Andrey Yu. Vinokurov

The balance between production and neutralization of reactive oxygen species (ROS) regulates macrophage polarization, which determines the progression and resolution of inflammation. Nonetheless, the redox profiles of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages remain controversial, and it is not clear if modulation of ROS production can reprogram macrophage phenotype. Using THP-1-derived macrophages polarized with IFN-γ/LPS (M1) or IL-4/IL-13 (M2), we characterized ROS production in different cellular compartments, NADPH oxidase activity, antioxidant capacity, nitric oxide (NO) production and lipid peroxidation. We found that M2 macrophages had higher rates of cytosolic and mitochondrial ROS production and higher reduced glutathione (GSH) content. While cytosolic ROS predominance on M2 macrophages is mostly linked to basal level of NADPH oxidases activity, M1 macrophages had a stronger PMA-inducible ROS production. Despite the lower oxidation of ROS-sensitive probes, M1 macrophages demonstrated increased NO production and lipid peroxidation, indicating higher possibility of oxidative and nitrosative stress. Incubation of the cells with IFN-γ/LPS-induced polarization with sodium aminodihydrophthalazinedione (ADPh) increased superoxide production and decreased NO levels without changing GSH content. Subsequently, ADPh reduced the proportion of CD80-positive cells and GLUT1-dependent glucose uptake, indciating the shift of macrophages polarization towards an M2-like phenotype even in the presence of IFN-γ/LPS. Thus, stimulation of ROS production, rather than only activation of antioxidant systems, can prevent pro-inflammatory macrophage polarization.

Mitochondria, acquired during the evolution of eukaryotic cells, have become essential organelles for cellular life. Beyond their well-established role in energy production, mitochondria are key regulators of intracellular signaling, acting as modulators of Ca²⁺ transport and major sources of reactive oxygen species. Mitochondrial metabolism is also closely linked to the metabolism of signaling molecules, including glutamate and monoamines. Another vital role of mitochondria is their involvement in the regulation of cell death. Disruption of mitochondrial functions contributes to the development of multiple pathological conditions, including neurodegenerative diseases. Here, we discuss how impairments in major mitochondrial processes contribute to neuronal death in neurodegenerative diseases and highlight the principal mitochondrial targets with potential pharmacological relevance.

Background: Atherosclerosis is a complex cardiovascular disease often associated with mitochondrial dysfunction, which can lead to various cellular and metabolic abnormalities. Within the mitochondrial genome, specific mutations have been implicated in contributing to mitochondrial dysfunction. Atherosclerosis-associated m.15059G>A mutation has been of particular interest due to its potential role in altering mitochondrial function and cellular health. Objective: This study aims to investigate the role of the atherosclerosis-associated m.15059G>A mutation in the development of mitochondrial dysfunction in monocyte-- like cells. Methods: Monocyte-like cytoplasmic hybrid cell line TC-HSMAM1, which contains the m.15059G>A mutation in mtDNA, was used. The MitoCas9 vector was utilized to eliminate mtDNA copies carrying the m.15059G>A mutation from TC-HSMAM1 cybrids. Mitochondrial membrane potential, generation of reactive oxygen species, and lipid peroxidation levels were assessed using flow cytometry. Cellular reduced glutathione levels were assessed using the confocal microscopy. The oxygen consumption rate was measured using polarographic oxygen respirometry. Results: The elimination of the m.15059G>A mutation resulted in a significant increase in mitochondrial membrane potential and improved mitochondrial efficiency while also causing a decrease in the generation of reactive oxygen species, lipid peroxidation, as well as cellular bioenergetic parameters, such as proton leak and non-mitochondrial oxygen consumption. At the same time, no changes were found in the intracellular antioxidant system after the mitochondrial genome editing. Conclusions: The presence of the m.15059G>A mutation contributes to mitochondrial dysfunction by reducing mitochondrial membrane potential, increasing the generation of reactive oxygen species and lipid peroxidation, and altering mitochondrial bioenergetics. Elimination of the mtDNA containing atherogenic mutation leads to an improvement in mitochondrial function.

Macrophage metabolic plasticity providing their polarization towards classically (M1) or alternatively (M2) activated cells is an important element of the initiation, development, and resolving or inflammation-linked pathologies. The prevalence of M1 or M2 types of macrophages during different stages of diseases supports increased inflammation and phagocytosis or tissue repair, respectively. An imbalance leading to a shift toward an M1- or M2-dominant state is associated with a chronic pathological process. This characterizes the regulation of macrophage phenotypes as a prospective strategy in the treatment of various diseases and makes it relevant to a deep understanding of the mechanisms defining cell polarization. According to the central role of calcium signaling in cell metabolism, changes in calcium homeostasis are closely linked to the regulation of polarization. The exact balance between calcium flows across plasma and intracellular membranes provided by a number of receptors and channels, as well as the differences in the calcium-buffering capability of endoplasmic reticulum and mitochondria, are able to influence macrophage polarization towards an M1 or M2 phenotype. This review focuses on the role of the calcium homeostasis system in macrophage functionality and calcium-induced changes in macrophage metabolism that forms the basis of target disease therapy.

The process of signal transmission and transformation in the central nervous system requires active energy metabolism with high consumption of glucose and oxygen. Reactive oxygen species (ROS) produced as a result of these processes participate in intracellular signaling, but their overproduction leads to oxidative stress. Oxidative stress and α-synuclein aggregation are recognized as activators of neuronal death in Parkinson’s disease. However, much less is known about the physiological role of monomeric synucleins. Using acute brain slices and primary co-cultures of cortical neurons and glial cells derived from transgenic animals with knockout of α-, β-, and γ-synuclein genes, we investigated the role of these proteins in ROS production and energy metabolism. We found that absence of synucleins leads to the reduced ROS production compared to the wild-type cells. The xanthine oxidase (XO) inhibitor led to the decrease in ROS production in the wild-type cells and the brain slices with β-synuclein knockout, whereas in the slices lacking α- or γ-synuclein, the XO inhibition was not observed, suggesting possible regulation of this enzyme by these proteins. Knockout of α- and γ-synucleins resulted in the decrease in mitochondrial membrane potential and reduction in energy capacity (in the form of ATP), which could be one of the mechanisms of XO regulation by synucleins.

