Application of genistein in preparation of medicine for improving brain cognitive impairment caused by low pressure and low oxygen

Abstract

The invention belongs to the technical field of medicines and disease treatment, and particularly relates to application of genistein to preparation of a medicine for improving brain cognitive impairment caused by low pressure and low oxygen. Research results show that low pressure and low oxygen can cause brain cognitive dysfunction of mice. The genistein can relieve brain dysfunction after low-pressure and low-oxygen exposure of mice by inhibiting neuroinflammation, recovering hippocampal neurological functions and improving hippocampal oxidative aggression. The invention provides a novel therapy for brain cognitive impairment caused by low pressure and low oxygen in various leguminous plant components, which is mainly derived from soy isoflavone by verifying a pharmaceutical active component, namely genistein.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine and disease treatment, and particularly relates to a use of genistein in preparation of medicine for improving brain cognitive dysfunction caused by low pressure and low oxygen. BACKGROUND

[0002] Low pressure (50%~70% of sea level) and low oxygen (oxygen content is reduced by 35%~40%) in high altitude areas are prone to cause central nervous damage, and induce memory decline, attention deficit and other high altitude cognitive dysfunction. Long-term exposure can reduce cerebral blood flow, induce neuron apoptosis and synaptic plasticity damage, and severe cases develop into chronic mountain sickness with cognitive decline. With the development of highland economy and tourism, the cognitive protection demand of people who rush in or live for a long time is urgent. The existing drugs (such as acetazolamide and dexamethasone) have many side effects, such as acetazolamide mainly causing electrolyte disorder, metabolic acidosis and paresthesia, and dexamethasone causing metabolic abnormalities, osteoporosis, immune suppression and multiple system organ adverse reactions. SUMMARY

[0003] In order to develop a new strategy applicable to brain cognitive dysfunction and solve the defect of many side effects of the existing drugs, the application provides a use of genistein in preparation of medicine for improving brain cognitive dysfunction caused by low pressure and low oxygen.

[0004] To achieve the above-mentioned purpose, the application adopts the following technical solutions.

[0005] Genistein is expected to become a potential natural intervention agent for improving cognitive dysfunction caused by low pressure and low oxygen through anti-inflammatory, antioxidant, estrogen-like effect and intestinal flora metabolic transformation and other multiple pathways. The research results of the application show that low pressure and low oxygen can cause brain cognitive dysfunction in mice. Genistein can inhibit neural inflammation, restore hippocampal neural function, improve hippocampal oxidative stress and thus relieve brain dysfunction in mice after low pressure and low oxygen exposure. Therefore, in order to develop a new strategy applicable to brain cognitive dysfunction and solve the defect of many side effects of the existing drugs, the application provides a use of genistein in preparation of medicine for improving brain cognitive dysfunction caused by low pressure and low oxygen. The structure of the genistein is as follows:

[0006] .

[0007] The application fundamentally solves the technical problem of many side effects of the existing chemical synthetic drugs by using natural plant-derived genistein as an active ingredient: the multi-target action mechanism (including antioxidant stress, inhibition of neural inflammation and anti-neuron apoptosis) of genistein, which is an isoflavone compound extracted from soybeans, is highly consistent with the complex pathophysiological process of brain cognitive dysfunction caused by low pressure and low oxygen, and can improve cognition while avoiding the common serious adverse reactions of single-target drugs; its LD50 The value is much higher than the therapeutic dose, long-term application is well tolerated, and there is no obvious endocrine interference effect; in addition, genistein can effectively cross the blood-brain barrier and accumulate in brain tissue, and under low pressure (50%~70% of sea level) and low oxygen (oxygen content is reduced by 35%~40%) environment, it plays a role by activating the Nrf2 / HO-1 pathway, inhibiting the NF-kB signal cascade reaction and other endogenous protection mechanisms, and this regulation mode in line with the body's own repair function further reduces the drug dependence and toxicity risk, so the natural drug scheme provided by the application significantly improves the drug safety while ensuring the efficacy, and is particularly suitable for long-term preventive application of officers and soldiers and the public in special environments such as highlands.

[0008] Further, the medicine takes the genistein as the active ingredient, and is supplemented with a pharmaceutically acceptable auxiliary material or carrier.

[0009] Further, the medicine is selected from any one of a tablet, a capsule, a granule, a powder, an oral liquid and an injection.

[0010] Further, the auxiliary material is selected from any one or more of starch, sodium carboxymethyl cellulose, distilled water, ethanol, microcrystalline cellulose and sodium sulfite.

