RU2757118C2 - Use of lactobacillus fermentum u-21 strain cells for prevention of pathologic tissue change caused by oxidative stress - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to the pharmaceutical industry, namely to use of the Limosilactobacillus fermentum U-21 strain cell culture. Use of a cell culture of the Limosilactobacillus fermentum U-21 strain, filed by RNCIM under the number B-12075, in an effective amount for prevention and elimination of pathological changes caused by the oxidative stress in vital organs and tissues, namely in the liver, lungs, brain and nerve tissue.

EFFECT: described application allows reducing the level of tissue and organ cell damage, especially in nerve tissue.

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Description

Technology area

The invention relates to pharmacology and medicine and relates to the use of cell culture of the strain Limosilactobacillus fermentum U21 (number in VKPM (Moscow) B-12075 from 08.10.14, number in GenBank: WGS PNBB00000000.1. ( Lactobacillus fermentum LfU21)) to prevent changes in tissues caused by oxidative stress.

The present invention can find application in the development of prophylactic and therapeutic drugs with antioxidant properties intended to prevent the development of oxidative stress and its consequences developing in infectious (including viral), neurodegenerative, cardiovascular diseases, in the development of ischemia-reperfusion syndrome, as well as in the development of drugs for the prevention and elimination of pathological changes caused by oxidative stress in vital organs, including the liver, lungs, brain and nerve tissues, when exposed to chemical agents - inducers of oxidative stress.

Background Art

In accordance with modern concepts, most of the pathological, inflammatory and degenerative processes in the body and the development of many "age-related" systemic diseases, in addition to genetic and environmental factors, are associated with the development of oxidative stress (OS) as a result of exposure to reactive oxygen species (ROS) formed in the process of cell life and free radical oxidation (FRO) of biomolecules - proteins, lipids, nucleic acids. (Oxidants, antioxidants and the current incurability of metastatic cancers. Watson J. // Open Biol 2013, 3 120-144; Neurodegeneration, neurogenesis, and oxidative stress. Santos R, Ruiz de Almodóvar C, Bulteau AL, Gomes CM. Oxid Med Cell Longev. 2013; 2013: 730581). Sies H. Oxidative Stress: Concept and Some Practical Aspects. Antioxidants (Basel). 2020; 9 (9): 852.

A shift in the redox balance between the formation of reactive oxygen species (ROS) and the ability of the biological system to detoxify them, leads to the development of OS, which is accompanied by the accumulation of damage to proteins, lipids and nucleic acids, interferes with normal physiological processes and, ultimately, leads to metabolic disorders cells and its death. The reasons for the formation of ROS can be external factors directly, or through a number of metabolic transformations and reactions (for example, negative environmental factors, the development of an immune cellular response to the ingress of various antigens, including microbial agents, etc.), which contribute to the formation of highly reactive particles. OS can also develop as a result of natural processes of cell life, including at the molecular level (implementation of cellular respiration by mitochondria, the work of various enzymes). The most common and active oxidants are ROS, such as superoxide anion, hydroxyl radical, and peroxides.

OS and human diseases

OS is a common pathogenetic mechanism of tissue damage and is one of the main factors influencing the development of many chronic and “age-related” diseases. Chronic lung diseases, including those provoked by infectious and toxic agents, develop under the influence of an excessive amount of reactive oxygen species formed as a result of excessive or chronic activation of the immune system (Domej W., Oettl K., Renner W. Oxidative stress and free radicals in COPD - implications and relevance for treatment. // Int J Chron Obstruct Pulmon Dis. 2014; 9: 1207-1224.doi: 10.2147 / COPD.S51226). Oxidative stress and oxidative modification of proteins are a common feature of almost all cardiovascular pathologies (Rana AK, Singh D. Targeting glycogen synthase kinase-3 for oxidative stress and neuroinflammation: Opportunities, challenges and future directions for cerebral stroke management. Review. Neuropharmacology. 2018 Sep 1 ; 139: 124-136. Doi: 10.1016 / j.neuropharm.2018.07.006], develop during surgical interventions and organ transplantation, in vascular surgery [Bejaoui M, Zaouali MA, Sakly R, Ben Abdennebi H. Olprinone protects the liver from ischemia-reperfusion injury through oxidative stress prevention and protein kinase Akt activation.Can J Physiol Pharmacol. 2018. Mar. 96 (3): 227-231.doi: 10.1139 / cjpp-2017-0153; Ferrari RS, Andrade CF. Oxidative Stress and Lung Ischemia-Reperfusion Injury. Review. Oxid Med Cell Longev. 2015; 2015: 590987. doi: 10.1155 / 2015/590987). OS serves as a trigger for neurodegenerative brain diseases such as Parkinson's Disease, Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS) and others (Oxidative Stress, Pro-Inflammatory Cytokines, and Antioxidants Regulate Expression Levels of MicroRNAs in Parkinson's Sci. Disease. Prasad Aging KN. 2017 ; 10 (3): 177-184).

The central nervous system of a person is one of the most metabolically active tissues of the body, the consumption of a large amount of oxygen by brain cells inevitably leads to the formation of a large number of its active forms. The blood-brain barrier (BBB), which protects the nervous tissue from the penetration of toxins, also restricts the diffusion of other compounds into neurons and glial cells. In the context of oxidative stress, the presence of the BBB prevents some of the naturally occurring antioxidants from reaching the brain cells. All of these features increase the brain's susceptibility to free radicals.

In response to oxidative stress, cytokines are produced - inflammatory mediators, which in turn provoke the production of reactive oxygen species. Those. inflammation and oxidative stress can both provoke and reinforce each other (Oxidative stress in the pathogenesis of neurodegenerative diseases. Treatment options. Vasenina EE, Levin O.S.// Modern therapy in psychiatry and neurology (STNN) 2013, 3-4, pp. 39-46) . Neuroinflammation can be the result of innate immunity - a cellular immune response to improperly folded, nitrosylated, phosphorylated forms of proteins or their aggregates, formed as a result of cellular metabolism failures, or to the penetration of endotoxins into the bloodstream.

