RU2821311C1 - Method of using superoxide dismutase 2 as an antioxidant in ultra-low dosages - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to medicine, biotechnology, biology and agriculture and concerns a method of using superoxide dismutase 2 in ultra-low doses from 10^-12 to 10^-200 as an activator of SOD2 gene expression with development of subsequent systemic reactions increasing synthesis of intrinsic intracellular SOD and effects of antioxidant protection stimulation.

EFFECT: invention enables effective use of superoxide dismutase 2 in ultra-low doses.

1 cl, 10 tbl

Description

Technical field

The invention relates to the field of biotechnology, biology and agriculture and concerns the use of superoxide dismutase 2 (SOD2) as an activator of SOD2 gene expression with the development of subsequent systemic reactions of increasing the synthesis of its own intracellular SOD2 and the effects of stimulating antioxidant defense in ultra-low dosages.

This drug can be effectively used for the treatment of inflammatory, infectious, cardiovascular, oncological and neurodegenerative diseases.

In agriculture, the drug can be used in livestock farming, increasing the immune resistance of animals. In crop production, the drug can increase stress resistance to environmental factors, activate the yield of green mass and cause an increase in crop yields.

The drug can be used in the form of aqueous solutions or dry form for enteral use. In the form of sterile solutions for parenteral administration. In agriculture in the form of a dry substance for the preparation of aqueous solutions for irrigation and use in solutions for hydroponic growing of plants.

Superoxide dismutase (SOD, EC 1.15.1.1) belongs to the group of antioxidant enzymes. It protects the human body from constantly formed highly toxic oxygen radicals.

Superoxide dismutase catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. Consequently, the enzyme plays a large role in the antioxidant protection of many cells that are in contact with oxygen.

The enzyme is present in all aerobic organisms. In mammals, SOD2 is localized in the mitochondrial matrix. In humans, superoxide dismutase 2 is encoded by the SOD2 gene on chromosome 6.

State of the art

The use of superoxide dismutase as an antioxidant is widely described in the literature. However, nowhere in the literature is there any mention of the effect of ultra-low doses on living organisms.

The use of the enzyme superoxide dimutase in various industries has been described.

Thus, the patent literature discloses the use of superoxide dismutase in medicine, cosmetology, agriculture, the food industry, etc.

Patent RU 2600843, published on October 27, 2016, discloses a composition for preventing or reducing the risk of developing metabolic dysfunctions associated with obesity, containing Saccharomyces cerevisiaevarboulardii in an amount between 106 and 109 colony forming units (CFU) per daily administration and the enzyme superoxide dismutase in an amount between 1 and 100 mg.

Patent RU 2557894, published on July 27, 2015, discloses a cosmetic product for rapid skin restoration, containing superoxide dismutase, B vitamin, succinic acid, characterized in that it additionally contains a water-soluble analogue of vitamin E, Trolox and L-arginine, the activity of superoxide dismutase is 200,000-300,000 IU/mg, B vitamin is represented by dexpanthenol, component ratio, wt. %: superoxide dismutase 0.01-32, dexpanthenol 0.1-20, succinic acid 0.5 - up to pH 6.0, Trolox 0.13-45, L-arginine 0.9-64, water for injection - the rest.

Patent RU 2789458, published on 02/03/2023, describes a method of increasing the immune status of bee colonies infected with nosematosis by feeding obtained by enriching sugar syrup 1:1 with a high-molecular-weight antioxidant drug S.O.D. - superoxide dismutase + catalase + glutathione peroxidase, pre-dissolved in water, intended for 3-fold feeding with a frequency of 12 days with the drug in dosages of 100 mg, 200 mg and 300 mg

Patent GB 2183658, published 06/10/1987, describes manganese dependent superoxide dismutase MnSOD (SOD2), which can be used to catalyze the reduction of superoxide radicals, reduce reperfusion injury, prolong the survival time of isolated organs or treat inflammation.

