Oral administration of the KATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis

Background Multiple Sclerosis (MS) is an acquired inflammatory demyelinating disorder of the central nervous system (CNS) and is the leading cause of nontraumatic disability among young adults. Activated microglial cells are important effectors of demyelination and neurodegeneration, by secreting cytokines and others neurotoxic agents. Previous studies have demonstrated that microglia expresses ATP-sensitive potassium (KATP) channels and its pharmacological activation can provide neuroprotective and anti-inflammatory effects. In this study, we have examined the effect of oral administration of KATP channel opener diazoxide on induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Methods Anti-inflammatory effects of diazoxide were studied on lipopolysaccharide (LPS) and interferon gamma (IFNy)-activated microglial cells. EAE was induced in C57BL/6J mice by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Mice were orally treated daily with diazoxide or vehicle for 15 days from the day of EAE symptom onset. Treatment starting at the same time as immunization was also assayed. Clinical signs of EAE were monitored and histological studies were performed to analyze tissue damage, demyelination, glial reactivity, axonal loss, neuronal preservation and lymphocyte infiltration. Results Diazoxide inhibited in vitro nitric oxide (NO), tumor necrosis factor alpha (TNF-¿) and interleukin-6 (IL-6) production and inducible nitric oxide synthase (iNOS) expression by activated microglia without affecting cyclooxygenase-2 (COX-2) expression and phagocytosis. Oral treatment of mice with diazoxide ameliorated EAE clinical signs but did not prevent disease. Histological analysis demonstrated that diazoxide elicited a significant reduction in myelin and axonal loss accompanied by a decrease in glial activation and neuronal damage. Diazoxide did not affect the number of infiltrating lymphocytes positive for CD3 and CD20 in the spinal cord. Conclusion Taken together, these results demonstrate novel actions of diazoxide as an anti-inflammatory agent, which might contribute to its beneficial effects on EAE through neuroprotection. Treatment with this widely used and well-tolerated drug may be a useful therapeutic intervention in ameliorating MS disease

Correction: KATP Channel Opener Diazoxide Prevents Neurodegeneration: A New Mechanism of Action via Antioxidative Pathway Activation.

[This corrects the article DOI: 10.1371/journal.pone.0075189.]

K(ATP) channel opener diazoxide prevents neurodegeneration: a new mechanism of action via antioxidative pathway activation.

Pharmacological modulation of ATP-sensitive potassium channels has become a promising new therapeutic approach for the treatment of neurodegenerative diseases due to their role in mitochondrial and cellular protection. For instance, diazoxide, a well-known ATP-sensitive potassium channel activator with high affinity for mitochondrial component of the channel has been proved to be effective in animal models for different diseases such as Alzheimer's disease, stroke or multiple sclerosis. Here, we analyzed the ability of diazoxide for protecting neurons front different neurotoxic insults in vitro and ex vivo. Results showed that diazoxide effectively protects NSC-34 motoneurons from glutamatergic, oxidative and inflammatory damage. Moreover, diazoxide decreased neuronal death in organotypic hippocampal slice cultures after exicitotoxicity and preserved myelin sheath in organotypic cerebellar cultures exposed to pro-inflammatory demyelinating damage. In addition, we demonstrated that one of the mechanisms of actions implied in the neuroprotective role of diazoxide is mediated by the activation of Nrf2 expression and nuclear translocation. Nrf2 expression was increased in NSC-34 neurons in vitro as well as in the spinal cord of experimental autoimmune encephalomyelitis animals orally administered with diazoxide. Thus, diazoxide is a neuroprotective agent against oxidative stress-induced damage and cellular dysfunction that can be beneficial for diseases such as multiple sclerosis

Natural Docosahexaenoic Acid in the Triglyceride Form Attenuates In Vitro Microglial Activation and Ameliorates Autoimmune Encephalomyelitis in Mice

