Metformin accelerates myelin recovery and ameliorates behavioral deficits in the animal model of multiple sclerosis via adjustment of AMPK/Nrf2/mTOR signaling and maintenance of endogenous oligodendrogenesis during brain self-repairing period.

BACKGROUND: Multiple sclerosis (MS) is a devastating autoimmune disorder characterized by oligodendrocytes (OLGs) loss and demyelination. In this study, we have examined the effects of metformin (MET) on the oligodendrogenesis, redox signaling, apoptosis, and glial responses during a self-repairing period (1-week) in the animal model of MS. METHODS: For induction of demyelination, C57BL/6 J mice were fed a 0.2% cuprizone (CPZ) for 5 weeks. Thereafter, CPZ was removed for 1-week and molecular and behavioral changes were monitored in the presence or absence of MET (50 mg/kg body weight/day). RESULTS: MET remarkably increased the localization of precursor OLGs (NG2+/O4+ cells) and subsequently the renewal of mature OLGs (MOG+ cells) in the corpus callosum via AMPK/mammalian target of rapamycin (mTOR) pathway. Moreover, we observed a significant elevation in the antioxidant responses, especially in mature OLGs (MOG+/nuclear factor erythroid 2-related factor 2 (Nrf2+) cells) after MET intervention. MET also reduced brain apoptosis markers and lessened motor dysfunction in the open-field test. While MET was unable to decrease active astrogliosis (GFAP mRNA), it reduced microgliosis by down-regulation of Mac-3 mRNA a marker of pro-inflammatory microglia/macrophages. Molecular modeling studies, likewise, confirmed that MET exerts its effects via direct interaction with AMPK. CONCLUSIONS: Altogether, our study reveals that MET effectively induces lesion reduction and elevated molecular processes that support myelin recovery via direct activation of AMPK and indirect regulation of AMPK/Nrf2/mTOR pathway in OLGs. These findings facilitate the development of new therapeutic strategies based on AMPK activation for MS in the near future.

Metformin-induced AMPK activation stimulates remyelination through induction of neurotrophic factors, downregulation of NogoA and recruitment of Olig2+ precursor cells in the cuprizone murine model of multiple sclerosis.

PURPOSE: Oligodendrocytes (OLGs) damage and myelin distraction is considered as a critical step in many neurological disorders especially multiple sclerosis (MS). Cuprizone (cup) animal model of MS targets OLGs degeneration and frequently used to the mechanistic understanding of de- and remyelination. The aim of this study was exploring the effects of metformin on the OLGs regeneration, myelin repair and profile of neurotrophic factors in the mice brain after cup-induced acute demyelination. METHODS: Mice (C57BL/6 J) were fed with chow containing 0.2% cup for 5 weeks to induce specific OLGs degeneration and acute demyelination. Next, the cup was withdrawn to allow one-week recovery (spontaneous remyelination). At the end of this period, mature OLGs markers, myelin-associated neurite outgrowth inhibitor protein A (NogoA), premature specific OLGs transcription factor (Olig2), anti-apoptosis marker (survivin), neurotrophic factors, and AMPK activation were monitored in the presence or absence of metformin (50 mg/kg body weight/day) in the corpus callosum (CC). RESULTS: Our finding indicated that consumption of metformin during the recovery period potentially induced an active form of AMPK (p-AMPK) and promoted repopulation of mature OLGs (MOG+ cells, MBP+ cells) in CC through up-regulation of BDNF, CNTF, and NGF as well as down-regulation of NogoA and recruitment of Olig2+ precursor cells. CONCLUSIONS: This study for the first time reveals that metformin-induced AMPK, a master regulator of energy homeostasis, activation following toxic demyelination could potentially accelerate regeneration and supports spontaneous demyelination. These findings suggest the development of new therapeutic strategies based on AMPK activation for MS in the near future. Graphical abstract An overview of the possible molecular mechanisms of action of metformin-mediated remyelinationa.

Effect of different doses of oxytocin on cardiac electrophysiology and arrhythmias induced by ischemia.

