Results shown are representative of three separate experiments E

Results shown are representative of three separate experiments. Expression of IL-8 mRNA was quantified by densitometry, and standardized by the β-actin level. *p < 0.05, **p < 0.01 compared with the level at 1 h or 2 h. PMA: phorbol 12-myristate 13-acetate. Induction of IL-8 release by PCN learn more in PMA-differentiated U937 cells Previous studies have identified that PCN stimulates IL-8 production by lung macrophage cells [23] and surface epithelial cells [8, 14, 24]. Based on the physical properties of PCN, we hypothesized that

it was able to stimulate differentiated U937 cells to produce IL-8. To test this hypothesis, we exposed differentiated human U937 cells to purified PCN and measured its effects on the release of IL-8. After 24 hours Selleck CUDC-907 of incubation with different concentrations of PCN (5 μM, 25 μM, or 50 μM) in PMA-differentiated U937cells, the supernatants were collected and IL-8 release detected by ELISA. The results showed that PCN increased IL-8 release in differentiated U937 cells in a concentration-dependent manner. An increase in IL-8 release was observed with PCN concentration at as low as 5 μM and the concentration of 50 μM produced the strongest stimulation as to the cellular response (Figure 2A and B). The increase in

IL-8 above SGC-CBP30 cost control levels was observed at as early as 8 h after PCN (50 μM) addition, and these levels continued to increase between 24 h and 48 h (data not shown). Longer periods of incubation were not tested. Figure 2 PCN increases IL-8 release in PMA-differentiated U937 cells. (A) Different concentrations of PCN (5 μM, 25 μM, or 50 μM) were added to the cell cultures for 24 h. Supernatants were harvested for measuring IL-8 secretion by ELISA. (B) A fixed concentration of PCN (50 μM) was added to the cell cultures PAK5 for 8, 16 or 24 h. Supernatants

were harvested for measuring IL-8 level by ELISA. Values represented are the mean ± SD of four independent experiments in triplicate. **p < 0.01 compared with PMA-differentiated U937 cells. PMA: phorbol 12-myristate 13-acetate. The oxidative effect of PCN on differentiated U937 cells A previous study has shown that PCN induces a concentration-dependent loss of cellular glutathione (GSH), an important cellular antioxidant, up to 50% in the tissues infected by P. aeruginosa [25]. N-acetyl cysteine (NAC) is the precursor of GSH. So we hypothesized that NAC may play a protective role in cells exposed to PCN. Thus, different concentrations of PCN (5, 25, and 50 μM) were added into differentiated U937 cells, and the supernatants were collected after 24 hours. We then detected the leakage of LDH, the content of MDA, and the activities of SOD and CAT using their respective detection kits.

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