Application of Lactobacillus johnsonii composite bacterial agent in the preparation of drugs for preventing and treating memory impairment caused by plateau hypoxia

Abstract

The invention discloses application of a Lactobacillus johnsonii composite bacterial agent in preparing a drug for preventing and treating memory impairment caused by plateau hypoxia, and belongs to the field of microorganisms. The composite bacterial agent of Lactobacillus johnsonii YH1136 and Bacillus subtilis BS01 provided by the invention can effectively prevent and treat brain memory dysfunction caused by exposure to a plateau hypoxic environment. The composition can restore spatial memory ability, significantly reduce the escape latency, significantly increase the expression level of hippocampal memory-related proteins, significantly increase the antioxidant capacity of the hippocampus in the brain, and alleviate oxidative damage to the hippocampus. The composite bacterial agent of Lactobacillus johnsonii YH1136 and Bacillus subtilis BS01 provided by the invention can be used to prepare a drug for preventing and treating brain memory dysfunction caused by exposure to a plateau hypoxic environment and a composition thereof, and can also be used as an auxiliary product of existing therapeutic drugs and treatment methods, and has very broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microbial preparations, and in particular to application of a Lactobacillus johnsonii composite bacterial agent in preparing a drug for preventing and treating memory impairment caused by plateau hypoxia. Background Art

[0002] The natural environment in the plateau is complex and special. Compared with the plains, its main feature is the lower air pressure and oxygen level. As the altitude of the plateau gradually increases, the atmospheric pressure level continues to decrease, resulting in a continuous decrease in the oxygen partial pressure in the atmosphere. For example, at an altitude of 5,000 meters, the oxygen partial pressure is only half of that at sea level. Therefore, even if the relative proportion of oxygen in the atmosphere remains unchanged, the thin atmosphere will lead to hypoxia, which is a huge challenge to the normal respiratory function of the human body. The amount of oxygen intake during normal breathing is reduced, which affects the normal physiological health of the human body. In addition, high-altitude exposure (i.e., low pressure, low oxygen and low temperature conditions) can lead to acute or chronic altitude sickness due to the lack of compensatory ability. The impact of the low oxygen environment on the plateau on the human body is mainly manifested in the decline of labor capacity, appetite, weight, physical strength, memory and other abilities. Severe impacts may lead to plateau intestinal stress and induce altitude sickness (including high-altitude pulmonary edema, high-altitude cerebral edema, high-altitude heart disease, etc.). The plateau hypoxic environment has different effects on the digestive, cardiovascular and nervous systems. The brain is the largest oxygen-consuming organ in the human body, and hypoxia has a significant adverse effect on its cognitive function. Studies have shown that high-altitude hypoxic environments can lead to changes in various cognitive processes in the brain, such as decreased attention, weakened will control, reduced correctness of executive function tasks, and prolonged reaction time. Studies have shown that plateau exposure increases the body's protein oxidation level and catalase activity, and reduces serum iron, ferritin, and transferrin saturation. The plateau environment affects the body's antioxidant capacity and iron homeostasis. In addition, the hypoxic environment in plateau areas is closely related to symptoms of brain dysfunction such as decreased memory. It is reported that the hippocampus of the brain is very important for learning and memory. Its function affects the spatial memory ability of the organism. It is one of the most studied brain nervous systems and is sensitive to pathological changes such as reduced antioxidant capacity. Oxidative damage can cause damage to working memory in humans and animals.

[0003] At present, the chemical drugs commonly used to prevent and treat altitude sickness mainly include acetazolamide, dexamethasone, nifedipine and aminophylline. These drugs can enhance the body's ability to resist hypoxia to prevent and treat altitude sickness by increasing lung ventilation, increasing blood oxygen saturation, and enhancing the toughness of capillaries and cell membranes. There is also a way to prevent the occurrence of altitude sickness by using oxygen cylinders to supply oxygen. In dealing with altitude sickness, these drugs and direct oxygen supply methods can effectively reduce the pressure of the plateau environment on the human body and make it easier for humans to adapt to the hypoxic environment of the plateau. But at the same time, these methods also have some limitations. According to reports, most drugs for anti-altitude reactions have some common side effects, which manifest as vomiting and diarrhea, dizziness, and affected stomach digestion. In addition, the special environment of the plateau will also affect the body's ability to metabolize and absorb drugs, so it is also necessary to be cautious when using drugs.