The onset of acute myocardial ischemia (MI) is accompanied by a rapid increase in electrical instability and often fatal ventricular arrhythmias. This study investigated that whether oxytocin (OT) can modulate ischemia-induced arrhythmias and considered relationships between the severity of arrhythmia and the electrocardiogram parameters during ischemia. OT (0.0001-1 µg) was administrated intraperitoneally 30 min before ischemia. To examine receptor involved, a selective OT-receptor antagonist, atosiban (ATO), was infused 10 min before OT. OT caused a significant and biphasic dose-dependent reduction in ectopic heart activity and arrhythmia score. OT doses that reduced ventricular arrhythmia elicited significant increase in QT interval. OT attenuated the electrophysiological changes associated with MI and there was significant direct relationship between QRS duration and arrhythmia score. ATO treatment reduced beneficial effects of OT on arrhythmogenesis. Nevertheless, ATO failed to alter OT effects on premature ventricular contractions. We assume that the ability of OT to modulate the electrical activity of the heart may play an important role in the antiarrhythmic actions of OT.

Differentiation of P19 embryonal carcinoma stem cells into insulin-producing cells promoted by pancreas-conditioned medium.

The ability of embryonal carcinoma )EC (stem cells to generate insulin-producing cells (IPCs) is still unknown. We examined the trophic effects of pancreas-conditioned medium (PCM) on in vitro production of IPCs. Initially, P19 EC cells were characterized by the expression of stem cell markers, Oct3/4, Sox-2 and Nanog. To direct differentiation, P19-derived embryoid bodies (EBs) were induced by selection of nestin-positive cells and treatment with different concentrations of PCM. Morphological studies documented the presence of islet-like cell IPCs clusters. The differentiated cells were immunoreactive for ß cell-specific proteins, including insulin, proinsulin, C-peptide and insulin receptor-ß. The expression of genes related to pancreatic ß cell development and function (PDX-1, INS1, INS2, EP300 and CREB1) was confirmed by qPCR. During differentiation, the expression of EP300 and CREB1 increased by 2.5 and 3.1 times, respectively. In contrast, a sharp decrease in the expression of Oct3/4, Sox-2 and Nanog by 4, 1.5 and 1.5 times, respectively, was observed. The differentiated cells were functionally active, synthesizing and secreting insulin in a glucose-regulated manner. Network prediction highlighted crosstalk between PDX-1 transcription factor and INS2 ligand in IPC generation and revealed positive regulatory effects of EP300, CREB1, PPARA, EGR, KIT, GLP1R, and PKT2 on activation of PDX-1 and INS2. This is the first report of the induction of IPC differentiation from EC cells by using neonate mouse PCM. Since P19 EC cells are widely available, easily cultured without feeders and do not require special growth conditions, they would provide a valuable tool for studying pancreatic ß cell differentiation and development. Copyright © 2016 John Wiley & Sons, Ltd.

Role of atrial natriuretic Peptide in oxytocin induced cardioprotection.

BACKGROUND: The purpose of this study was to determine whether endogenous atrial natriuretic peptide (ANP) contributes to the protective effect of neurohypophysial hormone oxytocin (OT) in heart preconditioning. METHODS: Sprague-Dawley male rats were subjected to 25 min regional ischaemia and 120 min reperfusion and were divided into eight groups. Oxytocin or an equivalent volume of saline was administrated intraperitoneally, 30 min before ischaemia. The OT receptor antagonist (atosiban), ANP receptor antagonist (anantin) and nitric oxide synthase inhibitor (L-NAME) were injected 10 min before OT. In other groups, atosiban, anantin and L-NAME were only administered 40 min prior to ischaemia. RESULTS: Compared with the ischaemia/reperfusion group (I/R), alterations in infarct size, biochemical parameters [LDH (lactate dehydrogenase), CK-MB (creatine kinase-MB), MDA (malondialdehyde) plasma levels] and severity of ventricular arrhythmia due to I/R injury were attenuated and VF was abolished by OT treatment. These OT effects were eliminated by OT and ANP receptor blockers and nitric oxide synthase inhibitor, but anantin did not reverse the effect of OT in lipid peroxidation. CONCLUSIONS: These findings demonstrate an important contributory role of ANP in the OT induced protection in myocardial ischaemia reperfusion. OT also reduced lipid peroxidation with a NO-dependent mechanism.

Efficient and simple production of insulin-producing cells from embryonal carcinoma stem cells using mouse neonate pancreas extract, as a natural inducer.

