Protective Effect Of Lycopene On Mitochondrial Damage Of PC12 Cells Induced By SOD1-G93A

Amyotrophic lateral sclerosis(ALS),which is a chronic progressive neurodegenerative disease, involving in upper neuron and lower neuron, characterized by muscle atrophy spasticity and weakness, can lead to death within three to five years of diagnosis. About 5-10% of ALS patients are familial ALS(f ALS), while other 90-95% of ALS patients are sporadic ALS(s ALS). The mechanism of ALS, which has not been fully understood, is considered to be related to gene mutations of Superoxide Dismutase 1(SOD1),oxidative stress, immunological mechanism, mitochondrial dysfunction, abnormal neurons surrounding glial cells, oxide toxicity and other relevant. SOD1-based mutation was considered to be the most common cause of ALS.A large number of SOD1 mutations has been reported, of which G93 A mutation is the most. Mice with SOD1-G93 A mutation, has become the world’s recognized animal model of ALS because of the similar clinical characteristics to human. With the development of various types of research, it is found that mitochondria dysfunction played a crucial role in the occurrence and development of ALS. Mitochondria dysfunction may be the initiating factor in the pathogenesis of ALS, while SOD1, which is closely related to the pathogenesis of ALS, is located in the mitochondria. Studies have illustrated that a large number of abnormal morphology of mitochondria and the mutant protein accumulation do exist in the motor neurons of ALS patients and animal models.The main function of SOD1 is to oxidate the toxic substances in the mitochondria by disproportionation reaction(such as ROS) oxidation. The reduction the binding force of zinc, caused by SOD1 gene mutation, can lead to motor neuron toxicity, and can cause the mitochondrial cavitation and inflation in ALS patients.Simultaneously, protein condensate caused by SOD1 mutation hinders the transport channel of mitochondria, and thereby inhibits the biological function of mitochondria, resulting in the injury of mitochondria,and activation of apoptosis, which leads to motor neuron death and the occurrence of ALS.Dynamin related protein 1(Drp1) is closely related to mitochondrial dynamics, it is a factor mediating division of mitochondria. Normally, most of the intracellular Drp1 locate in cytoplasmic matrix, only about 3% of which combine to the outer membrane of mitochondria. When mammalian mitochondria is splitting, split mitochondrial protein 1(Fis1) promotes Drp1 to aggregate in the outer membrane of mitochondria,and causes mitochondrial split, then Drp1 expression enhancement can cause continuing mitochondria split and lead to apoptosis.PC12 cells are rat pheochromocytoma cells, which are rich in tyrosine hydroxylase,monoamine oxidase necessary for dopamine synthesis and decomposition,and its chemical property is very close to the midbrain dopaminergic neurons, it has the property of tumor cell, therefore is widely used in the research of the central nervous system degenerative disease.Lycopene is a kind of carotenoids, a kind of natural pigment contained in plants, it mainly exists in fruits such as tomato, carrot, watermelon, guava etc, the highest contents are found in tomato.It is one of the most powerful antioxidants among all the nature plants. It is very remarkable in antioxidant properties and free radical scavenging, which make it very effective in preventing the heart disease, slowing the progression of atherosclerosis, preventing many types of cancer, protecting cardiovascular, anti-aging and protecting the skin. The protective effect of lycopene on neurodegenerative diseases such as AD, PD has been confirmed, but in terms of ALS has not been reported.Objective: The SOD1- G93 A transfected PC12 cells were used to build ALS cell model, aiming to investigate the protective effect of lycopene on mitochondrial damage of PC12 cells induced by SOD1- G93 A transfection.Method: Empty( E) plasmid, Wild Type( WT) plasmid,SOD1-G93 A plasmid were used to transfer PC12 cell.The transfection efficiency was observed under microscope,the cell holes with the transfection efficiency of 70% or more were selected to measure the cell vitality by CCK8 assay,and the cell damage against transfection were detected.Normal cells were administrated with lycopene(0、1、10、20、50、100 umol?L), cell viability was determined by CCK-8 assay.SOD1-G93 A transfected PC12 cells with the satisfied transfection efficiency were given lycopene(0、0.1、0.5、1、5、10、20 umol?L) for preincubation, cell viability was determined by CCK-8 assay and the optimal drug concentration was screened.Cells were divided into normal cells, transfection E plasmid group, transfection WT plasmid group, transfection SOD1-G93 A group, each group was divided into control group and lycopene group. In lycopene group,a culture solution containing the optimal concentration of lycopene was used,while in the control group, ordinary culture medium was given.After 24 h treatment, the flow cytometry was used to detect the level of ROS and Confocal microscope was used to observe the change of Drp1 to observe the protective effect of lycopene.Result:1 After transfection with E、WT、SOD1-G93 A plasmids, the transfection efficiency was observed under microscope, the cell holes with the transfection efficiency of 70% or more were selected to measure the cell vitality by CCK8 assay.Compared with control group, the cell vitality of E, WT, SOD1- G93 A group gradually decline, cell vitality had the greatest reduction in SOD1-G93 A group, difference was statistically significant(P﹤0.05).There was no significant difference in cell vitality between E group and WT group.The cell vitality in SOD1-G93 A group decreased significantly compared with E and WT groups. The cell model was successfully set up.2 CCK-8 assay showed that normal cells were given different concentra- tions of lycopene, the cell viability decreased obviously in 20μmol?L、50μmol?L、100μmol?L group compared with control group, the difference was statistically significant(P ﹤ 0.05).The results showed that a high concentration of lycopene can be harmful to cell growth, survival,and cannot be used in the experiment.Cell viability gradually increased after the pre-treatement of low concentration of lycopene, with the drug concentration gradually increased, the cell viability gradually increased when the concentration was 10 mol/L, then decreased, so 10μmol?L was the best protection concentration.3 Intracellular ROS levels increased in WT group compared with the E group(P﹤0.05), Compared with the cells in the E、WT group,intracellular ROS levels increased in the G93 A group(P﹤0.05). Compared with the cells in the E、WT group,the Drp1 levels obviously increased in the G93 A group, indicating the mitochondria damage in the PC12 cells transfected with SOD1-G93 A.4 Giving the optimal concentration of lycopene to the PC12 cells, in all the E、WT、SOD1-G93 A groups, the ROS content decreased obviously(P﹤ 0.05) the Drp1 content reduced in lycopene group compared with control group.Conclusions:1 The cell model of ALS was successfully prepared, there was mitochondrial damage in the PC12 cells transfected with SOD-G93 A.2 Lycopene with appropriate concentration can improve the cell vitality, and have a protective effect for the PC12 cells transfected with SOD1-G93 A.3 Lycopene can help remove excessive accumulation of ROS and reduce the content of Drp1, this could be one of the possible mechanisms of lycopene protecting mitochondria and reducing cellular damage.