[0004] Therefore, there is an urgent need for a method without side effects to prevent and treat brain memory dysfunction caused by exposure to high-altitude hypoxic environment. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art, and provides an application of a Lactobacillus johnsonii composite bacterial agent in preparing a drug for preventing and treating memory impairment caused by plateau hypoxia, belonging to the field of microorganisms. The composite bacterial agent of Lactobacillus johnsonii YH1136 and Bacillus subtilis BS01 provided by the present invention can effectively prevent and treat brain memory dysfunction caused by exposure to a plateau hypoxic environment, and the composition can restore spatial memory ability, significantly reduce the escape latency, significantly increase the expression level of hippocampal memory-related proteins, significantly increase the antioxidant capacity of the hippocampus in the brain, and alleviate oxidative damage to the hippocampus. The composite bacterial agent of Lactobacillus johnsonii YH1136 and Bacillus subtilis BS01 provided by the present invention can be used to prepare a drug for preventing and treating brain memory dysfunction caused by exposure to a plateau hypoxic environment and a composition thereof, and can also be used as an auxiliary product of existing therapeutic drugs and treatment methods, and has a very broad application prospect.

[0006] In order to achieve the above technical effects, the following technical solutions are adopted:

[0007] Application of a Lactobacillus johnsonii composite bacterial agent in the preparation of a drug for preventing and treating memory impairment caused by plateau hypoxia, wherein the Lactobacillus johnsonii composite bacterial agent is formed by combining a Lactobacillus johnsonii YH1136 strain and a Bacillus subtilis BS01 strain;

[0008] The deposit number of the Lactobacillus johnsonii YH1136 strain is: CCTCC NO: M 20221116, the deposit date is July 15, 2022, the deposit unit is China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, and the postal code is 430072;

[0009] The deposit number of the Bacillus subtilis BS01 strain is: CCTCC NO: M 2012485, the deposit date is November 28, 2012, the deposit unit is China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the postal code is 430072;

[0010] The number of Lactobacillus johnsonii YH1136 strains is 1-2×10 9 CFU / mL;

[0011] The number of the Bacillus subtilis BS01 strain is 1-2×10 9 CFU / mL;

[0012] The ratio of the number of the Lactobacillus johnsonii YH1136 strain to the number of the Bacillus subtilis BS01 strain is 1-2:1.

[0013] Furthermore, the composite bacterial agent of the Lactobacillus johnsonii YH1136 strain and the Bacillus subtilis BS01 strain is a powder or an aqueous solution.

[0014] Furthermore, the drug also contains a pharmaceutically acceptable carrier.

[0015] Furthermore, the application method is to intragastrically or orally administer the medicine or pharmaceutical composition prepared by the composite bacterial agent.

[0016] Furthermore, the method for preventing and treating brain memory impairment caused by plateau hypoxia includes at least one of (a) to (c):

[0017] (a) Restore the memory ability of mammals and significantly reduce the escape latency of mammals;

[0018] (b) Significantly increased the expression level of memory-related proteins in the mammalian hippocampus;

[0019] (c) Significantly improve the antioxidant capacity of the hippocampus in the mammalian brain and alleviate oxidative damage in the hippocampus.

[0020] Furthermore, the mammals include humans.

[0021] The beneficial effects of the present invention are:

[0022] The invention discloses application of a Lactobacillus johnsonii composite bacterial agent in preparing a drug for preventing and treating memory impairment caused by plateau hypoxia, and belongs to the field of microorganisms. The composite bacterial agent of Lactobacillus johnsonii YH1136 and Bacillus subtilis BS01 provided by the invention can effectively prevent and treat brain memory dysfunction caused by exposure to a plateau hypoxic environment. The composition can restore spatial memory ability, significantly reduce the escape latency, significantly increase the expression level of hippocampal memory-related proteins, significantly increase the antioxidant capacity of the hippocampus in the brain, and alleviate oxidative damage to the hippocampus. The composite bacterial agent of Lactobacillus johnsonii YH1136 and Bacillus subtilis BS01 provided by the invention can be used to prepare a drug for preventing and treating brain memory dysfunction caused by exposure to a plateau hypoxic environment and a composition thereof, and can also be used as an auxiliary product of existing therapeutic drugs and treatment methods, and has very broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. The drawings in the following description are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0024] Figure 1 This is a comparison diagram of the effects of hypoxia exposure and different bacteria treatments on the spatial memory of mice on the first day in an embodiment of the present invention;

[0025] Figure 2 This is a comparison diagram of the effects of hypoxia exposure and different bacteria treatment on the spatial memory of mice on the second day in an embodiment of the present invention;

