Application of lactobacillus rhamnosus in preparation of product for relieving cognitive impairment caused by plateau hypoxia

Abstract

The invention provides application of lactobacillus rhamnosus in preparation of a product for relieving cognitive impairment caused by plateau hypoxia, and belongs to the technical field of prevention and treatment of brain injury caused by plateau hypoxia. According to the application disclosed by the invention, the fact that the lactobacillus rhamnosus can be used for preventing and / or treating cognitive impairment and / or brain injury caused by plateau hypoxia is proposed for the first time, the limitation of the prior art is broken, and a brand new research direction and a brand new product development idea are provided for the field. The lactobacillus rhamnosus is used as a probiotic, has good biological safety and tolerance, is used for food processing, provides a new natural, safe and effective choice for relieving altitude stress and brain injury for people who travel and work in plateau regions, avoids side effects of drug prevention and treatment, and has a good application prospect. The effectiveness of food intervention is improved, and the acceptability and compliance of related products are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of prevention and treatment of brain damage caused by plateau hypoxia, and specifically relates to the use of Lactobacillus rhamnosus in the preparation of a product for alleviating cognitive dysfunction caused by plateau hypoxia. Background Art

[0002] Plateau refers to an area with an altitude of more than 1,000 meters and a relative altitude of more than 500 meters, and a vast area with relatively flat terrain or certain undulations, such as the Qinghai-Tibet Plateau and the Yunnan-Guizhou Plateau. The notable characteristics of plateau areas are low oxygen, low pressure, cold, strong ultraviolet radiation, etc., among which the low oxygen environment is a key factor leading to physiological and pathological changes in the human body. Long-term hypoxia in the plateau will cause serious damage to the central nervous system, cause cognitive dysfunction (such as memory loss, inattention, decreased executive function, etc.), and even lead to irreversible brain damage (such as neuronal apoptosis, glial cell damage, white matter lesions, etc.). These problems not only seriously affect the quality of life and work efficiency of people in plateau areas, but may also threaten their lives. Therefore, the research and development of products that effectively prevent and treat cognitive dysfunction or brain damage caused by plateau hypoxia is of great practical significance and urgent need.

[0003] Currently, research on the prevention and treatment of cognitive impairment or brain damage caused by high-altitude hypoxia primarily focuses on medication, physical intervention, and oxygen therapy. Commonly used medications include drugs that improve cerebral circulation (such as nimodipine), neurotrophic drugs (such as methylcobalamin), and nootropics (such as piracetam). While these medications can alleviate symptoms to a certain extent, they have significant limitations: Vasodilators such as nimodipine can cause blood pressure fluctuations, leading to adverse reactions such as dizziness and hypotension; neurotrophic drugs are relatively slow-acting and have limited effectiveness in repairing nerve cell damage caused by severe hypoxia; long-term use of nootropics can lead to drug resistance, and some patients experience insignificant results. Regarding physical interventions, techniques such as transcranial magnetic stimulation (TMS) and hyperbaric oxygen therapy (HBO) can improve symptoms by regulating brain nerve excitability and increasing blood oxygen levels. However, TMS equipment is expensive and requires specialized personnel to operate, making it difficult to promote in remote areas of the plateau; HBO therapy requires specialized equipment and a specific environment, requiring patients to frequently travel to treatment sites, making it inconvenient to use, and long-term, multiple treatments carry certain risks. Oxygen inhalation is the most direct way to alleviate high-altitude hypoxia, but it only alleviates acute hypoxia symptoms and cannot fundamentally repair damaged nerve cells and nerve function. Long-term reliance on oxygen therapy can also limit patients' range of motion. Therefore, exploring new methods to effectively prevent or treat cognitive dysfunction or brain damage caused by high-altitude hypoxia is of great significance. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the use of Lactobacillus rhamnosus in the preparation of a product for alleviating cognitive dysfunction and / or brain damage caused by plateau hypoxia, thereby providing a new option for the prevention and treatment of cognitive dysfunction and / or brain damage caused by plateau hypoxia.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of Lactobacillus rhamnosus in preparing a product for preventing, treating or alleviating cognitive dysfunction caused by plateau hypoxia.

[0007] The present invention also provides the use of Lactobacillus rhamnosus in preparing a product for preventing, treating or alleviating brain damage caused by plateau hypoxia.

[0008] Preferably, the Lactobacillus rhamnosus includes Lactobacillus rhamnosus SHA113.

