Anti-drug Reward Effects And Mechanisms Of Targeting Hypoxia-inducible Factor-1α

Objective: To investigate the anti-drug reward effects and molecular mechanisms behind modulation of hypoxia-inducible factor-1α(HIF-1α).Methods: The anti-drug reward effects of roxadustat(a HIF prolyl hydroxylase inhibitor approved for the treatment of renal anemia)were evaluated by conditioned place preference(CPP).The expression of phospharylated-c AMP response element binding protein(p-CREB),dopamine transporter and Δ fos B in response to roxadustat treatment were detected by immunoblot and immunofluorescence,respectively.Iron content in cells and tissues were measured by inductively coupled plasma-mass spectrometry(ICP-MS).Expression of iron metabolism-related molecules after roxadustat treatment were measured through quantitative real-time PCR(Q-RT-PCR)and enzyme-linked immunosorbent assay(ELISA).The iron-deficient mouse model was established via iron-deficient diet,and the influence of systematic iron deficiency on CPP formation was evaluated.The effects of iron deficiency on mice basal fear levels,spatial learning and memory were tested by fear conditioning and Morris water maze.Furthermore,by local infusion of deferiprone(DFP)in anterior cingulate cortex(ACC),the influence of brain iron deficiency on CPP formation was investigated.Results: Morphine treatment can down-regulate the expression of HIF-1α in mice prefrontal cortex(PFC)and striatum(STR).Roxadustat can inhibit the formation,expression and reinstatement of CPP and promote the extinction of CPP without affecting spontaneous activity,anxiety and basal fear levels,spatial learning and memory of mice.Similarly,roxadustat can inhibit morphine-induced upregulation of p-CREB and Δ fos B.Mechanism studies have shown that roxadustat can promote HIF-1α accumulation and nuclear translocation and thereby decrease hepcidin expression and intracellular iron content,which further inhibit ubiquitinated degradation of DAT,leading to the up-regulation of DAT expression on plasma membrane and decrease extracellular dopamine concentration.To explore the deeper relationship between iron and drug reward effects,we established a systematic iron deficient mouse model,and found that iron deficiency can significantly inhibit the formation of morphine and METH-induced CPP formation without affecting the basal fear levels,spatial learning and memory of mice.To further clarify the role of brain iron content in drug reward,we injected DFP to chelate iron ion in ACC area and found DFP-induced iron deficiency in local brain region could also inhibit the formation of morphine-induced CPP in mice.Conclusion: Roxadustat has both preventive and therapeutic effects on the reward effects of addictive drugs.Mechanically,roxadustat promotes HIF-1α nuclear translocation,downregulates hepcidin expression,contributes to the iron-dependent ubiquitinated degradation disorder of DAT and a reduced endocytosis of DAT,which further increases the expression of functional DAT on plasma membrane,reduces the excessive dopamine in the synaptic clefts caused by addictive drugs and ultimately inhibits the reward effects of addictive drugs.Our study provides direct evidence that iron homeostasis plays a key role in the reward effects of drugs.Regulation of HIF-1α-mediated iron metabolism may be a new therapeutic strategy for drug reward effects and roxadustat may operate as a potential anti-reward drug.