Probucol reverses homocysteine induced inflammatory monocytes differentiation and oxidative stress

Reactive oxygen species have been demonstrated to involve in homocysteine-induced Ly-6C^hi monocytes differentiation. Probucol is an anti-oxidant agent that has been used to treat atherosclerosis. We sought to evaluate the effect and potential mechanism of probucol on homocysteine-induced inflammatory monocytes differentiation. The primary mouse splenocytes suspensions were initiated by recombinant interferon-γ and cultured with L-homocysteine in the presence or absence of probucol. The cells were co-incubated with monoclonal antibodies to CD11b-PE and Ly-6C FITC. Flow cytometry analysis was performed on BD FACS caliber. Data were analyzed using the FlowJo software. Mononuclear cells were gated according to the lower granular and larger size, distinguished with granulocytes and lymphocytes. Monocytes were defined as CD11b⁺ mononuclear cells and further divided into three groups based on their Ly-6C expressions, Ly-6C^hi, Ly-6C^mid and Ly-6C^low subsets. The productions of reactive oxygen species in monocytes subsets were detected by 2',7'-dichlorofluorescein-diacetate (DCFH-DA) containing monocytes were marked as DCFH-DA⁺ cells in both Ly-6C⁺ and Ly-6C^- subsets. The activity of nicotinamide adenine dinucleotide phosphate oxidase in THP-1 cells was measured by assay kit on enzyme-labelling instrument. L-homocysteine promoted inflammatory monocytes differentiation and its reactive oxygen species productions in dose-dependent manner. Probucol dose-dependently suppressed the differentiation and reactive oxygen species productions of inflammatory monocytes induced by L-homocysteine. Furthermore, the increased NADPH oxidase activity induced by L-homocysteine was significantly reversed by probucol in THP-1 cells. Probucol prevented L-homocysteine-induced inflammatory monocytes differentiation and its reactive oxygen species generation probably through inhibiting NADPH oxidase activity.