Figure 2.

Effect of Agm on two main enzymes of arginine metabolism: iNOS and ARG1 and markers of a microglial activation state. BV-2 cells were pretreated with Agm (100 µm) for 30 min and then stimulated with Lps (1 µg/mL) for an additional 4 or 6 h (qPCR analysis) or 24 h (Western blot analysis and fluorescent microscopy). Data represent gene expressions, protein levels, and immunofluorescence of iNOS (a), ARG1 (b), COX-2 (c), and CD206 (d). Levels of target genes (Nos2, Arg1, Ptgs2, and Mrc1) are expressed relative to the expression of the Gapdh gene. The ratios of iNOS, ARG1, COX2, and CD206 to β-actin are expressed relative to the control group (fold change). Data are presented as mean ± SEM, from three separate determinations. The results were analyzed by Kruskal–Wallis test followed by Dunn’s multiple comparisons tests (qPCR) or by two-way ANOVA followed by Bonferroni’s post hoc tests (Western blot). iNOS protein level was analyzed by t test for comparing the mean values between two groups (Lps vs. LpsAgm) because Ctrl and Agm groups were not detectable. * p < 0.05 compared with the control group; # compared with Lps group. Photomicrographs on the right represent immunofluorescence labeling against target protein (red) counterstained with Hoechst (blue). If not otherwise specified, in this and following figures experimental groups are: Ctrl—untreated control cells; Agm—agmatine sulfate (100 µm) treated cells; Lps—cells stimulated with 1 µg/mL Lps; LpsAgm—cells pretreated with 100 µm Agm and then stimulated with Lps. Plotted scale bar applies to all micrographs.