SGLT2 Inhibitor Empagliflozin Modulates Ion Channels in Adult Zebrafish Heart.

Empagliflozin, an inhibitor of sodium-glucose co-transporter 2 (iSGLT2), improves cardiovascular outcomes in patients with and without diabetes and possesses an antiarrhythmic activity. However, the mechanisms of these protective effects have not been fully elucidated. This study aimed to explore the impact of empagliflozin on ion channel activity and electrophysiological characteristics in the ventricular myocardium. The main cardiac ionic currents (INa, ICaL, ICaT, IKr, IKs) and action potentials (APs) were studied in zebrafish. Whole-cell currents were measured using the patch clamp method in the isolated ventricular cardiomyocytes. The conventional sharp glass microelectrode technique was applied for the recording of APs from the ventricular myocardium of the excised heart. Empagliflozin pretreatment compared to the control group enhanced potassium IKr step current density in the range of testing potentials from 0 to +30 mV, IKr tail current density in the range of testing potentials from +10 to +70 mV, and IKs current density in the range of testing potentials from -10 to +20 mV. Moreover, in the ventricular myocardium, empagliflozin pretreatment shortened AP duration APD as shown by reduced APD50 and APD90. Empagliflozin had no influence on sodium (INa) and L- and T-type calcium currents (ICaL and ICaT) in zebrafish ventricular cardiomyocytes. Thus, we conclude that empagliflozin increases the rapid and slow components of delayed rectifier K+ current (IKr and IKs). This mechanism could be favorable for cardiac protection.

Dynamics of the Glycogen ß-Particle Number in Rat Hepatocytes during Glucose Refeeding.

Glycogen is an easily accessible source of energy for various processes. In hepatocytes, it can be found in the form of individual molecules (ß-particles) and their agglomerates (α-particles). The glycogen content in hepatocytes depends on the physiological state and can vary due to the size and number of the particles. Using biochemical, cytofluorometric, interferometric and morphometric methods, the number of ß-particles in rat hepatocytes was determined after 48 h of fasting at different time intervals after glucose refeeding. It has been shown that after starvation, hepatocytes contain ~1.6 × 108 ß-particles. During refeeding, their number of hepatocytes gradually increases and reaches a maximum (~5.9 × 108) at 45 min after glucose administration, but then quickly decreases. The data obtained suggest that in cells there is a continuous synthesis and degradation of particles, and at different stages of life, one or another process predominates. It has been suggested that in the course of glycogenesis, pre-existing ß-particles are replaced by those formed de novo. The main contribution to the deposition of glycogen is made by an increase in the glucose residue number in its molecules. The average diameter of ß-particles of glycogen during glycogenesis increases from ~11 nm to 21 nm.

Color-Coding Method Reveals Enhancement of Stereotypic Locomotion by Phenazepam in Rat Open Field Test.

One of the most important tasks in neuroscience is the search for theoretical foundations for the development of methods for diagnosing and treating neurological pathology, and for assessing the effect of pharmacological drugs on the nervous system. Specific behavioral changes associated with exposure to systemic influences have been invisible to the human eye for a long time. A similar pattern of changes is characteristic of phenazepam, a drug with a wide range of effects on the brain. In this study, we used a color-coding method, which consists of combining three time positions in one image, the present (0 s), the near future (0.33 s) and the far future (1.6 s). This method made it possible to identify movement patterns, such as the initialization of ahead movements, side turns and 180° turns (back), and also to determine the degree of predictability of future movements. The obtained data revealed a decrease in the number of turns to the sides while maintaining ahead movement, as well as an increase in the predictability of movements in rats under the influence of phenazepam. Thus, sedative doses of phenazepam do not exhibit general depression of brain functions, but the inhibition of specific centers, including the medial prefrontal cortex and postsubiculum, which are involved in stereotypic locomotive behavior.

Principles of Rational COVID-19 Therapy in Pediatrics.

The purpose of this review was to conduct a comparative assessment of the concepts of therapy for pediatric patients with COVID-19 in the framework of global clinical practice. A structural analysis of the range of drugs and treatment strategies in the context of etiotropic, pathogenetic, and symptomatic therapy has shown that in the global context and in real clinical practice, the etiotropic-pathogenetic approach based on information about the effectiveness of individual medical technologies prevails today. It has been established that eight international nonproprietary/grouping names are present in international practice as means of etiotropic therapy for pediatric patients with COVID-19, and 18 positions are used for pathogenetic therapy. In terms of frequency of occurrence, the leading positions are occupied by remdesivir and the combination of nirmatrelvir with ritonavir, as well as dexamethasone and tocilizumab. The paper emphasizes the relevance of research in the field of evaluating the effectiveness of individual treatment regimens as well as the analysis of the delayed consequences of pathology suffered in childhood under the conditions of using various approaches to pharmacotherapy.