The cause of the ingress of endotoxins into the blood can be a disturbed intestinal barrier (BC), the normal functioning of which primarily depends on the activity of the intestinal microbiota (The role of oxidative stress in parkinson's disease. Dias, V., Junn, E., Mouradian, MM / / Journal of Parkinson's Disease. 2013.3, 461-491). Increased intestinal permeability and disrupted intestinal barrier promote the translocation of microorganisms and microbial products, which, through stimulation of Toll-like receptors (TLR), initiate a cellular immune response, activation of macrophages and provoke inflammation and oxidative stress. (Increased intestinal permeability correlates with sigmoid mucosa alpha-synuclein staining and endotoxin exposure markers in early Parkinson's disease. Forsyth, CB; Shannon, KM; Kordower, JH; Voigt, RM; Shaikh, M; Jaglin, JA; Estes, JD; Dodiya , HB; Keshavarzian, A. // PLoS ONE 2011, 6). The same processes can disrupt the integrity of the blood-brain barrier (BBB), contributing to neuroinflammation and damage to the central nervous system (CNS) (The blood-brain barrier and immune function and dysfunction. Banks, WA; Erickson, MA // Neurobiol. Dis. 2010, 37 , 26-32; Microbiome-Gut-Brain Axis and Toll-Like Receptors in Parkinson's Disease. Caputi V, Giron MC. Int J Mol Sci. 2018 Jun 6; 19 (6)). The main component of the cell wall of gram-negative bacteria, lipopolysaccharide (LPS), causes inflammation and neuronal death.

Parkinson's disease (PD) is a chronic progressive neurodegenerative disorder characterized by a number of motor (motor) and non-motor symptoms (autonomic and cognitive impairments, sleep disturbances, olfactory dysfunction and depression) - a consequence of the progressive loss of dopaminergic neurons in the Substantia nigra pars compacta (SNp ), which plays an important role in the regulation of motor and many autonomic functions: respiration, cardiac activity, and the tone of blood vessels. Non-motor symptoms occur at an early stage of the disease in the form of sleep disturbances, constipation, dysphagia, bloating, abdominal pain, bacterial overgrowth in the small intestine, inflammatory bowel disease, and others, may be due to the spread of pathology beyond the basal ganglia (Shulman et al., 2011 ). (Gut dysfunction in Parkinson's disease. Mukherjee A., Biswas A., Das SK // World J Gastroenterol. 2016 Jul 7; 22 (25): 5742-52) and are clearly associated with intestinal microbiota (CM) (Idiopathic Parkinson's disease: Possible routes by which vulnerable neuronal types may be subject to neuroinvasion by an unknown pathogen. Braak H., Rüb U., Gai WP, Del Tredici K. // J. Neural Transm. 2003.110, 517-536).

The presence of reactive oxygen species and other oxidants in the cytosol of a neuron leads to irreversible oligomerization of α-synucleins, the formation of its insoluble form and the formation of so-called Lewy bodies (Interaction between α-synuclein and tau genotypes and the progression of Parkinson's disease. Huang Y, Rowe DB, Halliday GM. J Parkinsons Dis. 2011; 1 (3): 271-6; Role of cytochrome c in α-synuclein radical formation: implications of α-synuclein in neuronal death in Maneb- and paraquat-induced model of Parkinson's disease Kumar A, Ganini D, Mason RP. // Mol Neurodegener. 2016 Nov 24; 11 (1): 70), composed mainly of nitrosylated and phosphorylated synuclein protein oligomers.

Traditional therapies for PD include a variety of replacement therapy options that increase the level of available dopamine in the brain. More modern methods can combine traditional drugs with modulators of TLRs activity, with correction of the diet and composition of nutrients that normalize the state of the intestinal microbiota. The use of probiotic bacteria with a certain antioxidant activity for the preparation of functional food increases the effectiveness of diet therapy (Fetissov SO, Averina OV, Danilenko VN. Neuropeptides in the microbiota-brain axis and feeding behavior in autism spectrum disorder. Review // Nutrition. 2018 . Oct. 27; 61: 43-48). This intervention reduces dysbiosis, relieves symptoms of gastrointestinal dysfunction, positively affects the microbiota-gut-brain axis, and protects the complex network of neuroglia in both the ENS and CNS [Cryan, JF; Dinan, TG Mind-altering microorganisms: The impact of the gut microbiota on brain and behavior. // Nat. Rev. Neurosci. 2012, 13, 701-712; Clemente, JC; Ursell, LK; Parfrey, LW; Knight, R. The impact of the gut microbiota on human health: An integrative view. // Cell 2012, 148, 1258-1270; Maslowski, KM; Mackay, CR Diet, gut microbiota and immune responses. // Nat. Immunol. 2011, 12, 5-9; Perez-Pardo, P .; Dodiya, HBet.al. Gut-brain and brain-gut axis in Parkinson's disease models: Effects of a uridine and fish oil diet. // Nutr. Neurosci. 2017, 1–12; Perez-Pardo, et.al. PD The gut-brain axis in Parkinson's disease: Possibilities for food-based therapies .. // Eur. J. Pharmacol. 2017, 817, 86–95).