[0009] Preferably, the Lactobacillus rhamnosus is inactivated selenium-containing Lactobacillus rhamnosus.

[0010] Preferably, the method for preparing the inactivated selenium-containing Lactobacillus rhamnosus comprises the following steps: expanding and culturing Lactobacillus rhamnosus, adding selenium-rich raw materials to the culture medium for continued cultivation, and crushing the precipitate to obtain the inactivated selenium-containing Lactobacillus rhamnosus.

[0011] Preferably, the selenium-rich raw material includes at least one of sodium selenite, sodium selenate, selenium yeast, selenium polysaccharide, selenomethionine, selenium-rich malt powder and selenium-rich edible fungus powder.

[0012] Preferably, the selenium concentration in the inactivated selenium-containing Lactobacillus rhamnosus is greater than 2 mg / g.

[0013] Preferably, the plateau is a high altitude area with an altitude of more than 1000m.

[0014] Preferably, the brain damage includes hippocampal damage and / or amygdala damage.

[0015] The present invention also provides a product for preventing, treating or alleviating brain damage or cognitive dysfunction caused by plateau hypoxia. The active ingredient of the product includes inactivated selenium-containing Lactobacillus rhamnosus, and the selenium concentration in the inactivated selenium-containing Lactobacillus rhamnosus is greater than 2 mg / g.

[0016] Beneficial effects of the present invention:

[0017] Traditional means of preventing and treating cognitive dysfunction and / or brain damage caused by plateau hypoxia, such as drug therapy, physical intervention and oxygen therapy, may have side effects, be inconvenient to operate, or be unable to fundamentally repair damaged nerve cells. The present invention proposes for the first time that Lactobacillus rhamnosus can be used to prevent and / or treat cognitive dysfunction and / or brain damage caused by plateau hypoxia, breaking the limitations of the existing technology and providing a new research direction and product development idea for this field. As a probiotic, Lactobacillus rhamnosus has good biosafety and tolerance. By developing it into a prevention and treatment product, a natural, safe and effective new prevention and treatment option is provided for people in plateau areas, avoiding the side effects of drug therapy, improving the effectiveness of food intervention, and improving user acceptance and compliance. The promotion of the application of the present invention can not only fill the gap in food products for the prevention and treatment of cognitive dysfunction or brain damage caused by plateau hypoxia on the market, meet the urgent needs of residents, tourists and stationed personnel in plateau areas, but also drive the development of probiotic-related industries and create significant economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of the cognitive function test of mice are shown, where (a) is the ratio of the number of alternating stops in the spontaneous alternating space Y maze, (b) is the time the mouse stays in the learning arm of the Y maze, and (c) is the time the mouse stays in the novel arm of the Y maze. The results are expressed as mean ± standard error (n = 7), *P < 0.05, **P < 0.01, ****P < 0.0001;

[0019] Figure 2 HE staining results of mouse hippocampus, where A, B and C are mice gavaged with normal saline for 6 days and under normal conditions (SPSS 6d +NN group) hippocampal CA1, CA3 and DG areas; D, E and F are the animals that were gavaged with sterile saline for 6 consecutive days and then placed in a hypobaric hypoxic animal experimental chamber for 7 days (SPSS 6d +HH 7d The results of the CA1, CA3 and DG regions of the hippocampus of mice in the group (G, H and I) were respectively gavage with Lactobacillus rhamnosus raw materials obtained from selenium-added medium for 6 days and then exposed to high altitude hypoxia for 7 days (SeLRS 6d +HH 7d (group) Results of the CA1, CA3, and DG regions of the hippocampus of mice;

[0020] Figure 3 Figure 3 shows the counting results of neurons in the CA region of the hippocampus of mice, where (a) is the total neuron density, (b) is the normal neuron density, and (c) is the pyknotic neuron density. The results are the mean ± standard error of four replicates. *P < 0.05, **P < 0.01, ***P < 0.001.

