Musk is a dried secretion of the preputial gland of musk deer, widely used since the middle of the 4th century as a biostimulant in traditional Arabic, Indian, Tibetan, and Chinese medicine. Currently, within the transition to evidence-based medicine, relationships between the composition of musk and its pharmacological properties is being studied. Musk is a multicomponent system consisting of substances of different classes, the content and relative amounts of which are directly affected by the technology of musk extraction. The development of a new method – electropulse extraction – significantly increased the yield of the extract and affected the composition of musk by increasing the relative content of the peptide-protein fraction. This ensured the emergence of new properties of musk, namely the property to increase physical performance while increasing the safety of physical activity by suppressing the state of inflammation and oxidative stress associated with extreme stress. In general, progress in the development of new deer musk-based drugs is associated with technological innovations in this industry.

Sirtuin 1 (SIRT1) is a class III histone deacetylase that plays a key role in resolving inflammation through known epigenetic mechanisms involving histone and nuclear factor κB (NF-κB) deacetylation. Deacetylation reduces the transcriptional activity of NF-κB and the associated production of pro-inflammatory cytokines such as interleukin 1β (IL-1β), tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). In the present study, we show for the first time that biomodeling of acute lung inflammation by a single injection of lipopolysaccharide (LPS) induces synchronous oscillations of mRNA levels of cytokines IL-1β, TNF-α, IL-6 and SIRT1 deacetylase in the lungs, the maximum amplitudes of cytokine mRNA oscillations are observed between 1.5 and 5 hours, whereas high levels of SIRT1 mRNA are observed up to 24 hours, when cytokine mRNA oscillations have already faded, which is consistent with the hypothesis about the role of SIRT1 as a factor acting in the phase of inflammation resolution. The study shows that the mechanism of anti-inflammatory action of inhaled hexapeptide Leutragin, a δ-opioid receptor agonist, is related to its ability to increase SIRT1 mRNA expression and decrease the amplitudes of IL-1β, TNF-α, and IL-6 mRNA oscillations in the lungs, which generally leads to the resolution of inflammation in the conditions of biomodeling of acute lung inflammation.

Human leukocyte antigen plays a primary role in the formation of immune response and pathogenesis of diseases of various etiologies, including the development of negative side effects induced by pharmacological agents. Modern pharmacosafety standards require improvement of existing test systems to conduct high-quality preclinical studies. A number of humanized transgenic mouse lines with hybrid HLA I class molecules on the cell surface, which correspond to the human allelic variants HLA-A*02:01, HLA-B*07:02 , and HLA-C*07:02 , were developed at the Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia. In this article, we present experimental data on quantitative determination of β2-microglobulin protein and HLA by the “sandwich” ELISA method in mice with different alleles of HLA I class genes. The results obtained confirm the presence of target functional proteins (transgenicity) in humanized transgenic mice, which is consistent with our previous data obtained when determining the primary sequence of the transgene using Sanger sequencing. We also discuss the scientific and practical significance of such biomodels, as well as the scope of their application.

The introduction of a transgene can impact negatively the functioning of vital systems in biomodels. We carried out a comparative analysis of the immune response of mice of the HLA-A*02:01 humanized transgenic line, mice with mouse β2-microglobulin gene knockout, and wild-type mice to the introduction of horse immunoglobulin as an antigen. The biomodel lines were created at the Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia. The maximum immune response was achieved on the 30th day from the onset of immunization in animals of the HLA-A*02:01 line and wild-type mice. Antibody titers in these groups increased sharply and approached 1:8,000,000 and 1:4,000,000, respectively. This indicates that genome modification in HLA-A*02:01 transgenic humanized mice did not affect functioning of the immune system. No similar dynamics of the increase in antibody titers was observed in the mice line with mouse β2-microglobulin gene knockout. On the 7th and 30th day, the antibody titer in this group increased to a value of 1:400 and 1:6,400, respectively. The weak immune response in mice with mouse β2-microglobulin gene knockout confirms the undeniably important role of this protein in immune response formation.

