氨溴索对α-突触核蛋白寡聚体诱导鼠多巴胺能神经元细胞株MES23.5损伤的预防作用观察

Preventive effect of ambroxol onα-synuclein oligomer-induced injury in mouse dopaminergic neuronal cells MES23.5

目的观察氨溴索(AMB)对α-突触核蛋白(α-syn)寡聚体诱导鼠多巴胺能神经元细胞株MES23.5(帕金森病模型细胞)损伤的预防作用。方法将MES23.5细胞分为A、B、C、D组4组,A组细胞加入二甲基亚砜,B组细胞加入终浓度为5μmol/L的α-syn寡聚体,C组细胞加入终浓度为100μmol/L的AMB,D组细胞先加入终浓度为100μmol/L的AMB预处理12 h后再加入终浓度为5μmol/L的α-syn寡聚体。各组继续培养24 h后,采用ELISA法测算细胞β-葡萄糖脑苷脂酶(GCase)活性和α-syn寡聚体水平,采用MTT法检测细胞增殖能力(以OD_(450)值表示),使用JC-1探针检测线粒体膜电位,采用Western Blotting法检测酪氨酸羟化酶(TH)磷酸化水平。结果B组细胞GCase活性均低于其余三组(P均<0.05),C组细胞GCase活性均高于其余三组(P均<0.05)。B组细胞中α-syn寡聚体水平均高于其余三组(P均<0.05),C组细胞中α-syn寡聚体水平均低于其余三组(P均<0.05)。与A组相比,B组细胞培养24、48、72 h的OD_(450)值均降低(P均<0.05),D组细胞培养72 h的OD_(450)值降低(P<0.05);与B组相比,C、D组细胞培养24、48、72 h的OD_(450)值均升高(P均<0.05);与C组相比,D组细胞培养24、72 h的OD_(450)值均降低(P均<0.05)。B组细胞线粒体膜电位均低于其余三组(P均<0.05),D组细胞线粒体膜电位均低于A、C组(P均<0.05)。B组细胞TH磷酸化水平均低于其余三组(P均<0.05),C组细胞TH磷酸化水平均高于其余三组(P均<0.05)。结论AMB可通过升高GCase活性、降低α-syn寡聚体水平,恢复MES23.5细胞增殖能力、线粒体膜电位、TH磷酸化水平,对α-syn寡聚体诱导的细胞损伤起预防作用。

Objective To observe the preventive effect of ambroxol(AMB)onα-synuclein(α-syn)oligomer-induced damage in mouse dopaminergic neuronal cells MES23.5(a Parkinson's disease cell model).Methods MES23.5 cells were divided into four groups:groups A,B,C,and D.Cells in the group A received dimethyl sulfoxide(DMSO),cells in the group B were treated with a final concentration of 5μmol/Lα-synuclein oligomers,cells in the group C were treated with a final concentration of 100μmol/L AMB,and cells in the group D were pretreated with a final concentration of 100μmol/L AMB for 12 h before adding 5μmol/Lα-synuclein oligomers.After 24 h of incubation,the activity ofβ-glucocerebrosidase(GCase)and the level ofα-synuclein oligomers were detected by ELISA.Cell proliferation capability was assessed using the MTT assay(represented by OD_(450) values).Mitochondrial membrane potential was measured by the JC-1 probe,and the phosphorylation level of tyrosine hydroxylase(TH)was detected by Western blotting.Results The GCase activity was lower in the group B than in the other three groups(all P<0.05),and the GCase activity was higher in the group C than in the other three groups(all P<0.05).The level ofα-syn oligomer in the group B was higher than those in the other three groups(all P<0.05),and the level ofα-syn oligomer in the group C was lower than those in the other three groups(all P<0.05).Compared with the group A,the OD_(450) values of cells in the group B at 24,48,and 72 h of culture decreased(all P<0.05),and the OD_(450) value of cells in the group D at 72 h of culture decreased(P<0.05).Compared with the group B,the OD_(450) values of cells in the groups C and D at 24,48,and 72 h of culture increased(all P<0.05).Compared with the group C,the OD_(450) values of cells in the group D at 24 and 72 h of culture decreased(all P<0.05).Mitochondrial membrane potential of cells in the group B was lower than that in the other three groups(all P<0.05),and mitochondrial membrane potential of cells in the group D was lower than that in the groups A and C(all P<0.05).The phosphorylation level of TH in the group B was lower than those in the other three groups(all P<0.05),and the phosphorylation level of TH in the group C was higher than those in the other three groups(all P<0.05).Conclusion AMB can preventα-synuclein oligomer-induced cell damage by increasing GCase activity,reducingα-synuclein oligomer levels,and restoring cell proliferation capability,mitochondrial membrane potential,and TH phosphorylation levels in MES23.5 cells.