Rther reduce Evans blue leakage in gp91phox KO mice. *p < 0.05 versus NA WT group, n = 7.Transmembrane protein occludin and claudins are the key molecules forming the seal between adjacent endothelial cells of the BBB. Using gp91 phox knockout mice, we tested the effect of gp91phox containing NADPH oxidase on tight junction protein occludin and claudin-5. Consistent with our results obtained from ischemic stroke rats [4], ischemia and reperfusion induced a reduction in occludin protein, but not claudin-5, in wildtype mice, and NBO treatment significantly reversed this reduction (Figure 5). Considering the fact that occludin is substrate of MMP-9 and reduced MMP-9 6-Methoxybaicalein clinical trials induction in the ischemic brain of gp91phox knockout mice (Figure 4), we speculated that gp91 phox knockout could lead to a reduction in occludin loss in ischemic brain tissue. Indeed, 90-min MCAO with 22.5 hrs of reperfusion induced occludin degradation to much less degree in gp91phox knockout mice than normoxic PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26240184 wild-type mice (Figure 5). Similar to their effect on MMP-9 induction (Figure 4), the combination of NBO and gp91phox knockout led to a further, but not significant, reduction in occludin protein loss in the ischemic tissue compared to each manipulation alone (Figure 5). No significant effects were observed for NBO or gp91phox knockout on claudin-5 protein. These results suggest that gp91phox containing NADPH oxidase is implicated in occludin degradation in the ischemic brain.Discussion Using gp91phox knockout mice, the present study unambiguously demonstrates that Nox2-containing NADPHLiu et al. Medical Gas Research 2011, 1:22 http://www.medicalgasresearch.com/content/1/1/Page 5 ofFigure 4 NBO treatment or gp91phox knock-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 gp91phox knock-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 gp91phox KO. The combination of NBO and gp91phox KO 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. b-actin served as a protein loading control. Bottom panel: the relative quantity of MMP-9 protein was calculated after normalization to b-actin. *p < 0.05 versus Non-I, n = 6; #p < 0.05 versus NA-WT, n = 6.Liu et al. Medical Gas Research 2011, 1:22 http://www.medicalgasresearch.com/content/1/1/Page 6 ofFigure 5 NBO treatment or gp91 phox knock-out significantly reduces occludin degradation in ischemic brain tissue after 90min MCAO with 22.5 hrs of reperfusion. Hemispheric brain tissue was homogenized for analyzing tigh.