An attractive approach to replace the destroyed insulin-producing cells (IPCs) is the generation of functional ß cells from stem cells. Embryonal carcinoma (EC) stem cells are pluripotent cells which can differentiate into all cell types. The present study was carried out to establish a simple nonselective inductive culture system for generation of IPCs from P19 EC cells by 1-2 weeks old mouse pancreas extract (MPE). Since, mouse pancreatic islets undergo further remodeling and maturation for 2-3 weeks after birth, we hypothesized that the mouse neonatal MPE contains essential factors to induce in vitro differentiation of pancreatic lineages. Pluripotency of P19 cells were first confirmed by expression analysis of stem cell markers, Oct3/4, Sox-2 and Nanog. In order to induce differentiation, the cells were cultured in a medium supplemented by different concentrations of MPE (50, 100, 200 and 300 µg/ml). The results showed that P19 cells could differentiate into IPCs and form dithizone-positive cell clusters. The generated P19-derived IPCs were immunoreactive to proinsulin, insulin and insulin receptor beta. The expression of pancreatic ß cell genes including, PDX-1, INS1 and INS2 were also confirmed. The peak response at the 100 µg/ml MPE used for investigation of EP300 and CREB1 gene expression. When stimulated with glucose, these cells synthesized and secreted insulin. Network analysis of the key transcription factors (PDX-1, EP300, CREB1) during the generation of IPCs resulted in introduction of novel regulatory candidates such as MIR17, and VEZF1 transcription factors, as well as MORN1, DKFZp761P0212, and WAC proteins. Altogether, we demonstrated the possibility of generating IPCs from undifferentiated EC cells, with the characteristics of pancreatic ß cells. The derivation of pancreatic cells from EC cells which are ES cell siblings would provide a valuable experimental tool in study of pancreatic development and function as well as rapid production of IPCs for transplantation.

Role of central oxytocin in stress-induced cardioprotection in ischemic-reperfused heart model.

BACKGROUND AND PURPOSE: There is growing evidence that stress contributes to cardiovascular disease and triggers the release of oxytocin. Moreover previous studies confirmed oxytocin mimics the protection associated with ischemic preconditioning. The present study was aimed to assess the possible cardioprotective effects of the centrally released oxytocin in response to stress and intracerebroventricular (i.c.v.) administration of exogenous oxytocin in ischemic-reperfused isolated rat heart. METHODS AND SUBJECTS: Rats were divided in two main groups and all of them were subjected to i.c.v. infusion of vehicle or drugs: unstressed rats [control: vehicle, oxytocin (OT; 100 ng/5 µl), atosiban (ATO; 4.3 µg/5 µl) as oxytocin antagonist, ATO+OT] and stressed rats [St: stress, OT+St, ATO+St]. After anesthesia, hearts were isolated and subjected to 30 min regional ischemia and 60 min reperfusion (IR). Acute stress protocol included swimming for 10 min before anesthesia. Myocardial function, infarct size, coronary flow, ventricular arrhythmia, and biochemical parameters such as creatine kinase and lactate dehydrogenase were measured. Ischemia-induced ventricular arrhythmias were counted during the occlusion period. RESULTS: The plasma levels of oxytocin and corticosterone were significantly elevated by stress. Unexpectedly hearts of stressed rats showed a marked depression of IR injury compared to control group. I.c.v. infusion of oxytocin mimicked the cardioprotective effects of stress, yet did not elevate plasma oxytocin level. The protective effects of both stress and i.c.v. oxytocin were blocked by i.c.v. oxytocin antagonist. CONCLUSIONS: These findings suggest that i.c.v. infusion of exogenous oxytocin and centrally released endogenous oxytocin in response to stress could play a role in induction of a preconditioning effect in ischemic-reperfused rat heart via brain receptors.

Effects of selegiline, a monoamine oxidase B inhibitor, on differentiation of P19 embryonal carcinoma stem cells, into neuron-like cells.

