| Objective: ALS is amongst neurodegenerative diseases characterized by selectively invading anterior horn cells of spinal cord, motor neurons of brain stem, cortical pyramidal cells as well as pyramidal tract leading to progressive degeneration of upper and lower motor neurons. Clinical features of damaging in both upper and lower motor neurons are observed, with clinical manifestations of clumsy finger activities either unilateral or bilateral. Bulbar paralysis emerges at advanced stage of ALS, with fully conscious, patients are driven into great misery suffering the company of tongue atrophy, bucking, dysphagia, dysarthria and dysphonia. With disease progress continuing, patients mostly end up in respiratory failure or pulmonary infection. Nevertheless, international pathogenesis researches never stop advancing, mainly focus on the mechanism of mutant protein h SOD1-G93 A. By all accounts, great significance has been granted to the exploration of this particular protein. Lycopene, as one of the carotinoid, gain strong antioxidative quality from large amount of carbon-carbon double bond existing in the structure. Current studies already confirmed that lycopene confronting oxidative damage by increasing the expression of phase Ⅱ detoxification enzyme through Nrf2-ARE signal transduction pathway. At least 14 genes controlled by Nrf2 have been found down regulating expression in SOD1 mutant models yet, including NQO1, GST, HO-1, cytochrome P450 and G-6-PD. However, there is still something remains to be explored up till now, for instance the in vitro evidence of therapeutic effect of lycopene on neurodegenerative disease. This experiment inquiry into possible impact and mechanism of lycopene on NSC34 cells transfected with h SOD1-G93 A from aspect of antioxidation. As noted above, the aim of this study is laying a preliminary foundation of widespread therapeutic methods for amyotrophic lateral sclerosis.Methods:1 Study Group DesignThe experiment set blank control group, empty plasmid group,h SOD1-G93 A group and three different concentrations of lycopenegroup(1.5μmol/L,2.5μmol/L,7.5μmol/L). 24 hours after transfection, NSC34cells were grown with DMSO or different concentrations of lycopene foranother 24 hours.2 Cell Culture and TransfectionAfter defrosting, NSC34 cells were grown with DMEM containing 10%FBS, with penicillin G and streptomycin for at least three generations. Pricisely based on LipofectamineTM 2000 instructions, transfection only operated when cells reach logarithmic phase, with apparent processes, fine morphology and 80%~90% cell density. 24 hours after transfection, cells were removed into fresh DMEM medium with different amounts of lycopene stock solution(1mmol/L)added, resulting in final concentrations of 1.5 μmol/L, 2.5 μmol/L, 7.5μmol/L. Samples were gathered 48 hours after transfection for further study.3 MDA DetectionCells of each group were collected for MDA detection 24 hours after treated with lycopne. MDA detection was operated under 532 nm wavelength using TBA method based on official instructions.4 Western BlotTotal cell protein was extracted from each group for Western Blot(60μg), with protein concentration measured by BCA method. After that, target proteins on PAGE gel were transferred by electric methods onto PVDF membranes. PVDF membranes were then incubated with primary antibodies at 4℃ for at least 12 hours before incubating with fluorescent dyes binding secondary antibodies. To establish the level of protein expression, PVDF membranes were scanned by Odyssey infrared image scanning system and analyzed by corresponding software Image J.5 Apoptosis Rate DeterminationAfter 500 rpm, 5minutes centrifuging, cells were resuspended with pre-cooled PBS before counting. 5μl PI and 500μl 1x Binding buffer were added into 1~5*105 cells after centrifuging for resuspending. Cells were incubated at room temperature for 30 minutes in dark before FCM detection.Result:1 Successful NSC34 cell culture and plasmid transfection.Observation with fluorescence microscope, NSC34 cells were in regular shape, adherent nicely with apparent processes. Green fluorescentlight were also confirmed in empty plasmid group, h SOD1-G93 A group and three different concentrations of lycopene groups. As for h SOD1-G93 A group and three lycopene groups, exogenous SOD1 with EGFP tag was confirmed by Western Blot.Transfection efficiency of those groups were23.30% and 26.21% detected by flow cytometry. In short, plasmid transfection was a success.2 MDA detectionThe content of MDA represents the degree of cellular lipid peroxidation, while indirectly reflected the severity of oxidative stress. MDA content in h SOD1-G93 A group is 2.25nmol/mgprot, obviously higher than that of empty plasmid group. MDA result showed significant differences between h SOD1-G93 A group and each lycopene group( P<0.05) with statistical significance. MDA level of each lycopene group(1.5μmol/L, 2.5μmol/L, 7.5μmol/L)are 1.94 nmol/mgprot, 1.46 nmol/mgprot, and 1.30 nmol/mgprot, higher than control group in various degree but lower than that of h SOD1-G93 A group, both in a dose related manners. This result confirmed that h SOD1-G93 A did aggravated lipid peroxidation. However lycopene can inhibit this effect in a dose related manner.3 Expression of NQO-1 proteinNQO-1 is the key enzyme participates in the process of defeating ROS damaging, providing multiple protection under oxidative stress by over expression. Lycopene(7.5μmol/L) were added in DMEM culture medium 24 hours after transfection. After transfected for 48 hours, cells in each group were gathered for protein exraction and Western Blot for NQO-1 detection. As Western Blot results showed, compare to control group, relative NQO-1 protein expression of h SOD1-G93 A groupis 29.85%. Relative NQO-1 protein expression of lycopene group(7.5μmol/L) is 67.01%, higher than that of h SOD1-G93 A group, with statistical significance(P<0.05).4 Apoptosis RateCell membrane integrity no longer remains perfection in middle stage and late stage of apoptosis, as well as dead cells. That fact provides chances for PI entering. PI enters cells, stains nuleus into red. Thus apoptosis rate can be detected objectively. Different concentrations of lycopene(1.5μmol/L,2.5 μmol/L,7.5 μmol/L)were given 24 hours after transfection. Cells of each group were gathered for detection 48 hours after transfection. FCM results demonstrated that lycopene inhibited apoptosis in a does related manner compared with h SOD1-G93 A samples(P<0.05). Apoptosis rates of each concentration are 36.55%,30.15%,24.8%,repectively.Conclusion:Lycopene provides cellular protection to h SOD1-G93 A transfected NSC34 cells mainly marked by:1 Reducing lipid preoxidation in does related manners2 Up regulating protein expression of NQO-13 Reducing apoptosis rate... |
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