Many neurodegenerative diseases are associated, at least in part, to an inflammatory process in which microglia plays a major role. The effect of the triglyceride form of the omega-3 polyunsaturated fatty acid docosahexaenoic acid (TG-DHA) was assayed in vitro and in vivo to assess the protective and anti-inflammatory activity of this compound. In the in vitro study, BV-2 microglia cells were previously treated with TG-DHA and then activated with Lipopolysaccharide (LPS) and Interferon-gamma (IFN-γ). TG-DHA treatment protected BV-2 microglia cells from oxidative stress toxicity attenuating NO production and suppressing the induction of inflammatory cytokines. When compared with DHA in the ethyl-ester form, a significant difference in the ability to inhibit NO production in favor of TG-DHA was observed. TG-DHA inhibited significantly splenocyte proliferation but isolated CD4+ lymphocyte proliferation was unaffected. In a mice model of autoimmune encephalomyelitis (EAE), 250 mg/kg/day oral TG-DHA treatment was associated with a significant amelioration of the course and severity of the disease as compared to untreated animals. TG-DHA-treated EAE mice showed a better weight profile, which is a symptom related to a better course of encephalomyelitis. TG-DHA may be a promising therapeutic agent in neuroinflammatory processes and merit to be more extensively studied in human neurodegenerative disorders

Diazoxide increases Nrf2 nuclear translocation in NSC-34 motoneurons and prevents endogenous oxidative damage.

<p>Western blot showed an increase of Nrf2 signaling in the nuclear extracts from NSC-34 neurons treated with different doses of diazoxide for 24 h. The higher increase of Nrf2 was found at lower doses (10 and 1 µM) (A). Cell viability of NSC-34 cells was measured after 24 h AAPH oxidative stress activation and results demonstrated that diazoxide treatment effectively ameliorates cell viability at low doses (B). Results expressed as mean ± SEM. n≥4 experiments. *: p < 0.05, ** p<0.01. Scale bar  =  30 µm</p

Diazoxide effects in NMDA-induced neurodegeneration in hippocampal slice culture.

<p>At 7 DIV, treatment with 10 µM NMDA for 4 h induced a region-specific increase neuronal cell death of the CA1 layer compared to undamaged slices, as determined as PI uptake (PI, red) in colocalization with the neuronal nuclear marker NeuN (green) (A, B). Treatment 30 min before NMDA lesion with diazoxide prevented cell death, significantly at 100 µM but also at lowest dose 1 µM (C, D). Upon quantification, results showed a significant decrease at 100 µM (65±5% in PI uptake compared to control) and 1 µM (67±7% in PI uptake compared to control (E). When microglia was removed from the slices, cell protection remained at the lowest dose 1 µM (62±10% in PI uptake compared to control) (F-J). Data are a summary of four to seven individual experiments with 6 slices per conditions analyzed in each experiment. Results are shown as mean ± SEM. * p<0.05, ** p<0.01. Scale bar 300 µm</p

Diazoxide prevents neuronal damage after different neurotoxic insults.

<p>Representative images of differentiated NSC-34 motoneuron cells line after glutamate damage and diazoxide treatment (A). Diazoxide inhibited glutamate mediated cell death by 24 h in differentiated NSC-34 motoneuron cell line (B). Diazoxide inhibited H₂O₂ mediated cell death by 24 h (C) and also when used as pretreatment during 2 h (survival analysis performed 24 h after toxic insult) (D). Diazoxide inhibited inflammatory BV2 microglial mediated cell death by 24 h in NSC-34 motoneuron. Conditioned Medium (CM): Neurons damaged with activated BV2 medium. CM (Dxz to NSC-34): Neurons treated with diazoxide and damaged with activated BV2 medium afterwards. CM (Dzx to BV2): Neurons damaged with diazoxide treated BV2 activated medium (E). Diazoxide treatment 100 µM. Results expressed as mean ± SEM. n≥4 experiments. *: p < 0.05. Scale bar =  30 µm</p