Figure 4

NBO treatment or gp91^phoxknock-out significantly reduces MMP-9 induction in ischemic brain tissue after 90-min MCAO with 22.5 hrs of reperfusion. A)Hemispheric brain tissue was homogenized for analyzing MMP-2 and 9 levels with gel gelatin zymography. Upper panel: representative gelatin zymograms showing the expression of proforms of MMP-2 and 9 in Nonischemic (Non-I) and ischemic (I) brain tissues in normoxic wild-type (NA-WT), NBO-treated WT (NBO-WT), normoxic gp91^phoxknock-out (NA-KO) and NBO-KO mice. STD is a mixture of human standard MMP-2 and 9. Bottom panels: the band intensities of MMP-2 and 9 were quantified. MMP-9 was significantly induced in the ischemic brain of NA-WT mice, and this induction was significantly inhibited by NBO or gp91^phoxKO. The combination of NBO and gp91^phoxKO led to a further, but not significant, reduction in MMP-9 compared each modulation alone (Left bottom panel). No significant changes were observed in MMP-2 for all groups (Right bottom panel). *p < 0.05 versus Non-I, n = 6; ^#p < 0.05 versus NA-WT, n = 6. B)Hemispheric brain tissue was homogenized for analyzing MMP-9 protein level with western blot. Upper panel: representative blots of MMP-9 protein in hemispheric brain tissue obtained from NA-WT, NBO-WT, NA-KO and NBO-KO mice. β-actin served as a protein loading control. Bottom panel: the relative quantity of MMP-9 protein was calculated after normalization to β-actin. *p < 0.05 versus Non-I, n = 6; ^#p < 0.05 versus NA-WT, n = 6.