Paraquat-induced OS

Paraquat (PQ) is a herbicide, a systemic toxicant, and is one of the standard drugs for simulating intracellular and systemic oxidative stress. In cells, in the presence of oxygen, PQ acts as a redox cycling agent and is a potent inducer of oxidative stress. At the cytosolic level, paraquat produces massive oxidative stress: increases lipid peroxidation, decreases levels of antioxidants such as reduced glutathione (GSH), damages mitochondrial function, increases production and aggregation of α-synuclein (Kuter K, Nowak P, Golembiowska K, Ossowska K. Increased reactive oxygen species production in the brain after repeated low-dose pesticide paraquat exposure in rats. A comparison with peripheral tissues. // Neurochem. Res. 2010.35: 1121–30). The main active metabolite in the induction of OS by paraquat is the superoxide anion radical - the most active and unstable particle that causes a cascade of oxidative and radical processes in the cell.

Paraquat-induced OS is a recognized model for studying various pathologies, provoked or accompanied by increased ROS production and development of OS. The lungs are one of the main targets - in the lung tissues, paraquat is actively absorbed and accumulated in epithelial cells of alveolar types I and II, due to the mechanism of monoamine transport, due to which it is used in modeling pulmonary diseases associated with inflammation (Chakraborti S. et al. (Eds.) . Paraquat-Induced Oxidative Stress and Lung Inflammation Oxidative Stress in Lung Diseases. 2020. https://doi.org/10.1007/978-981-32-9366-3_11)

When administered acutely (once in large doses), paraquat causes pulmonary and systemic inflammation, increases the levels of expression of inflammatory response genes, such as interleukin IL-1, IL-6, IL-8, tumor necrosis factor TNF-α, TNF-β, interferon -1; recruits inflammatory cells such as neutrophils, macrophages and lymphocytes, which contribute to the development of OS and inflammation by generating ROS. Transcription factors are activated: transforming growth factor (TGF) -β NF-kB and protein-1 activator (ap-1), a change in the inflammatory status, measured by the total number of lymphocytes, the level of TNF-α and C-reactive protein) (Tyagi N., Singh R. Paraquat-Induced Oxidative Stress and Lung Inflammation. // Oxidative Stress in Lung Diseases, pp 245-270, 2019. doi: 10.1007 / 978-981-32-9366-3_11; Meng Z., Dong Y ., Gao H., Yao D., Gong Y., Meng Q., Zheng T., Cui X., Su X., and Tian Y. The effects of ω-3 fish oil emulsion-based parenteral nutrition plus combination treatment for acute paraquat poisoning. // J Int Med Res. 2019 Feb; 47 (2): 600-614. doi: 10.1177 / 0300060518806110). These changes are largely similar to the specific response of the immune system to the activity of the SARS-CoV2 virus, the so-called cytokine storm. A cytokine storm (cytokine cascade) is a potentially lethal immune system response that occurs in some cases when infected with the SARS-CoV-2 virus. At the same time, elevated levels of ferritin, interleukin IL-6 and C-reactive protein (CRP) correlate with viral-mediated hyperinflammation and in the clinic serve as a predictor of mortality. The implementation of a paraquat-induced OS model on a BEAS-2B lung cell culture obtained from normal bronchial epithelium was successfully carried out by Podder B. (Podder B., Song KS, Song H.-Y., Kim Y.-S. "Cytoprotective Effect of Kaempferol on Paraquat-Exposed BEAS-2B Cells via Modulating Expression of MUC5AC ". J-STAGE, 2014 Volume 37, Issue 9, P. 1486-1494. doi: 10.1248 / bpb.b14-00239), which may also be a surrogate OS development model for COVID-19.

Small, regular doses of paraquat provoke oxidative damage to the nervous tissue. Due to its ability to penetrate the blood-brain barrier, paraquat, along with other toxic substances (such as rotenone, maneb, MPTP and MPTP) capable of causing corresponding changes in the brain, is used in models for studying Parkinson's disease (PD), since its administration to model animals causes the same changes that are observed with PD. (Dias V., Junn E., Mouradian M.M. The role of oxidative stress in parkinson’s disease. // Journal of Parkinson’s Disease. 2013. 3. 461–491). In mice treated with paraquat, a decrease in motor activity, a dose-dependent loss of dopaminergic nerve fibers in the striatum, and an increased expression and aggregation of a-synuclein in the substantia nigra of the compact brain region (Substancia Nigra pars compacta, SNpc) (Manning-Bog AB, McCormack AL, Li J, Uversky VN, Fink AL & Di Monte DA. The herbicide paraquat causes up-regulation and aggregation of alphasynuclein in mice: paraquat and alpha-synuclein. // J. Biol. Chem. 2002 277, 1641-1644). In addition, there is documented evidence that prolonged exposure to paraquat significantly increased the incidence of PD in humans. (Pouchieu C, Piel C, Carles C, Gruber A, Helmer C, Tual S, Marcotullio E, Lebailly P, Baldi I. Pesticide use in agriculture and Parkinson's disease in the AGRICAN cohort study. // Int J Epidemiol. 2018 Feb 1 ; 47 (1). 299-310; Colle D, Farina M, Ceccatelli S, Raciti M. Paraquat and Maneb Exposure Alters Rat Neural Stem Cell Proliferation by Inducing Oxidative Stress: New Insights on Pesticide-Induced Neurodevelopmental Toxicity. // Neurotox Res . 2018 Jun 1)

Preventing oxidative stress

Antioxidants (AOs) are molecules that interact with free radicals generated in cells and stop the ROS-induced chain reaction that disrupts the functioning of the cell. Substances with antioxidant properties play a key role in maintaining the overall health of a person, prolonging his life and youth (Firuzi, O .; Miri, R .; Tavakkoli, M .; Saso, L. Antioxidant Therapy: Current Status and Future Prospects. Current Medicinal Chemistry , 2011, 18, 3871-3888 3871 0929-8673 / 11 Bentham Science Publishers Ltd). A huge number of antioxidants of various origins, chemical nature and mechanism of action are known (A review on antioxidants, prooxidants and related controversy: Natural and synthetic compounds, screening and analysis methodologies and future perspectives. Carocho M., Ferreira ICFR // Food and Chemical Toxicology 51 (2013) 15-25). In this case, the composition of AO, the amount and form of intake into the body are of great importance.