[0011] Further, the carrier is selected from any one or more of a lipid nanocarrier, a polymer nanocarrier, a cyclodextrin inclusion and a probiotic bacteria powder.

[0012] Further, the probiotic bacteria in the probiotic bacteria powder include Bifidobacterium and Lactobacillus.

[0013] Further, when the medicine is an injection, the concentration of genistein in the medicine is 9.8mg / mL~10.2mg / mL.

[0014] Further, the concentration of genistein in the medicine is 10mg / mL.

[0015] Further, the medicine is used for inhibiting neuroinflammation, improving oxidative stress and restoring hippocampal nerve function. The medicine reduces the levels of pro-inflammatory cytokines such as TNF-α, IL-6, IL-1 and MCP-1 in serum, enhances the body's antioxidant stress capacity, and promotes the improvement of hippocampal neuron morphology.

[0016] Compared with the prior art, the application has the following beneficial effects: 1. In order to develop a new strategy suitable for brain cognitive dysfunction, solve the defect that the existing drugs have many side effects, the present application provides a use of genistein in the preparation of a drug for improving brain cognitive dysfunction caused by low pressure and low oxygen, and provides a new therapy of a drug active ingredient for brain cognitive dysfunction caused by low pressure and low oxygen. The use provided by the present application is a new therapy of soy isoflavones for brain cognitive dysfunction caused by low pressure and low oxygen. It is found through research that genistein is used for inhibiting neuroinflammation, improving oxidative stress and restoring hippocampal nerve function. Brain function is regulated, and then it can be applied to the preparation of a drug for improving brain cognitive dysfunction caused by low pressure and low oxygen.

[0017] The present application fundamentally solves the technical problem of many side effects of existing chemical synthetic drugs by using natural plant-derived genistein as an active ingredient: genistein is an isoflavone compound extracted from soybeans, and its multi-target mechanism of action (including anti-oxidative stress, inhibition of neuroinflammation, anti-neuron apoptosis, and ) is highly consistent with the complex pathophysiological process of brain cognitive dysfunction caused by low pressure and low oxygen, which can improve cognition while avoiding the common serious adverse reactions of single-target drugs; the LD 50 value is much higher than the therapeutic dose, long-term application is well tolerated, and there is no obvious endocrine interference effect; in addition, genistein can effectively cross the blood-brain barrier and accumulate in brain tissue, and play a role in low pressure and low oxygen environment by activating the Nrf2 / HO-1 pathway, inhibiting the NF-kB signal cascade reaction and other endogenous protection mechanisms. This regulation method in line with the body's own repair function further reduces the risk of drug dependence and toxicity, so the natural drug solution provided by the present application significantly improves the safety of drug use while ensuring efficacy, and is particularly suitable for long-term preventive application of officers and soldiers and the public in special environments such as highlands.

[0018] 2. The research results of the present application show that low pressure and low oxygen can cause brain cognitive dysfunction in mice. Genistein can alleviate brain cognitive dysfunction in mice after low pressure and low oxygen exposure by inhibiting neuroinflammation, improving oxidative stress and restoring hippocampal nerve function.

[0019] 3. The present application simulates low pressure and low oxygen conditions in the laboratory, and determines the changes in brain function of mice under low pressure and low oxygen exposure through behavioral experiments. The protective effect of genistein is evaluated from the behavioral and histological levels through HE staining and ELISA methods. The present application provides a theoretical basis and potential treatment strategy for preventing brain cognitive function changes caused by low pressure and low oxygen environment based on soy isoflavones-genistein with potential protective effect.

[0020] 4, The research results of the application are: the behavior data shows that, compared with the Model group, the spontaneous alternation rate of the Y maze of the Genistein group mice increases, the water maze experiment shows that the number of crossing the platform after administration increases, the latency shortens, and the residence time in the target quadrant prolongs. The levels of pro-inflammatory cytokines such as TNF-α, IL-6, IL-1 and MCP-1 in the serum of the Genistein (genistein) group mice decrease, the HE staining result shows that the neurons of the Genistein group mice are arranged in order, and the neuron necrosis decreases. The oxidative stress level of the hippocampus of the Genistein group mice improves. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The figure is a low-pressure and low-oxygen-induced mouse cognitive dysfunction model constructed in the application.

[0022] Figure 2 The figure is the influence of low-pressure and low-oxygen on the Y maze exploration function of mice, wherein: A is the spontaneous alternation rate; B is the Y maze trajectory diagram; Control: control group; Model: low-pressure and low-oxygen group, Genistein: genistein group; p <0.05;** p <0.01; p <0.001; p <0.0001.