Pharmacotherapy for Non-Alcoholic Fatty Liver Disease: Emerging Targets and Drug Candidates.

Non-alcoholic fatty liver disease (NAFLD), or metabolic (dysfunction)-associated fatty liver disease (MAFLD), is characterized by high global incidence and prevalence, a tight association with common metabolic comorbidities, and a substantial risk of progression and associated mortality. Despite the increasingly high medical and socioeconomic burden of NAFLD, the lack of approved pharmacotherapy regimens remains an unsolved issue. In this paper, we aimed to provide an update on the rapidly changing therapeutic landscape and highlight the major novel approaches to the treatment of this disease. In addition to describing the biomolecules and pathways identified as upcoming pharmacological targets for NAFLD, we reviewed the current status of drug discovery and development pipeline with a special focus on recent evidence from clinical trials.

Novel Chromone-Containing Allylmorpholines Induce Anxiolytic-like and Sedative Effects in Adult Zebrafish.

Chromone-containing allylmorpholines (CCAMs) are a novel class of compounds that have demonstrated acetyl- and butyryl-cholinesterase-inhibiting and N-methyl-D-aspartate (NMDA) receptor-blocking properties in vitro, but their in vivo pharmacological activity remains underexplored. In this work, we evaluated the psychotropic activity of five different CCAMs (1 (9a), 2 (9j), 3 (9l), 4 (33a), and 5 (33b)) using the novel tank test (NTT) and light/dark box (LDB) test in adult zebrafish. The CCAMs were screened in the NTT at a range of concentrations, and they were found to induce a dose-dependent sedative effect. Compound 4 (33a) was also evaluated using the LDB test, and it was found to have anxiolytic-like properties at low concentrations. To assess the potential contribution of the glutamate and cholinergic mechanisms in the effects of the CCAMs, we conducted experiments with pre-exposure to putative antagonists, NMDA and biperiden. Neither biperiden nor NMDA were able to diminish or cancel the effects of the CCAMs, countering the in vitro data obtained in previous studies. The apparent discrepancy could be related to the specifics of CCAM metabolism or to the interspecies differences between the putative target proteins, possibly due to the relatively low identity percentage of their sequences. Although further research in mammals is required in order to establish their pharmacological properties, novel CCAMs may represent an appealing group of psychoactive drug candidates.

Changes in Brain Electrical Activity after Transient Middle Cerebral Artery Occlusion in Rats.

Objectives. Ischemic stroke is a leading cause of death and disability worldwide. To search for new therapeutic and pharmacotherapeutic strategies, numerous models of this disease have been proposed, the most popular being transient middle cerebral artery occlusion. Behavioral and sensorimotor testing, biochemical, and histological methods are traditionally used in conjunction with this model to assess the effectiveness of potential treatment options. Despite its wide overall popularity, electroencephalography/electrocorticography is quite rarely used in such studies. Materials and methods. In the present work, we explored the changes in brain electrical activity at days 3 and 7 after 30- and 45-min of transient middle cerebral artery occlusion in rats. Results. Cerebral ischemia altered the amplitude and spectral electrocorticogram characteristics, and led to a reorganization of inter- and intrahemispheric functional connections. Ischemia duration affected the severity as well as the nature of the observed changes. Conclusions. The dynamics of changes in brain electrical activity may indicate a spontaneous partial recovery of impaired cerebral functions at post-surgery day 7. Our results suggest that electrocorticography can be used successfully to assess the functional status of the brain following ischemic stroke in rats as well as to investigate the dynamics of functional recovery.

Postprandial Glycogen Content Is Increased in the Hepatocytes of Human and Rat Cirrhotic Liver.

Chronic hepatitises of various etiologies are widespread liver diseases in humans. Their final stage, liver cirrhosis (LC), is considered to be one of the main causes of hepatocellular carcinoma (HCC). About 80-90% of all HCC cases develop in LC patients, which suggests that cirrhotic conditions play a crucial role in the process of hepatocarcinogenesis. Carbohydrate metabolism in LC undergoes profound disturbances characterized by altered glycogen metabolism. Unfortunately, data on the glycogen content in LC are few and contradictory. In this study, the material was obtained from liver biopsies of patients with LC of viral and alcohol etiology and from the liver tissue of rats with CCl4-induced LC. The activity of glycogen phosphorylase (GP), glycogen synthase (GS), and glucose-6-phosphatase (G6Pase) was investigated in human and rat liver tissue by biochemical methods. Total glycogen and its labile and stable fractions were measured in isolated individual hepatocytes, using the cytofluorometry technique of PAS reaction in situ. The development of LC in human and rat liver was accompanied by an increase in fibrous tissue (20- and 8.8-fold), an increase in the dry mass of hepatocytes (by 25.6% and 23.7%), and a decrease in the number of hepatocytes (by 50% and 28%), respectively. The rearrangement of the liver parenchyma was combined with changes in glycogen metabolism. The present study showed a significant increase in the glycogen content in the hepatocytes of the human and the rat cirrhotic liver, by 255% and 210%, respectively. An increased glycogen content in cells of the cirrhotic liver can be explained by a decrease in glycogenolysis due to a decreased activity of G6Pase and GP.

