Background: 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 (iNOS^highCD206^low) to quiescent anti-inflammatory M2 phenotype (iNOS^lowCD206^high) 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.

Microglial cells are resident macrophages in the brain that have been implicated in the pathophysiology of schizophrenia. There is a lack of studies covering the effects of antipsychotics on microglial cells. The current literature points to a possible anti-inflammatory action without clear mechanisms of action. The aim of this study is to characterize the effects of haloperidol, risperidone and aripiprazole on BV-2 microglial cells in in vitro conditions. We have used immunofluorescence and flow cytometry to analyze the classical pro and anti-inflammatory markers, while a real-time metabolic assay (Seahorse) was used to assess metabolic function. We analyzed the expression of p70S6K to evaluate the mTOR pathway activity with Western blot. In this study, we demonstrate the varying effects of haloperidol, risperidone and aripiprazole administration in BV-2 microglial cells. All three tested antipsychotics were successful in reducing the pro-inflammatory action of microglial cells, although only aripiprazole increased the expression of anti-inflammatory markers. Most significant differences in the possible mechanisms of action were seen in the real-time metabolic assays and in the mTORC1 signaling pathway activity, with aripiprazole being the only antipsychotic to reduce the mTORC1 activity. Our results shed some new light on the effects of haloperidol, risperidone and aripiprazole action in microglial cells, and reveal a novel possible mechanism of action for aripiprazole.

With the progress of medicine, especially in the last century, life expectancy increased considerably. As a result, age-related diseases also increased, especially malignancies and degenerative diseases of the central nervous system. The incidence and prevalence of neurodegenerative diseases steadily increased over the years, but despite efforts to uncover the pathophysiological processes behind these conditions, they remain elusive. Among the many theories, oxidative stress was proposed to be involved in neurodegenerative processes and to play an important role in the morbidity and progression of various neurodegenerative disorders. Accordingly, a number of studies discovered the potential of natural plant constituents to have significant antioxidant activity. This review focused on several plant-based antioxidants that showed promising results in the prevention and treatment of neurodegenerative diseases. Laurus nobilis, Aronia melanocarpa, and celastrol, a chemical compound isolated from the root extracts of Tripterygium wilfordii and T. regelii, are all known to be rich in antioxidant polyphenols.

Extracellular vesicles (EVs) are a versatile group of cell-secreted membranous nanoparticles present in body fluids. They have an exceptional diagnostic potential due to their molecular content matching the originating cells and accessibility from body fluids. However, methods for EV isolation are still in development, with size exclusion chromatography (SEC) emerging as a preferred method. Here we compared four types of SEC to isolate EVs from the CSF of patients with severe traumatic brain injury. A pool of nine CSF samples was separated by SEC columns packed with Sepharose CL-6B, Sephacryl S-400 or Superose 6PG and a ready-to-use qEV10/70 nm column. A total of 46 fractions were collected and analysed by slot-blot followed by Ponceau staining. Immunodetection was performed for albumin, EV markers CD9, CD81, and lipoprotein markers ApoE and ApoAI. The size and concentration of nanoparticles in fractions were determined by tunable resistive pulse sensing and EVs were visualised by transmission electron microscopy. We show that all four SEC techniques enabled separation of CSF into nanoparticle- and free protein-enriched fractions. Sepharose CL-6B resulted in a significantly higher number of separated EVs while lipoproteins were eluted together with free proteins. Our data indicate that Sepharose CL-6B is suitable for isolation of EVs from CSF and their separation from lipoproteins.