We studied the effect of reprogrammed CD8+ T cells (rT cells) from the bone marrow of intact mice on tumor cells and neovasculogenesis in mice with orthotopic Lewis lung carcinoma (LLC). Reprogramming of T cells was carried out using a MEK inhibitor and a PD-1 blocker; the targeting of rT cells to tumor cells was achieved by preincubation with LLC cell lysate. It was shown that the antitumor effect of rT cells was based on apoptosis of tumor cells. In addition, cell therapy reduced the number of endothelial cells (CD45—CD309+) and angiogenic cell precursors (CD45—CD117+CD309+), mesenchymal stem cells (CD45—CD31—CD34—CD44+), myeloid (CD45+CD34+CD31—) and non-myeloid (CD45+CD34—CD31—) fibrocytes, and leukocytes (CD45+) in the lungs and increased their number in the blood. Thus, rT cells impaired the recruitment of neovasculogenic cells to the lung. The antitumor effects of rT cells are superior to those of naive CD8+ T cells. The proposed reprogramming method can be useful in developing effective approaches to the therapy of lung cancer, as it allows obtaining cytotoxic rT cells capable of reducing the activity of neovasculogenesis.

We propose a model of combination treatment of Lewis lung carcinoma (LLC) in C57BL/6 mice that includes tumor resection and chemotherapy. A single injection of 5×106 LLC cells into the right lateral subcostal region caused the growth of the primary tumor and its metastasis to the lung. For reducing metastasis and mortality after resection, the primary tumor should be removed with subcutaneous fat on day 8 after inoculation. Antitumor and antimetastatic effects and reduced mortality were achieved by intraperitoneal injection of carboplatin (63.3 mg/kg) and paclitaxel (13.3 mg/kg); chemotherapy was administered twice. The combination of the two approaches increased the survival: the antitumor and antimetastatic effects were observed in 60

Circadian rhythm disruption induced by exposure to light—excessive in duration and intensity (dark deprivation)—and the impact of hepatotoxins are both significant risk factors for liver pathology. The purpose of this research was to evaluate the potentially synergistic effects of continuous lighting and carbon tetrachloride (CCl4) toxicity on the structural and functional organization and daily (circadian) rhythmicity of the liver in rats, as well as to look at the corrective capability of exogenous melatonin under such influences. The experiment was conducted on 200 outbred 6-month-old Wistar rat males, which were distributed into five groups, including a control (normal light/dark cycle), dark deprivation (constant light), CCl4 intoxication, and combined exposure to CCl4 and dark deprivation with or without melatonin administration (0.3 mg/kg). Histological, immunohistochemical (Ki-67, Per2, and Bmal1), biochemical, and ELISA methods were used. Circadian rhythms were analyzed using cosinor. It was shown that dark deprivation and CCl4 intoxication act synergistically, potentiating liver damage. The most severe necrosis (54.17 ± 9.13%), steatosis (57.85 ± 12.14%), and suppression of regenerative potential (decreased proportion of binucleated hepatocytes to 2.17 ± 0.21%) were observed in the group with combined exposure. This correlated with a substantial decline in melatonin content in blood plasma (7.85 ± 2.1 pg/mL) and a profound disruption in circadian rhythms. Administration of exogenous melatonin exerted pronounced hepatoprotective and chronotropic effects: it significantly reduced pathological changes (necrosis reduced to 16.35 ± 6.17%), stimulated regeneration (binucleated hepatocytes increased to 13.57 ± 0.81%), and restored the circadian rhythms of the studied parameters to levels close to those of the control. The key pathogenetic link in the potentiation of CCl4 hepatotoxicity under dark deprivation is light-induced deficiency of endogenous melatonin. Exogenous melatonin demonstrated high efficacy in correcting both structural and functional damage and liver desynchronosis, confirming its therapeutic potential under conditions of combined exposure to chronodisruptors and toxins.

