Figure 8.

Involvement of NO in the LCN2 effects in astrocytes. After primary astrocytes were treated with SNP (0.5 mm) for 24 h, GFAP/vimentin mRNA or GFAP protein was detected by RT-PCR (A) or Western blot analysis (B), respectively. The expression of GFAP was significantly increased by SNP (0.5 mm) in primary astrocytes. Primary astrocytes were stimulated with recombinant LCN2 protein (10 μg/ml), LPS (100 ng/ml), IFN-γ (50 U/ml), or a combination of LPS (100 ng/ml) and IFN-γ (50 U/ml) for 24 h, and then nitrite production was assessed by Griess reagent (C). *p < 0.05 compared with no treatment. Primary astrocytes were also pretreated with polymyxin B (PB) (10 μg/ml) for 30 min before the treatment with LCN2 protein (10 μg/ml), GST protein (10 μg/ml) (D), or a combination of LPS (100 ng/ml) and IFN-γ (50 U/ml) (E) for 24 h, and then nitrite production was assessed by Griess reagent (D, E). *p < 0.05 compared with untreated control (C, D) or as indicated (E). Alternatively, primary astrocytes were treated with LCN2 protein (10 μg/ml) for 24 h or pretreated with NMMA (500 μm) for 30 min before the treatment with recombinant LCN2 protein (10 μg/ml) for 24 h. The expression of GFAP or vimentin was then detected by RT-PCR (F) or Western blot analysis (G) as indicated. After a similar treatment of primary astrocytes with LCN2 and NMMA in the presence of various cytotoxic agents [H₂O₂ (1 mm), paraquat (PQ) (100 μm), SNP (0.5 mm), and LPS (100 ng/ml) plus IFN-γ (50 U/ml) for 24 h], cell viability was assessed by MTT assay (H). NMMA (500 μm) alone did not exert any cytotoxicity (data not shown). Error bars indicate SD. The single asterisks indicate statistically significant differences between treatments with cytotoxic agents in the absence and presence of LCN2 (*p < 0.05; None vs LCN2; comparison 1). The double asterisks indicate statistically significant differences between treatments with cytotoxic agents in the presence of LCN2 and NMMA plus LCN2 (**p < 0.05; LCN2 vs NMMA plus LCN2; comparison 2).