Proteome Alterations Of SH-SY5Y Cells Treated With Valproic Acid

Valproic acid (VPA) is a short-chain fatty acid of histone deacetylases (HDACs) inhibitor family, whose molecular structure is simple, has been used for the treatment of epilepsy and bipolar disorder and by which epilepsy, manic and depressive symptoms can be effectively controlled. The side effects of VPA are relatively small, and VPA has a specificity of HDAC inhibition. Moreover, the bioavailability of VPA is higher than other types of HDACs. Routine clinical oral application of VPA is able to achieve an effective plasma concentration with good toleration. Recent studies have found that VPA has a protective role for the neurons, but the neuroprotection mechanism of VPA is not fully clear.The SH-SY5Y cells present many neuronal biochemical and functional characteristics, and are widely used in the research of neurological pathogenesis and mechanism of drug action. The clinical application of VPA is relatively extensive, and the molecular mechanisms of VPA actions may be various. The mechanism of VPA, particularly neuroprotective mechanism, are very difficult to clarify. Proteomics may be a powerful tool to help us to elucidate the neuroprotective mechanism of VPA. It may reveal the neuroprotective mechanism of VPA and may find the potential target for neurodegenerative therapies. This study may provide valuable new clues for the mechanisms of VPA and for the clinical application of VPA in neurodegenerative diseases.Objective:In this study, human SH-SY5Y neuroblastoma cells were used as a neuronal model to investigate the neuroprotective effects of VPA on changes of proteins of SH-SY5Y cells. Proteomic and Western blot methods were used to find and verify the mark/key proteins participated in the the neuroprotective effects of VPA. The aim of this study is to futher clarify the complex mechanisms of neuroprotection of VPA and provide more valuable clues for elucidating the neuroprotective mechanism of VPA and for the clinical applications of VPA in neurodegenerative diseases.Methods:SH-SY5Y cells were cultured with normal media at the same time and condition. Then each of these groups was treated with different concentrations of VPA (0.0, 0.25,0.5,1.0,2.0 and 4.0mM) for different time (12,24, and 48h), respectively. The dynamic morphological changes of SH-SY5Y cells were observated by the inverted optical microscope. Cell viability was calculated by the method of MTT assay, and cell apoptosis was observed via AO-EB staining method. Quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) and 2D-Western Blot analysis were used to determine and verify the changes of protein levels in SH-SY5Y cells.Results:Cell viability, proliferation and survival rate of the groups at lower concentrations (0.25,0.5 and 1.0mM) of VPA were higher than those in the control group, the cell viability of the group treated by 1.OmM of VPA for 24 hours is maximum, and higher concentrations (2.0 and 4.OmM) of VPA decreased the cell viability, proliferation rate and cell survival percentage. In addition, after treatment with different concentrations (0.25,0.5,1.0, 2.0 and 4.0mM) of VPA for 36 hours, the cell viability declined in a concentration-dependent manner in SH-SY5Y cells. The lower concentrations of VPA played a beneficial role on cell proliferation and nutrition, while higher concentrations of VPA and longer time induced cell damage. AO-EB staining showed apoptosis rates as follows:In the groups treated for 24 hours, apoptosis rate of normal control group reached to 4.2±0.9%, apoptosis rate of experimental group treated by 1.0mM VPA reached to 3.3±0.5%(compared with normal controls group P> 0.05), and apoptosis rate of experimental group treated by 4.OmM VPA reached to 19.3%±1.3%(compared with the normal control group P<0.05). In the groups treated for 36 hours, apoptosis rate of normal control group reached to 5.1±1.1%, apoptosis rate of experimental group treated by 1.0mM VPA reached to 7.4%±0.3%(compared with the normal control group P>0.05), and apoptosis rate of experimental group treated by 4.OmM VPA reached to 32.6%±1.7%(compared with the normal control group P<0.05).After VPA-induced SH-SY5Y cell neuroprotective model was built, a comparative proteomic approach (2D-DIGE coupled with MALDI-ToF MS) was performed to identify differentially expressed proteins in this model. Via comparing VPA-treated SH-SY5Y cells to controls, a total of 15 proteins displayed at least a 1.3-fold changes in relative abundance, including 11 proteins upregulated and 4 proteins downregulated. Among these 15 protein spots, three proteins were successfully identified as eukaryotic translation initiation factor 4A isoform 1 (eIF4A1), heterogeneous nuclear ribonucleoprotein K (hnRNP K) and ATP6V1B2 protein, which are all associated with oxidative stress. Changes in these three proteins further suggest that anti-oxidative stress may play an important role in neuroprotective mechanisms of VPA in SH-SY5Y cells.Conclusion:The results of this study show that oxidative stress and anti-ovidative stress pathways may be associated with the neuroprotective effects of VPA on SH-SY5Y cells. Significantly altered expression of three proteins in SH-SY5Y cells treated with VPA was first found and reported.Bioinformatics analysis showed that the neuroprotective mechanism of VPA may be involved in anti-oxidative stress and present studies provided new valuable clues for further clinical application of VPA as a neuroprotective drug.