[0026] Figure 3 This is a comparison diagram of the effects of hypoxia exposure and different bacteria treatment on the spatial memory of mice on the third day in an embodiment of the present invention;

[0027] Figure 4 This is a comparison diagram of the effects of hypoxia exposure and different bacteria treatments on the spatial memory of mice in the embodiments of the present invention;

[0028] Figure 5 This is a comparison chart of the effects of hypoxia exposure and different bacteria treatments on the relative mRNA expression of the hippocampal memory-related protein brain-derived neurotrophic factor BDNF in the embodiments of the present invention;

[0029] Figure 6 is a comparative diagram of the effects of hypoxia exposure and different bacteria treatments on the mRNA relative expression of the hippocampal memory-related protein recombinant synaptophysin SYP in the embodiments of the present invention;

[0030] Figure 7This is a comparison diagram of the effects of hypoxia exposure and different bacteria treatments on the mRNA relative expression of hippocampal memory-related protein postsynaptic density protein PSD95 in the embodiments of the present invention;

[0031] Figure 8 It is a comparative diagram of immunofluorescence sections of the effects of hypoxia exposure and different bacteria treatment on the hippocampal memory-related protein brain-derived neurotrophic factor (BDNF) in the embodiments of the present invention;

[0032] Fig. 9 It is a comparison diagram of immunofluorescence sections of the hippocampal memory-related protein recombinant synaptophysin SYP in the embodiment of the present invention under hypoxia exposure and different bacteria treatment;

[0033] Fig.10 This is a comparison of immunofluorescence sections of the postsynaptic density protein PSD95, a hippocampal memory-related protein, under hypoxia exposure and different bacterial treatments in the embodiments of the present invention;

[0034] Fig.11 is a comparison chart of the activity level of T-AOC in the antioxidant capacity of the hippocampus in the embodiments of the present invention under hypoxia exposure and different bacteria treatments;

[0035] Fig.12 is a comparison chart of the activity level of MDA in the antioxidant capacity of the hippocampus in the embodiments of the present invention under hypoxia exposure and different bacteria treatments;

[0036] Fig.13 is a comparison chart of the activity level of GSH-Px in the antioxidant capacity of the hippocampus in the embodiments of the present invention under hypoxia exposure and different bacteria treatments;

[0037] Fig.14 It is a comparison chart of the activity level of SOD in the antioxidant capacity of the hippocampus after hypoxia exposure and different bacteria treatments in the examples of the present invention. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0039] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0040] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0041] Embodiment 1:

[0042] The Lactobacillus johnsonii YH1136 involved in the present invention is isolated and screened from the feces of a healthy Tibetan girl in Bange County, Nagqu District, Tibet Autonomous Region (about 5000m above sea level). Lactobacillus johnsonii The preservation number of the YH1136) strain is: CCTCC NO: M 20221116, the preservation date is July 15, 2022, the preservation unit is China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, and the postal code is 430072.

[0043] The Bacillus subtilis BS01 involved in the present invention ( Bacillus subtilis The Bacillus subtilis BS01 strain was isolated and screened from soil. The deposit number of the Bacillus subtilis BS01 strain is: CCTCC NO: M 2012485, the deposit date is November 28, 2012, the deposit unit is China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, and the postal code is 430072.

[0044] Initial screening of Lactobacillus johnsonii YH1136 strain:

[0045] Weigh 0.5 g of feces into a 100 mL conical flask, add 49.5 mL of sterile saline and sterile small glass beads (the saline and glass beads were sterilized in a high-pressure steam sterilizer at 120°C for 15 min in advance), place on an air shaker at room temperature, and shake at 150 rpm / min for 30 min to fully homogenize the feces sample to obtain 10 -2 Then draw 1 mL of the homogenate from the conical flask and add it to a test tube containing 9 mL of sterile saline solution and shake to mix well to obtain 10 -3 Diluent, dilute to 10 -7. Take 100 μL and spread it on MRS solid culture medium, culture at 37℃ for 24h, pick colonies with typical lactobacillus characteristics (colonies are white and round, with smooth surface, slightly raised, and neat edges) for Gram staining microscopy, and select Gram-positive rod-shaped bacteria for streaking pure culture. Then pick the single colonies obtained by streaking and expand them in MRS broth culture medium, and select 30 single colonies with lactobacillus characteristics, named Lactobacillus YH1121-YH1150.