The natural antioxidant defense system of the body is represented by proteins and enzymes (thioredoxin, glutathione peroxidase, superoxide dismutase, catalase and glutathione reductase, etc.) and low molecular weight compounds (thiol compounds, uric acid, some peptides and amino acids, ascorbic acid, tocathione and others), and glut the same polyunsaturated fatty acids (Pre- and postweaning iron deficiency alters myelination in sprague-dawley rats. Beard JL, Wiesinger JA, Connor JR. // Developmental Neuroscience. 2003; 25: 308–315). Low molecular weight AOs, including thiols, play an important role in the cell: they participate in the maintenance of the cellular redox status, in the work of the detoxification system, in the synthesis of eicosanoids, in the regulation of many mechanisms of cellular signaling, regulation of the cell cycle, gene expression and apoptosis.

In the case of a violation of the redox balance under the influence of unfavorable factors, the antioxidant status of the body can be increased by the introduction of exogenous antioxidants, or by stimulating the body's own antioxidant system, for example, by suppressing microglial activation and decreasing the release of inflammatory factors (IL-6 and TNF) (Beamer, CA; Shepherd, DM Inhibition of TLR-ligand and interferon gamma-induced murine microglial activation by panax notoginseng. // J. Neuroimmune Pharmacol. 2012, 7, 465–476). In many cases, this method can be preferable, since premature degradation of the administered antioxidant and problems with the passage of the biological barrier between tissues and organs, for example, the blood-brain or intestinal barriers, are excluded.

The ability of individual strains of probiotic bacteria to reduce oxidative stress has been proven by various in vitro and in vivo experiments (Amaretti A., di Nunzio M., Pompei A., Raimondi S., Rossi M., Bordoni A. Antioxidant properties of potentially probiotic bacteria: in vitro and in vivo activities. Appl Microbiol Biotechnol. 2013. 97: 809-817. doi: 10.1007 / s00253-012-4241-7), including due to the ability to produce antioxidants (Mishra V., Shah C., Mokashe N ., Chavan R., Yadav H., and Prajapati J. Probiotics as potential antioxidants: A Systematic Review. J. Agric. Food Chem. 26 Mar 2015: 1-48. Doi: 10.1021 / jf506326t; Nowak A., Paliwoda A . and Błasiak J. Anti-proliferative, pro-apoptotic and antioxidative activity of Lactobacillus and Bifidobacterium strains: A review of mechanisms and therapeutic perspectives. Crit Rev Food Sci Nutr. 2018 Oct 16: 1-12. doi: 10.1080 / 10408398.2018.1494539 ; Marsova MV, Abilev SK, Poluektova EU, Danilenko VN A bioluminescent test system reve als valuable antioxidant properties of Lactobacillus strains from human microbiota. World Journal of Microbiology and Biotechnology. 2018.34:27. doi: 10.1007 / s11274-018-2410-2). Since systemic oxidative stress and inflammation (including those provoked by infectious agents, surgical operations and imbalance in the intestinal microflora) play an important role in the development of many age-related and chronic diseases, today an active search and study of probiotic agents that can prevent the development of OS and its consequences are underway. (The role of oxidative stress in Parkinson's disease. Review. Dias V, Junn E, Mouradian MM.//J Parkinsons Dis. 2013; 3 (4): 461-91; The bowel and beyond: the enteric nervous system in neurological disorders Review. Rao M., Gershon MD // Nat Rev Gastroenterol Hepatol. 2016 September; 13 (9): 517-528; Sun MF, Zhu YL, Zhou ZL, Jia XB, Xu YD, Yang Q, et al. Neuroprotective effects of fecal microbiota transplantation on MPTP-induced Parkinson's disease mice: Gut microbiota, glial reaction and TLR4 / TNF-alpha signaling pathway.// Brain Behav Immun. 2018, 70: 48-60; Wanchao S., Chen M., Zhiguo S., Futang X., Mengmeng S. Protec tive effect and mechanism of Lactobacillus on cerebral ischemia reperfusion injury in rats. Braz J Med Biol Res. 2018; 51 (7): e7172. doi: 10.1590 / 1414-431X20187172; Wang N., Song G., Yang Y., Yuan W., Qi M. Inactivated Lactobacillus promotes protection against myocardial ischemia – reperfusion injury through NF-κB pathway. Research Article // Bioscience Reports (2017) 37. https://doi.org/10.1042/BSR20171025).

Probiotic microorganisms and lactobacilli in particular, due to the active synthesis of biologically active compounds and bi-directional communication with the host organism, can actively influence the general condition and antioxidant status of the organism. Many studies have confirmed the antioxidant activity of some strains of lactobacilli. The influence on the AO status of the organism is carried out through a number of mechanisms described in the literature, which can be conditionally divided into three main types.

