Blockade of the inhibitory effect of hemin on NO production Pretreatment of human astrocytes with SnPP significantly selleck chem ameliorated hemin mediated inhibition of IL 1b induced NO production, although SnPP did not fully restore the NO level when 20 uM hemin was used suggesting involvement of additional mechanism. Pretreatment with SnPP appeared to enhance IL 1b induced NO pro duction suggesting that SnPP itself had no effect on NO production, but rather had exerted inhibition on induci ble HO 1 and the constitutive HO 2. Blockade of the inhibitory effect of hemin on iNOS expression Hemin treatment inhibited IL 1b induced iNOS expression in human astrocytes. Further more, hemin induced HO 1 expression was further enhanced in the presence of IL 1b.

Inhibitors,Modulators,Libraries The con stitutively expressed HO 2 was minimally changed by hemin and IL 1b treatment while no effect on Inhibitors,Modulators,Libraries b actin expression was found. Pretreatment with the HO 1 inhibitor SnPP significantly reversed the inhibitory effects of hemin on IL 1b induced iNOS expression. As mentioned above, SnPP also enhanced IL 1b induced iNOS indicating that SnPP not only inhibited HO 1, but also may have relieved the inhibitory effect of endogenous components, e. g, HO 2, exerted upon iNOS. Overexpression of HO Inhibitors,Modulators,Libraries 1 inhibits iNOS expression To further investigate the role of HO 1 in iNOS expres sion, human astroyctes were transfected with a pLEX expression vector containing human HO 1 sequences under a CMV promoter for 72 h. The transfection effi ciency was approximately 30% in human primary astro cytes and this treatment was associated with expression of HO 1, while treatment with a blank sequence con taining vector was not.

In combination with IL 1b, HO 1 expression was further enhanced. IL 1b induced iNOS expression was markedly downre gulated by overexpression of HO 1, further demonstrat ing the inhibitory effect of HO 1 on iNOS expression. No effect on b actin expression was found. Immunocytochemical reaction of IL 1b induced HO 1 expression Although Inhibitors,Modulators,Libraries IL 1b treatment induced undetectable HO 1 expression by western blot, induction of HO 1 was detectable by immunocytochemical reaction, possibly due to different detection sensitivities between these methods. Involvement of p38 MAPK Because IL 1b is known to trigger activation of both p38 and ERK12 MAPK signaling pathways in human astrocytes, we studied the effects of specific inhibitors of p38 and ERK12 MAPK on NO production.

As shown in Fig. 7A, we found Inhibitors,Modulators,Libraries that NO production was dependent on p38 but not p4442 MAPK activation. The inactive inhibitor of p38 MAPK had no effect on NO production. Treatment with these inhibitors alone did not induce astrocyte toxicity by MTT or alamarBlue assay. Because hemin treatment more information inhibited IL 1b induced NO production, we investigated the effect of hemin on IL 1b induced p38 MAPK activation.