Application of lactobacillus rhamnosus in preparation of product for relieving anxiety and / or depression caused by plateau hypoxia

Abstract

The invention provides application of lactobacillus rhamnosus in preparation of a product for relieving anxiety and / or depression caused by plateau hypoxia, and belongs to the technical field of prevention and treatment of diseases caused by plateau hypoxia. According to the application disclosed by the invention, the lactobacillus rhamnosus can be used for preventing and / or treating anxiety and / or depression caused by plateau hypoxia for the first time, the blank of research and product development under a specific application scene of plateau hypoxia is filled up, and a new choice is provided for solving mental health problems related to plateau hypoxia. The lactobacillus rhamnosus is used as a probiotic, has good biological safety and tolerance, is developed into a prevention and treatment product, provides a new natural, safe and effective prevention and treatment choice for tourists and working people in plateau regions, avoids side effects of drug treatment, overcomes defects of traditional modes such as oxygen uptake and the like, and has a good application prospect. And the acceptability and the compliance of a user are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of prevention and treatment of diseases caused by plateau hypoxia, and particularly relates to the application of Lactobacillus rhamnosus in the preparation of products for relieving anxiety and / or depression caused by plateau hypoxia. Background Art

[0002] The plateau refers to an area with an altitude of more than 1000 meters and a relative height of more than 500 meters, a relatively flat or gently undulating vast area, such as the Qinghai-Tibet Plateau, Yunnan-Guizhou Plateau, etc. In plateau areas, the air is thin, the atmospheric pressure is low, and the oxygen partial pressure is low. When the altitude increases to 3000m, the arterial oxygen partial pressure and arterial oxygen saturation decrease significantly, and the human body shows hypoxia. Hypobaric hypoxia is the main cause of plateau diseases. Upper respiratory tract infections, fatigue, cold, mental stress, hunger, pregnancy, etc. can all induce the onset and promote the occurrence and development of plateau diseases. Plateau diseases include acute mountain sickness, high altitude pulmonary edema, high altitude cerebral edema, chronic mountain sickness, high altitude polycythemia, high altitude heart disease, high altitude hypertension, and high altitude hypotension, etc. In severe cases, multiple organ damage may occur and the prognosis is poor.

[0003] In the plateau environment, in addition to the above-mentioned physiological plateau diseases, long-term exposure to plateau hypoxia will also have an adverse impact on the mental state of the human body, and anxiety and depression are common accompanying symptoms. Anxiety and depression not only reduce the quality of life of plateau residents and stationed personnel, but may also further affect their cognitive function and daily activity ability, and may even trigger a series of serious mental health problems, such as suicidal tendencies. Therefore, developing products that can effectively prevent and treat anxiety and / or depression caused by plateau hypoxia has important practical significance, which is crucial for protecting the physical and mental health of people in plateau areas, improving their quality of life, and promoting the stable development of plateau areas.

[0004] At present, the treatment of anxiety and / or depression caused by plateau hypoxia mainly focuses on drug treatment, psychological intervention, and oxygen therapy. Although drug treatment can relieve symptoms to a certain extent, it is often accompanied by various side effects such as drowsiness, dizziness, and dry mouth. Long-term use may also cause drug dependence and tolerance, imposing an additional burden on the patient's body. Psychological intervention requires long-term guidance from professional psychologists. In plateau areas, due to relatively scarce medical resources and a limited number of professional psychologists, it is difficult to meet the needs of a large number of patients, and the effect of psychological intervention takes effect relatively slowly, making it difficult to quickly relieve acute anxiety and depression symptoms. Although oxygen therapy can improve the hypoxia condition, its effect on improving neuropsychological damage caused by long-term chronic hypoxia is limited, and in actual application, continuous oxygen therapy has many inconveniences, such as difficult equipment carrying and the need for a stable oxygen supply source. Therefore, exploring a new method that can effectively prevent or treat anxiety and / or depression caused by plateau hypoxia is of great significance. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide the application of Lactobacillus rhamnosus in the preparation of products for preventing and treating or alleviating anxiety and / or depression caused by high altitude hypoxia, so as to provide a new direction for the prevention, treatment or alleviation of anxiety and / or depression caused by high altitude hypoxia.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the application of Lactobacillus rhamnosus in the preparation of products for preventing and treating or alleviating anxiety and / or depression caused by high altitude hypoxia.

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

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

[0010] Preferably, the preparation method of the inactivated selenium-containing Lactobacillus rhamnosus includes the following steps: after the Lactobacillus rhamnosus is expanded in culture, a selenium-rich raw material is added to the culture medium for continuous culture, and the precipitate is taken and broken 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 fungi.