[0046] Tolerance of Lactobacillus johnsonii YH1136 strain:

[0047] The expanded cultured lactobacilli YH1121-YH1150 were inoculated at a 2% inoculum size into sterile MRS liquid culture medium (10 g peptone, 8 g beef powder, 4 g yeast powder, 20 g glucose, 2 g dipotassium hydrogen phosphate, 2 g diammonium hydrogen citrate, 5 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1 g Tween 80, pH 5.7±0.2) with a pH of 3.0 and a cow bile salt concentration of 0.3%, and cultured at 37°C for 12 h. The changes in the number of viable bacteria in the culture medium were detected at 0 h and 12 h, and the survival rate of the test bacteria at the end of the culture was calculated (the number of viable bacteria at 12 h / the number of viable bacteria at 0 h, and the counting result was calculated as the logarithm with base 10. The results showed that 10 strains of bacteria, including YH1122, YH1125, YH1128, YH1130, YH1136, YH1137, YH1140, YH1143, YH1144, and YH1147, could maintain a high survival rate after 12 hours of culture under the conditions of pH=3.0 and bile salt concentration of 0.3%, indicating that these 10 strains of bacteria have good tolerance and possess the basic characteristics of probiotics. Next, antibacterial experiments were conducted on these 10 strains to explore their ability to inhibit common pathogens.

[0048] Table 1 Acid and bile salt resistance test of Lactobacillus YH1120-YH1150 (survival rate%)

[0049] strains pH 3.0 Bile salt 0.3% YH1121 21.44±1.61% 2.89±1.61% YH1122 59.50±2.54% 30.56±5.21% YH1123 27.44±1.74% 12.41±4.41% YH1124 28.94±4.16% 19.47±4.01% YH1125 45.12±5.78% 25.41±4.62% YH1126 18.94±4.41% 9.41±4.89% YH1127 37.84±4.06% 8.41±6.10% YH1128 40.85±4.23% 31.40±7.85% YH1129 29.84±7.64% 17.41±1.65% YH1130 50.23±1.25% 30.78±4.12% YH1131 10.26±5.48% 18.44±1.06 YH1132 9.27±1.02% 7.41±1.98% YH1133 24.79±4.98%. 5.98±1.87% YH1134 14.84±1.04% 13.84±4.10% YH1135 28.25±4.79% 6.44±3.15% YH1136 65.58±5.79% 34.81±3.10% YH1137 50.23±1.50% 28.48±6.49% YH1138 8.75±4.18% 23.10±3.15% YH1139 28.48±10.10% 13.89±7.43% YH1140 49.75±3.69% 43.47±1.90% YH1141 14.71±1.09% 28.48±3.10% YH1142 28.47±7.49% 8.43±1.94% YH1143 48.21±4.25% 20.14±8.74% YH1144 28.55±8.26% 30.47±4.87% YH1145 14.98±2.81% 14.74±10.04% YH1146 22.00±8.41% 7.56±3.01% YH1147 37.25±4.52% 40.00±4.78% YH1148 15.57±2.90% 9.85±4.19% YH1149 4.39±1.27% 21.11±8.94% YH1150 17.77±3.69% 19.98±1.30%

[0050] Antibacterial ability of Lactobacillus johnsonii YH1136 strain:

[0051] Activate the pathogens such as enterotoxigenic Escherichia coli (ETEC), Staphylococcus aureus and Salmonella stored in the laboratory using LB medium, and then dilute the viable count of these pathogens to 10 with sterile saline. 5CFU / mL, take 100μL of pathogenic bacteria liquid and spread it evenly on the LB solid culture medium with a coating rod. Then use a 6mm diameter puncher to evenly punch three holes on the LB plate, and the depth of the holes is about 3mm. Use a pipette to add 200μL of the supernatant of the test bacteria into the wells (inoculate the test bacteria into the MRS broth culture medium at a 2% inoculation rate, culture at 37℃ for 24h, take an appropriate amount of mixed bacterial liquid in a 15mL centrifuge tube and place it in a high-speed centrifuge, centrifuge at 8000r / min for 10min, and the supernatant is the supernatant required for the experiment). At the same time, use a culture medium with a hole but no supernatant as a control group, place it in a constant temperature incubator at 37℃ for 10h and then take it out. If the supernatant of the test bacterial solution has the ability to inhibit the growth of pathogens, a blank circle without pathogen growth will appear around the punch hole. Observe and record the size of the inhibition circle, and use a vernier caliper to measure the diameter of the inhibition circle. During the measurement, the same inhibition circle is measured from three different positions, and the average value is taken as the measured value. The antibacterial ability of the test strains can be evaluated by analyzing the size of the inhibition circle of all the test strains. The results showed that the supernatants of 5 strains of bacteria, including YH1122, YH1130, YH1136, YH1140, and YH1144, have strong antibacterial ability. These 5 strains were selected for the subsequent cholesterol-lowering ability test.

