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Naltrexone is an opioid receptor antagonist commonly used to treat opioid and alcohol dependence. The use of low dose naltrexone (LDN) was found to have anti-inflammatory properties for treatment of diseases such as fibromyalgia, Crohn’s disease, multiple sclerosis and regional pain syndromes. Related to its anti-neuroinflammatory properties, the mechanism of action is possibly mediated via Toll-like receptor 4 antagonism, which is widely expressed on microglial cells. The aim of the present study was to assess the immunometabolic effects of naltrexone on microglia cells in in vitro conditions. Methods: All experiments were performed in the BV-2 microglial cell line. The cells were treated with naltrexone at 100 μM concentrations corresponding to low dose for 24 h. Cell viability was assessed for every drug dose. To induce additional activation, the cells were pretreated with LPS and IFN-γ. Immunofluorescence was used to analyse the classical microglial activation markers iNOS and CD206, while Seahorse was used for real-time cellular metabolic assessments. mTOR activity measured over the expression of a major direct downstream target S6K was assessed using western blot. Results: LDN induced a shift from highly activated pro-inflammatory phenotype (iNOShighCD206low) to quiescent anti-inflammatory M2 phenotype (iNOSlowCD206high) in BV-2 microglia cells. Changes in the inflammatory profile were accompanied by cellular metabolic switching based on the transition from high glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). LDN-treated cells were able to maintain a metabolically suppressive phenotype by supporting OXPHOS with high oxygen consumption, and also maintain a lower energetic state due to lower lactate production. The metabolic shift induced by transition from glycolysis to mitochondrial oxidative metabolism was more prominent in cells pretreated with immunometabolic modulators such as LPS and IFN-γ. In a dose-dependent manner, naltrexone also modulated mTOR/S6K expression, which underlies the cell metabolic phenotype regulating microglia immune properties and adaptation. Conclusion: By modulating the phenotypic features by metabolic switching of activated microglia, naltrexone was found to be an effective and powerful tool for immunometabolic reprogramming and could be a promising novel treatment for various neuroinflammatory conditions.

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Recombinant human erythropoietin (rhEpo) is a multi-functional drug with antioxidant potential. However, the underlying molecular mechanisms of its action are still unclear. The purpose of this study was to investigate the effects of rhEpo on the brain infarct volume as well as on the levels of the neuronal damage, oxidative stress parameters and active caspase-3, nuclear factor erythroid 2-related factor 2 (Nrf2) and haemeoxygenase-1 (HO-1) expressions in the hippocampi of rats exposed to the right middle cerebral artery occlusion (MCAO) for 1 hr. Ischaemic animals received either vehicle or rhEpo (5000 IU/kg, i.p.) immediately or 3 hr after the induction of ischaemia. Sham-operated, vehicle-treated animals served as the control group. Rats were killed 24 hr after the onset of the ischaemic or sham experimental procedure. MCAO caused ipsilateral brain infarction within the striatum and cortex. In the CA1 region of the hippocampi, we did not find significant neuronal loss, but a statistically significant rise in the active caspase-3 and Nrf2 protein expressions was registered. We detected also significant increases in the hippocampal levels of oxidative stress parameters (thiobarbituric acid-reactive substances, superoxide dismutase, glutathione peroxidase). Post-ischaemic administration of rhEpo significantly reduced the brain infarct volume, decreased levels of all tested oxidative stress parameters and increased the Nrf2 expression level. These findings suggest that decrease in oxidative stress parameters in the hippocampus could be an early indicator of post-ischaemic neuroprotective effect of rhEpo in rats exposed to focal cerebral ischaemia and that this effect could be attributable to additional post-ischaemic activation of Nrf2 endogenous antioxidant system.

The aim of our study was to investigate blood pressure (BP) control and different factors with possible influence on BP control in Croatian hypertensive patients. In this cross-sectional investigation, a representative sample of target populations (primary care physicians and patients) from different parts of Croatia was included according to the study protocol. During December 2003 and January 2004, we included, according to correctly completed questionnaires, 141 physicians and 814 hypertensive patients. A controlled BP (BPv140/90 mmHg) in this hypertensive population treated with antihypertensive drugs was in 23% of patients. The analysis of BP control according to risk factors showed that significantly related with higher levels of systolic or diastolic BP were the age (poorer systolic BP control in patients older than 60 years), left ventricular hypertrophy, changes of the eye retina, smoking and diabetes mellitus. Furthermore, patients from towns closer to the hospital, from urban centers, with higher education and employed had significantly lower average BP. According to our results of hypertension control in Croatia, there is a need and a possibility for the improvement of the quality of hypertension care. The relationship between demographic and cardiovascular risk factors with poor BP control should be taken into account when treating patients.