[0021] Figure 4 The HE staining results of the amygdala of mice are shown in Figure 1. A, B and C are mice that were gavaged with normal saline for 6 days and mice that were placed under normal conditions (SPSS 6d The results of the BLA, CeA, and MeA regions of the amygdala of mice in the +NN group; D, E, and F are the results of the BLA, CeA, and MeA regions of mice in the low-pressure hypoxic animal experimental chamber after continuous gavage of sterile saline for 6 days (SPSS 6d +HH 7d (A) The results of the BLA, CeA and MeA regions of the amygdala of mice in the control group (G, E and F) were respectively obtained by gavage with Lactobacillus rhamnosus raw materials obtained by selenium-added medium for 6 days and then exposed to high altitude hypoxia for 7 days (SeLRS 6d +HH 7d (group) Results of the BLA, CeA, and MeA regions of the amygdala of mice;

[0022] Figure 5 The results of Golgi-Cox staining of the hippocampus of mice, A, B and C are respectively the mice that were gavaged with normal saline for 6 days and under normal conditions (SPSS 6d +NN group) hippocampal CA1, CA3 and DG areas; D, E and F are the results of the animals that were gavaged with sterile saline for 6 consecutive days and then placed in a hypobaric hypoxic animal experimental chamber for 7 days (SPSS 6d +HH 7d The results of the CA1, CA3 and DG regions of the hippocampus of mice in the group (G, H and I) were respectively gavage with Lactobacillus rhamnosus raw materials obtained from selenium-added medium for 6 days and then exposed to high altitude hypoxia for 7 days (SeLRS 6d +HH 7d (group) Results of the CA1, CA3, and DG regions of the hippocampus of mice;

[0023] Figure 6 The Golgi-Cox staining results of the amygdala of mice, A, B and C are mice that were gavaged with normal saline for 6 days and in normal environment (SPSS 6d The results of the BLA, CeA and MeA regions of the amygdala of mice in the +NN group; D, E and F were gavaged with sterile saline for 6 consecutive days and then placed in a hypobaric hypoxic animal experimental chamber for 7 days (SPSS 6d +HH 7d The results of the BLA, CeA and MeA regions of the amygdala of mice in the G, H and I groups were respectively gavage of Lactobacillus rhamnosus raw materials obtained by selenium-added medium for 6 days and then exposed to high altitude hypoxia for 7 days (SeLRS 6d +HH 7d The results of the BLA, CeA and MeA regions of the amygdala of mice in group (P<0.05) were obtained. DETAILED DESCRIPTION

[0024] The present invention provides the use of Lactobacillus rhamnosus in preparing a product for preventing and treating cognitive dysfunction and / or brain damage caused by plateau hypoxia.

[0025] The present invention has no particular limitation on the specific source of Lactobacillus rhamnosus. In some embodiments of the present invention, the Lactobacillus rhamnosus is Lactobacillus rhamnosus SHA113, which was deposited in the China Center for Type Culture Collection on December 25, 2017, with a deposit number of CCTCCNO: M2017838.

[0026] In the present invention, the Lactobacillus rhamnosus is inactivated selenium-containing Lactobacillus rhamnosus. The method for preparing the inactivated selenium-containing Lactobacillus rhamnosus preferably comprises the following steps: after expanding the culture of the Lactobacillus rhamnosus, adding a selenium-rich raw material to the culture medium for continued culture, and crushing the precipitate to obtain the inactivated selenium-containing Lactobacillus rhamnosus. In the present invention, the culture medium used for the expanded culture is preferably MRS liquid culture medium. The specific source of the MRS liquid culture medium is not particularly limited in the present invention; conventional commercially available products in the art can be used. The temperature of the expanded culture is preferably 37°C, and the expanded culture duration is preferably 48 hours. In the present invention, the selenium-rich raw material preferably includes at least one of sodium selenite, sodium selenate, selenium yeast, selenium polysaccharides, selenomethionine, selenium-enriched malt powder, and selenium-enriched edible fungus powder. In the present invention, the incubation duration after adding the selenium-rich raw material is preferably 24 hours. After the continued culture is completed, the culture is preferably centrifuged, preferably at a speed of 8000 rpm, and the centrifugation duration is preferably 3 minutes. In the present invention, the Lactobacillus rhamnosus is actually a crushed and inactivated selenium-containing Lactobacillus rhamnosus, i.e., a dead bacterium. In the present invention, the selenium concentration in the inactivated selenium-containing Lactobacillus rhamnosus is preferably greater than 2 mg / g, more preferably 35 mg / g. In the present invention, the plateau is preferably a plateau area with an altitude of 1000 meters or more. In the present invention, the brain damage preferably includes hippocampal damage and / or amygdala damage.