The introduction of a transgene can impact negatively the functioning of vital systems in biomodels. We carried out a comparative analysis of the immune response of mice of the HLA-A*02:01 humanized transgenic line, mice with mouse β2-microglobulin gene knockout, and wild-type mice to the introduction of horse immunoglobulin as an antigen. The biomodel lines were created at the Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia. The maximum immune response was achieved on the 30th day from the onset of immunization in animals of the HLA-A*02:01 line and wild-type mice. Antibody titers in these groups increased sharply and approached 1:8,000,000 and 1:4,000,000, respectively. This indicates that genome modification in HLA-A*02:01 transgenic humanized mice did not affect functioning of the immune system. No similar dynamics of the increase in antibody titers was observed in the mice line with mouse β2-microglobulin gene knockout. On the 7th and 30th day, the antibody titer in this group increased to a value of 1:400 and 1:6,400, respectively. The weak immune response in mice with mouse β2-microglobulin gene knockout confirms the undeniably important role of this protein in immune response formation.

Human leukocyte antigen plays a primary role in the formation of immune response and pathogenesis of diseases of various etiologies, including the development of negative side effects induced by pharmacological agents. Modern pharmacosafety standards require improvement of existing test systems to conduct high-quality preclinical studies. A number of humanized transgenic mouse lines with hybrid HLA I class molecules on the cell surface, which correspond to the human allelic variants HLA-A*02:01, HLA-B*07:02, and HLA-C*07:02, were developed at the Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia. In this article, we present experimental data on quantitative determination of β2-microglobulin protein and HLA by the “sandwich” ELISA method in mice with different alleles of HLA I class genes. The results obtained confirm the presence of target functional proteins (transgenicity) in humanized transgenic mice, which is consistent with our previous data obtained when determining the primary sequence of the transgene using Sanger sequencing. We also discuss the scientific and practical significance of such biomodels, as well as the scope of their application.

In this work, we investigate the effect of a number of pharmaceutical preparations on the expression of genes, which are used as molecular targets under the conditions of debilitating physical exercise in minipigs. In the conducted experiment, the effectiveness of the following preparations was compared: “Mio-Activ-Sport” dietary supplement, a liposomal deer musk extract, intranasal insulin, and a liposomal ginseng preparation. The selected biomarkers included the leukocyte blood fraction and mRNA expression of NFE2L2 and HMGB1 genes in lymphocytes. Among the studied preparations, the liposomal deer musk extract showed the highest effectiveness. Thus, during seven days of therapy, this preparation almost completely prevented the pro-inflammatory effect of physical load. At the same time, the liposomal deer musk extract led to a manyfold increase in the expression of NFE2L2 gene, which is responsible for antioxidant defense of the organism. In terms of action, the liposomal deer musk extract outperformed insulin – the reference drug, whose protective effects are well known from the scientific literature. These findings confirm the prospects of deer musk preparations for medical rehabilitation.