[0052] Table 2 Determination of antibacterial ability of lactobacilli (diameter of inhibition zone, mm)

[0053] strains ETEC Staphylococcus aureus salmonella YH1122 20.46±1.56mm 25.08±1.99mm 18.52±0.79mm YH1125 10.86±0.68mm 12.92±1.08mm 8.71±0.15mm YH1128 9.79±0.73mm 10.10±1.23mm 9.13±0.86mm YH1130 22.85±2.06mm 23.88±0.75mm 17.22±0.43mm YH1136 25.68±2.33mm 25.01±1.21mm 19.47±1.20mm YH1137 15.19±0.23mm 18.33±0.62mm 12.75±0.96mm YH1140 18.75±0.78mm 24.79±3.25mm 20.02±3.67mm YH1143 9.88±0.28mm 13.77±0.44mm 10.43±1.05mm YH1144 17.58±1.87mm 25.83±2.45mm 18.66±0.69mm YH1147 13.83±1.06mm 14.89±0.28mm 11.13±2.79mm

[0054] Determination of cholesterol degradation ability of Lactobacillus johnsonii YH1136 strain:

[0055] The activated 5 strains of lactobacillus were inoculated into MRS-CHO (MRS liquid medium with a cholesterol concentration of 0.1 mg / mL) at a 2% inoculation rate. After culturing at 37°C for 24 hours, the mass fraction of cholesterol in the supernatant, washing liquid and cell disruption liquid of the bacterial liquid was determined according to the national standard GB5009.128-2016 "Determination of cholesterol in food". At the same time, the MRS-CHO medium without lactobacillus inoculation was used as a control, and the degradation rate of cholesterol in the medium was calculated according to the cholesterol content in the supernatant. The specific method is as follows: 1. Weigh 0.10g (accurate to 0.1mg) of cholesterol standard, dissolve it with glacial acetic acid and dilute to 100mL to prepare a cholesterol standard stock solution, and store it at 4°C. 2. Take 10mL of cholesterol standard stock solution and dilute to 100mL with glacial acetic acid, and prepare it before use. 3. Weigh 4.463g of ammonium ferric sulfate [FeNH4(SO4)2·H2O] in 100mL of phosphoric acid (fully dissolve, perform ultrasonic treatment to promote dissolution if necessary) to prepare a stock solution, and store it at room temperature for later use. Pipette 10mL of the stock solution and dilute to 100mL with concentrated sulfuric acid to obtain the ammonium ferric sulfate colorimetric solution, and store it in a dry container for later use. 4. Draw a standard curve: Pipette 2mL of 0, 0.5, 1.0, 1.5, and 2.0mL of the cholesterol standard working solution into a 10mL test tube, add glacial acetic acid to each tube to make the total volume reach 4mL, add 2mL of ammonium ferric sulfate colorimetric solution along the tube wall, mix well, and compare colors at a wavelength of 560-575nm within 15-90min. Use the cholesterol standard concentration as the horizontal axis and the absorbance as the vertical axis to make a standard curve to obtain the regression equation. 5. Determine the cholesterol concentration of the test bacterial solution: Take 2 mL of the supernatant, washing solution and bacterial cell disruption solution of the test bacterial solution respectively, add glacial acetic acid to make the total volume 4 mL, slowly add 2 mL of ammonium ferric sulfate colorimetric solution along the tube wall, mix well, leave at room temperature for 15 minutes, use an ELISA instrument to compare the color at a wavelength of 560 nm, obtain the corresponding absorbance OD value, and calculate the cholesterol concentration based on the OD value. 6. Calculation of cholesterol degradation rate:

[0056] Degradation rate (%) = (C blank - C determination) / C blank;

[0057] Cblank = cholesterol concentration of the supernatant fermentation broth of the blank control group;

[0058] C determination = cholesterol concentration of supernatant fermentation broth in the determination group;

[0059] The results showed that Lactobacillus johnsonii YH1136 had the best cholesterol degradation rate. Next, the strain of Lactobacillus johnsonii YH1136 was identified.

