BAY11-7082 treatment suppresses the severity of EAN in mice
BAY11-7082 was applied by two different patterns to treat EAN mice (Fig. 3a). For the preventative group, BAY11-7082 was administered from immunized day to the onset of clinical signs of EAN (day 8 p.i). For the therapeutic group, the same dose of BAY11-7082 was administered from the onset of clinical signs to the peak phase. Our results showed that the scores of severity of EAN were decreased in both treated groups with BAY11-7082 compared to control EAN groups. The clinical scores of preventative group were more significant. However, the onset of EAN was delayed in the preventative group, and the duration of symptoms was shorter than both therapeutic and control groups. (Figure 3a).
According to the results of Western blotting, the expression of p-NF-κB p65 was inhabited in the preventative group treated by BAY11-7082 compared to the control group. (Figure 3b).
BAY11-7082 promotes M2 macrophagepolarization in EAN mice
The proportion of M1 and M2 cells in spleens of EAN mice received BAY11-7082 in treatment/preventative groups was investigated via flow cytometry analysis. The results indicated that the proportion of M2 was elevated significantly in the preventative group compared to the control group (P﹤0.01). Interestingly, the proportion of M1 was reduced significantly in the preventative group compared to the control group (P﹤0.05), suggesting that BAY11-7082 is able to promote macrophage polarization to M2 type (Fig. 3c).
To explore the mechanisms behind the effects of BAY11-7082 suppressing EAN, M1 and M2 macrophages were cultured in vitro and half of them were added by BAY11-7082 as experimental group. The activity and proliferation of M1 and M2 macrophages were evaluated by MTT method. The results showed that the BAY11-7082 inhibited the activity and proliferation of M1 macrophages significantly compared to the control cultures without BAY11-7082 ( (P﹤0.001), whereas, the BAY11-7082 upregulated the activity and proliferation of M2 in spite of the differences is not significant compared to control culture group.(Fig. 4d-e)