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

[0013] Preferably, the high altitude is a high altitude area above 1000 m above sea level.

[0014] The present invention also provides a product for preventing and treating or alleviating anxiety and / or depression caused by high altitude hypoxia, and the active ingredient of the product includes inactivated selenium-containing Lactobacillus rhamnosus.

[0015] Preferably, the selenium concentration in the inactivated selenium-containing Lactobacillus rhamnosus is greater than 2 mg / g.

[0016] Preferably, the Lactobacillus rhamnosus includes Lactobacillus rhamnosus SHA113.

[0017] The beneficial effects of the present invention:

[0018] The present invention first proposes that Lactobacillus rhamnosus can be used for preventing and / or treating anxiety and / or depression caused by hypoxia at high altitudes, filling the research and product development gaps in specific application scenarios of hypoxia at high altitudes, and providing a new direction for solving mental health problems related to hypoxia at high altitudes. As a probiotic, Lactobacillus rhamnosus has good biosafety and tolerance. By developing it into a prevention and treatment product, it provides a natural, safe and effective new option for people in high-altitude areas, avoiding the disadvantages of traditional treatment methods and improving the acceptance and compliance of users. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Results of the open field test for mice, where (a) is the total moving distance in the open field, (b) is the cumulative duration of the non-moving state in the open field, (c) is the proportion of the cumulative duration of the non-moving state in the open field, (d) is the frequency of entering the central area of the open field, (e) is the cumulative duration in the central area of the open field, and (f) is the proportion of the cumulative duration in the central area of the open field; the results are presented as mean ± standard error (n = 7), *P < 0.05, **P < 0.01, ****P < 0.0001;

[0020] Figure 2 Results of the elevated plus maze test for mice, where (a) is the total moving distance in the maze, (b) is the total moving speed in the maze, (c) is the proportion of the frequency of entering the open arms of the maze, (d) is the proportion of the cumulative duration of entering the open arms of the maze, (e) is the proportion of the frequency of entering the closed arms of the maze, (f) is the proportion of the cumulative duration of entering the closed arms of the maze, n = 7, and the results are presented as mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.001;

[0021] Figure 3 Results of the depression-like behavior test for mice, where (a) is the cumulative immobility time in the tail suspension test, (b) is the cumulative immobility time in the forced swimming test, (c) is the sucrose preference percentage, n = 7, and the results are presented as mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention provides the application of Lactobacillus rhamnosus in the preparation of products for preventing and treating anxiety and / or depression caused by hypoxia at high altitudes.

[0023] The present invention has no special 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 at the China Center for Type Culture Collection on December 25, 2017, with the deposit number CCTCC NO: M2017838.

[0024] In the present invention, the Lactobacillus rhamnosus is preferably inactivated Lactobacillus rhamnosus containing selenium, and the preparation method of the inactivated Lactobacillus rhamnosus containing selenium preferably includes the following steps: after the Lactobacillus rhamnosus is expanded in culture, a selenium-rich raw material is added to the culture medium for continuous culture, and the precipitate is taken and broken to obtain the inactivated Lactobacillus rhamnosus containing selenium. In the present invention, the culture medium used for the expanded culture is preferably MRS liquid medium. The present invention has no special limitation on the specific source of the MRS liquid medium, and conventional commercially available products in the art can be used. The temperature of the expanded culture is preferably 37 °C, and the time of the expanded culture is preferably 48 h. In the present invention, the selenium-rich raw material preferably includes at least one of sodium selenite, sodium selenate, selenium yeast, selenium polysaccharide, selenomethionine, selenium-rich malt powder, and selenium-rich edible fungi. In the present invention, the time for continuous culture after adding the selenium-rich raw material is preferably 24 h. After the continuous culture is completed, centrifugal separation is preferably carried out. The rotation speed of the centrifugation is preferably 8000 r / min, and the time of the centrifugation is preferably 3 min. In the present invention, the inactivated Lactobacillus rhamnosus containing selenium is actually the broken Lactobacillus rhamnosus containing selenium, that is, the dead bacteria of Lactobacillus rhamnosus containing selenium. In the present invention, the selenium concentration in the inactivated Lactobacillus rhamnosus containing selenium is preferably greater than 2 mg / g, and more preferably 35 mg / g. In the present invention, the plateau preferably refers to a high-altitude area with an altitude of more than 1000 m.