Введение. Согласно данным литературы Аппарат лазерной диагностики «ЛАЗМА СТ» не был ранее использован для доклинических исследований тканевых нарушений на мелких лабораторных животных. Цель исследования – изучение возможности использования аппарата лазерной диагностики «ЛАЗМА СТ» на мелких лабораторных животных – мутантных мышах линии С57BL/KsJYLeprdb/+ (db/db) – в качестве новой тест системы, для оценки тканевых изменений при сахарном диабете 2 типа (СД 2). Методика. Патологические изменения у мышей с СД изучали на генетической модели СД 2 у мутантных мышей С57BL/KsJYLeprdb/+ (db/db) (n=40); для контроля использовали группу фенотипически здоровых гетерозиготных мышей той же линии (db/+m) (n=16). Общее количество животных составляло 56 голов. Исследование проводили на аппарате лазерной диагностики «ЛАЗМА СТ», который адаптирован приспособлением, ограничивающим подвижность мышей во время измерений. Аппарат «ЛАЗМА СТ» позволяет осуществлять одновременный неинвазивный контроль состояния микроциркуляци крови и лимфы, а также определять уровень активности митохондриальных окислительных коферментов в тканях. Дополнительно измеряли уровень глюкозы в крови фотометрическим методом на приборе Accu-Chek (Швейцария). Результаты. Использование аппарата ЛАЗМА СТ позволило в реальном времени неинвазивно изучить в динамике нарушения микроциркуляции (крови и лимфы) и окислительного метаболизма (НАДН и ФАД) при СД 2, сопоставить их с нарушениями глюкозы в крови, а также прогнозировать тяжесть выявленных нарушений в условиях компенсации, субкомпенсации и декомпенсации. Аппарат ЛАЗМА СТ позволил одновременно выявить начавшиеся нарушения липидного и белкового обмена (пигменты-липофусцин и порфирин), которые свидетельствуют о тяжести прогноза заболевания. Выводы. Аппарат ЛАЗМА СТ, адаптированный для применения у мышей с генетической моделью СД 2 типа, представляет собой новую неинвазивную, информативную и безопасную тест-систему, позволяющую в динамике контролировать и прогнозировать тяжесть нарушений микроциркуляции и тканевых окислительно-восстановительных коферментов – НАДН и – ФАД. The aim of this study was to evaluate a possibility of using the LAZMA ST laser diagnostic apparatus in small laboratory animals, C57BL/KsJYLeprdb/+ (db/db) mutant mice, as a new test system for assessing changes in tissues in type 2 diabetes mellitus (DM2). Methods. Pathological changes were studied on a genetic model of DM2 in C57BL/KsJYLeprdb/+ (db/db) mutant mice (n=40). Phenotypically healthy heterozygous mice of the same strain (db/+m) (n=16) were used as a control group. The study was performed with a LAZMA ST laser diagnostic apparatus that was adapted for mice with a device limiting their mobility during measurements. LAZMA ST allows simultaneous noninvasive monitoring of blood and lymph microcirculation along with measurements of tissue activity of mitochondrial oxidative coenzymes. Additionally, blood glucose was measured photometrically with an Accu-Chek (Switzerland) glucometer. Results. The use of LAZMA ST allowed noninvasive, real-time evaluation of disorders in blood and lymph microcirculation and oxidative metabolism (NADH and FAD) in DM2, comparing them with glycemic disorders, and also predicting severity of these disorders in the conditions of DM2 compensation, subcompensation and decompensation. The LAZMA ST device provided simultaneous detection of the onset of lipid and protein metabolic disorders (lipofuscin and porphyrin pigments) to predict severity of the disease. Conclusion. The LAZMA ST device adapted for using in a murine genetic model of DM2 is a new, noninvasive, informative, and safe test system that allows to monitor and predict the dynamics of severity of disorders in microcirculation and tissue redox coenzymes, NADH and FAD.

We studied the effect of infrared radiation (IR) course exposure on pathological changes in the tissues of C57BL/KsJYLeprdb/+ (db/db) mutant mice with type 2 diabetes mellitus using a LASMA ST device. This apparatus allows simultaneous non-invasive monitoring of blood and lymph microcirculation, as well as determining the activity of mitochondrial oxidative coenzymes in tissues before and after IR therapy. IR waves (690 nm) were found to have a positive therapeutic effect on the functional state of db/db mice. This included a rapid healing of skin wounds caused by maceration and prolonged normalization of oxidative metabolism parameters (NADH, FAD, POM) in body tissues. IR therapy has a cumulative effect, leading to a 2.3–2.5-fold increase in life expectancy and life quality, compared to the control group.

We studied the effect of infrared radiation (IR) course exposure on pathological changes in the tissues of C57BL/KsJYLepr db/+ (db/db) mutant mice with type 2 diabetes mellitus using a LASMA ST device. This apparatus allows simultaneous non-invasive monitoring of blood and lymph microcirculation, as well as determining the activity of mitochondrial oxidative coenzymes in tissues before and after IR therapy. IR waves (690 nm) were found to have a positive therapeutic effect on the functional state of db/db mice. This included a rapid healing of skin wounds caused by maceration and prolonged normalization of oxidative metabolism parameters (NADH, FAD, POM) in body tissues. IR therapy has a cumulative effect, leading to a 2.3–2.5-fold increase in life expectancy and life quality, compared to the control group.