Selegiline, the irreversible inhibitor of monoamine oxidase B (MAO-B), is currently used to treat Parkinson's disease. However, the mechanism of action of selegiline is complex and cannot be explained solely by its MAO-B inhibitory action. It stimulates gene expression, as well as expression of a number of mRNAs or proteins in nerve and glial cells. Direct neuroprotective and anti-apoptotic actions of selegiline have previously been observed in vitro. Previous studies showed that selegiline can induce neuronal phenotype in cultured bone marrow stem cells and embryonic stem cells. Embryonal carcinoma (EC) cells are developmentally pluripotene cells which can be differentiated into all cell types under the appropriate conditions. The present study was carried out to examine the effects of selegiline on undifferentiated P19 EC cells. The results showed that selegiline treatment had a dramatic effect on neuronal morphology. It induced the differentiation of EC cells into neuron-like cells in a concentration-dependent manner. The peak response was in a dose of selegiline significantly lower than required for MAO-B inhibition. The differentiated cells were immunoreactive for neuron-specific proteins, synaptophysin, and ß-III tubulin. Stem cell therapy has been considered as an ideal option for the treatment of neurodegenerative diseases. Generation of neurons from stem cells could serve as a source for potential cell therapy. This study suggests the potential use of combined selegiline and stem cell therapy to improve deficits in neurodegenerative diseases.

Oxytocin protects rat heart against ischemia-reperfusion injury via pathway involving mitochondrial ATP-dependent potassium channel.

Cardiac preconditioning represents the most potent and consistently reproducible method of rescuing heart tissue from undergoing irreversible ischemic damage. One of the major goals of the current cardiovascular research is to identify a reliable cardioprotective intervention that can salvage ischemic myocardium. The aim of the present study is to evaluate the oxytocin (OT)-induced cardioprotection and the signaling pathway involved with mitochondrial ATP-dependent potassium (mitoKATP) channel in the anesthetized rat heart. Animals were divided into six groups (n=6): (1) IR; hearts were subjected to 25 min ischemia and 120 min reperfusion, (2) OT; oxytocin was administered (0.03 microg/kg i.p.) 25 min prior to ischemia, (3) ATO+OT; atosiban (ATO) was used as an OT-selective receptor antagonist (1.5 microg/kg i.p.) 10 min prior to OT administration, (4) ATO; atosiban was used 35 min prior to ischemia, (5) 5HD+OT; 5-hydroxydecanoic acid (5HD) was used as a specific inhibitor of mitoKATP channel (10mg/kg i.v.) 10 min prior to OT administration, (6) 5HD; 5HD was used 35min prior to ischemia. Then infarct size, ventricular arrhythmia and creatine kinase-MB isoenzyme (CK-MB) plasma level were measured. Hemodynamic parameters were recorded throughout the experiment. OT administration significantly decreased infarct size, CK-MB plasma level, severity and incidence of ventricular arrhythmia as compared to IR group. Administration of atosiban and 5HD abolished the cardiopreconditioning effect of OT. This study demonstrates that cardioprotective effects of OT are mediated through opening the mitoKATP channels.

Biphasic protective effect of oxytocin on cardiac ischemia/reperfusion injury in anaesthetized rats.

Oxytocin (OT) is well known for its role in reproduction. However, evidence has emerged suggesting a role in cardiovascular system. The aim of this study was to investigate the cardioprotective effect of oxytocin on ischemia/reperfusion (I/R) injury in an in vivo rat. Myocardial ischemia, was surgically induced by means of left anterior descending coronary artery occlusion for 25 min followed by reperfusion for 120 min. Infarct size was evaluated using the staining agent 2,3,5-triphenyltetrazolium chloride. Creatine kinase-MB isoenzyme (CK-MB) and lactate dehydrogenase (LDH) levels in plasma were analyzed to assess the degree of cardiac injury. Intraperitoneal administration of OT 0.001, 0.01 and 0.1 microg significantly reduced infarct size, LDH and CK-MB levels as compared to control (I/R) group and it had a biphasic effect on the reduction of ischemia/reperfusion injury. This biphasic effect was revealed as a U-shaped curve in which efficacy was optimal between very low and very high doses. Furthermore there were no significant differences in mean arterial pressure or heart rate between the OT treatment groups and control group during I/R. Blockade of specific OT receptors by atosiban (10(-6)M) abolished or attenuated the effect of OT preconditioning. The result of this study shows that OT possess a dose-dependent cardioprotective effect against ischemia/reperfusion injury and so study of OT preconditioning may provide a new target site for therapeutic exploitation.