DAPK1/MAPK1 Axis Regulates Neuronal Damage in Parkinson's Disease Models
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摘要:
目的 阐明死亡相关蛋白激酶1（death-associated protein kinase 1，DAPK1）/有丝分裂蛋白激酶1（mitogen-activated protein kinase 1，MAPK1）轴在帕金森病（parkinson’ s disease，PD）致病机制中的机制。 方法 采用1-甲基-4-苯基-1，2，3，6-四氢吡啶（1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine，MPTP）和1-甲基-4-苯基吡啶（1-methyl-4-phenylpyridinium，MPP+）构建体内PD模型和体外PD细胞模型，构建si-DAPK1和oe-MAPK1质粒转染至细胞中，RT-qPCR和Western blot用于评估转染效率。通过CCK-8、流式细胞术、试剂盒检测和酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）检测评估细胞活力、细胞凋亡水平、Caspase-3和Caspase-9 活性以及乳酸脱氢酶（lactate dehydrogenase，LDH）、超氧化物变形酶（superoxide dismutase，SOD）、活性氧（reactive oxygen species，ROS）、肿瘤坏死因子α（tumor necrosis factor α，TNF-α）、白介素1β（interleukin 1β，IL-1β）和白介素6（interleukin 6，IL-6）水平。利用数据库STRING进行蛋白互作预测，并采用免疫共沉淀（co-immunoprecipitation，CO-IP）进行验证。 结果 在体内PD模型和体外PD细胞模型中DAPK1和MAPK1的表达水平显著升高。沉默DAPK1减轻MN9D细胞中MPP+诱发细胞凋亡和半胱天冬酶-3（Caspase-3）和Caspase-9水平，降低了LDH、ROS、TNF-α、IL-1β和IL-6的含量，增加了SOD的含量。细胞中DAPK1与MAPK1的相互作用。过表达MAPK1后抑制了沉默DAPK1的作用，促进了MPP+诱发神经炎症。 结论 沉默DAPK1通过与MAPK1相互作用，缓解了MPP+诱发的细胞毒性发挥神经保护作用，是PD治疗的潜在靶点。 Abstract:Objective To elucidate the mechanism of the death-associated protein kinase 1 （DAPK1）/mitogen-activated protein kinase 1 （MAPK1） axis in the pathogenesis of Parkinson's disease （PD）. Methods In vivo and in vitro PD models were established using 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine （MPTP） and 1-methyl-4-phenylpyridinium （MPP+）, respectively. Plasmids encoding si-DAPK1 and oe-MAPK1 were transfected into cells, and transfection efficiency was assessed using RT-qPCR and Western blotting. Cell viability, apoptosis levels, caspase-3 and caspase-9 activities, and levels of lactate dehydrogenase （LDH）, superoxide dismutase （SOD）, reactive oxygen species （ROS）, tumor necrosis factor α （TNF-α）, interleukin 1β （IL-1β）, and interleukin 6 （IL-6） were assessed using CCK-8 assay, flow cytometry, commercial kits, and enzyme-linked immunosorbent assay （ELISA）. Protein-protein interactions were predicted using the STRING database and verified by co-immunoprecipitation （CO-IP）. Results DAPK1 and MAPK1 expression levels were significantly elevated in both in vivo and in vitro PD models. Silencing DAPK1 attenuated MPP+-induced apoptosis and reduced caspase-3 and caspase-9 levels in MN9D cells, decreased LDH, ROS, TNF-α, IL-1β, and IL-6 levels and increased SOD content. Direct interaction between DAPK1 and MAPK1 was demonstrated in cells. Overexpression of MAPK1 reversed the inhibitory effects of DAPK1 silencing promoted MPP+-induced neuroinflammation. Conclusion Silent DAPK1 exerts neuroprotective effects against MPP+-induced cytotoxicity through interaction with MAPK1, representing a potential therapeutic target for PD. -
Key words:
- Parkinson's disease /
- DAPK1 /
- MAPK1 /
- Apoptosis /
- Neuroinflammation
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图 1 DAPK1/MAPK1在体内和体外PD模型中高表达
A：免疫荧光检测各组SNpC区域TH荧光强度（Scale bar=50 μm）；B：RT-qPCR检测PD体内模型中DAPK1、MAPK1的表达水平；C：Western blot检测PD体内模型中DAPK1、MAPK1的蛋白表达水平；D：RT-qPCR检测PD细胞模型中DAPK1、MAPK1的表达水平；E：Western blot检测PD细胞模型中DAPK1、MAPK1的蛋白表达水平；nsP > 0.05；*P < 0.05；***P < 0.001；****P < 0.0001。
Figure 1. High expression of DAPK1/MAPK1 in vivo and in vitro PD models
图 2 沉默DAPK1减轻MN9D细胞中MPP+诱发的神经元损伤
A：RT-qPCR检测转染效率；B：Western blot检测转染效率；C：CCK-8检测细胞活力；D：流式细胞术检测细胞凋亡；E：试剂盒Caspase-3和Caspase-9 活性检测；F：ELISA检测LDH、ROS、SOD、TNF-α、IL-1β和IL-6水平；nsP > 0.05；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 2. DAPK1 silencing attenuates MPP+-induced neuronal injury in MN9D cells
图 3 DAPK1与MAPK1相互作用
A：PPI网络；B：CO-IP检测；C：RT-qPCR检测MAPK1的表达水平；D：Western blot检测MAPK1的蛋白表达水平；nsP > 0.05；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 3. Interaction between DAPK1 and MAPK1
图 4 沉默DAPK1与MAPK1相互作用减轻MN9D细胞中MPP+诱发的神经元损伤
A：RT-qPCR检测转染效率；B：Western blot检测转染效率；C：RT-qPCR检测MAPK1的表达水平；D：Western blot检测MAPK1的蛋白表达水平；E：CCK-8检测细胞活力；F：流式细胞术检测细胞凋亡；G：试剂盒Caspase-3和Caspase-9 活性检测；H：ELISA检测LDH、ROS、SOD、TNF-α、IL-1β和IL-6水平；nsP > 0.05；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 4. Silencing DAPK1 and MAPK1 interaction attenuates MPP+-induced neuronal damage in MN9D cells
表 1 RT-qPCR引物序列
Table 1. RT-qPCR Primer Sequences
基因 引物序列 DAPK1 正向 5'-ACCTCGCCAAGGCAGAAC-3' 反向 5'-AGACGGAGCCACGAACAC-3' MAPK1 正向 5'-ATCTGTGACTTTGGCCTTGC-3' 反向 5'-GAGCCTGTTCAACTTCAATCCT-3' GAPDH 正向 5'-CATCACTGCCACCCAGAAGACTG-3' 反向 5'-ATGCCAGTGAGCTTCCCGTTCAG-3' 
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