A Quantitative Proteomic Analysis to Reveal Effects of N-acetylcysteine on H2O2-induced Cytotoxicity

Abstract

Aims: This study aimed to perform a quantitative analysis of the proteomic changes in neural cells under H2O2 exposure and N-acetylcysteine (NAC) treatment. Background: NAC is a potent antioxidant and an effective free radical scavenger that has been used as a potential treatment of several neurological disorders. Although the molecular mechanisms of neuroprotective action by NAC have been studied, more efforts are still needed for the elucidation of the mechanisms through a quantitative proteomic analysis. Objective: This study aimed to identify differentially expressed proteins among control, H2O2-treated group, and NAC+H2O2-treated group as well as reveal proteins involved in the protection of neural cells from H2O2-induced toxicity. Methods: SK-N-MC cells were untreated (control), treated with H2O2 (disease group), pretreated with NAC, and then treated with H2O2 (NAC group). Proteins were digested to peptides and analyzed using liquid chromatography-tandem mass spectrometry with the data-independent acquisition. Skyline was used to quantify peptides and proteins. MSstats was used for statistical analysis. Gene ontology and protein-protein interactions were performed using the Differentially Expressed Proteins (DEPs). Results: Cytoprotective effects of NAC on the cell against H2O2-induced toxicity were first proven using a cell viability study and lactate dehydrogenase assay. The proteomic analysis found 93 DEPs in three comparisons. Among them, 37 proteins were differentially expressed under H2O2 exposure. Only 10 DEPs were rescued in the case of NAC pretreatment. Aspartate aminotransferase and L-lactate dehydrogenase B chain were two DEPs involved in the cysteine and methionine metabolism pathway, which might relate to the mechanisms of NAC protective effects. Conclusion: The findings of cell studies and proteomic analysis were in agreement with previous results, confirming the cytoprotective effects of NAC on neural cells against oxidative stress.