沉默信息调节因子1与Toll样受体4信号通路在1-甲基-4-苯基-吡啶离子帕金森病细胞模型炎症反应中的作用

Role of the silent information regulator 1 - Toll-like receptor 4 signaling pathway in inflammatory response in a cell model of Parkinson disease induced by 1-methyl-4-phenyl pyridine ion

摘要:

目的 探究沉默信息调节因子1（Sirt1）与Toll样受体4（TLR4）信号通路在1-甲基-4-苯基-吡啶离子（MPP⁺）帕金森病（PD）细胞模型炎症反应中的作用。方法 使用MPP⁺作用于SH-SY5Y细胞进行干预以构建PD细胞模型，使用白藜芦醇（RV）和EX527干预细胞模型中Sirt1的表达水平。将SH-SY5Y细胞分为对照组（control组）、模型组（MPP⁺组）、激活剂组（RV+MPP⁺组）、抑制剂组（EX527+MPP⁺组）置于高糖培养基中培养。采用细胞计数试剂盒-8（CCK-8）进行MPP⁺、RV和EX527的细胞毒性检测，最终选用MPP⁺（0.75 mmol/L）、RV（20 μmol/L）、EX527（80 μmol/L）作为干预浓度，均培养48 h。采用蛋白质印迹法检测酪氨酸羟化酶（TH）、Sirt1、TLR4炎症相关蛋白的表达情况。采用酶联免疫吸附分析（ELISA）检测细胞上清液中白细胞介素（IL）-1β、IL-6、肿瘤坏死因子-α（TNF-α）水平。采用免疫荧光检测TH、Sirt1、TLR4以及相关炎症因子在细胞上的表达情况。结果 与control组相比，MPP⁺组TH、Sirt1表达降低，TLR4表达增高（P<0.05）；与MPP⁺组相比，RV+MPP⁺组TH、Sirt1表达增高，TLR4表达降低（P<0.05）；EX527+MPP⁺组TH、Sirt1表达较control组和RV+MPP⁺组降低，TLR4表达增高（P<0.05）。ELISA结果显示，MPP⁺组和EX527+MPP⁺组IL-1β、IL-6、TNF-α水平较control组和RV+MPP⁺组显著增高（P<0.05），与MPP⁺组比较，EX527+MPP⁺组IL-1β、IL-6、TNF-α表达水平增高（P<0.05）。免疫荧光结果显示，与control组相比，MPP⁺组TH和Sirt1的表达水平减少，TLR4 及炎症因子IL-1β、TNF-α的表达增高（P<0.01）；与MPP⁺组相比，RV+MPP⁺组TH、Sirt1的表达增高，TLR4、IL-1β及TNF-α的表达水平减少（P<0.01）；与MPP⁺组相比，EX527+MPP⁺组TH、Sirt1的表达水平降低，TLR4及炎症因子IL-1β、TNF-α的表达增高（P<0.01）。结论 Sirt1-TLR4信号通路与PD神经炎症反应过程有一定的相关性，可能在疾病的发生、发展中发挥了作用。

Abstract:

Objective To investigate the role of the silent information regulator 1 （Sirt1） - Toll-like receptor 4 （TLR4） signaling pathway in inflammatory response in a cell model of Parkinson disease （PD） induced by 1-methyl-4-phenyl pyridine ion （MPP⁺）.Methods SH-SY5Y cells were treated with MPP⁺ to establish a cell model of PD， and resveratrol （RV） and EX527 were used to modulate the expression of Sirt1 in the cell model. SH-SY5Y cells were divided into control group， model group （MPP⁺ group）， activator group （RV+MPP⁺ group）， and inhibitor group （EX527+MPP⁺ group）， and the cells were cultured in a high-glucose medium. CCK-8 assay was used to assess the cytotoxicity of MPP⁺， RV， and EX527， and finally MPP⁺ （0.75 mmol/L）， RV （20 μmol/L）， and EX527 （80 μmol/L） were selected for 48 hours of intervention. Western blotting was used to measure the expression levels of tyrosine hydroxylase （TH）， Sirt1， and the inflammation-related protein TLR4； ELISA was used to measure the levels of interleukin-1β （IL-1β）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） in cell supernatant； immunofluorescence assay was used to measure the expression of TH， Sirt1， TLR4， and related inflammatory factors in cells.Results Compared with the control group， the MPP⁺ group had significant reductions in the expression levels of TH and Sirt1 and a significant increase in the expression level of TLR4 （P<0.05）； compared with the MPP⁺ group， the RV+MPP⁺ group had significant increases in the expression levels of TH and Sirt1 and a significant reduction in the expression level of TLR4 （P<0.05）； compared with the control group and the RV+MPP⁺ group， the EX527+MPP⁺ group had significant reductions in the expression levels of TH and Sirt1 and a significant increase in the expression level of TLR4 （P<0.05）. ELISA showed that compared with the control group and the RV+MPP⁺ group， the MPP⁺ group and the EX527+MPP⁺ group had significant increases in the levels of IL-1β， IL-6， and TNF-α （P<0.05）， and compared with the MPP⁺ group， the EX527+MPP⁺ group had significant increases in the levels of IL-1β， IL-6， and TNF-α （P<0.05）. Immunofluorescence assay showed that compared with the control group， the MPP⁺ group had significant reductions in the expression levels of TH and Sirt1 and significant increases in the expression levels of TLR4 and the inflammatory factors IL-1β and TNF-α （P<0.01）； compared with the MPP⁺ group， the RV+MPP⁺ group had significant increases in the expression levels of TH and Sirt1 and significant reductions in the expression levels of TLR4， IL-1β， and TNF-α （P<0.01）； compared with the MPP⁺ group， the EX527+MPP⁺ group had significant reductions in the expression levels of TH and Sirt1 and significant increases in the expression levels of TLR4 and the inflammatory factors IL-1β and TNF-α （P<0.01）.Conclusions The Sirt1-TLR4 signaling pathway is significantly associated with the process of neuroinflammation in PD and may play a role in the development and progression of the disease.