The effect of para-tyrosol (PT), a hydroxyalkylphenol exhibiting antioxidant properties, on the level of DNA damage in testicular tissue cells and sperm of rats treated with doxorubicin was studied using the DNA comet assay. N-acetylcysteine (NAC) was used as a reference drug. It was found that both drugs reduced the number of DNA breaks in rat testicular tissue cells (by 46-48%). The antigenotoxic effect, judging by %DNA in tail, in relation to spermatozoa was detected only in PT. The number of DNA damage in male germ cells after treatment with PT was reduced by 52% from the control (administration of doxorubicin alone). The results suggest that it is advisable to use PT in order to reduce the genotoxicity of doxorubicin, in the therapy of Hodgkin lymphoma (HL) in treatment regimens containing this anthracycline antibiotic.

The effect of dark deprivation on the morphofunctional state and rhythmostasis of the liver under CCl4 toxic exposure has been studied. The relevance of this study is due to the fact that the hepatotoxic effect of carbon tetrachloride on the liver is well studied, but there are very few data on the relationship between CCl4 intoxication and circadian biorhythms, and most of the studies consider the susceptibility of the organism in general and of the liver in particular to the influence of CCl4 in some separate periods of the rhythm, but not the influence of this chemical agent on the structure of the whole rhythm. In addition, earlier studies indicate that light disturbance causes certain changes in the morphofunctional state of the liver and the structure of the circadian rhythm of a number of parameters. As a result of this study, we found that the effect of CCl4 in conditions of prolonged dark deprivation causes more significant structural and functional changes in hepatocytes, as well as leading to significant changes in the circadian rhythms of a number of parameters, which was not observed in the action of CCl4 as a monofactor. We assume that the severity of structural and functional changes is due to the light-induced deficiency of melatonin, which has hepatoprotective properties. Thus, the mechanisms of CCl4 action on CRs under conditions of light regime violations leave a large number of questions requiring further study, including the role of melatonin in these processes.

Regenerative processes in the liver were studied in 3-month-old (young) and 9-month-old (aged) male Wistar rats on day 1 after 30 and 70% hepatectomy. Regardless of the resected liver volume, shifts in the biochemical parameters of the serum in aged rats were more pronounced than in young animals. After 30% hepatectomy, no age differences in the rate of hepatic regeneration were found, while after 70% liver resection this parameter was higher in young rats. Hepatectomy in young rats led to recruitment of MSC, hepatocyte precursors, endothelial and epithelial progenitor cells into the liver parenchyma and increased fluidity of the plasma and mitochondrial membranes of hepatocytes. In aged rats, the recruitment of MSC, hepatocyte precursors, and endothelial progenitor cells into the injured liver was impaired and the rigidity of the mitochondrial membranes of hepatocytes increased.

The antitumor and antimetastatic activity of dopamine D2 receptor antagonists spiperone was studied in C57BL/6 mice in a model of combined pathology (emphysema and lung cancer). Emphysema was induced by administration of LPS and cigarette smoke extract. Lung cancer was induced by injection of Lewis lung carcinoma cells into the lung. It has been shown that under conditions of combined lung pathology, spiperone prevents inflammatory infiltration and emphysematous expansion of the lungs and reduces the size of the primary tumor node, the number of metastases, and the area of the lungs affected by metastases. Spiperone reduces the number of cancer stem cells (CSCs) in the lungs and blood of mice with combined pathology. CSCs isolated from the lungs and blood of mice with combined pathology treated with spiperone had a significantly lower potential to form a tumorosphere in vitro than CSCs from untreated mice with emphysema and lung carcinoma. Thus, blockade of dopamine D2 receptors is a promising approach for correcting combined lung pathology and can be used in the development of a method for treating lung cancer in patients with emphysema.