Patent CN 101420973, published 04/29/2009, relates to compositions adapted for pharmaceutical administration containing at least one superoxide dismutase and at least one prolamine-based peptide fragment.

Disclosure of the invention.

Unexpectedly, the authors of the present invention discovered the ability of the superoxide dismutase enzyme in ultra-low doses to be used as an activator of SOD2 gene expression with the development of subsequent systemic reactions of increasing the synthesis of its own intracellular SOD and the effects of stimulating antioxidant defense, i.e. with preservation of antioxidant properties even in dilutions from 10 ^-12 to 10 ^-200 .

Thus, the present invention relates to a method of using superoxide dismutase 2 as an activator of SOD2 gene expression with the development of subsequent systemic reactions of increasing the synthesis of its own intracellular SOD and the effects of stimulating antioxidant defense, which consists in introducing superoxide dismutase 2 into a living organism in a dilution of 10 ^{- 12} to 10 ^-200 .

The technical result of the present invention lies in the effectiveness of using superoxide dismutase 2 as an activator of SOD2 gene expression with the development of subsequent systemic reactions of increasing the synthesis of its own intracellular SOD and the effects of stimulating antioxidant defense, in ultra-low dosages in dilutions from 10 ^-12 to 10 ^-200 .

The mechanism of action of the drug in this dilution is based on the ability to restore and maintain at an optimal level the expression of the Superoxide Dusmutase 2 gene, thereby increasing its own intracellular synthesis of the enzyme, ensuring pronounced systemic biological reactions.

Recombinant human mitochondrial superoxide dismutase 2 (SOD2) protein (NM_001024465) MVPro, produced by OriGeneTechnologies Inc, USA, was used as superoxide dismutase 2. The dose used was 1.0 mg. The indicated dose is dissolved in a 3 ml glass bottle with a screw cap in 99 drops of ethyl alcohol. The initial dynamization of the matrix solution is carried out by striking the bottle, clenched in a fist, on a leather pad 10 times. 1 ml of the matrix solution is taken and transferred to a bottle with a screw stopper with a capacity of 200 ml, dissolved with bidisitylated water in a volume of 99 ml, mixed and shaken on a pillow 11 times. the process is repeated in new disposable bottles until the entire spectrum of hundredth potencies is obtained from matrix to CH 200 (domestic potency C200), each subsequent potency adds one hit to 40. Thus, the enzyme was obtained in the required dilution from 10 ^-12 to 10 ^-200 .

Carrying out the invention

Experiments were carried out with dilution of superoxide dismutase 2, preparation B1 - dilution 10 ^-12 and preparation B2 - 10 ^-200 , test object of ceriodaphnia, species Ceriodaphnia dubia.

The object of the experiment is ceriodaphnia, the species Ceriodaphnia dubia (Since 2016, this has been the designation for a group of hard-to-distinguish ceriodaphnia species, which includes C.dubia itself, C.affinis and several other species. In all domestic methods, this species is called C.affinis, but according to WoRMS and opinion Doctor of Biological Sciences A.A. Kotova, it is correct to call it S.dubia. They are smaller than daphnia, which makes working with them more difficult, but their lifespan is shorter, only about 2 months, and therefore we were able to get results faster than when conducting experiments on daphnia, fish or mice.

In each line, 20 ceriodaphnia were used, individually seeded in 10 ml penicillin vials.

The following lines participated in the experiment:

KO Clean control (with the addition of clean water 2 times a week for uniformity of manipulations);

K Control with saline supplements 2 times a week for a month (for comparison with B1, B2);

K+ Control with saline supplements 2 times a week throughout the experiment (for comparison with B1+, B2+);

B1 Experimental repetitions with the addition of drug B1 2 times a week for a month;

B2 Experimental repetitions with the addition of drug B2 2 times a week for a month;

B1+ Experimental repetitions with additions of drug B1 2 times a week throughout the entire experiment (58 days);

B2+ Experimental repetitions with additions of the drug B2 2 times a week throughout the entire experiment (58 days).

Total: 7 lines of 20 penicillin vials = 140 penicillin vials = 140 ceriodaphnia.