[0060] Table 3 Determination of cholesterol degradation ability of lactobacilli (degradation rate %)

[0061] strains Supernatant Washing liquid Bacteria disruption liquid YH1122 20.75±1.43% 15.33±1.47% 10.03±1.20% YH1130 30.17±2.32% 22.69±1.33% 15.57±0.77 YH1136 37.65±1.25% 30.09±0.78% 25.75±1.79% YH1140 28.68±1.86% 22.96±1.06% 18.61±1.23% YH1144 25.96±2.66% 21.96±0.98% 17.36±1.55%

[0062] Identification of Lactobacillus johnsonii strain YH1136:

[0063] YH1136 genomic DNA was extracted using Rapid Bacterial Genomic DNA Isolation Kit (Shanghai Sangon Biotechnology Co., Ltd.). The full length of its 16S rRNA was then amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3'). The amplification system was 25 μL: 1 μL DNA template, 1 μL of upstream and downstream primers (10 nmol / L), 12.5 μL 2×TapPCR Master Mix, and the rest was made up with deionized water. The PCR amplification reaction program was: 95 ℃ pre-denaturation for 5 min; 94 ℃ for 1 min, 58 ℃ for 30 s, 72 ℃ for 1 min, for a total of 30 cycles; 72 ℃ extension for 10 min. The PCR product was detected by 1.5% agarose gel, and then the amplified product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the BLAST homology sequence on the NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov / ) website. The results showed that YH1136 had the highest homology with Lactobacillus johnsonii 15QC3AN (99.79%), so YH1136 was identified as Lactobacillus johnsonii and named Lactobacillus johnsonii YH1136. After the identification, its 16S rRNA gene sequence was uploaded to the Genbank database of NCBI with the accession number OP049985.1.

[0064] Embodiment 2:

[0065] Animal experiment design:

[0066] For a more concise description later, the definition is as follows:

[0067] The Lactobacillus johnsonii YH1136 strain involved in the embodiment is defined as a probiotic A strain.

[0068] The Bacillus subtilis BS01 strain involved in the embodiment is defined as a probiotic B strain.

[0069] Mice were used as research subjects to simulate the hypoxic environment of high altitude areas in a hypobaric oxygen chamber, and probiotics were supplemented by gavage during the experiment. There were 7 groups in the experiment, namely plateau exposure + low-concentration probiotic A group (HAP-A1), plateau exposure + low-concentration probiotic B group (HAP-B1), plateau exposure + high-concentration probiotic A group (HAP-A2), plateau exposure + high-concentration probiotic B group (HAP-B2), plateau exposure + probiotic complex AB group (HAP-AB), plateau exposure group (HA) and normal control group (control). After a period of modeling, behavioral experiments were performed on mice to evaluate the spatial memory ability of mice, and further detect the expression of memory-related proteins (such as BDNF) in the hippocampus of the mouse brain and the antioxidant level.

[0070] 63 C57BL / 6J mice (purchased from Beijing Sibeifu Biotechnology Research Institute) were randomly divided into 7 groups. Before the formal experiment began, all mice were fed a normal diet for one week to stabilize the initial physiological state of the mice. During the experiment, all mice were kept in a room with a temperature of 20-22°C and a humidity of 40-60%, with a day and night cycle of 12 hours, from 8 am to 8 pm. The air pressure in the control group was maintained at 94.5kPa. Other conditions such as temperature and humidity were consistent with those in the room. The low-pressure oxygen chamber was opened for about 1 hour every day for gavage and replenishment of water and food.

[0071] The seven groups were plateau exposure + low-concentration probiotic A group (HAP-A1), plateau exposure + low-concentration probiotic B group (HAP-B1), plateau exposure + high-concentration probiotic A group (HAP-A2), plateau exposure + high-concentration probiotic B group (HAP-B2), plateau exposure + probiotic complex AB group (HAP-AB), plateau exposure group (HA) and normal control group (control). HA, HAP-A1, HAP-B1, HAP-A2, HAP-B2 and HAP-AB groups were always exposed to a hypoxic oxygen chamber simulating an altitude of 4000m during the experiment, and the pressure of the hypoxic oxygen chamber was set to 60-65kPa. The concentration was 10 9 The preparation method of the bacterial suspension of Lactobacillus johnsonii YH1136 with a CFU / mL is as follows: according to the live bacterial count result of the Lactobacillus johnsonii YH1136 seed liquid placed in a sterile MRS liquid medium at 37°C for 24 hours, the bacterial cells are prepared into 10 CFU / mL suspension using a PBS buffer with a concentration of 1X and a pH of 7.0. 9 CFU / mL of bacterial solution was stored at 4°C for future use. A vortex was used to mix the bacterial suspension before intragastric administration. Fresh bacterial solution was prepared once a week on average. 9The preparation method of the bacterial suspension of Bacillus subtilis BS01 with a concentration of 10 CFU / mL is as follows: according to the viable bacterial count result of Bacillus subtilis BS01 seed liquid placed in a sterile LB liquid medium at 37°C for 24 hours, the bacterial cells are prepared into 10 CFU / mL suspension using a PBS buffer with a concentration of 1X and a pH of 7.0. 9 CFU / mL of bacterial solution was stored at 4°C for future use. A vortex instrument was used to mix the bacterial suspension before gavage. Fresh bacterial solution was prepared once a week on average.