[0023] Figure 3 The figure is the influence of low-pressure and low-oxygen on the water maze exploration function of mice, wherein: A is the platform latency after 5 days of training; B is the latency after 3 days of low-pressure and low-oxygen exposure; C is the residence time percentage in the target quadrant; D is the number of crossing the platform; E is the movement trajectory diagram; Control: control group, Model: low-pressure and low-oxygen group, Genistein: genistein group; p <0.05;** p <0.01.

[0024] Figure 4 The figure is the influence of low-pressure and low-oxygen on the levels of inflammatory factors IL-1, IL-6, MCP-1 and TNF of mice, wherein: A is the influence of low-pressure and low-oxygen on the level of inflammatory factor IL-1 of mice; B is the influence of low-pressure and low-oxygen on the level of inflammatory factor IL-6 of mice;​​ C represents the effect of low pressure and hypoxia on the level of the inflammatory factor MCP-1 in mice; D represents the effect of low pressure and hypoxia on the level of the inflammatory factor TNF in mice. Control: control group; Model: hypobaric hypoxia group; Genistein: genistein group. * p <0.05;** p <0.01; *** p <0.001; *** p <0.0001.

[0025] Figure 5 The effects of low-pressure hypoxia on SOD, MDA, and CAT in the hippocampus of mice in this invention are as follows: A represents the effect of hypobaric hypoxia on SOD in the hippocampus of mice; B represents the effect of hypobaric hypoxia on MDA in the hippocampus of mice; C represents the effect of hypobaric hypoxia on CAT levels in the hippocampus of mice. Control: control group; Model: hypobaric hypoxia group; Genistein: genistein group. * p <0.05.

[0026] Figure 6 The image shows the effect of low pressure and hypoxia on HE staining of mouse hippocampal tissue in this invention, where black arrows indicate damaged cells. Control: control group; Model: hypobaric hypoxia group; Genistein: genistein group.

[0027] Figure 7 This is the structural formula of genistein in this invention. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0029] Example 1 I. Methods 1. Establishment of experimental animals and models Male C5 7BL / 6 mice aged 6 to 8 weeks were purchased from the Experimental Animal Center of Air Force Medical University (Xi'an, China) and were raised under strict hygiene and good ventilation conditions, with free access to food and water.

[0030] The feeding conditions are as follows: temperature 23℃±2℃, humidity 50%±2%, 12h light / 12h darkness.

[0031] All animal experiments in the present application are approved by the Animal Welfare Committee of Air Force Military Medical University (Xi'an, China).

[0032] The above mice are divided into a control group, a model group and a drug administration group.

[0033] The control group (normal feeding group, Control group): 6-8-week-old male C57BL / 6 mice are given a solvent (1.25% (v / v) DMSO and 98.75 PEG mixed solution) by gavage, and the gavage dose is 0.01 ml / g.

[0034] The model group (Model group): 6-8-week-old male C57BL / 6 mice are exposed to a low-pressure and low-oxygen cabin at an altitude of 7000 meters for 3 days, and are given a 1.25% (v / v) DMSO and 98.75 PEG mixed solution by gavage, and the gavage dose is 0.01 ml / g.

[0035] The drug administration group (Genistein group): 6-8-week-old male C57BL / 6 mice are given a genistein solution by gavage, and the administration (gavage) dose is 100 mg / kg / day, and the gavage is continuously performed for 5 days, and the mice are subjected to cabin.

[0036] The preparation method of the genistein solution is as follows: 0.02 g of genistein powder is dissolved in 2 mL of 1.25% (v / v) DMSO and 98.75 PEG mixed solution, and the preparation method of the 1.25% (v / v) DMSO and 98.75 PEG mixed solution is as follows: DMSO and PEG stored at room temperature are taken according to the gavage solution required by the number of mice, and the respective doses of the above different groups are calculated according to the proportion (gavage dose), and a homogenizer is used for homogenization for standby use. The experimental design is shown in Figure 1 .

[0037] The genistein powder is purchased from Xi'an Bide Biological Technology Co., Ltd., and the catalog number is PC-00333. The structural formula of genistein is shown in Figure 7 .

[0038] 2. Behavior detection 2.1, Y maze Adaptation period: the mice are placed in the house for Y maze experiment for 2 hours.

[0039] Test period: the mice are placed in the center of the Y maze with three equal-length arms, and the A arm, B arm and C arm are sequentially marked, the order of the mice entering the three arms is observed, and the spontaneous alternation rate is calculated.