[0027] In the present invention, the intervention with inactivated selenium-containing Lactobacillus rhamnosus can effectively protect the cognitive function of mice after exposure to a low-pressure, low-oxygen plateau environment for 7 days. The main manifestation is that compared with uninterventional mice, the mice that were fed with inactivated selenium-containing Lactobacillus rhamnosus in advance had a significantly increased shuttle time in the spontaneous alternating space Y maze; the residence time in the learning arm of the Y maze was significantly reduced, and the residence time in the novel arm was significantly increased.

[0028] In the present invention, intervention with inactivated selenium-containing Lactobacillus rhamnosus can effectively alleviate neuronal damage in the hippocampus-amygdala brain region of mice after 7 days of hypobaric and hypoxic exposure.

[0029] The present invention also provides a product for preventing and treating brain damage or cognitive dysfunction caused by plateau hypoxia. The active ingredient of the product includes inactivated selenium-containing Lactobacillus rhamnosus, and the selenium concentration in the inactivated selenium-containing Lactobacillus rhamnosus is greater than 2 mg / g.

[0030] In the product of the present invention, the selenium-enriched Lactobacillus rhamnosus is the only active ingredient of the product. In the present invention, the Lactobacillus rhamnosus is preferably Lactobacillus rhamnosus SHA113.

[0031] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] In the following examples, unless otherwise specified, all methods are conventional.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] Example 1

[0035] 1. Experimental animals:

[0036] Specific pathogen-free (SPF) 8-week-old male C57BL / 6 mice were purchased from Xi'an Keoke Biotechnology Co., Ltd. Animals were housed in an SPF-grade animal room, with 4 to 6 mice per cage, under a standard temperature (22 ± 2°C), humidity (50 ± 10%), and a 12-h circadian cycle (8:00–20:00 light cycle). Animals had free access to water and food. Behavioral experiments were conducted in a quiet, open experimental room. All animal procedures adhered to the ethical standards of laboratory animals and to the regulations of the National Animal Use and Care Committee and the Peking University Health Science Center Animal Care Committee.

[0037] 2. Mouse model preparation:

[0038] The preparation process of the inactivated selenium-containing Lactobacillus rhamnosus suspension is as follows: Lactobacillus rhamnosus SHA113 (i.e., Lactobacillus rhamnosus SHA113) is inoculated into MRS liquid culture medium at 1% (volume ratio) and cultured at 37°C for 48 hours. Afterwards, 3.0mM sodium selenite is added to the culture medium and cultured at 37°C for another 24 hours. The entire culture is centrifuged at 8000 rpm for 3 minutes, and the precipitate is washed three times with sterile water and then collected. The collected precipitate is ultrasonically broken in an ice bath for 2 seconds, 2 seconds, and continued this process for 60 minutes at an ultrasonic power of 500 watts. It is then freeze-dried to obtain an inactivated selenium-containing Lactobacillus rhamnosus powder for subsequent use. Testing shows that the selenium concentration in the resulting bacterial powder is 35mg / g. Before use, it is prepared into an inactivated selenium-containing Lactobacillus rhamnosus suspension with sterile physiological saline and used in animal experiments.

[0039] After one week of adaptive feeding, C57BL / 6 male mice were randomly divided into three groups, with 12 mice in each group.

[0040] A group of animals were gavaged with sterile saline for 6 consecutive days and then placed in an SPF animal room to establish a control group (SPSS 6d +NN); one group was gavaged with sterile saline for 6 consecutive days and then placed in a low-pressure hypoxic animal experimental chamber (simulating an altitude of 5000m) for 7 days to establish a 7-day low-pressure hypoxic exposure model in mice (SPSS 6d +HH 7d ); a group of mice were gavaged with an inactivated selenium-containing Lactobacillus rhamnosus suspension in sterile saline for 6 consecutive days (the gavage dose was calculated based on the selenium content, 0.12 mg selenium / kg body weight / day, and the total gavage volume was 10 mL / kg body weight) and then placed in a low-pressure and low-oxygen animal experimental chamber (simulating an altitude of 5000 m) for 7 days to establish a low-pressure and low-oxygen exposure mouse model induced by selenium-containing Lactobacillus rhamnosus (SeLRS) 6d +HH 7d The three groups of mice received the same total oral volume. A hypobaric, hypoxic chamber was set up to simulate experimental conditions at a plateau altitude of 5,000 meters. The pump pressure was set at 405 mmHg. The chamber maintained a temperature of 22 ± 2°C, a humidity of 50 ± 10%, and a 12-hour circadian rhythm (8:00 AM to 8:00 PM). The animals had free access to water and food.