[0025] In the present invention, the intervention with inactivated Lactobacillus rhamnosus containing selenium can effectively alleviate the anxiety-like behavior of mice caused by 7-day exposure to high-altitude hypoxia. The main manifestations are that, compared with the mice exposed to low pressure and low oxygen for 7 days, the cumulative duration and proportion of the mice exposed for 7 days after the intervention with inactivated Lactobacillus rhamnosus containing selenium in the central area of the open field test show an upward trend; the total moving distance and moving speed in the elevated plus maze test are significantly increased, the frequency proportion of entering the open arms of the maze is significantly increased, the cumulative duration and proportion in the open arms of the maze are significantly increased, the frequency proportion of entering the closed arms of the maze is significantly decreased, and the cumulative duration and proportion in the closed arms of the maze are significantly decreased.

[0026] In the present invention, the intervention with inactivated Lactobacillus rhamnosus containing selenium can effectively alleviate the depressive-like behavior of mice caused by 7-day exposure to low pressure and low oxygen. The main manifestations are that, compared with the mice without intervention exposed to low pressure and low oxygen for 7 days, the cumulative immobility time of the mice exposed for 7 days after the intervention with inactivated Lactobacillus rhamnosus containing selenium in the tail suspension test and the forced swimming test in the last 4 min is significantly reduced; the sucrose preference index in the sucrose preference test is significantly increased.

[0027] The present invention also provides a product for preventing and treating anxiety and / or depression caused by high-altitude hypoxia, and the active ingredient of the product includes inactivated Lactobacillus rhamnosus containing selenium.

[0028] In the product of the present invention, the inactivated Lactobacillus rhamnosus containing selenium is used as the sole active ingredient, and the selenium concentration in the inactivated Lactobacillus rhamnosus containing selenium is preferably greater than 2 mg / g, more preferably 35 mg / g. In the present invention, the Lactobacillus rhamnosus is preferably Lactobacillus rhamnosus SHA113.

[0029] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] In the following embodiments, unless otherwise specified, all are conventional methods.

[0031] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0032] Example 1

[0033] 1. Experimental animals:

[0034] Specific pathogen-free (SPF) grade 8-week-old male C57BL / 6 mice were used, purchased from Xi'an Ke'ao Co., Ltd. The animals were housed in an SPF-grade animal house, and 4 - 6 mice were housed in each cage in an animal house with standard temperature (22 ± 2 °C), humidity (50 ± 10%), and a 12-hour day-night rhythm cycle (8:00 - 20:00 was the light cycle). The animals could drink water and eat freely, and the behavioral experiments were carried out in a quiet and empty experimental room. All operations received by the animals complied with the requirements of laboratory animal ethics and followed the regulations promulgated by the national and Peking University Health Science Center Animal Use and Protection Committee.

[0035] 2. Preparation of mouse model:

[0036] The preparation process of the inactivated Lactobacillus rhamnosus containing selenium suspension was as follows: Lactobacillus rhamnosus SHA113 (i.e., Lactobacillus rhamnosus SHA113) was inoculated into MRS liquid medium at 1% (volume ratio) and cultured at 37 °C for 48 h. Then, 3.0 mM sodium selenite was added to the medium, and the culture was continued at 37 °C for 24 h. The whole culture was centrifuged at 8000 r / min for 3 min, and the precipitate was washed three times with sterile water and then collected. The precipitate was ultrasonically broken in an ice bath for 2 s, stopped for 2 s, and this was continued for 60 min with an ultrasonic power of 500 watts, and then it was freeze-dried to obtain inactivated selenium-containing powdered bacteria, which was reserved. After detection, the selenium concentration in the inactivated selenium-containing powdered bacteria was 35 mg / g. Before use, it was formulated into an inactivated Lactobacillus rhamnosus containing selenium suspension with sterile normal saline for animal experiments.

[0037] After one week of adaptive feeding, male C57BL / 6 mice were randomly divided into three groups of 12 mice each. One group was intragastrically administered sterile normal saline continuously for 6 days and then placed in a SPF-class animal room to establish a control group (SPSS 6d +NN); one group was intragastrically administered sterile normal saline continuously for 6 days and then placed in a hypobaric hypoxia animal experimental chamber for 7 days to establish a mouse model of 7-day hypobaric hypoxia exposure (SPSS 6d +HH 7d ); one group was intragastrically administered an inactivated suspension of Lactobacillus rhamnosus containing selenium in sterile normal saline as a solvent (the intragastric administration dose was calculated according to the selenium content, which was 0.12 mg selenium / kg body weight / day, and the total intragastric administration volume was 10 mL / kg body weight) and then placed in a hypobaric hypoxia animal experimental chamber for 7 days to establish a mouse model of hypobaric hypoxia exposure intervened by inactivated Lactobacillus rhamnosus containing selenium (SeLRS 6d +HH 7d ). The total intragastric administration volume of the above three groups was the same. During this period, the animal hypobaric hypoxia animal experimental chamber was set to simulate the experimental conditions at an altitude of 5000 m on the plateau. The pump pressure was shown as 405 mmHg, and the temperature (22±2 °C), humidity (50±10%), and 12-h day-night rhythm cycle (8:00 - 20:00 was the light cycle) in the hypobaric hypoxia chamber were maintained. The animals could drink water and eat freely.