1. The action directly in the gastrointestinal tract and the reduction of inflammation in the intestine is carried out through the regulation of the composition of the microbiota due to antagonistic activity against pathogenic bacteria and a decrease in the number of their derivatives (Hill, C .; Guarner, F Salminen, S .; et al. Expert consensus document. The international scientific association for probiotics and prebiotics consensus statement on the scope and appropriate use of the term probiotic. // Nat. Rev. Gastroenterol. Hepatol. 2014, 11, 506-514; Wang Y, Wu Y, Wang Y, Fu A , Gong L, Li W, Li Y. Bacillus amyloliquefaciens SC06 alleviates the oxidative stress of IPEC-1 via modulating Nrf2 / Keap1 signaling pathway and decreasing ROS production. // Appl Microbiol Biotechnol. 2017 Apr; 101 (7): 3015-3026 ]; through the restoration of the intestinal barrier (reducing the permeability of KB, reducing the amount of toxins in the blood and inflammation induced by pathogens) [Shieh MJ, Shang HF, Liao FH, Zhu JS, Chien YW. Lactobacillus fermentum improved intestinal bac teria flora by reducing Clostridium perfringens. Eur. J. Clin. Nutr. 2011; 6: e59 – e63; Westfall S., Lomis N., Kahouli I., Dia SY, Singh SP, Prakash S. Microbiome, probiotics and neurodegenerative diseases: deciphering the gut brain axis. Cell Mol Life Sci. 2017 Oct; 74 (20): 3769-3787. doi: 10.1007 / s00018-017-2550-9; Fung TC The microbiota-immune axis as a central mediator of gut-brain communication. // Neurobiol Dis. 2019 Dec 14; 136: 104714. doi: 10.1016 / j.nbd.2019.104714; Benakis C, Martin-Gallausiaux C, Trezzi JP, Melton P, Liesz A, Wilmes P. The microbiome-gut-brain axis in acute and chronic brain diseases. Review.// Curr Opin Neurobiol. 2019 Dec 5; 61: 1-9. doi: 10.1016 / j.conb.2019.11.009).

2. Direct elimination of ROS, possible due to the production of substances with antioxidant properties (SCFA, peptides, amino acids, vitamins C, B1, B12, E, etc.). For example: synthesis of proteins and antioxidant enzymes (superoxide dismutase, glutathione, thioredoxin, catalase, etc.); chelation of toxic ions, incl. iron ions (Fe + 2) and copper (Cu + 2); fermentation of prooxidant substances and toxins; adhesion of prooxidant substances and toxins by exopolysaccharides of the cell wall. Also, many low molecular weight derivatives with antioxidant activity are able to enter the bloodstream through the intestinal barrier and directly reduce the level of oxidative stress in various parts of the body (Kerksick, C .; Willoughby, D. The Antioxidant Role of Glutathione and N-Acetyl-Cysteine Supplements and Exercise-Induced Oxidative Stress Journal of the International Society of Sports Nutrition 2005, 2: 38-44; Pophaly, SD; Singh, R .; Kaushik, JK; Tomar, SK Current status and emerging role of glutathione in food grade lactic acid bacteria Microbial Cell Factories 2012, 11: 114).

3. Direct effect on the host organism, through the implementation of a signaling function through stimulation of intestinal neurons, stimulation of the immune system using microbe-associated patterns through TLR receptors of epithelial cells. Such a mechanism of communication with the host organism has been described, including for lactobacilli. The emission of signaling molecules and hormones stimulates the regulation of the internal systems of signal transduction of eukaryotic cells and the activation of the transcription of enzymes that neutralize free radicals (activating the metabolic pathways Nrf2-Keap1-ARE, NFκB, MAPK and PKC) (Tkachev V.O., Menshchikova E.B., Zenkov N.K. The mechanism of the signaling system Nrf2 / Keap1 / ARE. Review. // Biochemistry, 2011, volume 76, issue 4, pp. 502 - 519; Yunes RA, Poluektova EU et.al. GABA production and structure of gadB / gadC genes in Lactobacillus and Bifidobacterium strains from human microbiota // Anaerobe, 2016. - No. 42. - P. 197-204; Sun MF, Zhu YL, Zhou ZL, Jia XB, Xu YD, Yang Q, et al. Neuroprotective effects of fecal microbiota transplantation on MPTP-induced Parkinson's disease mice: Gut microbiota, glial reaction and TLR4 / TNF-alpha signaling pathway. // Brain Behav Immun. 2018, 70: 48-60; Laiño J., Villena J., Kanmani P., Kitazawa H. Immunoregulatory Effects Triggered by Lactic Acid Bacteria Exopolysaccharides: New Insights i nto Molecular Interactions with Host Cells. // Microorganisms. 2016 Sep; 4 (3): 27. Published online 2016 Aug 15. doi: 10.3390 / microorganisms4030027; Kim DH, Kim S., Lee JH, Kim JH, Che X., Ma HW, Seo DH, Kim TI, Kim WH, Kim SW, Cheon JH Lactobacillus acidophilus suppresses intestinal inflammation by inhibiting endoplasmic reticulum stress.// J Gastroenterol Hepatol ... 2019. Jan; 34 (1): 178-185. doi: 10.1111 / jgh.14362).

There are a number of patents for probiotic organisms with antioxidant activity, including probiotics for prophylactic use in neurodegenerative disorders including Parkinson's disease, Alzheimer's disease (Ho L, Ono K, Tsuji M, Mazzola P, Singh R, Pasinetti GM. Protective roles of intestinal microbiota derived short chain fatty acids in Alzheimer's disease-type beta-amyloid neuropathological mechanisms. // Expert Rev Neurother. 2018 Jan; 18 (1): 83-90), amyotrophic lateral sclerosis and others (WO 2013190068 A1; US 20160199425 A1 ; WO 2011083354 A1; CN 102858948 B; US 9056123 B2; WO 2003002131 A1).

It is important to note the high strain-specificity of the antioxidant and immunomodulatory properties of lactobacilli. The strains described in them demonstrate antioxidant properties and antimicrobial properties against pathogenic microorganisms and are the closest analogue of the invention. However, in contrast to the present invention, the effectiveness of previously presented substances, microorganisms and their extracts was shown only in vitro in models of individual proteins or cell culture, or in a model of the life span of invertebrates, for example, the soil nematode C. elegans . The question remains whether it is possible to extrapolate with certainty the results obtained in an in vivo mammalian model. There are no antioxidant probiotic products on the market.

Disclosure of the essence of the invention

The aim of the present invention is to present a cell culture of the microorganism strain Limosilactobacillus fermentum U21 ( Lactobacillus fermentum U21 ) as a component with antioxidant and protective activity for preventing and eliminating pathological changes under the influence of oxidative stress in a model of paraquat-induced oxidative stress in a mouse body.