[0041] 3. Mouse behavioral experiments

[0042] Within one week after the end of the hypobaric and hypoxic exposure, the mice were tested in the spontaneous alternating space Y-maze test and the Y-maze test using the EthoVisionXT small animal behavioral system to observe the behavioral phenotypes of the mice.

[0043] (1) Spontaneous alternating space Y-maze experiment in mice

[0044] The test apparatus consisted of a trisected radial maze consisting of three arms (30 cm × 10 cm × 15 cm) and a connecting area. The arms were angled 120° relative to each other, each with a removable central partition. The test apparatus was constructed entirely of opaque, dark acrylic. The experiment required that the mice be prevented from prior exposure to or other familiarization with the Y-maze. Mice were placed in the connecting area in the center of the maze and allowed to freely explore the maze for 5 minutes. The total number of arm entries and the number of consecutive alternations between different maze entries were recorded. The evaluation metric was the percentage of spontaneous alternations: percentage of spontaneous alternations = number of alternations / (total number of arms - 2) × 100%. Between each test, the test box was cleaned of any remaining feces and wiped with 75% alcohol to remove any residual odor.

[0045] The results are as follows Figure 1 As shown in (a) in .

[0046] (2) Mouse Y-maze experiment

[0047] The Y-maze experiment was divided into two phases. The three arms of the Y-maze were designated as the novel arm, the start arm, and the learned arm, with icons of different shapes marking the end points of each arm. Between each test, the maze was cleaned of any remaining feces, and the testing box was wiped with 75% alcohol to remove any residual odor.

[0048] The mouse Y-maze test is divided into two stages:

[0049] Learning phase: After the mice became familiar with the Y-maze, the novel arm was blocked with a baffle and the mice were placed in the starting arm. The mice were allowed to freely explore between the starting arm and the learning arm for 15 minutes. The testing phase was carried out 6 hours after the end of this phase.

[0050] Testing phase: During the testing phase, the baffle blocking the novel arm was removed and the mouse was placed in the starting arm. The mouse was allowed to move freely in the Y-maze for 10 minutes, and the time and distance the mouse explored the three arms were recorded.

[0051] The results are as follows Figure 1 As shown in (b) and (c) in .

[0052] Depend on Figure 1 It can be seen that SeLRS 6d +HH 7d Group and SPSS 6d +HH 7dCompared with the control group, the mice's rotation ratio in the spontaneous alternating space Y maze was significantly increased; the cumulative residence time in the learning arm of the Y maze was significantly reduced, and the cumulative residence time in the novel arm was significantly increased. These results show that after 6 days of intervention with inactivated selenium-containing Lactobacillus rhamnosus and then 7 days of hypobaric hypoxia exposure, the mice's spatial memory and short-term working memory were significantly improved, and the mice's cognitive function was protected. Moreover, SeLRS 6d +HH 7d Group and SPSS 6d There was no statistically significant difference in the alternating ratio of mice in the spontaneous alternating space Y-maze or the cumulative time spent in the learning arm of the Y-maze, and the cumulative time spent in the novel arm was significantly reduced in the +NN group. These results suggest that intervention with inactivated selenium-containing Lactobacillus rhamnosus protects mice from cognitive impairment under high-altitude hypoxia to a level close to that under normal conditions.

[0053] 4. Hematoxylin-eosin (HE) staining method verified that the intervention of inactivated selenium-containing Lactobacillus rhamnosus can alleviate the hippocampal body-amygdala damage in mice under high altitude hypoxia environment

[0054] 4.1 Sample Preparation

[0055] (1) Cardiac perfusion: After anesthetizing the mouse by intraperitoneal injection of an overdose of 1% sodium pentobarbital, fix the mouse in a supine position, cut the chest cavity to fully expose the heart, remove the pericardium around the heart, insert the syringe into the left ventricle, and cut the right ventricle to drain the fluid. First perfuse PBS, and maintain a uniform and slow flow rate during perfusion. After observing that the liver turns white and no blood or water flows out of the right ventricle, replace it with 4% paraformaldehyde for perfusion. The mouse conjunctiva is bloodless and the tongue and head become hard as signs of successful perfusion. Remove the intact brain tissue, immerse it in 4% paraformaldehyde, and place it in a 4°C refrigerator for fixation for 48 hours;

[0056] (2) Gradient dehydration: The brain tissue of mice that had been externally fixed for more than 48 hours was removed, transferred to a 20% sucrose solution, and placed in a 4°C refrigerator to settle for 24 hours. Subsequently, the brain tissue was transferred to a 40% sucrose solution and placed in a 4°C refrigerator. Dehydration was complete when the brain tissue was observed to sink to the bottom of the sucrose solution tube.