[0040] 2.2, Water maze Position navigation test: after setting the platform in one of the quadrants, each mouse was put into the water from different quadrant, and the monitoring time was 1 minute. If the mouse did not successfully find the platform, it was guided to find the platform and stay for 20 seconds. Continuous training for 5 days.

[0041] Exploration training: on the 9th day, the platform in the quadrant was removed, and the exploration training of 60 seconds was started. The time, distance and number of times of crossing the platform of the mouse staying in the target quadrant were recorded to detect the spatial memory ability of the mouse.

[0042] Among them, the mice detected in this part of the experiment are Control group, Model group, Genistein group.

[0043] 3, Inflammatory factor detection The ELISA kit of Zhucai Biology was used to detect the levels of TNF-α, IL-6, IL-10 and MCP-1 in the serum of mice. The specific steps are as follows:

[0044] (1) Sample addition of standard product: set standard product holes and sample holes, and add 50 μL of standard product of different concentrations to each standard product hole.

[0045] (2) Sample addition: set blank holes (also known as blank control holes, which do not add test samples and enzyme-labeled reagents, and the remaining steps are the same) and sample holes. Add test samples (which refer to the serum supernatant obtained after centrifugation at 3000 rpm for 10 min at 4℃, and the solution obtained after dilution by 10 times) to the sample holes.

[0046] (3) Add test samples 50 μL to the sample holes on the enzyme-labeled coating plate, and add the test samples to the bottom of the enzyme-labeled plate holes as much as possible without touching the hole wall, and mix gently.

[0047] (4) Add enzyme: add 100 μL of enzyme-labeled reagent to each hole, except for the blank holes.

[0048] (5) Incubation: after sealing the plate with sealing film, incubate at 37℃ for 60 minutes.

[0049] (6) Liquid preparation: dilute the 20-fold concentrated washing solution with distilled water by 20 times for standby.

[0050] (7) Washing: carefully remove the sealing film, discard the liquid, and shake dry. Add washing solution to each hole, stand for 30 seconds, then discard, repeat for 5 times, and pat dry.

[0051] (8) Color development: add color developing agent A 50 μL to each hole, then add color developing agent B 50 μL, mix gently, and develop color at 37℃ for 15 minutes.

[0052] (9) Termination: add 50 μL of termination solution to each hole to terminate the reaction (at this time, the blue color turns to yellow color).

[0053] (10) Measurement: Zero with blank hole, 450 nm wavelength, measure the absorbance (OD value) of each hole in turn, measurement should be within 15 minutes after adding the stop solution.

[0054] In this part of the experiment, the mice detected are Control group, Model group and Genistein group.

[0055] 4. Oxidative stress detection Accurately weigh the hippocampus of the mouse, add physiological saline according to the ratio of weight (g) to volume (mL) of 1:9, grind at 60HZ, centrifuge for 1 minute, centrifuge twice, and take the homogenate supernatant (10% homogenate, v / v) for detection.

[0056] The protein concentration is measured by BCA method. The hydrogen peroxidase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) determination kit from Nanjing Jiancheng is used to detect according to its operation procedure table.

[0057] In this part of the experiment, the mice detected are Control group, Model group and Genistein group.

[0058] 5. HE staining Put the brain tissue slices of the mouse into the environment-friendly dewaxing transparent liquid I.20 min-environment-friendly dewaxing transparent liquid II.20 min-anhydrous ethanol I.5 min-anhydrous ethanol II.5 min-75% (v / v) alcohol 5 min, and then wash with tap water.

[0059] The slice is treated with high-definition constant dyeing pretreatment liquid for 1 minute.

[0060] Dyeing in hematoxylin dyeing liquid for 3 minutes, and then wash with water for 1 minute.

[0061] Differentiate in constant dyeing differentiation liquid for 4 seconds, and then wash with water for 20 seconds.

[0062] Treat in constant dyeing blue-reverting liquid for 1 minute, and then wash with water for 20 seconds.

[0063] Put into 95% (v / v) ethanol for 60 seconds.

[0064] Dyeing in eosin Y dyeing liquid for 25 seconds.

[0065] Dehydration and mounting: put the slice into anhydrous ethanol I 5 min, anhydrous ethanol II 5 min, anhydrous ethanol III 5 min, xylene I 5 min, and xylene II 5 min in turn, and then transparently mount with neutral balsam.

[0066] In this part of the experiment, the mice detected are Control group, Model group and Genistein group.

[0067] The method for obtaining the section is: using conventional paraffin section method.

