Lon蛋白酶1抑制脂质过氧化缓解多巴胺能神经细胞铁死亡的机制研究

Study on mechanism of Lon protease 1 inhibition lipid peroxidation and alleviating ferroptosis in dopaminergic nerve cells

目的探讨铁死亡脂质过氧化损伤在帕金森病(Parkinson′s disease,PD)细胞模型中的作用机制,并研究Lon蛋白酶1(Lon Protease 1,LONP1)抑制脂质过氧化缓解多巴胺(Dopamine,DA)能神经细胞铁死亡的可能机制。方法采用6-羟基多巴胺(6-OHDA)干预人神经母细胞瘤细胞(SH-SY5Y)构建PD细胞模型,CCK-8法筛选适宜造模浓度及测定细胞存活率;使用LONP1基因过表达质粒转染SH-SY5Y细胞,设立正常对照(NC)组、PD组、铁死亡抑制剂(Ferrostatin-1,Fer-1)+PD组、LONP1过表达+PD组及LONP1基因空载组;倒置荧光显微镜下观察细胞形态和质粒转染情况;丙二醛(MDA)试剂盒和谷胱甘肽(GSH)试剂盒检测5组细胞内脂质过氧化产物MDA和GSH含量;Western blot检测LONP1和Nrf2/Keap1/GPX4通路蛋白表达水平。结果(1)根据CCK-8结果选择80μM的6-OHDA干预SH-SY5Y细胞24 h制备PD细胞模型;(2)与NC组相比,PD组细胞存活率下降,MDA含量上升和GSH含量下降;与PD组相比,LONP1过表达+PD组MDA含量下降和GSH含量上升;(3)与NC组比较,PD组Keap1蛋白表达上升,Nrf2和GPX4蛋白表达下降(P<0.05);与PD组相比,LONP1过表达+PD组Keap1蛋白表达下降,Nrf2和GPX4蛋白表达上升(P<0.05)。结论LONP1能够改善DA能神经元铁依赖的脂质过氧化损伤,可能是通过影响Keap1/Nrf2/GPX4信号通路活化水平,抑制脂质过氧化缓解了DA能神经元铁死亡损伤。

Objective To investigate the mechanism of ferroptosis and lipid peroxidation damage in cell model of Parkinson′s disease(PD),and to investigate the possible mechanism of inhibition of lipid peroxidation by Lon Protease 1(LONP1)to alleviate ferroptosis in dopaminergic nerve cells.Methods Using 6-OHDA to interfere with SH-SY5Y cells to establish a PD cell model,CCK-8 method was used to screen the appropriate modeling concentration and determined the cell survival rate;SH-SY5Y cells were transfected with a LONP1 gene overexpression plasmid,and normal control(NC)group,PD group,Ferrostatin-1(Fer-1)+PD group,LONP1 over-expression+PD group,and LONP1 gene empty group was established.Cell morphology and plasmid transfection was observed under inverted fluorescence microscopy.Malondialdehyde(MDA)and Glutathione(GSH)kits were used to detect the levels of intracellular lipid peroxidation products MDA and GSH.Western blot was used to detect the expression levels of LONP1 and Nrf2/Keap1/GPX4 pathway proteins.Results(1)Based on CCK-8 results select 80μM′s 6-OHDA interfered with SH-SY5Y cells for 24 hours to establish a PD cell model.(2)Compared with NC group,cell survival rate in PD group was decreased,MDA level was increased,and GSH level was decreased.Compared with the PD group,the level of MDA in the LONP1 overexpression+PD group was decreased,and the level of GSH was increased.(3)Compared with NC group,the expression of Keap1 protein in PD group was increased,while the expression of Nrf2 and GPX4 protein was decreased(P<0.05).Compared with the PD group,the expression of Keap1 protein was decreased,and the expression of Nrf2 and GPX4 protein was increased in the LONP1 overexpression+PD group(P<0.05).Conclusion LONP1 can improve iron dependent lipid peroxidation damage in DA neurons,possibly by affecting the activation level of Keap1/Nrf2/GPX4 signaling pathway,and inhibit lipid peroxidation to alleviate the injury of ferroptosis in DA neurons.