Study Of Mitochondrial Ferritin On Protective Mechanism To SH-SY5Y Cell Damage Induced By Rotenone

Currently, the Parkinson’s disease in the elderly has become a common neurodegenerative disease, severely impact on the lives of elderly health and quality of life. Studies have shown that some toxins can cause neurological disorders in brain iron metabolism and oxidative stress, and the occurrence of PD and brain iron metabolism and oxidative stress are closely related. Neurotoxin rotenone,a natural mitochondrial respiratory chain complex I inhibitor,can cause a-synuclein and ubiquitin in some specific cells such as the appearance of inclusion to promote substantia nigra excessive activity of brain oxygen and iron deposition, which led to substantia nigra selective striatal degeneration and death of dopaminergic neurons.The brain iron metabolism and neurodegenerative disease research has become the field of neurobiology and neurochemistry in a very hot research topic.Intracellular iron overload can damage cells hardly, while cytosolic ferritin can capture excess free iron which reduce the iron damage. Mitochondrial ferritin (MtFt),another iron-related protein, was found in 2001 and mature MtFt in the structure and function is similar with the H-ferritin. The results show that MtFt incorporated iron even more efficiently than the cytosolic ferritins. Many experimental results show that the overexpression of MtFt caused a dose-dependent iron deficiency in the cytosol that was revealed by increased RNA-binding activity of iron regulatory proteins (IRPs) along with an increase in transferrin receptor levels and decrease in cytosolic ferritin. Consequently, the induction of MtFt resulted in a dramatic increase in cellular iron uptake from transferrin,most of which was incorporated into MtFt. The induction of MtFt caused a shift of iron from cytosolic ferritin to MtFt. In addition, several experiments confirmed that the MtFt protected the damage from mitochondrial iron overload caused by frataxin gene defect through efficient combination of free iron within the mitochondria. Rotenone can cause cell damage from iron deposition and might be the cause of dopaminergic neurons damage in PD. Whether Mtft has a protective effect on cell damage induced by Rotenone has not been reported. We chose MtFt overexpressed SH-SY5Y cell as model, pcDNA3.1-SY5Y (empty plasmid control) and SH-SY5Y cells as experimental control group, with rotenone as the drug induced injury in this paper. Meanwhile we use MTT, transmission electron microscopy, flow cytometry and Western-Blot to study the overexpression MtFt of the rotenone-induced cell injury mechanisms. the results are as follows:1. With a increasing concentration of rotenone treatment, SH-SY5Y cells, cell vitality show a dose-dependent decrease, when the rotenone concentration was 500nM/L, the cell viability decreased by 50%.2. At the 0-1000 nM/L level of rotenone treatment, MtFt-SY5Y cells viability was higher than that of pcDNA3.1-SY5Y cells and SH-SY5Y cells.It has significant change.3. Transmission electron microscope revealed that after 24 hours of 500nM/L rotenone treatment, pcDNA3.1-SY5Y cells and SH-SY5Y had a serious mitochondrial damage, almost cristae completely broken, but integrity of mitochondria in MtFt-SY5Ycells, mitochondrial cristae almost has no damage.4.500nM/L rotenone treated cells 24 hours, pcDNA3.1-SY5Y cells labile iron pool (,LIP) was significantly increased; while the MtFt-SY5Y cells LIP has little increased and there was no significant difference; LIP of MtFt-SY5Y cells treated with rotenone is lower than that pcDNA3.1-SY5Y cells. There was significantly different.5. pcDNA3.1-SY5Y cells with Rotenone treatment for 24 hours, compared with the control group, Ferritin-L and iron transport membrane protein (FPN1) increased expression, transferrin receptor 1 (TfRl) protein Expression was significantly decreased. MtFt-SY5Y cells in iron-related proteins of these changes are not obvious.6. Cells treated with 500nM/L rotenone for 24 hours, cellular ROS (reactive oxygen species, ROS) levels in SH-SY5Y and pcDNA3.1-SY5Y cells increased nearly 3-fold, while the MtFt-SY5Y cells ROS did not have significantly increase.7. Meanwhile after 24 hours of 500nM/L rotenone treatment, the mitochondrial membrane potential (mitochondria membrane potential, MMP) of SH-SY5Y cells and pcDNA3.1-SY5Y cells were significantly decreased, while the MtFt-SY5Y cells MMP decreased little and it has no significantly difference.8.Western Bolt showed that overexpression MtFt inhibited cytochrome C release rotenone-induced Conclusion:In this study we used the Rotenone as a oxidative stress model to investigate the function of MtFt and the relationship between iron metabolism and neuron oxidative stress.1.This study showed that rotenone can cause dopaminergic neurons SH-SY5Y cell Ferritin-L and LIP increased. Iron metabolism disorder is one of the reasons in degeneration of neurons.2. Our result showed that played an impotant role in oxidative damage of mitochondrial induced by Rotenone. Its mechanism was to regulate the generation of ROS, then to prevente the reduction of mitochondrial membrane potential and to inhibite cell apoptosis. MtFt protected cells from oxidative stress damage.3. Regulating the expression of MtFt can effectively prevent the progress of neurodegenerative diseases, inhibit cell apoptosis caused by oxidative stress. According to our recently published results, we think MtFt may plays a ctitical neuroprotective role.