外源性三价铁离子对SH-SY5Y细胞tau蛋白磷酸化和聚集的影响

Effect of exogenous trivalent iron ions on tau phosphorylation and aggregation in SH-SY5Y cells

[背景]脑内大量的铁沉积可通过氧化应激、神经炎症、线粒体功能异常引发神经元损害。铁沉积与阿尔茨海默病(AD)的病理发生也密切相关,tau蛋白过度磷酸化导致的神经原纤维缠结是AD的重要病理特征之一。[目的]探讨外源性三价铁离子对人神经母细胞瘤细胞(SH-SY5Y细胞)活性及tau过度磷酸化和聚集的影响。[方法]采用Fe^(3+)浓度为10、100、200和400 mg·L^(−1)的三氯化铁(FeCl_(3))处理SH-SY5Y细胞,通过CCK8法检测细胞存活率。用10和200 mg·L^(−1)染毒24 h,通过普鲁士蓝(Perl's)铁染色法测定胞内铁含量。转染tau-P301L质粒构建tau过表达的AD样细胞模型,10、200 mg·L^(−1)染毒SH-SY5Y细胞及tau过表达的SH-SY5Y细胞24 h后,采用免疫印迹检测磷酸化tau(p-tau)蛋白表达的差异。将磷酸缓冲盐溶液(PBS)稀释后的FeCl_(3)、人源性tauR3及FeCl_(3)和tauR3共同在37℃孵育,通过硫磺素T(ThT)荧光定量法在12、24、36、48、60、72、84和96 h检测荧光强度,反映tau蛋白聚集水平。同时,FeCl3和tauR3共同孵育96 h后,在透射电子显微镜(TEM)下观察tau聚集形成纤维的情况。[结果]FeCl_(3)染毒SH-SY5Y细胞24 h后,各组间细胞存活率的差异有统计学意义(F=8.63,P<0.01)。200、400 mg·L^(−1)染毒组细胞存活率分别是对照组的80.1%、68.7%(P<0.05)。与对照组细胞相比,胞内铁染色后200 mg·L^(−1)染毒组细胞胞浆主要呈黄褐色,且200 mg·L^(−1)染毒组阳性细胞率增加12.9%(P<0.01)。FeCl3染毒SH-SY5Y细胞24 h后,各组间p-tau(Ser396)表达有统计学意义(F=11.6,P<0.01)。与对照组相比,200 mg·L^(−1)染毒组p-tau(Ser396)蛋白表达量上调72.7%(P<0.01)。FeCl_(3)染毒tau过表达的SH-SY5Y细胞24 h后,各组间p-tau(Ser396)表达有统计学意义(F=27.8,P<0.01)。与tau组相比,tau+200 mg·L^(−1)染毒组p-tau(Ser396)蛋白表达量上调44.6%(P<0.05)。tauR3和FeCl_(3)共同孵育84和96 h后tauR3+FeCl_(3)组荧光强度比tauR3组增加49.9%和53.7%(P<0.01)。TEM下也观察到孵育96 h后,与tauR3组相比,FeCl_(3)+tauR3组聚集加重,且形成tau纤维沉积。[结论]外源性三价铁离子可能抑制SH-SY5Y细胞活性,促进转染tau-P301L质粒的SH-SY5Y细胞tau蛋白磷酸化,且加重了tauR3的聚集和纤维的产生。

[Background]A large amount of iron deposition in the brain can cause neuronal damage by inducing oxidative stress,neuroinflammation,and abnormal mitochondrial function.In addition,iron deposition is also reported to be closely related to the pathogenesis of Alzheimer's disease(AD).The neurofibrillary tangles aggregated by tau hyperphosphorylation are one of the important pathological features of AD.[Objective]To investigate potential effect of exogenous trivalent iron ions on neuronal activity in human neuroblastoma(SH-SY5Y)cells and tau hyperphosphorylation and aggregation.[Methods]SH-SY5Y cells were treated with ferric chloride(FeCl_(3))at four concentrations(10,100,200,and 400 mg·L^(−1)).Cell survival rate was then detected by CCK8 assay.Intracellular iron content was determined prussian blue(Perl's)by iron staining after 24 h exposure to FeCl_(3) at 10 or 200 mg·L^(−1).Transfection of tau-P301L plasmid was conducted to construct an AD-like cell model for tau overexpression.The differences in the expression of the phosphorylated tau(p-tau)protein in SH-SY5Y cells and SH-SY5Y cells with tau overexpression were detected by Western blotting after 24 h exposure to FeCl_(3) at 10 and 200 mg·L^(−1).After dilution with phosphate buffered saline(PBS),FeCl_(3),human tauR3,and FeCl_(3)+tauR3 were incubated at 37℃,and the fluorescence intensity reflecting tau aggregation level was measured by thioflavin T(ThT)method at 12,24,36,48,60,72,84,and 96 h,respectively.Meanwhile,after 96 h coincubation of FeCl_(3) and tauR3,the fibers formed by tau aggregation were observed under a transmission electron microscope(TEM).[Results]After 24 h of FeCl_(3) exposure,the cell survival rate of SH-SY5Y cells among all groups was statistically different(F=8.63,P<0.01).The cell survival rates in the 200 and 400 mg·L^(−1)groups were 80.1%and 68.7%of the control group,respectively(P<0.05).Compared with the control group,the nuclei of the 200 mg·L^(−1)FeCl_(3) group were mainly yellowish-brown after iron staining and the positive cell rate was up-regulated by 12.9%(P<0.01).After 24 h of FeCl_(3) exposure,the p-tau(Ser396)protein expression was statistically different among all groups(F=11.6,P<0.01).Compared with the control group,the p-tau protein expression level of SH-SY5Y cells in the 200 mg·L^(−1)group was up-regulated by 72.7%(P<0.01).After FeCl_(3)-treated SH-SY5Y cells with tau overexpression for 24 h,the p-tau(Ser396)protein expression was statistically different among all groups(F=27.8,P<0.01).Compared with the tau group,the p-tau(Ser396)protein expression level of SH-SY5Y cells in the tau+200 mg·L^(−1)group was up-regulated by 44.6%(P<0.05).Compared with the tauR3 group,the fluorescence intensities in the 84 and 96 h tauR3+FeCl_(3) groups were up-regulated by 49.9%and 53.7%(P<0.01)respectively.After 96 h of coincubation,compared with the tauR3 group,FeCl_(3)+tauR3 aggravated tau aggregation and formed fiber deposition under TEM.[Conclusion]Exogenous trivalent iron ions may inhibit SH-SY5Y cell viability,promote the phosphorylation of tau in SH-SY5Y cells transfected with tau-P301L plasmid,and aggravate tauR3 aggregation and fiber production.