The source of isolation of the strain, its main characteristics and antioxidant activity in in vitro experiments are described in detail in patent No. 2705250 dated November 06, 2019 "Strain of Lactobacillus fermentum U21, producing a complex of biologically active substances that neutralize the superoxide anion induced by chemical agents." It should be noted that in April 2020, an article was published on the reclassification of the genus Lactobacillus , and the current name of the species Lactobacillus fermentum is Limosilactobacillus fermentum [Zheng et al., A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae , International Journal of Systematic and Evolutionary Microbiology, 2020]. Thus, the Limosilactobacillus fermentum U21 strain is the Lactobacillus fermentum U21 strain described by us earlier in patent No. 2705250. The L. fermentum U21 strain was deposited in the international collection VKPM (Moscow) under the number B-12075 from 08.10.14., The nucleotide sequence of the genome ( L. fermentum LfU21) was deposited in GenBank under the number WGS PNBB00000000.1.

Technical result

Application of a cell culture strainLimosilactobacillus fermentum U21 (Lactobacillus fermentumU21) promotes prevention and elimination of pathological changes in the vital organs of mice, including nerve tissues, lungs, liver and heart, caused by oxidative stress when exposed to paraquat. Application of a cell culture strainL.fermentum U21 in the development of drugs for the prevention and concomitant therapy of diseases accompanied or induced by oxidative stress seems to be a promising strategy for increasing the effectiveness of these drugs in reducing and preventing damage to tissues and cells of vital organs, including the brain, nervous tissue, heart lungs and liver.

Brief Description of Drawings

Figure 1. Changes in the internal organs of mice when exposed to paraquat. A - organs of mice simultaneously receiving paraquat and L. fermentum U21 strain (group "U21"); B - organs of mice treated with paraquat (group "Toxin").

Figure 2. Median and quartiles (25 - 75%) of the maximum percentage of the duration of vertical descent from the pole (%) in groups of mice: "Toxin" - intraperitoneal injections of paraquat; "U21" - paraquat and L. fermentum strain U21; "Control" - saline solution orally using a probe and intraperitoneal injections.

Implementation of the invention.

The L. fermentum U21 strain was selected from the collection of lactobacillus strains due to its unique antioxidant properties (Marsova MV, Abilev SK, Poluektova EU, Danilenko VN A bioluminescent test system reveals valuable antioxidant properties of Lactobacillus strains from human microbiota // World Journal of Microbiology and Biotechnology . 2018 / DOI 10.1007 / s11274-018-2410-2). The culture fluid of the strain reduced the level of bioluminescence induced by paraquat in test systems based on transgenic E. coli MG1655 strains. Live cell culture strain prolonged the median life expectancy in the model of soil nematodes paraquat-induced oxidative stress (RU №2705250 from. 02/05/2018. "The strain Lactobacillus fermentum U21, producing the biologically active agents, neutralizing superoxide anion induced by chemical agents" Danilenko V.N., Marsova M.V., Poluektova E.U., Odorskaya M.V., Younes R.A.).

To identify and characterize the antioxidant properties of L. fermentum U21 cell culture in vivo, we used the methods and systems of paraquat-induced OS in rodents, which are successfully used and scientifically substantiated in patents and articles (Attia HN, Maklad YA Neuroprotective effects of coenzyme Q10 on paraquat-induced Parkinson's disease in experimental animals.Behav Pharmacol. 2018; 29 (1): 79-86.doi: 10.1097 / FBP.0000000000000342; Bastías-Candia S, Zolezzi JM, Inestrosa NC. Revisiting the Paraquat-Induced Sporadic Parkinson's Disease -Like Model. Mol Neurobiol. 2019; 56 (2): 1044-1055.doi: 10.1007 / s12035-018-1148-z Brooks AI, et.al. (1999) Paraquat elicited neurobehavioral syndrome caused by dopaminergic neuron loss. Brain Res 823: 1-10. Https://doi.org/10.1016/S0006-8993(98)01192-5; Dinis-Oliveira RJ, et.al. Paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment. Crit Rev Toxicol. 2008; 38 (1): 13-71; Matsuura K, Kabuto H, Makino H, Ogawa N. Pole test is a useful method for evaluating the mouse movement disorder caused by striatal dopamine depletion. // J Neurosci Methods. 1997 Apr 25; 73 (1): 45-8; Smeyne RJ, et.al. Assessment of the Effects of MPTP and Paraquat on Dopaminergic Neurons and Microglia in the Substantia Nigra Pars Compacta of C57BL / 6 Mice. PLoS One. 2016; 11 (10): e0164094. doi: 10.1371 / journal.pone.0164094).

Model of oxidative stress induced by regular injections of paraquat solution

The studies were carried out on a model of chronic oxidative stress induced by regular injections of paraquat solution in C57 / BL6 mice. (Neuroprotective effects of coenzyme Q10 on Paraquat-induced Parkinson's disease in experimental animals. Attia H.N., Maklad Y.A. // Behav. Pharmacol. 2018.29, 79-86;).

Male C57 / BL6 mice at the age of 6 weeks were adapted to the vivarium for 2 weeks. The animals were housed in standard cages and kept under a 12 hour light regime and a temperature of ~ 21 ° C. One week before the start of the study, mice were randomly assigned to one of three experimental groups, 10 animals each. To minimize the effect of diurnal variation, tests and procedures were conducted between 08:00 and 13:00 hours.

To induce oxidative stress, an aqueous solution of paraquat (1,10-dimethyl-4,40-bipiridiniydihlorid, Sigma Aldrich, St Louis, MO , USA), study medication - freeze-dried cultures of the probiotic strain L. fermentum U21 at 10 ^8th.