[0057] (3) Frozen sections: After dehydration, the brain tissue was removed and placed on filter paper to absorb the surface sucrose. The entire brain tissue was wrapped with OCT freezing embedding medium, and it was placed vertically on the injection head. Then it was immediately placed in the freezing microtome. After the embedding medium solidified, it could be sliced. Serial coronal sections were cut from the hippocampus and amygdala. The freezing microtome temperature was set to -20 to -23 ° C, and the slice thickness was 20 μm. The slices were placed in a 48-well plate containing brain slice preservation solution and temporarily stored at -4 ° C.

[0058] 4.2 HE staining

[0059] Frozen sections were stained with HE to observe morphological changes in the hippocampus-amygdala region. HE staining mainly involves six steps: rinsing, fixation, staining, dehydration, transparency, and sealing.

[0060] (1) Rinse. Pour PBS buffer into the wells and place on a shaker to shake slowly for 10 minutes. Repeat three times to remove as much embedding agent and impurities as possible. Cut the tip of the rubber dropper and transfer 1-2 brain slices from the well plate to the slide. Gently move the brain slice with the needle tip to fully unfold it. Use dust-free paper to absorb excess liquid on the slide.

[0061] (2) Fixation. Add 0.5 mL of 4% paraformaldehyde fixative to the brain slices. After fixing for 10 minutes, remove the paraformaldehyde fixative and add 0.5 mL of distilled water to soak for 2 minutes. Then, remove all the distilled water.

[0062] (3) Staining. Take an appropriate amount of hematoxylin staining solution and drop it on the brain slice to cover the entire surface. After staining for 6 minutes, aspirate the staining solution. Place the slide gently in running water and rinse slowly for 10 minutes to remove excess staining solution. After that, wash it with distilled water for a few seconds. Add immunostaining differentiation solution and differentiate it for 1 minute. Place it in running water and rinse slowly for 10 minutes before removing it. Add an appropriate amount of eosin staining solution to cover the entire surface of the brain slice and stain it for 1 minute.

[0063] (4) Dehydration: Soak the slides in 70%, 80%, 90% and anhydrous ethanol for 10 seconds.

[0064] (5) Clear. Then clear in xylene for 5 minutes, replace with fresh xylene and clear again for 5 minutes.

[0065] (6) Seal the slices. Add neutral resin to the slices and cover with a coverslip, avoiding the formation of bubbles during the process.

[0066] The specimens were placed under a microscope and panoramic brain slices were photographed. CaseViewer software was used to compare the presence of cell atrophy, degeneration, or necrosis at the same anatomical location in the brain slices of the NN and HH1d groups, thereby determining whether 7 days of hypobaric hypoxia exposure caused brain tissue damage and the extent of the damage.

[0067] The HE staining results of mouse hippocampus were as follows Figure 2 and Figure 3 shown.

[0068] Depend on Figure 2 It can be seen that SeLRS 6d +HH 7d Group and SPSS 6d +HH 7d Compared with the control group, the CA1 pyramidal cells in the hippocampus of mice were arranged in a dense and orderly manner ( Figure 2The structure is intact, no obvious pathological changes in cell morphology were observed, and the number of vacuoles and inflammatory cells inside the cell layer was significantly less than that outside ( Figure 2 The pyramidal cell layer in the CA3 region of the hippocampus of mice was tightly arranged in an orderly manner, with plump cells and a significant decrease in the number of cells that had pyknosis, deformation, and necrosis (red arrow in Figure G). Figure 2 Black arrows in Figure H), the number of vacuoles and inflammatory cells in the cell layer decreased ( Figure 2 Red arrow in Figure H), and no angiogenesis-like phenomenon was observed.

[0069] Depend on Figure 2 It can be seen that SeLRS 6d +HH 7d Group and SPSS 6d Compared with the +NN group, the interstitial space between CA1 pyramidal cells in the hippocampus of mice was enlarged, the number of vacuoles and inflammatory cells inside and outside increased, and angiogenesis-like phenomena were seen outside. 6d +HH 7d Group and SPSS 6d +HH 7d Compared with the control group, the granule cell layer in the DG region of the hippocampus of mice was arranged in a tight and orderly manner and was closely connected with the inner polymorphic cell layer ( Figure 2 The size and shape of multiple cell lines returned to normal, and the number of vacuoles and inflammatory cells in the outer cell layer of the granular cell layer decreased, and no angiogenesis-like phenomenon was observed.

