Induction of hemeoxygenase-1 expression after inhibition of hemeoxygenase activity promotes inflammation and worsens ischemic brain damage in mice

• Ischemia down-modulates HO-1 gene repressor Bach-1 and induces HO-1 expression.
• HO inhibitor ZnPP induces HO-1 in brain vessels 24 h after systemic administration.
• Treatment with ZnPP 24 h before ischemia exacerbates inflammation and brain damage.
• Treatment with ZnPP 30 min before ischemia does not increase infarct volume.
• A detrimental action of HO-1 in the absence of HO activity is proposed.

Hemeoxygenase (HO) is an enzymatic system that degrades heme. HO-1 is an inducible isoform whereas HO-2 is constitutive. Stroke strongly induces HO-1 expression but the underlying mechanisms are not fully elucidated. Cytokines that are up-regulated after ischemia, like interleukin (IL)-10, can induce HO-1 gene expression, which is positively regulated by the transcriptional activator nuclear factor erythroid 2-related factor 2 (Nrf2) and negatively regulated by the transcriptional repressor breast cancer type 1 susceptibility protein (BRCA1) associated C-terminal helicase 1 (Bach-1). While Nrf2 is activated after ischemia and drugs promoting Nrf2 activation increase HO-1 and are beneficial, the involvement of Bach-1 is unknown. Here we investigated mechanisms involved in HO-1 induction and evaluated the effects of HO activity inhibition in mouse permanent middle cerebral artery occlusion (pMCAO). HO-1 was induced after ischemia in IL-10-deficient mice suggesting that post-ischemic HO-1 induction was IL-10-independent. Attenuation of Bach-1 gene repression after ischemia was associated to enhanced HO-1 induction. Administration of the HO activity inhibitor zinc proto-porphyrin IX (ZnPP) i.p. 24 h before pMCAO exacerbated ischemia-induced tumor necrosis factor-α (TNF-α) and IL-1β, nitro-oxidative stress, and the presence of neutrophils at 8 h, and increased infarct volume at day 4. However, ZnPP did not worsen ischemic damage when given 30 min before pMCAO. ZnPP induced HO-1 expression in the cerebral vasculature at 24 h, when it was still detected by high-performance liquid chromatography (HPLC) in plasma. While ZnPP was not found in brain tissue extracts of controls, it could be detected after ischemia, supporting that a small fraction of the injected drug can reach the tissue following blood–brain barrier breakdown. The deleterious effect of inhibiting HO activity in ischemia became apparent in the presence of ZnPP-induced HO-1, which is known to exert effects independent of its enzymatic activity. In conclusion, HO-1 induction after ischemia was associated to down-regulation of transcriptional repressor Bach-1, and induction of HO-1 when HO enzymatic activity was inhibited was related to worst outcome after brain ischemia.