Ndfip1对神经细胞铁超载损伤的保护作用及机制探讨
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摘要
目的探讨Ndfip1过表达对神经细胞SH-SY5Y铁超载损伤的保护作用及机制。方法以SH-SY5Y细胞为细胞模型,并成功构建Ndfip1质粒。应用Ndfip1质粒和空质粒分别转染SH-SY5Y细胞,建立实验组和对照组。MTT法检测不同浓度FeSO_4作用下,SH-SY5Y细胞存活率的变化,确定铁超载浓度;Ndfip1质粒转染SH-SY5Y细胞,确定转染效率;Ndfip1质粒和空质粒分别转染SH-SY5Y细胞,MTT法检测2组细胞存活率的变化;应用Calcein AM法处理细胞,荧光显微镜下观察2组细胞的荧光强度,以确定细胞内铁离子的含量;荧光分光光度计检测2组细胞的荧光强度变化,以确定细胞铁摄入能力的改变。结果随着FeSO_4浓度增加,SH-SY5Y细胞存活率逐渐降低。200μmol/L FeSO_4可导致SH-SY5Y细胞存活率明显下降。Ndfip1质粒转染可明显增加SH-SY5Y细胞Ndfip1蛋白表达量,提高SH-SY5Y细胞铁超载损伤后的存活率,减少细胞内铁离子含量及细胞对铁离子的摄入。结论铁超载对神经细胞造成损伤,导致其存活率降低;Ndfip1可减少铁离子进入神经细胞,减轻神经细胞内铁蓄积,降低铁超载对神经细胞的损伤作用,提高细胞存活率,从而发挥其保护作用。
Objective To investigate the protective mechanism of Ndfip1 overexpression on iron overload injury in nerve cells(SH-SY5Y cells).Methods The SH-SY5Y cells were used as the cell model, and the Ndfip1 plasmid was successfully constructed. SH-SY5Y cells were transfected with Ndfip1 plasmid or empty plasmid, respectively, and the experimental group and the control group were established. The MTT assay was used to detect changes of survival rates of SH-SY5Y cells under different concentrations of FeSO_4, and the iron overload concentration was determined. SH-SY5Y cells were transfected with Ndfip1 plasmid to determine the transfection efficiency. Ndfip1 plasmid and empty plasmid weretransfected into SH-SY5Y cells, respectively. The survival rates of the two groups were detected by MTT method. Thefluorescence intensity was observed by fluorescence microscopy using Calcein AM method to determine the intracellular ironion contents in two groups of cells. The fluorescence intensity was detected by fluorescence spectrophotometer to determinethe changes of iron uptake in two groups of cells.ResultsAs the concentration of FeSO_4 increased, the survival rates ofSH-SY5Y cells gradually decreased. The 200 μmol/L FeSO_4 can cause a significant decrease in survival rate of SH-SY5Ycells. The transfection of SH-SY5Y cells with Ndfip1 plasmid significantly increased the expression of Ndfip1. Theoverexpression of Ndfip1 can increase the survival rate of iron overload injury, reduce intracellular iron content, and reducethe uptake of iron ions in SH-SY5Y cells.Conclusion Iron overload causes nerve cells to damage, resulting in thedecrease in survival rate. Ndfip1 can reduce iron ions into nerve cells, reduce iron accumulation in nerve cells, reduce irondamage to nerve cells, improve survival rate, and play its protective role.
Objective To investigate the protective mechanism of Ndfip1 overexpression on iron overload injury in nerve cells(SH-SY5Y cells).Methods The SH-SY5Y cells were used as the cell model, and the Ndfip1 plasmid was successfully constructed. SH-SY5Y cells were transfected with Ndfip1 plasmid or empty plasmid, respectively, and the experimental group and the control group were established. The MTT assay was used to detect changes of survival rates of SH-SY5Y cells under different concentrations of FeSO_4, and the iron overload concentration was determined. SH-SY5Y cells were transfected with Ndfip1 plasmid to determine the transfection efficiency. Ndfip1 plasmid and empty plasmid weretransfected into SH-SY5Y cells, respectively. The survival rates of the two groups were detected by MTT method. Thefluorescence intensity was observed by fluorescence microscopy using Calcein AM method to determine the intracellular ironion contents in two groups of cells. The fluorescence intensity was detected by fluorescence spectrophotometer to determinethe changes of iron uptake in two groups of cells.ResultsAs the concentration of FeSO_4 increased, the survival rates ofSH-SY5Y cells gradually decreased. The 200 μmol/L FeSO_4 can cause a significant decrease in survival rate of SH-SY5Ycells. The transfection of SH-SY5Y cells with Ndfip1 plasmid significantly increased the expression of Ndfip1. Theoverexpression of Ndfip1 can increase the survival rate of iron overload injury, reduce intracellular iron content, and reducethe uptake of iron ions in SH-SY5Y cells.Conclusion Iron overload causes nerve cells to damage, resulting in thedecrease in survival rate. Ndfip1 can reduce iron ions into nerve cells, reduce iron accumulation in nerve cells, reduce irondamage to nerve cells, improve survival rate, and play its protective role.