The main indicators of changes in the state of the body were the parameters of survival, fertility and life expectancy in comparison with control animals. These parameters were measured 2-3 times a week. Twice a week the medium in the experiment was replaced with a new one, and, accordingly, the drug was added. The experiment continued until the death of the last ceriodaphnia in the observed samples. The total duration of the experiment was 58 days.

Results obtained during the experiment

During the first two weeks of the experiment, the best survival rate was observed in lines supplemented with B ₂ (95-100%) (Table 2). On the 31st day of the experiment, the number of crustaceans in all studied lines dropped to 50% and below (Table 2;). At the same time, the survival rate in replicates with the additions of B ₁ and B ₂ was higher than with the addition of saline solution.

On the 29th day of the experiment, survival rate with Dubianpm supplemented with pure saline solution was higher than in the zero control without its addition (Table 3).

In terms of the average life expectancy of Ceriodaphnia dubia in the studied lines, by the 30th day of the experiment, we found a statistically significant stimulating effect of 13.7 and 12.4%, respectively, for preparations B1 and B2 compared with additives of pure saline solution.

The maximum lifespan of individuals during the entire experiment was 58 days for the B1+ preparation, and 47 days for pure saline solution. Thus, the effect of increasing the maximum duration of Ceriodaphnia individuals in a 58-day experiment was discovered.

Thus, we studied preparations B1 and B2 using ceriodaphnia Cyeriodaphnia dubia as a test object during 1- and 2-month experiments when adding them to the medium in a supplement mode 2 times a week for 1 and 2 months.

According to the results of biotesting on the 29th day of the experiment, the effect on the survival of C. dubianpn crustaceans with the addition of preparation B2 and B1 was more pronounced than with the addition of pure saline solution.

In terms of the average life expectancy of Ceriodaphnia dubia, during a 1-month experiment, a statistically significant stimulating effect of 13.7 and 12.4%, respectively, was found for preparations B1 and B2 compared with additives of pure saline solution.

It should be noted that for such an important general biological integral indicator as the average lifespan, the increase we noted in the lifespan of the crustaceans Ceriodaphnia dubia by 12-14%) is very significant.

In terms of the maximum lifespan of Ceriodaphnia individuals, in a 58-day experiment for the B1+ preparation, an effect was found to increase the maximum lifespan of individuals by 10 days compared to pure saline solution.

The next experiment was carried out with microalgae.

B1-superoxide dismutase 2 at a dilution of 10 ^-12

B2-superoxide dismutase 2 at a dilution of 10 ^-200

The test object of the study was an algologically pure culture of the green chlorococcal microalgae Scenedesmus quadricauda (Typhoid) Breb. (=Desmodesmuscommunis (E.Hegew.)E. Hegew.), widespread in fresh water bodies of the Southern and Northern Hemispheres and is an important link in their trophic chains.

S. quadricauda was obtained from the collection of algal cultures of the Department of Microbiology, Faculty of Biology, Moscow State University named after M.V. Lomonosov (DMMSU, strain S-3). This type of algae belongs to coenobial organisms. 2- and 4-celled coenobia are more common, and 8- and 16-celled ones are less common. During reproduction, autospores are formed in each cell, which form a young colony inside the mother cell. This species is widely used in world practice in biotesting to assess the quality of the aquatic environment and the toxicity of various compounds and materials.

The culture was grown in Uspensky medium No. 1 (composition, g/l: 0.025 KNO3, 0.025 MgSO4, 0.1 KH2PO4, 0.025 Ca(NO3)2, 0.0345 K2CO3, 0.002 Fe2(SO4)3; pH 7.0-7 ,3) vluminostat at illumination of 3.5 klx with a change of day and night (12:12 hours), temperature 22±2°C and stirred 2 times a day to avoid cell sedimentation.