Effects of Alpha-2 Adrenergic Agonist Mafedine on Brain Electrical Activity in Rats after Traumatic Brain Injury.

The search for and development of new neuroprotective (or cerebroprotective) drugs, as well as suitable methods for their preclinical efficacy evaluation, are priorities for current biomedical research. Alpha-2 adrenergic agonists, such as mafedine and dexmedetomidine, are a highly appealing group of drugs capable of reducing neurological deficits which result from brain trauma and vascular events in both experimental animals and human patients. Thus, our aim was to assess the effects of mafedine and dexmedetomidine on the brain's electrical activity in a controlled cortical-impact model of traumatic brain injury (TBI) in rats. The functional status of the animals was assessed by electrocorticography (ECoG), using ECoG electrodes which were chronically implanted in different cortical regions. The administration of intraperitoneal mafedine sodium at 2.5 mg∙kg-1 at 1 h after TBI induction, and daily for the following 6 days, restored interhemispheric connectivity in remote brain regions and intrahemispheric connections within the unaffected hemisphere at post-TBI day 7. Animals that had received mafedine sodium also demonstrated an improvement in cortical responses to photic and somatosensory stimulation. Dexmedetomidine at 25 µg∙kg-1 did not affect the brain's electrical activity in brain-injured rats. Our results confirm the previously described neuroprotective effects of mafedine sodium and suggest that ECoG registration and analysis are a viable method evaluating drug efficacy in experimental animal models of TBI.

Pharmacological screening of a new alpha-2 adrenergic receptor agonist, mafedine, in zebrafish.

Pharmacological agents acting at alpha-2 adrenergic receptors are widely used in physiology and neuroscience research. Mounting evidence of their potential utility in clinical and experimental psychopharmacology, necessitates new models and novel model organisms for their screening. Here, we characterize behavioral effects of mafedine (6-oxo-1-phenyl-2- (phenylamino)-1,6-dihydropyrimidine-4-sodium olate), a novel drug with alpha-2 adrenergic receptor agonistic effects, in adult zebrafish (Danio rerio) in the novel tank test of anxiety and activity. Following an acute 20-min exposure, mafedine at 60 mg/L produced a mild psychostimulant action with some anxiogenic-like effects. Repeated acute 20-min/day administration of mafedine for 7 consecutive days at 1, 5 and 10 mg/L had a similar action on fish behavior as an acute exposure to 60 mg/L. Since mafedine demonstrated robust behavioral effects in zebrafish - a sensitive vertebrate aquatic model, it is likely that it may modulate rodent and human behavior as well. Thus, further studies are needed to explore this possibility in detail, and whether it may foster clinical application of mafedine and related alpha-2 adrenergic agents.

Metformin normalizes the structural changes in glycogen preceding prediabetes in mice overexpressing neuropeptide Y in noradrenergic neurons.

Hepatic insulin resistance and increased gluconeogenesis are known therapeutic targets of metformin, but the role of hepatic glycogen in the pathogenesis of diabetes is less clear. Mouse model of neuropeptide Y (NPY) overexpression in noradrenergic neurons (OE-NPYDßH) with a phenotype of late onset obesity, hepatosteatosis, and prediabetes was used to study early changes in glycogen structure and metabolism preceding prediabetes. Furthermore, the effect of the anti-hyperglycemic agent, metformin (300 mg/kg/day/4 weeks in drinking water), was assessed on changes in glycogen metabolism, body weight, fat mass, and glucose tolerance. Glycogen structure was characterized by cytofluorometric analysis in isolated hepatocytes and mRNA expression of key enzymes by qPCR. OE-NPYDßH mice displayed decreased labile glycogen fraction relative to stabile fraction (the intermediate form of glycogen) suggesting enhanced glycogen cycling. This was supported by decreased filling of glucose residues in the 10th outer tier of the glycogen molecule, which suggests accelerated glycogen phosphorylation. Metformin reduced fat mass gain in both genotypes, but glucose tolerance was improved mostly in wild-type mice. However, metformin inhibited glycogen accumulation and normalized the ratio between glycogen structures in OE-NPYDßH mice indicating decreased glycogen synthesis. Furthermore, the presence of glucose residues in the 11th tier together with decreased glycogen phosphorylase expression suggested inhibition of glycogen degradation. In conclusion, structural changes in glycogen of OE-NPYDßH mice point to increased glycogen metabolism, which may predispose them to prediabetes. Metformin treatment normalizes these changes and suppresses both glycogen synthesis and phosphorylation, which may contribute to its preventive effect on the onset of diabetes.