Mice received on a regular basis, twice a week for 3 weeks (6 injections in total), an intraperitoneal injection of paraquat (10 mg / kg) (groups "Toxin" and "Toxin + U21") or saline (group "Control ") In a volume of 100 μl. Daily (simultaneously with injections of paraquat), for 3 weeks, animals were injected parenterally using a probe in a volume of 0.5 ml of physiological saline (groups "Control" and "Toxin") or a lyophilized culture of the probiotic strain L. fermentum U21 in dose 10 ⁸ ("Toxin + U21"). Observations of the animals were carried out for 3 weeks. The animals were examined daily, deaths were recorded.

On the 21st and 23rd days of the study, a behavioral test "descent along a vertical pole" was carried out, which makes it possible to assess the coordination of mouse movements (Matsuura K, Kabuto H, Makino H, Ogawa N. Pole test is a useful method for evaluating the mouse movement disorder caused by striatal dopamine depletion. // J Neurosci Methods. 1997 Apr 25; 73 (1): 45-8) modified (Sleep-wakefulness cycle and behavior in pannexin1 knockout mice. Kovalzon VM, Moiseenko LS, Ambaryan AV, Kurtenbach S, Shestopalov VI, Panchin YV. Behav Brain Res. 2017 Feb 1; 318: 24-27).

On the 23rd day of the experiment, animals were randomly selected to study the safety of dopaminergic neurons in the substantia nigra of the brain.

Examples of determining the properties of the strain L. fermentum U21 according to the present invention.

Oral administration of a freeze-dried culture of the strain L. fermentum U21 at 10 ⁸ cfu significantly reduced mortality in mice line C57 / BL6 chronic paraquat poisoning. Daily use of L. fermentum U21 strain simultaneously with paraquat injections reduced the mortality of mice compared to control groups by 8% and 20%: in the group of intact mice, the mortality rate was 18%, in the group receiving paraquat - 30%, in the group simultaneously receiving paraquat and the strain L. fermentum U21 - mortality rate was less than 10%. (Example 1).

Oral administration of a freeze-dried culture of the strain L. fermentum U21 at 10 ⁸ cfu possible to considerably reduce or completely prevent the changes of the internal organs of experimental animals under the action of the herbicide paraquat. An autopsy of the animals from the paraquat group showed changes in the condition of the internal organs. The use of a culture of L. fermentum U21 strain prevented most of the pathological changes in organs and reduced the manifestations of such changes. (Example 2).

Oral administration of a freeze-dried culture of the strain L. fermentum U21 at 10 ⁸ cfu effectively prevent symptoms of toxic effects of paraquat in mice C57 / BL6 toxin on model of Parkinson's disease (PD), caused by regular injections of paraquat.

Daily use of L. fermentum U21 strain simultaneously with paraquat injections prevented the development of motor dysfunctions. The coordination of mouse movements was assessed according to the standard method in the "descent along a vertical pole" test. In this study, the analysis used the relative (versus total duration) duration of the “controlled” pole descent (vertically downward). According to the results of the study, it was revealed that the differences in the medians of the maximum percentage of the duration of vertical descent from the pole between mice from the "Control", "Toxin" and "Toxin + U21" groups were significantly different when evaluated by Mann-Whitney (p <0.05) and amounted to 54% and 100%, respectively, which indicates the prevention of motor dysfunctions in mice treated with the L. fermentum U21 strain preparation (Example 3).

Oral administration of a freeze-dried culture of the strain L. fermentum U21 at 10 ⁸ cfu showed relatively high protective activity on dopamine neuronal toxin model of Parkinson's disease caused by regular injections of paraquat to mice of C57 / BL6. Tyrosine hydroxylase (TG) is a key enzyme in the biosynthesis of catecholamines, including dopamine. With the development of parkinsonism, the number of TG-positive cells in the brain decreases, causing a number of characteristic dysfunctions. The number of TG-positive cells in the substantia nigra of the paraquat treated mice was determined by counting the frontal sections of the substantia nigra immunohistochemically stained with antibodies.

According to the results of the study, it was revealed that the number of TG-positive cells in the brain of mice from the Toxin and Toxin + U21 groups significantly differed (according to Mann-Whitney, p <0.05)) and, on average for the sample, amounted to 15, 1 and 21.6 thousand per sample, respectively (i.e. animals receiving the probiotic preparation retained 30% more dopaminergic neurons), which indicates the protective effect of the L. fermentum U21 strain against dopaminergic neurons of the substantia nigra. (Example 4).

Examples of the implementation of the method of using the substance according to the present invention.

Example 1. Influence of the strain L.fermentum U21 on the mortality of mice when exposed to oxidative stress induced by paraquat solution.

The studies were carried out on a model of oxidative stress induced by regular injections of a paraquat solution in C57 / BL6 mice. The use of a freeze-dried culture of the L. fermentum U21 strain in parallel with paraquat injections has significantly reduced the mortality of mice. The results of the study are presented in table 2.

Table 2. Changes in mortality in mice treated with paraquat when using the probiotic strain L. fermentum U21

Groups
(Conditions) Control
(NaCl / NaCl) Toxin (Paraquat /
NaCl) Toxin + U21
Paraquat /
L.fermentum Mortality,% eighteen thirty <10

Example 2. Changes in internal organs under the oxidative effects of paraquat.

The animals that died during the 15-21 days of the experiment were dissected. After 3 weeks, three individuals from each group were randomly selected to study the state of internal organs. The selected individuals were euthanized with ether. An autopsy was performed. The study revealed that the toxic effect of paraquat primarily affects the state of the lungs and liver.

In mice that received only paraquat (group "Toxin"), the liver is greatly enlarged and heterogeneous, the color is uneven, the surface with whitish spots, the edges of the lobes are smoothed, rounded; lungs of irregular shape, with traces of fibrotic changes, in particular, a tuberous surface, collapsed in size, brown spots, traces of hemorrhage; the heart is slightly enlarged, there is an effusion in the abdominal cavity (Figure 1).