[0068] 6. Statistical analysis Student's t test (unpaired) was used for comparison between two groups, one-way ANOVA was used for comparison between multiple groups, if there was a significant difference between groups, P <0.05 was considered to be statistically significant difference between groups, P <0.05, P <0.01, P <0.001; at the same time, the data was processed and plotted by GraphPad Prism 8 software, and the experimental results were expressed in the form of mean ± standard error (mean ± SEM).

[0069] II. Results 1. Genistein improves the cognitive impairment of mice induced by low pressure and low oxygen 1.1. Y maze experiment results Figure 2 Figure A in the middle and Figure 2 Figure B in the middle, the spontaneous alternation rate of the model group mice decreased, and the trajectory was messy, and the spontaneous alternation rate of the mice increased significantly after genistein intervention, indicating that genistein improved the short-term working memory ability of mice.

[0070] 1.2. Water maze experiment results Figure 3 Figure A in the middle is the 5-day training latency of the three groups. Figure 3 Figure B in the middle, Figure 3 Figure A in the middle and Figure 3 Figure D in the middle, respectively, the test period of mice, compared with the Model group, the Genistein group mice test period latency was shortened ( P <0.05), the number of crossing the platform increased (P<0.05), and the time spent in the platform quadrant was prolonged ( P <0.05). It is shown that genistein improves the spatial learning and cognitive ability of mice.

[0071] 2. The effect of genistein on the inflammatory level of low pressure and low oxygen induced cognitive impairment of mice Figure 4 Figure A in the middle Figure 4 The results of Figure E in the middle show that: compared with the Control group, the levels of pro-inflammatory factors TNF-α, IL-6, IL-1β and MCP-1 in the hippocampus of the Model group mice increased, and compared with the Model group, the contents of pro-inflammatory factors MCP-1 and IL-1 in the serum of the Genistein group mice decreased ( P <0.001), and IL-1 decreased P(<0.05) and TNF reduction (<0.001). It is shown that genistein can inhibit the nerve inflammation of mice in the low pressure and low oxygen model.

[0072] 3, the influence of genistein on the oxidative stress level of low pressure and low oxygen induced cognitive impairment of mice Figure 5 The MDA of the model group is higher than that of the blank group, and the intervention group significantly reduces the MDA increase caused by low pressure and low oxygen, and enhances the antioxidant capacity of the body.

[0073] 4, the influence of genistein on the neuron morphology of low pressure and low oxygen induced cognitive impairment of mice Figure 6 The hippocampal tissue HE staining structure is directly shown, the hippocampal structure of the model group is in disorder, cell damage occurs, and the neuron morphology structure of the genistein intervention group is obviously improved, which proves that genistein has a direct protective effect on neurons.

[0074] From the above experimental results, it can be known that genistein can inhibit nerve inflammation, improve oxidative stress and restore hippocampal nerve function, so as to relieve the cognitive dysfunction of mice after low pressure and low oxygen exposure.

[0075] It should be noted that when the numerical range is involved in the present application, it should be understood that each numerical range of the two endpoints and any numerical value between the two endpoints can be selected, and in order to prevent repetition, the present application describes the preferred embodiments.

[0076] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic creative concept, and these changes and modifications all fall within the scope of all changes and modifications of the present application.

Claims

1. Use of genistein in the manufacture of a medicament for ameliorating brain cognitive dysfunction caused by low pressure and low oxygen, characterized in that, The structure of the genistein is shown as follows: 。 2. Use according to claim 1, characterized in that, The medicine takes the genistein as an active ingredient and is supplemented with a pharmaceutically acceptable adjuvant or carrier.

3. Use according to claim 2, characterized in that, The medicine is selected from any one of a tablet, a capsule, a granule, a powder, an oral liquid and an injection.

4. Use according to claim 3, characterized in that, The adjuvant is selected from any one or more of starch, sodium carboxymethyl cellulose, distilled water, ethanol, microcrystalline cellulose and sodium sulfite.

5. Use according to claim 3, characterized in that, The carrier is selected from any one or more of a lipid nanocarrier, a polymer nanocarrier, a cyclodextrin inclusion and a probiotic bacterial powder.

6. Use according to claim 5, characterized in that, The probiotic bacteria in the probiotic bacterial powder include Bifidobacterium and Lactobacillus.

7. Use according to claim 3, characterized in that, When the medicine is an injection, the concentration of the genistein in the medicine is 9.8 mg / mL to 10.2 mg / mL.

8. Use according to claim 1, characterized in that, The medicine is used for inhibiting neuroinflammation, improving oxidative stress and restoring hippocampal nerve function.

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