[0070] Figure 3 The counting results showed that SeLRS 6d +HH 7d Group and SPSS 6d +HH 7d Compared with the SeLRS group, the density of pyknotic neurons decreased significantly; 6d +HH 7d Group and SPSS 6d Compared with the +NN group, there was no statistical difference in any of the indicators. These results indicate that the use of inactivated selenium-containing Lactobacillus rhamnosus SHA113 can significantly alleviate the changes in hippocampal morphology in mice after 7 days of hypobaric and hypoxic exposure, and the effect is close to the level when mice have not been exposed to a hypobaric and hypoxic environment.

[0071] HE staining results of mouse amygdala Figure 4 The results showed that feeding inactivated selenium-containing Lactobacillus rhamnosus could significantly alleviate the changes in amygdala morphology in mice after 7 days of hypobaric and hypoxic exposure. 6d +HH 7d Group and SPSS 6d +HH 7dCompared with the control group, the number of dense neurons in the BLA region of the mouse amygdala that were pyknotic, deformed, or even necrotic was reduced, while the number of normal neurons increased. The neuronal cells in the CeA region of the mouse amygdala were evenly distributed (yellow arrows), the cell shape was plump, and the number of vacuoles and inflammatory cells did not change significantly. There was no obvious pathological change in cell morphology, and there was still a phenomenon similar to angiogenesis (red arrows). The neuronal cells at the bottom underwent aggregated pyknotic, deformed, and even necrotic, while the number of normal neuronal cells with plump shapes increased away from the bottom (black arrows).

[0072] 5. Inactivated selenium-containing Lactobacillus rhamnosus alleviates hippocampal-amygdala damage

[0073] 5.1 Sample Preparation

[0074] (1) Prepare the Golgi staining working solution: Strictly follow the instructions in the FD Rapid Golgi Staining Kit. Mix Solution A and Solution B in a 1:1 ratio the day before sampling and let stand overnight to allow them to mix naturally.

[0075] (2) Cardiac perfusion: After anesthetizing the mouse by intraperitoneal injection of an overdose of 1% sodium pentobarbital, fix the mouse in the supine position, cut the chest cavity to fully expose the heart, remove the pericardium surrounding the heart, insert the syringe into the left ventricle, and cut the right ventricle to drain the fluid. First, perfuse PBS at a constant and slow flow rate. When the liver turns white and no blood or fluid flows out of the right ventricle, replace the perfusion with 4% paraformaldehyde. Successful perfusion is indicated by the absence of blood in the mouse conjunctiva and the hardening of the tongue and head. Remove the intact brain tissue.

[0076] (3) Soaking the sample: Rinse the blood stains on the surface of the brain tissue quickly in double-distilled water, then soak the brain tissue completely in the AB solution prepared the day before and store it at room temperature and away from light for 24 hours. Perform subsequent solution changes according to the instructions in the FD Rapid Golgi Staining Kit. Transfer the complete brain tissue to the new AB solution and soak it at room temperature and away from light for 2 weeks. During this period, use a glass rod to gently stir the solution every three days to prevent precipitation. After 2 weeks, transfer the complete brain tissue to C solution and let it stand at room temperature and away from light for 1 week for dehydration. During this period, change the C solution every 24 hours.

[0077] (4) Frozen sections: Remove the brain tissue from liquid C, freeze it in OCT embedding medium at -80°C and embed the brain tissue for preservation or direct sectioning. Fix the brain tissue on the freezing head with OCT and cut the coronal sections of the hippocampus and amygdala. Do not use a baffle when slicing, set the temperature of the freezing microtome to -23°C, the temperature of the freezing head to -20°C, and the slice thickness to 100 μm. Wrap the slides with 1% gelatin in advance. When collecting the slices, add solution C in the kit to the slides. Gently unfold the cut tissue slices and place them on the slides. Aspirate excess liquid C. Finally, place the slices in a dark environment to dry naturally, and store the slices in a dark room temperature environment for up to 3 days.

