Effect of 5-fluorouracil on lipopolysaccharide-induced apoptosis of macrophages in vitro

Objective To investigate the effects of 5-fluorouracil (5-Fu) on lipopolysacchaxide (LPS)-induced apoptosis of abdominal macrophages in rats and the possible mechanism.Methods Abdominal macrophages extracted from SD rats were identified,their vitality was estimated.The abdominal macrophages in rats were stimulated with LPS (1 mg/L) to establish inflammatory model in vitro.Macrophages were randomized into blank control group,LPS group and LPS (different concentrations) + 5-Fu group.Cell counting kit-8 (CCK-8) was used to test cell viability.Hoechst dyeing was introduced to observe cell apoptosis,and cysteinyl aspartate-specific protease (Caspase)-3 kit was applied to measure Caspase-3 activity.The expression of B lymphocytes/leukemia-2 (bcl-2),bax and p53 genes was detected by using real-time polymerase chain reaction (real-time PCR),and that of bcl-2,bax,p-Caspase-3 and p53/p-p53 proteins by using Western blotting.Results Abdominal macrophages were successfully extracted from rats.CCK-8 revealed that 5-Fu could promote apoptosis of LPS-stimulated macrophages (0.749 ±0.011,0.694 ±0.009,0.549 ±0.009,0.525±0.010,0.441 ±0.014,0.336 ±0.012 vs.1.036±0.47,P ＜ 0.05).Hoechst dyeing exhibited that 5-Fu could increase the apoptosis of cells activated with LPS.The expression of Caspase-3 mRNA and protein in LPS + 5-Fu groups was higher than that in LPS group (12.29 ± 0.34,12.75 ± 0.44,15.25 ± 0.58,17.01 ± 0.54 vs 10.92 ± 0.24,P ＜ 0.05).The expression of bax and p53 mRNA and protein was significantly higher in LPS + 5-Fu groups than that in LPS group (38.32±1.70,63.24 ±4.82,73.83 ±5.79,166.00 ±7.00 vs 3.19 ±0.29,P＜0.05,and 3.60 ±0.18,5.47 ± 0.25,6.30 ± 0.13,8.49 ± 0.38 vs 3.16 ± 0.06,P ＜ 0.05),and the expression of bcl-2 mRNA and protein was the opposite (1.99 ± 0.11,1.14 ± 0.11,0.76 ± 0.04,0.37 ± 0.05 vs 4.14 ±0.27,P ＜ 0.05).Conclusion 5-Fu could obviously accelerate the apoptosis of LPS-stimulated macrophages,which might be related to the p53 pathway by regulating the activation of Caspase-3,bcl-2 and bax. Key words: 5-fluorouracil ;  Lipopolysaccharide;  Macrophages;  Apoptosis