This literature review is devoted to the urgent problem of organ-saving surgery for kidney cancer: hypoxic-ischemic changes in the spared renal tissue and possibilities of their prevention through the use of the succinate-forming drug sodium fumarate 15 %.

Inhibition of mitophagy is one of the signs of chronic disease pathogenesis. Detection and measurement of mitophagy levels under in vitro and in vivo models provide a better understanding of the role of mitophagy disorder in disease development and serve as prerequisites for creating a clinically applicable system test. The development of such a system is potentially feasible, but taking into account a number of factors that will be discussed in detail in this article. Here it is considered the main models of mitophagy-based test systems and an analysis is carried out showing their advantages and disadvantages. The future potential for the development of mitophagy-based diagnostic test systems is also discussed here.

Skeletal muscles constantly consume energy, and this consumption level increases correspondingly to the levels of physical activity. Mitochondrial energy metabolism requires constant supplementation with oxygen and substrates for ATP production. Limitation of the mitochondrial substrate supply leads to energy deprivation, which may be followed by sarcopenia and weight loss. Activation of mitochondrial energy metabolism can also stimulate the production of reactive oxygen species and oxidative stress. Here, we studied the effect of various mitochondrial substrates on the energy metabolism of primary skeletal myotubes and how it affects redox balance. We found that as individual components—glutamate, succinate, nicotinamide (NAM) as well as in combination—dicholine succinate (DISU) plus NAM, they increase mitochondrial membrane potential, alter NADH and FAD redox indices, which leads to an increased energy capacity of the skeletal myotubes. Changes in mitochondrial metabolism increased ROS production in mitochondria and cytosol but induced only a minor decrease in the level of the endogenous antioxidant reduced glutathione. Supplementation of young and aged rats with DISU + NAM through the drinking water for 7 days significantly increased myotube diameter in both age groups. Thus, provision of the myotubes with mitochondrial metabolism substrates activates energy metabolism and increases energy capacity but has no effect on oxidative stress. Moreover, it increases myotubes' diameters in young and aged rodent sarcopenia models in vivo.

The study was conducted using the MDCK renal epithelial cell line with sodium dithionite at a concentration of 5 mM to create hypoxic conditions. The parameters of cellular metabolism (including the value of mitochondrial membrane potential, the state of mitochondrial NADH and FAD, the content of Ca²⁺ and Mg²⁺ and the pH level in the cytosol, the rate of glucose absorption by cells, and cell death) were assessed by means of confocal and wide-field fluorescence microscopy. The concentration of dissolved oxygen was established using the polarographic method with a Clark electrode. It was demonstrated that the use of sodium dithionite allows modeling acute hypoxia in vitro with a rapid decrease in the oxygen concentration in the cell incubation medium, which resulted in a change in mitochondrial function and the apoptosis progression. At that, sodium fumarate reduces the level of cell death, which is associated not with the restoration of the ATP-producing ability of mitochondria, but rather with an increase in the contribution of alternative sources of high-energy compounds. At the cellular level, using an optimized hypoxia model, the study revealed the mechanism of the protective role of sodium fumarate, which explained the antihypoxant effectiveness in assisted ischemia of organs and tissues.

Background: Premature ovarian insufficiency is a syndrome characterized by secondary hypergonadotropic ovarian insufficiency and a decrease in the ovarian function in women under 40 years of age, thereby leading to impaired reproductive function, metabolic changes, and a decrease in the quality of life. Increased production of reactive oxygen species inhibits the mitochondrial respiration chain and leads to mitochondrial dysfunction and oxidative stress, which are considered as the triggers of premature ovarian insufficiency. To date, there is no universal method of premature ovarian insufficiency prevention, and accepted treatment methods can only compensate for clinical symptoms, but not restore the lost ovarian reserve. Aim: The aim of this study was to develop a new method of premature ovarian insufficiency treatment based on an experimental ovarian granulosa cell model using omaveloxolone (RTA 408). Materials and methods: The cell model of premature ovarian insufficiency used in the study was realized according to the Patent RU 2 815 539 C1, 2023 (by M.I. Yarmolinskaya, K.A. Zakuraeva, A.Yu. Vinokurov, M.Yu. Pogonyalova). Ovarian granulosa cells isolated from three-month Wistar rats were subcultivated five times with subsequent seeding on coverslips. The cells on the coverslips were divided into three groups (three coverslip in each group). In Group 1 (comparison group — premature ovarian insufficiency model without treatment), cells were treated with cyclophosphamide (0.1 mg/ml) during 6 h. In Group 2 (main group — premature ovarian insufficiency model with experimental treatment) cells were pretreated with RTA 408 (2 µl/ml) during 1 h with subsequent addition of cyclophosphamide (0.1 mg/ml) and cultivation during 6 h. And in Group 3 (control group), cells were added no substances. Results: Compared to Groups 2 and 3, the use of omaveloxolone (RTA 408) led to an increase in reduced glutathione level and a decrease in reactive oxygen species production rate, which indicates the antioxidant and anti-inflammatory effects of the drug and may be considered as a perspective strategy of premature ovarian insufficiency treatment. The claimed method expands the number of premature ovarian insufficiency treatment strategies and avoids the use of hormonal medications and surgical procedures, thus reducing the risk of side effects and complications associated with their use.