引文
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[6]Howitt J,Gysbers AM,Ayton S,et al.Increased Ndfip1 in the substantia nigra of Parkinsonian brains is associated with elevated iron levels[J].PLoS One,2014,9(1):e87119.doi:10.1371/journal.pone.0087119.
[7]Peng J,Liu H,Liu C.MiR-155 promotes uveal melanoma cell proliferation and invasion by regulating NDFIP1 expression[J].Technol Cancer Res Treat,2017,16(6):1160-1167.doi:10.1177/1533034617737923.
[8]Foot NJ,Dalton HE,Shearwin-Whyatt LM,et al.Regulation of the divalent metal ion transporter DMT1 and iron homeostasis by a ubiquitin-dependent mechanism involving Ndfips and WWP2[J].Blood,2008,112(10):4268-4275.doi:10.1182/blood-2008-04-150953.
[9]Foot NJ,Leong YA,Dorstyn LE,et al.Ndfip1-deficient mice have impaired DMT1 regulation and iron homeostasis[J].Blood,2011,117(2):638-646.doi:10.1182/blood-2010-07-295287.
[10]Howitt J,Low LH,Putz U,et al.Ndfip1 represses cell proliferation by controlling Pten localization and signaling specificity[J].J Mol Cell Biol,2015,7(2):119-131.doi:10.1093/jmcb/mjv020.
[11]Cheli VT,Santiago González DA,Marziali LN,et al.The divalent metal transporter 1(DMT1)is required for iron uptake and normal development of oligodendrocyte progenitor cells[J].J Neurosci,2018,38(43):9142-9159.doi:10.1523/JNEUROSCI.1447-18.2018.2018-10-172018-12-13
[2]Tian Q,Wu S,Dai Z,et al.Iron overload induced death of osteoblasts in vitro:involvement of the mitochondrial apoptotic pathway[J].Peer J,2016,4:e2611.doi:10.7717/peerj.2611.
[3]Wagle MV,Marchingo JM,Howitt J,et al.The ubiquitin ligase adaptor NDFIP1 selectively enforces a CD8+T cell tolerance checkpoint to high-dose antigen[J].Cell Rep,2018,24(3):577-584.doi:10.1016/j.celrep.2018.06.060.
[4]Xing LF,Guo HP,Wang DT,et al.Protective effects and mechanisms of Ndfipl on SH-SY5Y cell apoptosis in an in vitro Parkinson′s disease model[J].Genet Mol Res,2016,15(2).doi:10.4238/gmr.15026963.
[5]Hasegawa I,Takeda A,Hatsuta H,et al.An autopsy case of globular glial tauopathy presenting with clinical features of motor neuron disease with dementia and iron deposition in the motor cortex[J].Neuropathology,2018.doi:10.1111/neup.12457.[Epub ahead of print].
[6]Howitt J,Gysbers AM,Ayton S,et al.Increased Ndfip1 in the substantia nigra of Parkinsonian brains is associated with elevated iron levels[J].PLoS One,2014,9(1):e87119.doi:10.1371/journal.pone.0087119.
[7]Peng J,Liu H,Liu C.MiR-155 promotes uveal melanoma cell proliferation and invasion by regulating NDFIP1 expression[J].Technol Cancer Res Treat,2017,16(6):1160-1167.doi:10.1177/1533034617737923.
[8]Foot NJ,Dalton HE,Shearwin-Whyatt LM,et al.Regulation of the divalent metal ion transporter DMT1 and iron homeostasis by a ubiquitin-dependent mechanism involving Ndfips and WWP2[J].Blood,2008,112(10):4268-4275.doi:10.1182/blood-2008-04-150953.
[9]Foot NJ,Leong YA,Dorstyn LE,et al.Ndfip1-deficient mice have impaired DMT1 regulation and iron homeostasis[J].Blood,2011,117(2):638-646.doi:10.1182/blood-2010-07-295287.
[10]Howitt J,Low LH,Putz U,et al.Ndfip1 represses cell proliferation by controlling Pten localization and signaling specificity[J].J Mol Cell Biol,2015,7(2):119-131.doi:10.1093/jmcb/mjv020.
[11]Cheli VT,Santiago González DA,Marziali LN,et al.The divalent metal transporter 1(DMT1)is required for iron uptake and normal development of oligodendrocyte progenitor cells[J].J Neurosci,2018,38(43):9142-9159.doi:10.1523/JNEUROSCI.1447-18.2018.2018-10-172018-12-13
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