Proteomic Studies Of SH-SY5Y Cells Treated By Resveratrol

Resveratrol (3,4',5-trans-trihydroxystilbene) is a polyphenol phytoalexin. This compound has been shown to have a wide range of biochemical and physiological properties, including antiplatelet aggregation, anti-inflammatory actions, antitumor activity and antioxidant functions, which means resveratrol may have protective effect of myocardial preservation, anti-cancer, anti-inflammation and neuroprotection. So it have many health benefits, such as its actions of protecting heart and nerves, antitumor, anti-inflammation, etc. Althought there is growing concern about the neuroprotective effect of resveratrol, the neuroprotective mechanisms of resveratrol are not completely understood.In this study, we used 2D-DIGE to identify the different protein levels in SH-SY5Y cells treated by resveratrol. These results provides a new clue to explore the neuroprotective effect of resveratrol.Objective:To observe the effect of different-dose resveratrol on SH-SY5Y cells, and the dose-dependence of resveratrol was preliminarily discussed. Furthermore, SH-SY5Y cells were treated by resveratrol (25μM) for 24 hours, to study the differential proteomic changes in SH-SY5Y cells treated by resveratrol, and to clarifiy the neuroprotective effect of Resveratrol.Methods:SH-SY5Y cells were treated respectively with different concentrations of resveratrol for 24 hours (5μM,10μM,25μM,50μM,100μM), cell morphology was observed under inverse microscopy, and the viability of resveratrol-treated SH-SY5Y cells were calculated by MTT assay, cell apoptosis was observed via AO/EB staining. Then SH-SY5Y cells were treated by resveratrol (25μM) for 24 hours, Quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to determine the changing protein levels. Western Blot was used to test the expression of the protein (Erol-La), which MS had identified. Results:SH-SY5Y cells were treated with resveratrol of different concentrations for 24 hours, the low-dose group (5μM,10μM,25μM) showed no obvious morphological changes, and the SH-SY5Y cells in high-dose group became round significantly, and the dendrites disappeared, which showed apoptotic morphology. MTT assay showed that, with the increase of resveratrol concentration, the cell vitality was decreased gradually. Comparison of cell viability, high- dose group and low- dose group had significant differences. Cells in the low-dose group were showed no apoptotsis via AO/EB staining, but the high-dose group showed a large number of apoptotic cells.A comparative proteomic approach (2D-DIGE coupled with MALDI-ToF MS) was performed to identify differentially expressed proteins in Resveratrol-treated SH-SY5Y cells (25 uM), a total of 22 proteins displayed at least a 1.3-fold change in relative abundance. Among with, five proteins were identified successfully as Erol-like protein alpha, (Erol-La), p21-activated kinase 1 (PAK1), ARCN1 protein, T-complex protein 1 (TCP-1), T cell receptor beta chain (TCR), which function are associated respectively with endoplasmic reticulum stress, signal transduction, vesicle transport, chaperone proteins and immune regulation. The results in present study provide further evidence for a role of oxidative stress protection, vesicle transport regulation and signal transduction regulation in the molecular mechanism of the neuroprotective effect of resveratrol. Western Blot was successfully validated the Erol-La protein identified by mass spectrometry.Conclusion:These results indicated that multiple adjustment mechanism may participate in the underlying mechanism of the neuroprotective effect ofresveratrol. In the present study, Erol-Lα,ARCN1 protein,T cell receptor beta chain were reported firstly in the SH-SY5Y cells treated by resveratrol. These results may provide new valuable clues for the further exploration of the mechanism of the neuroprotective effect of resveratrol and for the discovery of the new drug targets. In this study, mass spectrometry and immunology was used to determine the Erol-Lαprotein expression. The result also proved the reliability of mass spectrometry.