[0038] 3. Mouse behavioral experiments

[0039] Within 1 week after the end of the mouse hypobaric hypoxia exposure, the EthoVision XT small animal behavioral system was used to conduct open field test (OFT), elevated plus maze test (EPM), tail suspension test (TST), forced swimming test (FST), and sucrose preference test (SPT) on the mice to observe the behavioral phenotypes of the mice after modeling.

[0040] 3.1. Anxiety-like behavior

[0041] (1) Mouse open field test

[0042] The experiment was carried out in an opaque acrylic box with an open top of 50 cm×50 cm×50 cm, and the central 25×25 cm area was used as the central area. The mice were placed in the center of the open field, and the moving distance (cm) and moving speed (cm / s) of the mice within 10 min in the open field were recorded as the evaluation criteria for the spontaneous activity level of the animals. The cumulative duration (s) and proportion (%) of the non-moving state of the mice within 10 min in the open field, the frequency (times) of entering the central area, and the cumulative duration (s) and proportion (%) in the central area were recorded as the indicators for evaluating anxiety-like behavior. The excrement left in the test box was cleaned during the test for each mouse, and the test box was wiped with 75% alcohol to remove the residual odor.

[0043] The results are as Figure 1As shown, SeLRS 6d +HH 7d group and the SPSS 6d +HH 7d group, the total moving distance of mice in the open field test was significantly increased; the cumulative duration and proportion of the non - moving state were significantly decreased; the frequency of entering the central area was not affected; the cumulative duration and proportion in the central area showed an upward trend. It indicates that heat - inactivated Lactobacillus rhamnosus containing selenium can significantly improve the spontaneous activity ability of mice in the open field after 7 - day exposure to low - pressure and low - oxygen, and relieve the anxiety - like behavior of mice in the open field.

[0044] (2) Elevated plus - maze test in mice

[0045] The test device consists of two open arms (30 cm×8 cm), two closed arms (30 cm×8 cm, 23 cm high) and a central area (8 cm×8 cm). The open arms are 75 cm above the ground. At the start of the test, the mice are gently placed in the central area with their heads facing the open arms, and the mice freely explore in the elevated plus - maze for 6 min. Record the total moving distance in the maze, the total moving speed in the maze, the proportion of the frequency of entering the open arms of the maze, the proportion of the cumulative duration of entering the open arms of the maze, the proportion of the frequency of entering the closed arms of the maze, and the proportion of the cumulative duration of entering the closed arms of the maze. Among them, the time (s) and the number of times of entering the open arms of the mice are used as indicators to evaluate anxiety - like behavior. During the test of each mouse, the excrement left in the test box is cleaned, and the test box is wiped with 75% alcohol to remove the residual smell.

[0046] The results are as Figure 2 shown, SeLRS 6d +HH 7d group and the SPSS 6d +HH 7d group, the total moving distance and moving speed of mice in the elevated plus - maze test were significantly increased; the proportion of the frequency of entering the open arms of the maze was significantly increased; the cumulative duration and proportion of the mice in the open arms of the maze were significantly increased; the proportion of the frequency of entering the closed arms of the maze was significantly decreased; the cumulative staying time and proportion of the mice in the closed arms of the maze were significantly decreased. These results indicate that heat - inactivated Lactobacillus rhamnosus containing selenium can significantly relieve the anxiety - like behavior of mice in the elevated plus - maze after 7 - day exposure to low - pressure and low - oxygen.

[0047] 3.2, Depressive - like behavior

[0048] (1) Tail - suspension test in mice

[0049] The tail suspension test device is 55 cm high. Medical tape needs to be attached to the mouse's tail (at a distance of 3 / 4 from the bottom of the mouse's tail), and the mouse is suspended. After suspension, the mouse is about 35 cm above the ground. There are partitions between the mice being tested simultaneously to block the line of sight. The test lasts for 6 minutes, and the total time that the mouse remains stationary in the last 4 minutes is recorded as an indicator of depressive-like behavior and despair behavior.