[0072] The formal experiment lasted for 19 days. The HAP-A1 group was gavaged with 0.1 mL of 10 9 The HAP-B1 group was gavaged with 0.1 mL of 10 CFU / mL suspension of Lactobacillus johnsonii YH1136 every day. 9 CFU / mL of Bacillus subtilis BS01 suspension; HAP-A2 group was gavaged with 0.2 mL of 10 9 The HAP-B2 group was gavaged with 0.2 mL of 10 CFU / mL suspension of Lactobacillus johnsonii YH1136 every day. 9 CFU / mL of Bacillus subtilis BS01 suspension, the HAP-AB group was gavaged with 0.2 ml of bacterial suspension made of 0.1 ml probiotic A and 0.1 ml probiotic B in a fixed ratio (1:1) every day. The HA group and the control group were gavaged with the same dose of normal saline. From the 15th to the 19th day, 63 mice were subjected to a 5-day Morris water maze experiment and behavioral experimental data were measured. After the behavioral experiment on the 19th day, the mice were killed by dislocation and the hippocampus was taken. The left hippocampus of 6 mice in each group was used for antioxidant capacity determination, and the right hippocampus was used for RT-qPCR analysis of the relative expression level of the mRNA of the target gene. The hippocampus of the remaining 3 mice was used for immunofluorescence determination.

[0073] Specific detection method:

[0074] Behavioral experiments

[0075] The Morris water maze experiment consists of a circular pool with a diameter of 120 cm and a height of 50 cm and a transparent escape platform with a diameter of 65 mm. The depth of the pool is set to 15-18 cm. Titanium dioxide is added to the water to ensure that the water is milky white and opaque. The inner wall of the pool is equipped with four different color markers, and the pool is divided into four quadrants according to these four markers. The escape platform is placed 0.8 cm below the water level to ensure that the mice cannot find it. The Morris water maze experiment is divided into two parts: (1) Positioning and navigation experiment: The mice are placed in the water facing the pool wall and allowed to explore freely. If the mice climb onto the platform within 90 seconds, they are allowed to stay on it for 15 seconds; if they fail to climb onto the platform, the experimenter guides the mice to climb onto the platform and stay there for 15 seconds. The time it takes for each mouse to find the escape platform is recorded. This stage of the experiment lasts for a total of five days, and each mouse performs one cruise exploration every day. The first two days are behavioral pre-experimental exercises, and the experimental data starting from the third day are included in the formal experiment and recorded as positioning cruise (Day1-Day3). (2) Spatial exploration experiment: 30 minutes after the end of the navigation experiment on the fifth day, the escape platform was removed and the mice were placed back in the water maze for 90 seconds. The time when the mice first reached the escape platform was recorded. The data from the free exploration experiment on the fifth day were included in the formal experiment.

[0076] Tissue biochemical testing:

[0077] The antioxidant capacity of the hippocampus, including total antioxidant (T-AOC), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malondialdehyde (MDA), was determined using commercial kits (Nanjing Jiancheng Bioengineering Institute).

[0078] Immunofluorescence analysis:

[0079] Hippocampal tissue was taken for immunofluorescence analysis. The sections were subjected to heat-induced antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) at 121°C for 10 min. The sections were blocked with 8% skim milk in TBST at 37°C for 40 min and then immunostained with primary antibodies for PSD95 (1:100, BA3304, ServicebioS), SYP (1:100, BA3279, Servicebios), and BDNF (1:200) at 4°C. The sections were washed and incubated with secondary fluorescent antibodies for 60 min at 37°C. Images were captured using a Nikon Eclipse C1 fluorescence microscope (Nikon, Tokyo, Japan).