In male C57BL/6 mice (8-12 weeks) and male Wistar rats (12 weeks), the effect of dibornol (2,6-diisobornyl-4-methylphenol) on the level of spontaneous DNA damage in cells of the bone marrow, liver, kidneys, and rectum of mice (series I) and genotoxic effects in rat testicular cells after administration of cytostatic drugs with different mechanisms of action (series II) were studied using the DNA comet assay. In series I, dibornol was intragastrically administered to mice once at doses of 200, 400, and 2000 mg/kg; in series II, dibornol was intragastrically administered to rats at a dose of 10 mg/kg for 5 days before and 5 days after the cytostatic treatment (methotrexate, doxorubicin). It was found that dibornol in all studied doses did not produce the genotoxic (carcinogenic) effect and reduced the level of spontaneous DNA damage in the bone marrow. After combined administration of cytostatic drugs (doxorubicin, methotrexate) and dibornol, the level of DNA breaks was reduced to 38.5 and 49% of the control, respectively.

The responses of tumor stem cells and various populations of CD4 and CD8 T cells of young and aged C57BL/6 mice were studied in a lung cancer model. Using Lewis lung carcinoma cell line, an orthotopic model of lung cancer was modeled. Cancer stem cells, circulating tumor cells, and various populations of CD4 and CD8 T cells in the blood and lung tissue were studied by cytometry. We revealed age-related differences in the content of various populations of CD4 and CD8 T cells in the blood and lungs of intact young and aged mice. Age-related features of the reaction of various populations of cancer stem cells and CD4 and CD8 T cells in the blood and lungs of animals in the Lewis lung carcinoma were shown.

The pharmacological activity of granulocyte CSF (G-CSF) immobilized using electron-beam synthesis nanotechnology (imG-CSF) was evaluated in an experimental model of ovarian reserve depletion. The effectiveness of the drug was compared with that of its unmodified form. Depletion of the ovarian follicular pool in female Sprague-Dawley rats was caused by a single intravenous injection of the antitumor drug etoposide in the maximum tolerated dose. The effectiveness of the studied drugs was assessed by serum concentration of anti-Mullerian hormone (AMH) measured by ELISA and by the number of primordial, two-layer, multilayer, and atretic follicles counted on serial sections of the ovaries (5-μm thick; through the entire organ) stained with hematoxylin and eosin. It was found that imG-CSF prevents depletion of the ovarian reserve in the model used, which was confirmed by high AMH concentration and higher numbers of primordial, two- and multilayer follicles in comparison with the corresponding parameters in the control (etoposide), and by a decrease in the severity of atretic processes. Unmodified form of the drug demonstrated lower efficiency.

The effect of ketanserin on inflammation, liver fibrosis, and microviscosity of the plasma and mitochondrial membranes of hepatocytes was studied on young (3 months) and old (9 months) male Wistar rats with experimental liver cirrhosis. Ketanserin reduced inflammation, area of the connective tissue, and liver damage and improved serum biochemical parameters in rats of both age groups; in old rats, the effects were more pronounced than in young animals. In old rats, ketanserin reduced polarity of hepatocyte plasma and mitochondrial membranes in the area of protein-lipid contacts, which determined higher effectiveness of ketanserin during the treatment of liver cirrhosis in aged animals.

The effect of humic acids and substances with similar action - derivatives of succinic acid (ethylmethylhydroxypyridine succinate) and combined agent consisting of succinic acid, nicotinamide, riboflavin, and riboxin on the performance and stress resistance of experimental rats was studied. Performance was assessed in the test of exhaustive forced swimming with a load, stress resistance was evaluated by the serum level of corticosterone and open field behavior, and the state of anaerobic metabolism was estimated by the serum level of lactate after swimming test. Humic acids from peat showed anti-stress activity comparable to that of the officinal preparation and preventive effect on fatigue during physical exercise. They can be recommended as a component for the development of drugs that increase human performance and stress resistance.