The test culture was previously adapted to substances B1 (enzyme superoxide dismutase 2 at a dilution of 10 ^-12 ) and B2 (enzyme superoxide dismutase 2 at a dilution of 10 ^-200 ). For this moment of sowing the culture on a nutrient medium with an initial amount of 40 thousand/ml substances were added to the culture according to the following scheme:

B1 was added to culture flasks (volume 50 ml) every working day (on days 0, 1, 2, 3, 4, 7, 8, 9, 10, 11, 14 of adaptation) 0.1 ml.

B2 was added in the same volume, but twice a week, on Monday and Friday (0.4, 7, 11, 14 days of adaptation).

Data on culture growth during the addition of substances B1 and B2 are presented in table. 6.

As can be seen from Table 6, the growth of the culture during adaptation to substances B1 and B2 practically did not differ from the control unadapted culture during the entire 14-day adaptation period. Moreover, in all variants, including control, the absolute number of cells in cells/ml during 14 days of adaptation increased approximately 60 times from 40 thousand/ml (on the day of sowing) to 2.5 million cells/ml.

According to fluorescence microscopy, the proportion of living cells in all variants and all observation periods was at the control level and amounted to 97-99%.

On the seventh day of adding substances, stimulation of the photosynthesis efficiency indicator was recorded compared to the control by 10% for substance B2 and by 24% for substance B1 (Table 7).

Experiment with the reference toxicant potassium bichromate.

On the 14th day of adaptation, an inoculum was taken from flasks with pure control and flasks with algae cells adapted to substances B1 and B2 to conduct the main part of the experiment with the toxicant. On the same day, an experiment was carried out to study the responses of a pure culture of Scenedesmus quadricaudcma adapted to substances B1 and B2. The standard toxicant is potassium bichromate. The toxicant was taken in concentrations of 0.1; 1 and 5 mg/l (low, medium and highly toxic concentrations) of potassium dichromate. The number of algae cells in each flask was 40 thousand/ml. The volume of nutrient medium with culture in each flask is 50 ml. For each studied concentration of toxicant (0.1, 1 and 5 mg/l), and each type of culture (pure, adapted to B1, adapted to B2), three replicates were taken, a total of 27 flasks. Three flasks with a pure culture without the addition of a toxicant were taken as a control. Total 30 flasks.

Measurements were carried out on days 1, 3, 7, 10 and 14 of the chronic experiment.

The studied indicators are the number of cells, the efficiency of photosynthesis and the proportion of living cells in the culture.

As can be seen from Table 8, a significant stimulation of photosynthesis of the crop (by 15-76%) was noted for all cases of experiment with different concentrations of the toxicant in cultures adapted to substances B1 and B2 already on the first day of the experiment. At the same time, in cultures adapted to B 2, the stimulating effect was more pronounced. Thus, the difference with the control in cultures adapted to B2 with different concentrations of the toxicant was 41-76%. The controls in this case were photosynthesis in the presence of appropriate concentrations of the toxicant in crops unadapted to the substance. And the difference with the control in cultures adapted to B1 at different concentrations of the toxicant was 15-29%.

Thus, we have identified the effect of reducing toxicity at all studied concentrations of potassium bichromate in crops adapted to substances B1 and B2 according to a physiological indicator - the magnitude of photosynthesis efficiency, which may indicate a rapid (already on the first day of the experiment) process of intensification of metabolic processes in plants adapted to crop substances for detoxification of the heavy metal chromium (as part of potassium dichromate). In toxicology, this indicator makes it possible to identify an earlier response to a toxic effect compared to integral indicators of the number or survival of individuals composing a population (in this case, a population of algae cells).

It is known that stimulation of the photosynthesis process can serve as an indicator of accelerating the removal of excess toxic metal from the cell. This process is one of the important intracellular mechanisms for the detoxification of toxic metals.

Thus, preparations B1 and B2 have antioxidant properties, reducing the toxic effect of the heavy metal chromium and reducing its toxicity for the test object studied at different levels of intoxication.

Experimentally, in a long-term experiment on a large number of generations of test object cells, a decrease in the toxic effect of potassium bichromate at different levels of its exposure under the influence of drugs B1 and B2 was shown.

