HUVECs were cultured in a glass culture chamber slide and fixed for 30 min in 10% neutral buffered formalin solution at room temperature. A TUNEL assay system was used, according to the manufacturer’s instructions, for examination

under a fluorescence microscope, with excitation Selleckchem INK128 at 488 nm and emission at 525 nm [26]. Cell death was detected by annexin V–fluorescein isothiocyanate (FITC) (BD PharMingen, San Diego, CA, USA) and propidium iodide (PI) staining of necrotic and apoptotic cells. Cells were washed in PBS, resuspended in 100 μL binding buffer containing 5 μL annexin V–FITC and 1 μg/mL PI, and incubated for 10 min at room temperature in the dark. Cells were analyzed using a FACScan (Becton Dickinson). Data were analyzed using CELLQuest software (Becton Dickinson). Positioning of quadrants on the Annexin V/PI dot

plots was performed as previously described [25]. Data were expressed as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance (GraphPad Prism version 4; GraphPad Software, San Diego, CA, USA) followed by Bonferroni’s multiple comparison test. Upregulation of COX-2 expression plays a key role in inflammation. A previous study found that acrolein in CS induces COX-2 expression in human endothelial cells [24]. We demonstrated the effect of KRG on COX-2 induction in acrolein-stimulated HUVECs. KRG inhibited acrolein-induced COX-2 protein expression in a concentration-dependent manner (Fig. 1A). Selleckchem GSK-3 inhibitor KRG also inhibited the COX2 mRNA level ( Fig. 1B). After pretreatment of acrolein-stimulated cells with KRG, the cells were fixed, and COX-2 localization in HUVECs was observed by immunofluorescence staining with an anti-COX-2 antibody followed by a fluorescence-tagged Methocarbamol secondary antibody. Immunofluorescence analysis showed that acrolein-induced COX-2 protein levels were inhibited in HUVECs after treatment with KRG (Fig. 1C). The induction of COX-2 expression is known to be responsible for PGE2 release in the culture medium of cells stimulated with acrolein. Acrolein increased PGE2 secretion, which was dramatically reduced by KRG (Fig. 2).

This result indicates that KRG leads to the reduction of COX-2 protein expression and subsequently PGE2 biosynthesis in acrolein-stimulated HUVECs. Elevation of intracellular ROS levels causes cellular dysfunction. Thus, we examined the effect of KRG on ROS production in acrolein-stimulated cells. The shift to the right of the curve due to increased fluorescence indicates an increase in the intracellular levels of ROS. The results indicate that ROS generation in cells treated with acrolein increased compared to untreated cells, whereas KRG inhibited acrolein-induced ROS generation (Fig. 3A and B). These results indicate that KRG may play a role in the inhibition of COX-2 expression via reduction of acrolein-generated ROS in acrolein-stimulated HUVECs.