The internal organs of mice, which received paraquat simultaneously with the L. fermentum U21 strain (group "Toxin + U21") and euthanized with ether, basically corresponded to the physiological norm: the liver was slightly enlarged, uniform in color, regular shape, smooth, the edges of the lobes were sharp; lungs of normal shape and size, evenly colored over the entire surface, without visible inclusions and signs of pathological changes. Heart without signs of pathological changes, normal size, no effusion in the abdominal cavity.

Example 3. Prevention of motor dysfunctions.

The studies were carried out on a model of oxidative stress induced by regular injections of a paraquat solution in C57 / BL6 mice.

A healthy intact mouse, when placed on a vertical pole, reorients vertically downward and descends using all four paws while moving. In this case, descent is carried out and controlled by the motor activity of the mouse itself, which requires a high degree of coordination. The method of sliding off the pole "sideways" is characteristic of mice exposed to the toxin and is poorly controlled by their own motor activity. In this study, the analysis used the relative (versus total duration) duration of the “controlled” pole descent (vertically downward).

According to the results of the study, it was revealed that the differences in the medians of the maximum percentage of the duration of vertical descent from the pole between mice from the Control group (95%) and the Toxin group (54%) were close to significant. This indicates a limitation of the ability of mice to vertical ("controlled") descent from the pole. The medians of the maximum percentage of the duration of vertical descent from the pole in mice simultaneously receiving paraquat and L. fermentum U21 strain preparation (100%) and in mice from the Control group (95%) did not differ significantly, which indicates that the administration of in mice, the bacterial strain prevents motor dysfunctions in mice (Figure 2).

Table 3. Medians of the maximum percentage of the duration of the vertical descent from the pole.

Groups
(Conditions) Control
(NaCl / NaCl) Toxin (Paraquat /
NaCl) "Toxin + U21" (Paraquat /
L.fermentum ) Vertical descent,% 95 54 100

Example 4. Protective activity against dopamine neurons

The brain is extremely sensitive to oxidative stress. One of the most vulnerable areas to this effect is the substantia nigra of the midbrain. (Substantia nigra) playing an important role in the regulation of motor function, muscle tone, the implementation of statokinetic function, in many autonomic functions: respiration, cardiac activity, tone of blood vessels. The neurons of the substantia nigra of the brain are active producers of catecholamines and are most susceptible to oxidation processes due to the peculiarities of metabolism. Tyrosine hydroxylase (TG) is a key enzyme in the biosynthesis of catecholamines, including dopamine. With a decrease in the number of TG-positive cells in the brain, a number of dysfunctions characteristic of the early stage of parkinsonism are manifested, including impaired coordination of movements and decreased intestinal transit.

The number of TG-positive cells in the substantia nigra was determined by counting the frontal sections of the substantia nigra immunohistochemically stained with antibodies.

To determine the protective activity of the L. fermentum U21 cell culture, on the 23rd day of the experiment, the animals were anesthetized with chloral hydrate and perfused through the heart with physiological saline in phosphate buffer (PBS), and then 4% formaldehyde solution in PBS. The brain was removed and after an additional fixation in the perfusion solution during 12 hours at 4 ^{° C} was impregnated with a 30% sucrose solution in PBS for 24 hours. Coronal sections of the midbrain and striatum 40 micrometers thick were obtained on a freezing microtome and collected in PBS. 4 series of sections of each brain were collected. Sections were placed in antifreeze and stored at ^-20 ° C until staining procedure.

Each 4th section containing substantia nigra of the midbrain was immunohistochemically stained for tyrosine hydroxylase (TH) using monoclonal antibodies against it, diluted 1: 200 in PBS solution supplemented with 2% normal horse serum, 0.5% TritonX100 detergent and 0.01% sodium azide (all reagents - Sigma-Aldich, USA). The primary antibody solution free-floating sections were kept with stirring at ⁴ ° C for 24 hours. After three times washing in PBS, the material was incubated in a solution of donkey antibodies against mouse immunoglobulin conjugated with the Cy2 fluorescent dye (Jackson Immuno Research, USA) at room temperature for one hour. Stained sections were placed on glass slides, covered with 50% glycerol and a cover slip.

Quantitative analysis of TH-immunopositive (TH +) cells was performed using an OlympusIX81 microscope equipped with a motorized stage and computer controlled, and an Olympus DP72 digital camera. Cells were counted on a computer monitor using the Cell * software (Olympus Soft Imaging Solution GmbH, Germany). At low magnification (10x objective), an overview image of the section with substantia nigra (SNC) and ventral tegmental area (VTA ventral tegmental area). Then, at high magnification (40x objective), an uninformed operator counted the number of TH + cells using the optical fractionator method. The counting results are presented in Table 4.

Table 4. The number of TG-positive cells in the substantia nigra of the brain.

Groups
(Conditions) Control
(NaCl / NaCl) Toxin (Paraquat /
NaCl) Toxin + U21
(Paraquat /
L.fermentum ) Number of TG + cells, average 19 520 15104 21600 Number of TG + cells,% 90 70 100

Claims (4)

1. The use of a cell culture of the Limosilactobacillus fermentum U-21 strain deposited by VKPM under the number B-12075, in an effective amount to prevent and eliminate pathological changes caused by the effects of oxidative stress in vital organs and tissues, namely in the liver, lungs, brain and nerve tissues. 2. Use according to claim 1, wherein the prevention and elimination of pathological changes in vital organs and tissues includes neuroprotection. 3. Use according to claim 2, wherein the pathological changes in vital organs and tissues include degradation of dopaminergic neurons. 4. Use according to claims 1 to 3, characterized in that the pathological changes in vital organs and tissues include protection against oxidative stress induced by the chemical agent.

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