The following experiment was carried out on laying hens of the Lohmann white LSL cross at the age of 96-100 weeks. The experimental and control groups were formed according to the principle of analogous pairs, taking into account egg production and egg weight, the birds were divided into 2 groups of 41 birds each.

Chickens in the control and experimental groups received complete feed in accordance with the recommendations of the cross breed manufacturer. The birds in the experimental group received an experimental feed additive, preparation B2, 2 times a week, i.e. superoxide dismutase 2 at a dilution of ^10-200 , applied to sugar granules measuring 38-41 pieces per 1 gram, and the control group bird received a placebo.

At the end of the experiment, 5 individuals from each group were euthanized. Liver and myocardial tissue samples were collected to evaluate superoxide dismutase gene expression.

Molecular genetic studies were performed.

Total RNA from samples was isolated manually using the RNeasyMiniKit (QIAGEN, Germany) according to the protocol. Quantitative assessment of isolated RNA was performed on a Qubit 3.0 fluorometer (ThermoFisherScientific, USA) using the Qubit RNA HS (HighSensitivity) AssayKit (ThermoFisherScientific, USA). cDNA synthesis from the GC matrix was carried out using the iScriptcDNAsynthesiskit (Bio-Rad, USA) using thermostat “Gnome” (DNA-Technology, Russia). Real-time polymerase chain reaction was performed on a LightCyc1er 96 thermal cycler (Roche, Switzerland) using 96-well plates and using the MHKcaPowerUp SYBR GreenMasterMix (AppliedBiosystems, ThermoFisherScientific, USA). The temperature profile of the amplification reaction consisted of: 95°C at within 10 minutes; 40 cycles at 95°C for 15 seconds, including primer annealing at individual temperature for 30 seconds and 72°C for 30 seconds. Each sample was tested in triplicate on a PCR plate. The amount of DNA for each PCR reaction was 2 μl at a primer concentration of 0.32 μM. Calculation of SOD gene expression relative to the reference gene (housekeeping gene) was performed manually using Microsoft Office Excel using the 2 ^-ΔΔCt method.

The obtained zootechnical and molecular genetic data were considered reliable at p<0.05.

From the data in Table 9, we can conclude that the expression of the superoxide dismutase 2 gene in the myocardium in the experimental group is 4% higher than in the control group.

From the data in Table 10, we can conclude that the expression of the superoxide dismutase 2 gene in the liver parenchyma in the experimental group is 28% higher than in the control group.

Thus, as a result of the experiment, the following result was obtained: Expression of the superoxide dismutase 2 gene in the experimental group increased by 4% in myocardial tissue and by 28% in the liver parenchyma.

Thus, the use of superoxide dismutase 2 (SOD2) as an antioxidant in ultra-low doses, namely, dilutions from 10 ^-12 to 10 ^-200 , makes it possible to restore and maintain at an optimal level the expression of the superoxide dismutase 2 gene, thereby increasing the own intracellular synthesis of the enzyme, ensuring pronounced systemic biological reactions.

Summarizing the above, the experiments conducted suggest that superoxide dismutase 2 (SOD2) can be used as an activator of SOD2 gene expression with the development of subsequent systemic reactions increasing the synthesis of its own intracellular SOD and the effects of stimulating antioxidant defense in any living organisms in ultra-low doses, namely, in dilution from 10 ^-12 to 10 ^-200 .

Consequently, it has been proven that it is possible to exert external influence on the expression of superoxide dusmutase genes in a relatively simple way, not related to genetic engineering, which opens up new prospects in biology, medicine, veterinary medicine, and plant growing.

Claims (1)

A method of using superoxide dismutase 2 (SOD2) as an activator of SOD2 gene expression with the development of subsequent systemic reactions increasing the synthesis of its own intracellular SOD and the effects of stimulating antioxidant defense, which consists in introducing superoxide dismutase 2 into a living organism in a dilution from 10 ^-12 to 10 ^-200 .