substantia nigra-striatum  Dopaminergic neuron  Dyskinesia

摘要:

摘要：目的 探究补肾活血方苁蓉舒痉颗粒改善帕金森病(Parkinson’s disease，PD)模型小鼠黑质-纹状体神经炎症的作用机制。方法 采用腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine，MPTP)制备PD模型小鼠。实验分组为正常组，模型组，药物组。在造模第7 d后、给药治疗第7 d、第14 d后，对小鼠进行行为学测试；免疫组化检测小鼠黑质-纹状体酪氨酸羟化酶(Tyrosine hydroxylase，TH)阳性细胞的表达；应用透射电镜观察小鼠黑质-纹状体神经元细胞的线粒体形态；流式细胞术检测细胞活性氧(Reactive oxygen species，ROS)的变化；Western blot法检测小鼠黑质-纹状体中NLRP3、NOX2、Caspase-1(CASP1)、IL-1β、TNF-α蛋白的表达。结果 行为学研究发现，造模7 d后，模型组金属网悬挂、核心区域运动时间明显减少(P<0.001)，横木通行时间明显增加(P<0.001)。给药治疗7 d、14 d后，药物组悬挂、核心区域运动时间明显增加(P<0.001)，横木通行时间明显减少(P<0.001)。免疫组化检测发现，模型组小鼠黑质TH阳性细胞数明显减少；药物组小鼠黑质TH阳性细胞数明显增多(P<0.001)。透射电镜观察发现，模型组黑质-纹状体神经元细胞线粒体出现肿胀、空泡样，嵴断裂甚至消失；药物组的线粒体数量，形态有所改善。流式细胞术检测发现，模型组细胞内ROS的水平明显升高，药物组细胞内活性氧水平明显有所降低(P<0.001)。Western blot检测发现，模型组NLRP3、NOX2、CASP1、IL-1β、TNF-α蛋白表达量明显升高；药物组蛋白表达量明显降低(P<0.001)。结论 补肾活血方可以改善PD模型小鼠大脑中黑质-纹状体的氧化应激和神经炎症反应，这种保护作用可能是通过调节NOX2/NLRP3/CASP1通路以实现的。

Abstract:

ABSTRACT: OBJECTIVE To explore the mechanism of Kidney-tonifying and blood-activating Recipe (Congrong Shujing Granules) in ameliorating neuroinflammation in the substantia nigra-striatum of parkinson's disease (PD) model mice. METHODS PD model mice were established by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP). The experimental group was divided into normal group, model group and treatment group. Behavioral tests were conducted on mice after the 7th day of modeling, as well as on the 7th and 14th days of treatment. The expression of tyrosine hydroxylase (TH) positive cells in the substantia nigra-striatum of mice was detected by immunohistochemistry(IHC). Mitochondrial morphology in substantia nigra-striatum neurons was observed using transmission electron microscopy. Changes in intracellular reactive oxygen species (ROS) were analyzed by flow cytometry.Western blot was performed to detect the expression of NLRP3, NOX2, Caspase-1, IL-1β, and TNF-α proteins in the substantia nigra-striatum of mice. RESULTS The behavioral study revealed that after 7 days after modeling, the duration of metal mesh hanging and movement in the core area in the model group was significantly reduced (P<0.001), while the time required to traverse the cross bar was significantly increased (P<0.001). Following 7 and 14 days of treatment, the duration of hanging and movement in the core area in the treatment group was significantly increased (P<0.001), and the time required to traverse the cross bar was significantly reduced (P<0.001). IHC analysis showed a significant reduction in the number of TH-positive cells in the substantia nigra of model group mice, while the treatment group showed a significant increase in TH-positive cells(P<0.001). Transmission electron microscopy revealed that mitochondria of substantia nigra-striatum neurons of model group appeared swollen, vacuolated, and exhibited broken or absent cristae; these abnormalities were improved in the treatment group. Flow cytometry showed that intracellular ROS levels in the model group were significantly increased, but significantly decreased in the treatment group (P<0.001). Western blot showed that the expressions levels of NLRP3, NOX2, casp1, IL-1 β and TNF-α proteins in the model group were significantly increased, while their levels were significantly reduced in the treatment group (P<0.001). CONCLUSION The Kidney-tonifying and blood-activating Recipe (Congrong Shujing Granules) can ameliorate oxidative stress and neuroinflammatory responses in the substantia nigra-striatum of PD model mice, and This protective effect might be mediated via regulation of the NOX2/NLRP3/CASP1 signaling pathway.