We studied the effects of the extract of the terrestrial part of Aconitum baicalense in BALB/c female mice at the early stages after the injection of N-methyl-N-nitrosourea (MNU). The extract reduced inflammatory activity and tumor growth in the mammary gland. The antitumor and anti-inflammatory effects of the extract are based on the inhibition of cancer stem cells, hematopoietic stem cells, and hematopoietic progenitor cells that promote inflammation. The extract of A. baicalense disrupted the recruitment of epithelial progenitor cells and angiogenesis precursors to the mammary gland preventing neovascularization and transformation of epithelial cells into tumor cells.

BACKGROUND: There are evidences that light pollution, which causes melatonin deficiency and disruption of circadian rhythm, is associated with the development of malignant neoplasms of the liver, non-alcoholic fatty liver disease, biliary cirrhosis, and a number of other pathologies of this organ. AIM: The aim of research was to study the features of chronic influence of constant lighting on lability of morphofunctional state of liver of mature Wistar rats and the structure of circadian rhythms of its parameters. MATERIALS AND METHODS: The study was conducted on 80 rats divided into 2 groups: a control group kept under a fixed light regime (light/dark 12/12 h, lights on at 8:00 and off at 20:00), and an experimental group kept under constant lighting 24 h a day. The duration of the experiment was 3 weeks. RESULTS: Its established that influence of constant light led to an increase in the size of hepatocytes and a decrease in nuclear-cytoplasmic ratio, average ploidy and proportion of binuclear hepatocytes, and also to development of fatty degeneration, a decrease in the expression of Bmal1 and Clock, and an increase in the expression of per2 and p53 in hepatocytes. At the same time, there was a decrease in glycogen content in hepatocytes. Dark deprivation also caused an increase in glucose levels, AST activity, and a decrease in blood levels of total protein and albumin. Constant lighting caused a rearrangement of the circadian rhythms of the area of nuclei, the area of the hepatocyte and nuclear-cytoplasmic ratio, Bmal1, per2, Clock expression, and led to destruction of Ki67 and p53 circadian rhythms in hepatocytes. Under conditions of constant lighting, the circadian rhythms of the content of lipids and glycogen in hepatocytes, ALT activity in the blood, and the content of total and direct bilirubin were also destroyed. CONCLUSIONS: It has been established that constant illumination causes a restructuring of the circadian rhythms of a number of studied parameters against the background of morphological and functional changes, indicating a decrease in the adaptive capacity of the liver.

Various stem cells were studied in female BALB/c mice at the early terms after administration of N-methyl-N-nitrosourea to search early diagnostic markers and therapeutic targets. At these terms, damage to the epithelium and endothelium, inflammation, and fibrosis were observed in the mammary gland, but the tumor was not detected. Cancer stem cells, hematopoietic stem cells (HSC), hematopoietic progenitor cells, angiogenic precursors, and epithelial progenitor cells were found in the blood and mammary gland. Cancer stem cells (CD44(+)CD24(-)) are proposed as the early diagnostic marker of breast cancer, and short-living HSC, hematopoietic progenitor cells, and angiogenic precursors (CD45-CD117(+)FLK-1(+)) as predictors of the formation of tumor microenvironment.