Direkord is an original drug containing the active substance of dicholine succinate, which improves the sensitivity of insulin receptors in neurons to insulin. The aim of the work was to select an optimal dosage and to study the efficacy, safety, and tolerability of Direkord, a solution for intramuscular injection, in ischemic stroke patients in the early recovery period. In total, 132 patients after the first ischemic stroke in the carotid system, confirmed by computed or magnetic resonance imaging, with the stroke remoteness from 3 weeks to 2 months and the mean age of 64.35±8.03 years, were randomized into three treatment groups. Patients in the first (n=44) and second (n=44) groups received Direkord intramuscularly for two weeks at a dose of 400 mg/day and 600 mg/day, respectively. Patients in the third group received placebo. The treatment response was assessed in terms of improved neurological status, functional state, and cognitive functions, including at least a two-fold decrease in the total score on the NIHSS scale, the total score on the Barthel scale ≥95, and the total score on the MoCA scale ≥26. Four weeks after the onset of the study, 34.1, 43.2, and 18.2% of the patients responded to therapy in the first, second, and third group, respectively. An analysis based on the Fisher’s exact test revealed a statistically significant difference between the groups (p=0.036). These results suggest that Direkord is statistically and clinically significantly superior to placebo at a two-week intramuscular therapy at a dose of 600 mg/day in patients with ischemic carotid stroke in the early recovery period. The safety profile of Direkord when used in various therapy regimens does not differ from that of placebo. The phase III study should confirm the preliminary results obtained in the current work.

Direkord is an original drug containing the active substance of dicholine succinate, which enhances neuronal insulin sensitivity. In this work, we study the tolerability, safety, and pharmacokinetic parameters of dicholine succinate when administered intramuscularly in a phase I clinical trial in healthy volunteers. In total, 18 healthy volunteers –11 men and 7 women – with a mean age of 30.4±7.8 years, were recruited into a randomized study. At stage I, 6 volunteers (group 1) received dicholine succinate intramuscularly every other day with a dose escalation from 0.16 mg/kg/day to 600 mg/day. At stage II, 12 volunteers (group 2) received dicholine succinate intramuscularly at a single dose of 200 mg, and then, at stage III, the same 12 volunteers received dicholinesuccinate at a dose of 600 mg/day (3 x 200 mg at an interval of 8 hours) for seven days. The safety population in this study included all randomized volunteers. Data from 12 volunteers (group 2) were included in the calculation of the pharmacokinetic parameters. All volunteers completed all procedures of the three research stages in accordance with the protocol. According to clinical and laboratory monitoring data, no adverse events were registered during the study. The drug was well tolerated, with no signs of hyperemia, edema, and bruising being observed at the injection site. The volunteers did not complain of pain, itching, and burning. After a single injection of dicholine succinate, the concentration of choline in the bloodstream reached its maximum value after an average of 0.375±0.365 hours with the half-life of 1.271±1.071 hours. After repeated administration at a dose of 600 mg per day, no cumulation of the active substance was observed. The data obtained have confirmed a good safety profile of Direkord; therefore, the drug can be recommended for further investigation in a study involving patients.

For the first time, LASMA ST, a device for laser diagnostic, was adapted for preclinical studies on laboratory db/db mouse genetic models of type 2 diabetes. The proposed method for studying of tissue changes during diabetes mellitus consists in a simultaneous control of microcirculation compartments: blood and lymph flow and oxidative coenzymes. The presented approach is characterized by a high informational value, safety and objectivity, as well as by the possibility of dynamic monitoring and obtaining online data on tissue metabolism (reduced nicotinamide adenine dinucleotide - NADH and oxidized flavin adenine dinucleotide - FAD).

Glutamate excitotoxicity is involved in the pathogenesis of many disorders, including stroke, traumatic brain injury, and Alzheimer’s disease, for which central insulin resistance is a comorbid condition. Neurotoxicity of glutamate (Glu) is primarily associated with hyperactivation of the ionotropic N-methyl-D-aspartate receptors (NMDARs), causing a sustained increase in intracellular free calcium concentration ([Ca2+]i) and synchronous mitochondrial depolarization and an increase in intracellular superoxide anion radical (O2–•) production. Recently, we found that insulin protects neurons against excitotoxicity by decreasing the delayed calcium deregulation (DCD). However, the role of insulin in O2–• production in excitotoxicity still needs to be clarified. The present study aims to investigate insulin’s effects on glutamate-evoked O2–• generation and DCD using the fluorescent indicators dihydroethidium, MitoSOX Red, and Fura-FF in cortical neurons. We found a linear correlation between [Ca2+]i and [O2–•] in primary cultures of the rat neuron exposed to Glu, with insulin significantly reducing the production of intracellular and mitochondrial O2–• in the primary cultures of the rat neuron. MK 801, an inhibitor of NMDAR-gated Ca2+ influx, completely abrogated the glutamate effects in both the presence and absence of insulin. In experiments in sister cultures, insulin diminished neuronal death and O2 consumption rate (OCR).

