Abstract
Low-dose aspirin therapy has become a standard in the treatment of cardiovascular diseases. Aspirin has been shown to inhibit atherosclerosis in mouse models. To determine the mechanisms by which aspirin might inhibit atherosclerosis, we incubated HEPG2 cells and rat primary hepatocytes with aspirin or salicylic acid and noted an increase in paraoxonase 1(PON1) activity in the medium, together with an induction of PON1 and apolipoprotein A-I (apoA-I) gene expression. Mice treated with aspirin also showed a 2-fold increase in plasma PON1 activity and a significant induction of both PON1 and apoA-I gene expression in the liver. The induction of the PON1 gene in cell culture was accompanied by an increase in arylhydrocarbon receptor (AhR) gene expression. Accordingly, aspirin treatment of AhR(-/-) animals failed to induce PON1 gene expression. We previously suggested that aspirin might be hydrolyzed by serum PON1, which could account for its short plasma half-life of 10 min. Taken together with the current studies, we suggest that the antiatherosclerotic effects of aspirin might be mediated by its hydrolytic product salicylate and that the induction of PON1 and apoA-I might be important in the cardioprotective effects of aspirin.
Highlights
Low-dose aspirin therapy has become a standard in the treatment of cardiovascular diseases
We evaluated HEPG2 cells and primary rat hepatocytes as in vitro models of hepatic cells, because liver is the major source for circulatory paraoxonase 1 (PON1)
The secreted PON1 arylesterase activity was measured in the medium from HEPG2 cells and primary rat hepatocytes treated with increasing concentrations of aspirin (Fig. 1A)
Summary
Chemicals All chemicals, such as aspirin, paraoxon, r-NPA, and salicylate were purchased from Sigma-Aldrich Chemical Co., Saint Louis, MO. PON1 short interfering RNA (siRNA) was purchased from Sigma-Aldrich Chemical Co. HEPG2 cells were purchased from the American Type Culture Collection, Manassas, VA. The cell-associated and plasma PON1 arylesterase activity was measured using 1 mM rNPA as substrate, as assessed from the formation of r-nitrophenol at 410 nm at 37°C [26]. Aliquots of 10 ml of medium or plasma were placed in microtiter plate wells in triplicate; the reaction was initiated by the addition of the substrate rNPA, to yield a final concentration of 1 mM in PBS buffer containing calcium and magnesium. The plate was read immediately to establish 0 time values, and the reactions were incubated at 37°C. PON activity was expressed as percent of control, where 100% corresponds to the PON1 activity in the control alcohol-treated cells
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