Fig. 8

NOX2/ET-1 axis mediates microglia activation switching to M1 pro-inflammatory phenotype via ERK1/2 pathway contributing to iBRB inflammatory injury. (A) Representative images of ET-1 stained retinal cryosections from WT and Nox2^−/− after Pressure 60 mmHg 24 h. Scale bar, 50 μm. (B) Analysis of ET-1 fluorescence intensity in the GCL and INL. Data are presented as the percent fluorescence intensity of the groups versus control-WT (n = 6 in each group). (C) Representative images of ET-1 stained retinal flat-mount and cryosections after H-IOP with 500µM gp91ds-tat treatment. Scale bar, 50 μm. (D) Analysis of ET-1 fluorescence intensity in the GCL and INL. Data are presented as the percent fluorescence intensity of the groups versus Veh-control (n = 6 in each group). (E) Retinal extracts from WT and Nox2^−/− mice under Pressure 60 mmHg and H-IOP retinas with 500µM gp91ds-tat treatment were assayed for Western blot analysis of ET-1. Representative Western blots are presented. The results obtained from Western blot are expressed as the ratio to β-actin from 4–6 independent experiments. (F) Messenger RNA expression of Et-1 in retinas after H-IOP and with 500µM gp91ds-tat treatment. n = 6 in each group. (G) Representative images of IB4 and ERK1/2 stained microglia cultured by 100nM ET-1 and 20µM MEK 20–24 h. Scale bar, 50 μm. (H) Microglia lysis were assayed for Western blot analysis of p-JNK, p-ERK1/2 and p-p38 MAPK. Representative Western blots are presented. The results obtained from Western blot are expressed as the ratio to β-actin from 3 independent experiments. Data are shown as mean ± SEM, one-way ANOVA with Tukey’s multiple comparisons, *p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001). (I) ELISA assay was performed to determine the cell protein of IL-6, IL-1β, and TNF-α (n = 8 in each group). Data are shown as mean ± SEM (unpaired t-test, ***p < 0.001, ****p < 0.0001)