Effect Of VEGF-A 165 On ALS Model Of NSC34 Cell Transfected With TDP-25 Plasmid And The Underlying Mechanism

Background: Amyotrophic lateral sclerosis(ALS)is a disease of degeneration which influence motor neurons in the central nervous system. Most of the patients manifest muscle weakness, atrophy and paralysis. Age of onset is 35-50 and life span is 3 to 5 years. Now, riluzole is the only available drug licensed by American FDA to treat ALS, but it can prolong life span for 3-5 months.Approximately 90-95% of ALS patients are sporadic ALS, others are familial ALS.The main cause of familial ALS is the mutation of superoxide dismutase 1(SOD1) gene. Other virulence genes of familial ALS include the TARDBP gene which encode the mutational protein of TDP-43 protein and the FUS gene which encode the mutational protein of RNA binding protein. Six exons of TARDBP gene encode TDP-43 protein, which consists of 414 amino acid.TDP-43 contains 2 RNA recognition sequence and C-terminal which is abunbant of glycine.TDP-43 has the ability of binding protein like DNA and RNA. TDP-43 protein can be cleaved into two cytotoxic fragments of 25-k Da and 35-k Da and TDP25 protein is more cytotoxic.Although the nosogenesis of ALS is unclear, the studies have certificated that damage of mitochondrion, lactate dyscrasia, immune system alterations and protein aggregation are all related with ALS. In addition, the deficiency of neurotrophy is very important. The expression of various growth factors including VEGF,IGF-1 are different in ALS. But they have neuroprotection and promote proliferation in motor neurons. VEGF, which is angiogenetic, can promote cell division and protect neurons against brain injury. VEGF is very important for the vascular growth and accommodation. Moreover, VEGF has the direct effect on neural growth and the immigration of neurons. The latest researches show that, VEGF can avoid neuron death and promote neuron growth when neurocyte is under the disadvantage of hypoxia,low carbohydrates and oxidative stress.VEGF family include seven elements, VEGF-A,VEGF-B,VEGF-C,VEGF-D,PLGF,VEGF-E and sv VEGF. VEGF-A is a dimeric glycoprotein of 33-42 k Da.The expression of VEGF gene is influenced by various conditions for instance hypoxia, growth factor, NO, the mutation of p53, thyroid stimulation hormone and tumor growth factor. The high expression of VEGF-A is mainly in suprarenal gland, liver, heart and brain. VEGF-A consists of 8 exons and 7 introns, it can be devided into different subtypes according to the activity. Among VEGF-A, the activity of 165 is the best. Study showed that the neuron death decreased, the onset of symptom prolonged and survival increased in SOD-1 G93 A mice with high expression of VEGF-A compare with that of the single SOD-1 G93 A mice. Now, the therapeutic effect of VEGF-A 165 on the model of NSC34 cell transfected with TDP25 is unclear.Objective: To research the protection of VEGF-A 165 to the model of NSC34 cell transfected with TDP25 and the underlying mechanism.Methods: Flow cytometry was used to elect the NSC34 cells transfected with TDP25. TDP-25 transfected NSC34 cells were resuscitated, passaged and cultivated. Then the total protein was extracted and the TDP25 protein level was detected by western blot. The selected NSC34 cells were verified to be transfected with TDP25 plasmid and the cell model of ALS was set up. Various concentration of VEGF-A 165 were used to TDP-25 transfected NSC34 cell. The optimal concentration of VEGF-A 165 was determined by CCK8 detection. The optimal concentration of VEGF-A 165 was administrated to the NSC34 cell transfected with TDP25. The levels of LDH,MDA were detected by LDH kit and MDA kit. Then caspase3 in apoptosis pathway and HO-1 in oxidative stress pathway were measured by western blot in order to investigate the pathway of VEGF-A 165.Results:1 The expression of TDP25 protein was verified by western blot in the NSC34 cell screened by flow cytometry.It was indicating that the selected cell was NSC34 cell transfected with TDP-25, so the cell model of ALS was set up.2 The NSC34 cell situation and cell vitality by CCK8 were observed after administrated with various concentration of VEGF-A 165. Results showed that the cell count, process and situation were the best when the concentration of VEGF-A 165 was 5ng/ml, So the optimal concentration of VEGF-A 165 was determined to be 5ng/ml.3 After administration with optimal concentration of VEGF-A 165 to NSC34 cell, the level of LDH and MDA by LDH and MDA kits decreased compared with control(P<0.05).4 After administration with optimal concentration of VEGF-A 165 to NSC34 cell, the expression of caspase 3 and HO-1 by western blot decreased significantly compared with control(P <0.01).Conclusion: VEGF-A 165 can protect the TDP-25 transfected NSC34 cell by inhibiting the expression of caspase 3 and HO-1.