Введение. Тромболитическая терапия с применением активаторов плазминогена достаточно эффективна, но сопряжена с риском возникновения геморрагических осложнений и реализуется только внутривенным путём введения. Существует необходимость в пероральной тромболитической терапии. Среди возможных кандидатов для эффективного перорального фибринолиза/тромболизиса следует обратить внимание на сериновые протеиназы, не вызывающие сайт-специфического гидролиза факторов свёртывания крови. Необходимо изучить их энтеральную биодоступность и тромболитическую активность. Цель исследования: изучить путь поступления сериновой протеиназы субтилизина в системный кровоток при пероральном применении, а также сравнить тромболитическую активность протеолитических ферментов трипсина, химотрипсина и субтилизина. Материалы и методы. Проведено открытое сравнительное экспериментальное исследование в параллельных группах крыс по определению тромболитической активности изучаемых препаратов in vitro. Сформировано 4 группы животных: 3 для исследования лекарственных препаратов субтилизина, трипсина, химотрипсина и 1 контрольная. В каждой группе происходила инкубация фрагментированных тромбов с предполагаемым тромболитическим агентом. Для исследования всасывания иммобилизированного субтилизина (ИС) 30 крысам после срединной лапаротомии интубировали фрагмент тощей кишки для инфузии растворов. Для ИС, меченного флуоресцеин‑5‑изотиоцианатом (ФИТЦ), определены особенности всасывания из тощей кишки в систему кровь–лимфа. Меченный ИС экстрагировали из прилегающей к области интубации интерстициальной жидкости кишечника, слизистый слой которого был удален, и из паренхимы лимфатических узлов (I и II порядка). Для визуальной детекции абсорбции ИС на эпителии тощей кишки применили метод конфокальной микроскопии. Тромболитическая активность ИС, трипсина и химотрипсина исследована на фрагментированном тромбе. Результаты. In situ установлено, что ИС через 60 мин после начала инкубации в тощей кишке детектируется и в плазме крови и в лимфе (p < 0,01). Концентрация препарата в плазме крови из v. mesenterica (223,8 ± 39,8 мЕд/мл) более чем в 10 раз выше, чем в лимфе из цистерны грудного протока (19,27 ± 2,33 мЕд/мл). Снижение концентрации препарата в v. cauda (143,7 ± 24,4 мЕд/мл) по сравнению с v. mesenterica (p < 0,01) объясняется первичным метаболизмом в печени и последующим почечным клиренсом. При конфокальной микроскопии хорошо видно, что ИС активно абсорбируется на слизистой тонкого кишечника с накоплением в толще крипт и поступлением в сосудистую сеть. В экспериментах in vitro показано, что в условиях нормального или компенсированного кислотно-основного состояния крови достоверно наибольшую тромболитическую активность имеет препарат ИС (p < 0,05 по сравнению с контролем, трипсином и химотрипсином). За 30 мин инкубации ИС уменьшает площадь тромба более чем на 20%. Заключение. Энтеральная биодоступность ИС обеспечивается за счёт активной абсорбции на слизистой тонкого кишечника, прямого поступления в сосуды портальной системы и накопления в лимфатическом русле с дальнейшим дренажем в системный кровоток. Тромболитическое действие ИС существенно превосходит трипсин и химотрипсин, что позволяет считать малоперспективным разработку пероральных тромболитиков на основе трипсина и химотрипсина. Background. Thrombolytic therapy with plasminogen activators is quite effective, but it is associated with the risk of hemorrhagic complications and is implemented only by intravenous administration. Need for oral thrombolytic therapy exists. Among the possible options for effective oral fibrinolysis/thrombolysis, attention should be paid to serine proteinases that do notcause site-specific hydrolysis of blood coagulation factors. It is necessary to study their enteral bioavailability and thrombolytic activity. Objectives: to study the route of entry of serine proteinase subtilisin into the systemic circulation after oral administration, and also to compare the thrombolytic activity of the proteolytic enzymes trypsin, chymotrypsin, subtilisin. Materials/Methods. We carried out an open comparative experimental study in parallel groups of rats to determine the thrombolytic activity of the studied drugs in vitro. Four groups were formed: 3 groups to study subtilisin, trypsin, chymotrypsin and 1 control group. In each group, fragmented thrombi were incubated with the putative thrombolytic agent. To study the absorption of immobilized subtilisin (IS), 30 rats after midline laparotomy were intubated with a jejunum fragment for infusion of solutions. For IS labelled with fluorescein‑5‑isothiocyanate (FITC), the features of absorption from the jejunum into the blood-lymph system were determined. The labelled IS was extracted from the interstitial fluid adjacent to the intubation area, the mucous layer of which was removed, and from the parenchyma of the lymph nodes (sentinel and second-tier nodes). Confocal microscopy was used to detect visually the IS absorption on the jejunal epithelium. The thrombolytic activity of IS, trypsin, and chymotrypsin was studied on a fragmented thrombus. Results. It was found in situ that IS 60 min after the start of incubation in the jejunum was detected in both blood plasma and lymph (p < 0.01). Plasma drug concentration from the mesenteric vein (223.8 8 ± 39.8 mU/ml) is more than 10 times higher than that in the lymph from the thoracic duct cistern (19.27 ± 2.33 mU/ml). Reducing drug concentration in the caudal vena (143.7 ± 24.4 mU/ml) compared to the mesenteric vein (p < 0.01) is explained by primary hepatic metabolism and subsequent renal clearance. Confocal microscopy clearly shows that IS is actively absorbed at the mucous membrane of the small intestine with accumulation in the crypt and entering the vasculature. In vitro experiments have shown that under conditions of the normal or compensated acid-base blood state, the IS has significantly the highest thrombolytic activity (p < 0.05 compared to control, trypsin and chymotrypsin). After 30 minutes of incubation, IS reduces the thrombus area by more than 20%. Conclusions. IS enteral bioavailability is provided due to active absorption at the mucous membrane of the small intestine, direct entry into the vessels of the portal system and accumulation in the lymphatic bed with further drainage into the systemic circulation. IS thrombolytic effect is significantly superior to trypsin and chymotrypsin, which makes the development of oral thrombolytics based on trypsin and chymotrypsin unpromising.

