DYRK1A调控NLRP3激活在帕金森病模型中的研究

Dual specificity tyrosine-regulated kinase1a (DYRK1A) regulates NOD-like receptor thermal protein domain associated protein 3 (NLRP3) activation in a Parkinson′s disease model

目的 通过在帕金森病模型中分析双特异性酪氨酸调控激酶1A(DYRK1A)与NOD样受体热蛋白结构域相关蛋白3(NLRP3)激活的关系,探讨帕金森病中神经炎症的治疗靶点。方法 (1)采用MPP^(+)处理BV2细胞(小鼠小胶质细胞)后,使用免疫印迹法(Western blot)检测DYRK1A及NLRP3蛋白的表达水平;加用DYRK1A抑制剂(Harmine)处理帕金森细胞模型,使用CCK8法检测小胶质细胞活性,免疫印迹法检测NLRP3蛋白表达水平。(2)MPTP慢性PD动物模型中,腹腔注射DYRK1A抑制剂(Harmine),免疫组织荧光观察小鼠中脑黑质区小胶质细胞数目,免疫印迹法检测小鼠中脑黑质区NLRP3蛋白表达水平。结果 (1)不同浓度MPP^(+)处理小胶质细胞后,DYRK1A及NLRP3的蛋白水平较对照组均有升高(P<0.05);Harmine(浓度为10、15μmol/L)时可改善MPP^(+)对BV2细胞活性的损伤(P<0.05);相比MPP^(+)组,Harmine+MPP^(+)组NLRP3蛋白水平明显下降(P<0.05)。(2)与对照组相比,模型组小鼠中脑黑质的小胶质细胞数量增多;与模型组相比,模型中浓度抑制剂组小鼠中脑黑质的小胶质细胞数量减少;模型组小鼠中脑黑质区NLRP3的蛋白水平较对照组升高,与模型组比较,模型中浓度及高浓度抑制剂组小鼠中脑黑质中NLRP3蛋白表达下降明显(P<0.05)。结论 抑制DYRK1A可减轻帕金森病模型中NLRP3炎症蛋白的激活,为帕金森病的治疗和研究提供新的思路。

Objective To explore the therapeutic target of neuroinflammation in Parkinson′s disease(PD)by analyzing the relationship between dual specificity tyrosine-regulated kinase1a(DYRK1A)and NOD-like receptor thermal protein domain associated protein 3(NLRP3)activation in PD model.Methods(1)After microglia was treated with MPP^(+),the protein levels of DYRK1A and NLRP3 were detected by Western blot.Parkinson′s cell model was treated with DYRK1A inhibitor(Harmine),microglia activity was detected by CCK8 method,NLRP3 protein level was detected by Western blot.(2)In MPTP chronic PD animal model,intraperitoneal injection of DYRK1A inhibitor(Harmine)was used to observe the number of microglia in the substantia nigra region of mice by immunohist of luorescence,and NLRP3 protein level in the substantia nigra region of mice was detected by Western blot.Results(1)The protein levels of DYRK1A and NLRP3 in microglia treated with different concentrations of MPP^(+)were higher than those in control group(P<0.05).Harmine(10,15μmol/L)can improve the damage of MPP^(+)on BV2 cell activity(P<0.05).Compared with MPP^(+)group,NLRP3 protein level in Harmine+MPP^(+)group was significantly decreased(P<0.05).(2)Compared with the control group,the number of microglia in the substantia nigra of the model group was increased;Compared with model group,the number of microglia in the substantia nigra of mice in the model group was decreased.The level of NLRP3 protein in the substantia nigra of the model group was higher than that of the control group.Compared with the model group,the expression of NLRP3 protein in the substantia nigra of the model group and the high-concentration inhibitor group was significantly decreased(P<0.05).ConclusionInhibition of DYRK1A can reduce the activation of NLRP3 inflammatory protein in PD model,which is helpful for the treatment and research of PD.