[0078] 5.2 Golgi staining

[0079] Golgi staining was performed on frozen sections to observe morphological changes in the hippocampus-amygdala region. HE staining mainly includes six steps: rinsing, staining, washing, dehydration, transparency, and sealing:

[0080] (1) Rinse. Select appropriate sections and rinse them twice with distilled water for 4 minutes each time.

[0081] (2) Staining. Prepare a mixed working solution (liquid D: liquid E: distilled water = 1:1:2) in advance. Soak the slide in the mixed working solution until the liquid completely covers the brain tissue slices for 5 minutes. Replace with a new mixed working solution and repeat the soaking process.

[0082] (3) Washing: Rinse twice with distilled water, 5 minutes each time.

[0083] (4) Gradient dehydration. Place the sections in 50%, 75%, and 95% ethanol in sequence, soaking for 4 minutes in each gradient. Dehydrate with absolute alcohol 4 times, 4 minutes each time.

[0084] (5) Transparent. Transparent the tissue with xylene, repeat 3 times, 4 minutes each time.

[0085] (6) Seal the slides. Seal the slides with neutral resin.

[0086] The specimens were placed under a microscope and panoramic brain slices were photographed. CaseViewer software was used to compare the hippocampus and amygdala regions of different groups.

[0087] The results of Golgi-Cox staining of hippocampus Figure 5 As shown. It can be seen that SeLRS 6d +HH 7d Group and SPSS 6d +HH 7d Compared with the control group, the dendritic branches of neurons in the CA1 and CA3 regions of the hippocampus increased, and the dendritic complexity increased ( Figure 5 (red arrows in Figures G and H) to achieve the same results as SPSS 6d The levels of selenium in the rats were similar to those in the +NN group; neuronal loss and axonal fragmentation in the CA1 region disappeared. These results indicate that inactivated selenium-containing Lactobacillus rhamnosus can protect against the reduction in dendritic complexity of hippocampal neurons caused by 7 days of hypobaric hypoxia exposure, with an effect comparable to that of normal mice.

[0088] Golgi-Cox staining results of amygdala Figure 6 As shown. It can be seen that SeLRS 6d +HH 7d Group and SPSS 6d +HH 7d Compared with the control group, the dendritic branches of neurons in the MeA region of the mouse amygdala increased, and the dendritic complexity increased, reaching the same level as SPSS 6d These results indicate that inactivated selenium-containing Lactobacillus rhamnosus can effectively protect the dendritic complexity of neurons in the amygdala MeA region induced by 7-day hypobaric hypoxia exposure, and the alleviating effect is comparable to that of normal mice.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and such improvements and modifications should also be considered as within the scope of protection of the present invention.

Claims

1. Application of Lactobacillus rhamnosus in the preparation of products for preventing and treating cognitive dysfunction caused by plateau hypoxia.

2. Application of Lactobacillus rhamnosus in the preparation of products for preventing and treating brain damage caused by high altitude hypoxia.

3. The use according to claim 1 or 2, characterized in that The Lactobacillus rhamnosus includes Lactobacillus rhamnosus SHA113.

4. The use according to claim 1 or 2, characterized in that The Lactobacillus rhamnosus is inactivated selenium-containing Lactobacillus rhamnosus.

5. The use according to claim 4, characterized in that The preparation method of the inactivated selenium-containing Lactobacillus rhamnosus comprises the following steps: expanding and culturing the Lactobacillus rhamnosus, adding a selenium-rich raw material to the culture medium and continuing the cultivation, taking the precipitate and crushing it to obtain the inactivated selenium-containing Lactobacillus rhamnosus.

6. The use according to claim 5, characterized in that The selenium-rich raw material comprises at least one of sodium selenite, sodium selenate, selenium yeast, selenium polysaccharide, selenomethionine, selenium-rich malt powder and selenium-rich edible fungus powder.

7. The use according to claim 4, characterized in that The selenium concentration in the inactivated selenium-containing Lactobacillus rhamnosus is greater than 2 mg / g.

8. The use according to claim 1 or 2, characterized in that The plateau is a high altitude area with an altitude of more than 1000m.

9. The use according to claim 2, characterized in that The brain damage includes hippocampal damage and / or amygdala damage.

10. A product for preventing and treating brain damage or cognitive dysfunction caused by plateau hypoxia, characterized in that: The active ingredient of the product comprises inactivated selenium-containing Lactobacillus rhamnosus, and the selenium concentration in the inactivated selenium-containing Lactobacillus rhamnosus is greater than 2 mg / g.

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