N-acetylcysteine attenuates TNF-α-induced human vascular endothelial cell apoptosis and restores eNOS expression

• DOI: 10.1016/j.ejphar.2006.08.044
• Publication Date: 2006-09-08T19:25:10Z
• Authors: Zhengyuan Xia; Min Liu; Yong Wu; Vijay Sharma; Tao Luo; Jingping Ouyang; John H. McNeill
• Keywords: Glutathione Peroxidase; 0303 health sciences; L-Lactate Dehydrogenase; Nitric Oxide Synthase Type III; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Blotting, Western; Endothelial Cells; Nitric Oxide Synthase Type II; Tetrazolium Salts; Apoptosis; Flow Cytometry; Nitric Oxide; Glutathione; Acetylcysteine; Thiazoles; 03 medical and health sciences; Malondialdehyde; In Situ Nick-End Labeling; Humans; Endothelium, Vascular; Cells, Cultured

The circulatory inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is increased in pathological conditions, such as diabetes, which initiate or exacerbate vascular endothelial injury. Both nitric oxide (NO) and reactive oxygen species may play a dual role (i.e., inhibiting or promoting) in TNF-alpha-induced endothelial cell apoptosis. We investigated the effects of the antioxidant N-acetylcysteine on TNF-alpha-induced apoptosis in human vascular endothelial cell (cell line ECV304) apoptosis, NO production and lipid peroxidation. Cultured vascular endothelial cell (ECV304) were either not treated (control), or treated with TNF-alpha (40 ng/ml) alone or TNF-alpha in the presence of N-acetylcysteine at 30 mmol/l or 1 mmol/l, respectively, for 24 h. Cell viability was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was assessed by flow cytometry. TNF-alpha-induced endothelial cell apoptosis was associated with increased inducible NO synthase but reduced endothelial NO synthase (eNOS) protein expression. NO production and the levels of the lipid peroxidation product malondialdehyde were concomitantly increased. Treatment with NAC at 30 mmol/l restored eNOS expression and further increased NO production as compared to TNF-alpha alone, resulting in improved cell viability and reduced apoptosis. This was accompanied by increased superoxide dismutase activity, increased glutathione peroxidase production and reduced malondialdehyde levels. N-acetylcysteine at 1 mmol/l, however, did not have significant effects on TNF-alpha-induced endothelial cell apoptosis and cell viability despite it slightly enhanced glutathione peroxidase production. N-acetylcysteine attenuation of TNF-alpha-induced human vascular endothelial cell apoptosis is associated with the restoration of eNOS expression.