The novel prolyl hydroxylase-2 inhibitor caffeic acid upregulates hypoxia inducible factor and protects against hypoxia

Background & aims: Hypoxia inducible factor (HIF) is a hypoxia-associated transcription factor that has a pro-tective role against hypoxia-induced damage. Prolyl hydroxylase-2 (PHD2) is a dioxygenase enzyme that spe-cifically hydroxylates HIF targeting it for degradation, therefore, inhibition of the PHD2 enzyme activity acts to upregulate HIF function. This study was to identify novel PHD2 inhibitors.Methods: An established fluorescence-based PHD2 activity assay was used for inhibitors screening. Western blot and quantitative real-time PCR was used to detect the protein and mRNA levels respectively. Further animal experiment was carried out.Results: Caffeic acid was screened and identified as a novel PHD2 inhibitor. Caffeic acid treated PC12 and SH-SY5Y neuronal cell lines stabilized endogenous HIF-1 alpha protein levels and consequently increased mRNA levels of its downstream regulated genes VEGF and EPO. Caffeic acid treatment reduced hypoxia-induced cell apoptosis and promoted HIF/BNIP3-mediated mitophagy. Moreover, animal studies indicated that caffeic acid increased the level of HIF-1 alpha protein and mRNA levels of VEGF and EPO in the brain of mice exposed to hypoxia. Con-ventional brain injury markers including malondialdehyde, lactic acid and lactate dehydrogenase in the caffeic acid treated mice were shown to be reduced to the levels of the control group.Conclusions: This study suggests that caffeic acid inhibits PHD2 enzyme activity which then activates the hypoxia-associated transcription factor HIF leading to a neuroprotective effect against hypoxia.