[0050] The results are as Figure 3 shown in (a) of

[0051] (2) Mouse forced swimming test

[0052] The experimental device is a plexiglass cylinder with a diameter of 10 cm and a height of 25 cm. The water depth is 16 ± 1 cm, and the water temperature is 25 ± 1 °C. When the mouse swims, ensure that it cannot rely on its limbs or tail for support and that its nose tip can be exposed above the water surface for breathing. There are baffles between each glass cylinder to prevent interference in the test due to mutual learning among mice. The water in the glass cylinder is changed for every 3 mice to avoid interference from excrement.

[0053] The mouse forced swimming test is divided into two stages:

[0054] Forced swimming adaptation stage: At the same time on the day before the test, conduct adaptation under the same conditions. The adaptation swimming time is 15 minutes.

[0055] Forced swimming test: The swimming time is 6 minutes, and the floating immobile time of the mouse in the last 4 minutes is recorded as an indicator of depressive-like behavior and despair behavior.

[0056] The results are as Figure 3 shown in (b) of

[0057] (3) Mouse sucrose preference test

[0058] The test includes three stages:

[0059] Adaptation stage: Prepare a 1% sucrose solution with the mouse's daily drinking water. Pour the 1% sucrose solution and drinking water into two water bottles with the same size and appearance respectively, and place them on the mouse cage lid. The mouse can freely choose to drink the water in the two water bottles. Conduct a 48-hour double-bottle drinking adaptation, and exchange the positions of the two water bottles at the end of 24 hours.

[0060] Water and food deprivation stage: After the 48-hour adaptation stage, the mouse undergoes 24 hours of water and food deprivation.

[0061] Sucrose preference test: After 24 hours of water and food deprivation, weigh one bottle of 1% sucrose solution and one bottle of ordinary drinking water, and then carefully place them on the lid of the mouse cage so that the mice can freely choose between ordinary drinking water and sucrose solution. The test lasts for 24 hours. Exchange the positions of the two water bottles at the 12th hour. After the test, weigh the two water bottles again, calculate the intake of 1% sucrose and ordinary drinking water by the mice within 24 hours, and allow free feeding during the test. The evaluation indicators include: sucrose preference value and total intake, and the sucrose preference value is used as an indicator of anhedonia. Sucrose preference value (%) = sucrose intake / (sucrose intake + ordinary drinking water intake) × 100%.

[0062] The results are as Figure 3 shown in (c) of

[0063] As can be seen from Figure 3 it, the SeLRS 6d +HH 7d group compared with the SPSS 6d +HH 7d group, the cumulative immobility time of the mice in the tail suspension test and the forced swimming test in the last 4 minutes was significantly reduced; the sucrose preference index in the sucrose preference test was significantly increased. These results indicate that after 6 days of intervention with inactivated selenium-containing Lactobacillus rhamnosus and then exposure to a low-pressure and low-oxygen environment for 7 days, the severe despair behavior and strong anhedonia of the mice were significantly reduced, and the depressive-like behavior was significantly alleviated.

[0064] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of Lactobacillus rhamnosus in the preparation of products for preventing and treating anxiety and / or depression caused by high altitude hypoxia.

2. The application according to claim 1, characterized in that, The Lactobacillus rhamnosus includes Lactobacillus rhamnosus SHA113.

3. The application according to claim 1, wherein The Lactobacillus rhamnosus is an inactivated selenium-containing Lactobacillus rhamnosus.

4. The application according to claim 3, characterized in that, The preparation method of the inactivated selenium-containing Lactobacillus rhamnosus comprises the following steps: after the Lactobacillus rhamnosus is expanded in culture, a selenium-rich raw material is added to the culture medium for continuous culture, and the precipitate is taken and broken to obtain the inactivated selenium-containing Lactobacillus rhamnosus.

5. The application according to claim 4, characterized in that, 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 fungi.

6. The application according to claim 4, wherein The selenium concentration in the inactivated selenium-containing Lactobacillus rhamnosus is greater than 2 mg / g.

7. The application according to claim 1, characterized in that, The high altitude area is a high altitude area with an altitude of more than 1000 m.

8. A product for preventing and treating anxiety and / or depression caused by hypoxia on the plateau, characterized in that, The active ingredient of the product includes the inactivated selenium-containing Lactobacillus rhamnosus.

9. The product according to claim 8, characterized in that, The selenium concentration in the inactivated selenium-containing Lactobacillus rhamnosus is greater than 2 mg / g.

10. The product according to claim 8, wherein The Lactobacillus rhamnosus includes Lactobacillus rhamnosus SHA113.

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