The regenerative properties of p-tyrosol were investigated in a model of testicular insufficiency caused by a toxic effect on spermatogonial stem cells (single administration of paclitaxel in the maximum tolerable dose). Against the background of p-tyrosol administration, we observed an increase in the number of normal spermatogonia and Sertoli cells, stimulation of spermatogenesis, and renewal of the spermatogenic tissue. The treatment with p-tyrosol also led to a decrease in DNA damage in cells of the testicular tissue. These changes were accompanied by a decrease in the level of free radicals, an increase in antioxidant protection, and normalization of the redox potential.

The morphological and functional state of the reproductive system was studied in male outbred rats (SD stock) and male F1(CBA×C57BL/6) mice after long-term (3 months) methotrexate administration. The drug was administered subcutaneously once a week for 4 weeks, the dose for male rats was 1 mg/kg, for male mice 2.2 mg/kg. It was found that male rats retained the ability to conceive, their reproductive potential was not limited by increased risk of embryo death. At the same time, signs of astheno- and pathospermia were revealed. The testicular tissue was characterized by reduced content of the sources of the proliferative pool of spermatogenesis. In mice treated with methotrexate, increased content of DNA breaks was detected in the testicular cells.

Under conditions of steady-state hemopoiesis, nuclear factor NF-κB, in contrast to MAP kinase p38, plays an important role in the maintenance of the initial level of secretory activity of monocytes. The increase in the production of G-CSF under stress conditions (10-h immobilization) is mainly regulated by the alternative p38MARK signaling pathway via activation of p38 synthesis. It was shown that under conditions of cytostatic-induced myelosuppression, the production of protein kinase p38 in cells decreases, and it, like NF-κB, is not the main one in the production of hemopoietin by mononuclear phagocytes.

We studied the formation of injuries in lung endothelium and the response of angiogenesis cells during modeling of pulmonary emphysema in male and female C57BL/6 mice with metabolic disorders. Hemodynamic disturbances and reduction in the area of the microvasculature caused by combined pathology in male mice were more pronounced than in females. Mobilization and migration of angiogenic precursors were impaired in both male and female mice. In males, activity of recruiting endothelial progenitor cells, vascular smooth muscle cells, luminal cells of nascent vessels and pericytes into the lungs was additionally reduced. In females, accumulation of endothelial progenitor cells (CD45—CD31+CD34+), vascular smooth muscle cells, and pericytes in the lungs was observed, which indicated activation of endothelial regeneration. Sex differences in the reaction of the lung endothelium and angiogenesis cells can be explained by genetic factors of lipid and glucose metabolism.