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Objective@#To investigate the role of lysosomes in manganese-induced toxicity in human neuroblastoma SK-N-SH cells.@*Methods@#SK-N-SH cells were treated with MnCl2 at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L for 24 h, and the cell viability was detected by MTT assay. Cells were treated with MnCl2 at doses of 0.125, 0.25, 0.5 and 1.0mmol/L for 24 h, and lysosomes labeled with lysotracker red were observed by laser confocal microscopy, the expression levels of LAMP1 and CTSD were detected by western blot, and CTSD activity was detected by Cathepsin D Activity Fluorometric Assay Kit.@*Results@#Compared with the control group, the survival rates of SK-N-SH cells were decreased significantly in the 0.5-4.0 mmol/L MnCl2 treatment groups (P<0.01) , the relative fluorescence intensities of 0.5 and 1.0 mmol/L MnCl2 treatment groups were increased (P<0.01) . Compared with the control group, the 0.125-0.5 mmol/L MnCl2 treatment groups had significant increase in the the expression of LAMP1 (P<0.01) . Compared with the control group, the expression of m-CTSD was significantly increased at the does of 0.125-0.25 mmol/L MnCl2, while it was decreased at the does of 1.0 mmol/L (P<0.01) . Otherwise, it wasn’t observed significant difference of the activity of CTSD between different MnCl2 treatment groups.@*Conclusion@#MnCl2 could cause cytotoxicity in SK-N-SH cells. Lysosomes may play a normal function at low doses of manganese, but they may be damaged at high doses of manganese. As an organelle that can degradate substrates in autophagy, lysosomes participate in the neurotoxic mechanism of manganese.
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Objective@#To investigate the effect of manganese chloride (MnCl2) or 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) on the neurobehavioral and histopathology in C57BL/6 mice and provide evidence for the diagnosis, treatment and prevention of manganism.@*Methods@#Adult male C57BL/6 mice were treated with MnCl2 and MPTP respectively by intraperitoneal injection at the doses of 5, 10, 20mg Mn/kg and 30mg MPTP/kg. Controls were injected equivalent normal saline. All animals were administrated 5 times a week for 4 consecutive weeks and sacrificed after behavior tests on the fifth week. Balance ability, anxiety and depression level and cognitive function were tested respectively by vertical pole test, open field locomotion test and Morris swim task. The neuron pathological changes of striatum and substantia nigra were examined through HE-staining pathological section by using optical microscope.@*Results@#Compared with the control group, the high dose of MnCl2 reduced body weight obviously (P<0.01) . The results of vertical pole test showed that MnCl2 and MPTP lengthened the pole-climbing time and turnaround time. Open field locomotion test showed that movement distance, stand-up time and central field time were decreased after the exposure of MnCl2 or MPTP. In the Morris swim task, the escape latency time increased and the target quadrant activity time decreased significantly after the injection of MPTP as well as high-dose MnCl2, comparing with controls (P<0.05) . Moreover, the escape latency time of high dose MnCl2 prolonged prominently comparing with MPTP grou (P<0.05) . The results of histopathology showed that acidophilic changes elevated in MnCl2 and MPTP group, comparing with controls. Furthermore, in striatum the oxyphil cells number increased in MnCl2 high-dose group comparing with MPTP group (P<0.01) . On the contrary, there were more oxyphil cells in MPTP group comparing with MnCl2 groups in substantia nigra (P<0.01) .@*Conclusion@#Both manganese and MPTP can induce the impairment of dopaminergic neural system, but the symptons and injured location of manganism are inconsistent with PD models induced by MPTP.
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Objective@#To investigate the effect of manganese chloride (MnCl2) or 1-methyl-4-phenylpyridinium (MPP +) on oxidative stress and autophagy in human neuroblastomaSK-N-SH cells and the mechanism of the neurotoxicity of manganese.@*Methods@#SK-N-SH cells were treated with MnCl2 or MPP+ at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, and 2.0 mmol/L for 24 hours, and MTT assay was used to measure cell viability. The cells weretreated with MnCl2 or MPP+ at doses of 0.125, 0.25, and 0.5 mmol/L for 24 hours, and flow cytometry was used to measure the content of reactive oxygen species (ROS) in cells, a laser scanning confocal microscope was used to observe autophagosome in cells, and Western blot was used to measure the expression of autophagy-related proteins P62 and LC3-II/LC3-I.@*Results@#Compared with the control group, the 0.0625-2.0 mmol/L MnCl2 and 0.125-2.0 mmol/L MPP + treatment groups had significant reductions in the viability of SK-N-SH cells, and the 0.25-2.0 mmol/L MnCl2 treatment groups had significantly lower viability than the groups treated with the same doses of MPP+ (all P<0.05) . Compared with the control group, the 0.125-0.25 mmol/L MnCl2 and 0.125-0.5 mmol/L MPP+ treatment groups had significant increases in the content of ROS, and the 0.25-0.5 mmol/L MPP+ treatment groups had significantly higher content of ROS than the groups treated with the same doses of MnCl2 (all P<0.05) . Compared with the control group, the 0.25-0.5 mmol/L MnCl2 andMPP+ treatment groups had significant increases in autophagy-related proteins LC3-II/LC3-I and significant reductions in P62 expression; the 0.125-0.5 mmol/L MPP+ treatment groups had significantly higher LC3-II/LC3-I than the groups treated with the same doses of MnCl2, and the 0.125 and 0.25 mmol/L MPP + treatment groups had significantly lower P62 expression than the groups treated with the same doses of MnCl2 (all P<0.05) .@*Conclusion@#Both MnCl2 and MPP+ can induce oxidative stress and autophagy in SK-N-SH cells, and MPP+ has a significantly greater inductive effect on autophagy of SK-N-SH cells than MnCl2.
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OBJECTIVE To investigate the nuclear proteomes in mitochondrial DNA (mtDNA)-depleted A549 cells (Rho0 cells) and their parental cells (Rho+ cells),and to learn more about the nuclear responses to mitochondrial dysfunction.METHODS The nuclear proteomes of Rho and Rho + cells were characterized by two dimensional electrophoresis (2-DE) and SELDI-TOF ProteinChip technologies,the differentially expressed protein- spots were identified by MALDI-TOF mass spectrum (MS),the nucleophosmin and P53 expression were detected by Western blotting assay,and the mitochondrial memhrane potential (MMP) was measured by the laser scanning confocal microscope.RESULTS 2-DE results showed 11 protein-spots were down-regulated and 21 protein-spots were up-regulated in Rho0 cell nuclei.SELDI-TOF MS analysis with NP20 ProteinChips revealed 4 protein-peaks decreased in Rho0 cell nuclei.One down-regulated protein-spot was identified as elF-6,and 4 up-regulated proteinspots were identified as nucleophosmin,SFRS1,SFRS3 and hnRNP G,respectively.The increased expression of nucleophosmin in Rho0 cells was verified by Western blotting.Based on the clues from proteomic analysis,P53 expression in Rho0 cells was higher than in Rho + cells,and MMP was consistent in Rho + and Rho0 cells.CONCLUSION mtDNA-depletion induces nuclear proteome alteration.Rho0 cells can be used as a model to study the crosstalk between mitochondrion and nucleus.