Extreme physical exertion (work to failure) was simulated in untrained mini pigs. The results of complete blood tests, lactate measurements and clinical urine analysis revealed variations in the basic parameters of animals similar to those observed in people after extreme exertion.

Glutamate excitotoxicity is implicated in the pathogenesis of many disorders, including stroke, traumatic brain injury, and Alzheimer’s disease, for which central insulin resistance is a comorbid condition. Massive glutamate release primarily through ionotropic N-methyl-D-aspartate receptors (NMDARs) causes a sustained rise in [Ca2+]i, followed by mitochondrial depolarization and an increase in intracellular O2• (superoxide) production. Recently, we found that insulin protected neurons against excitotoxicity by diminishing the delayed calcium deregulation (DCD), However, a role of insulin in superoxide production in excitotoxicity still needs to be clarified. The present study is aimed to investigate the effects of insulin on glutamate-evoked superoxide generation and DCD using the fluorescent indicators dihydroethidium, MitoSOX Red, and Fura-FF in rats cultured cortical neurons. We found that insulin significantly diminished both the intracellular and mitochondrial superoxide production in neurons exposed to glutamate and there was a strong linear correlation between [Ca2+]i and intracellular superoxide. MK 801, an inhibitor of NMDAR-gated Ca2+ influx, completely abrogated the glutamate effects in both the presence and absence of insulin. In experiments on sister cultures, insulin diminishes neuronal death. Thus, collectively, data obtained suggest that insulin diminishes glutamate-induced superoxide production in neurons via fall of [Ca2+]i increased and thereby improves viability of neurons

Since the discovery of insulin and insulin receptors (IR) in the brain in 1978, numerous studies have revealed a fundamental role of IR in the central nervous system and its implication in regulating synaptic plasticity, long-term potentiation and depression, neuroprotection, learning and memory, and energy balance. Central insulin resistance has been found in diverse brain disorders including Alzheimer’s disease (AD). Impaired insulin signaling in AD is evident in the activation states of IR and downstream signaling molecules. This is mediated by Aβ oligomer-evoked Ca2+ influx by activating N-methyl-D-aspartate receptors (NMDARs) with Aβ oligomers directly, or indirectly through Aβ-induced release of glutamate, an endogenous NMDAR ligand. In the present opinion article, we highlight evidence that IR activity and free intracellular Ca2+ concentration [Ca2+]i form a double-negative regulatory feedback loop controlling insulin sensitivity, in which mitochondria play a key role, being involved in adenosine triphosphate (ATP) synthesis and IR activation. We found recently that the glutamate-evoked rise in [Ca2+]i inhibits activation of IR and, vice versa, insulin-induced activation of IR inhibits the glutamate-evoked rise in [Ca2+]i. In theory, such a double-negative regulatory feedback loop predicts that any condition leading to an increase of [Ca2+]i may trigger central insulin resistance and explains why central insulin resistance is implicated in the pathogenesis of AD, with which glutamate excitotoxicity is a comorbid condition. This model also predicts that any intervention aiming to maintain low [Ca2+]i may be useful for treating central insulin resistance.

Mechanical damage to the primary neuronal culture serves as a convenient in vitro model for studying the molecular and cellular mechanisms involved in the spread of the lesion in mechanical brain trauma. In this study, changes in the intracellular concentrations of Ca2+ ([Ca2+]i) and Na+ ([Na+]i), as well as mitochondrial potential (ΔΨm) in response to mechanical damage to the primary culture of rat cortical neurons were studied. Rapid (within 1–2 s) damage to the neuronal network in the form of an approximately 3 × 0.1-mm scratch caused an abrupt increase in [Ca2+]i and [Na+]i as well as a sharp drop in ΔΨm. There was a gradual recovery of these parameters to the basal level in ~78% of the cells that responded to the culture damage. The second phase of the [Ca2+]i rise (delayed calcium deregulation, DCD), synchronous with a marked drop in ΔΨm, occurred in 22% of the cells. In such cells, [Na+]i remained at the level of a high plateau. The addition of insulin (100 nM) 5 min before the mechanical damage reduced the proportion of neurons that had DCD and a sustained increase in [Na+]i. Thus, the presence of insulin contributed to the normalization of Ca2+ and Na+ homeostasis and the functioning of mitochondria, which were impaired under the in vitro simulation of mechanical brain trauma.