[0080] RT-qPCR analysis of the relative expression level of mRNA of the target gene:

[0081] Mouse hippocampal RNA was extracted using the EZNA kit according to the instructions, washed with ice-cold sterile saline without RNase (RNase), and immediately frozen in liquid nitrogen for gene expression analysis. The isolated RNA was quantitatively and qualitatively analyzed by 260 / 280 nm absorbance ratio and agarose gel electrophoresis. The extracted RNA was immediately transcribed into cDNA using a commercial DNA reverse transcription kit, and the reverse transcribed cDNA was stored at -30°C for future use. Using cDNA as a template, synaptic plasticity-related proteins in the hippocampus, BDNF (neurotrophic factor), SYP (synaptic vesicles), and PSD-95 (synaptophysin) were detected.

[0082] PCR system: template 1 μL, upstream and downstream primers 1 μL each, SYBR Mix 5 μL and ddH2O 2 μL. The relative expression of mRNA was calculated using the 2ΔΔCt method.

[0083] Result analysis:

[0084] Effects of different treatment groups on spatial memory:

[0085] like Figure 1-Figure 4 As shown:

[0086] The results of the three-day positioning navigation experiment (Day1-Day3) and free exploration experiment showed that the time required for escape latency in the HA group was significantly longer than that in the control group, indicating that exposure to the plateau hypoxic environment can lead to impaired spatial memory ability of mice; there was no significant difference between the HAP-A1 group, HAP-B1 group, HAP-A2 group, HAP-B2 group and the HA group, indicating that low- and high-concentration probiotic single bacterial suspensions could not significantly improve the spatial memory ability of mice; the time required for escape latency in the HAP-AB group was significantly lower than that in the HAP-A1 group, HAP-B1 group, HAP-A2 group, and HAP-B2 group, and was close to the control group, indicating that the probiotic mixture suspension had a significant improvement effect on the spatial memory ability of mice. The Morris water maze experiment is the first choice for researchers to conduct behavioral research, especially learning and memory research. In this experiment, it was used to evaluate the spatial memory ability of mice. Spatial memory ability is inversely proportional to the time required for mice to escape latency, that is, the stronger the spatial memory ability, the shorter the time required for mice to escape latency; the weaker the spatial memory ability, the longer the time required for mice to escape latency. In summary, Figure 1-Figure 4The results showed that exposure to high-altitude hypoxic environment can lead to impaired spatial memory function of mice, which is mainly manifested as a decrease in the ability to find a platform, that is, an increase in the escape latency time. While being exposed to high-altitude hypoxia, supplementing with the probiotic composite bacterial suspension provided by the present invention at a dose of 0.1+0.1ml can synergistically restore the spatial memory ability of mice, and significantly reduce the escape latency time. Among the three groups using 0.2ml probiotic bacterial suspension (HAP-A2, HAP-B2, HAP-AB), the group using the bacterial suspension prepared by probiotic A and probiotic B in a fixed ratio (HAP-AB) had the best effect.

[0087] Effects of different treatment groups on hippocampal memory-related proteins:

[0088] Changes in the mRNA levels of memory-related proteins in mice under hypoxia Figure 5-Figure 10 As shown:

[0089] in Figure 5-Figure 7 It can be seen that: in terms of the mRNA levels of BDNF (brain-derived neurotrophic factor), SYP (recombinant synaptophysin) and PSD95 (postsynaptic density protein-95), the HA group was significantly lower than the control group; and there was no significant difference between the four probiotic single-strain groups (HAP-A1, HAP-B1, HAP-A2, HAP-B2) and the HA group; the HAP-AB group was significantly higher than the four probiotic single-strain groups (HAP-A1, HAP-B1, HAP-A2, HAP-B2), and close to the control group.

[0090] Figure 8-Figure 10 It can be seen that: in terms of the mRNA levels of BDNF (brain-derived neurotrophic factor), SYP (recombinant synaptophysin) and PSD95 (postsynaptic density protein-95), the HA group was significantly lower than the control group; and there was no significant difference between the four probiotic single-strain groups (HAP-A1, HAP-B1, HAP-A2, HAP-B2) and the HA group; the HAP-AB group was significantly higher than the four probiotic single-strain groups (HAP-A1, HAP-B1, HAP-A2, HAP-B2), and close to the control group. Figure 8-Figure 10 Results and Figure 5-Figure 7 The results showed a consistent trend.

[0091] BDNF is a homodimeric protein that is believed to play a key role in learning, memory and behavior. PSD-95 and SYP are important proteins in neuronal synapses and play a decisive role in controlling synaptic strength and activity-dependent synaptic plasticity. In summary, Figure 5-Figure 10The results showed that exposure to high-altitude hypoxic environment can lead to impaired memory function in mice, which is specifically manifested by a decrease in the expression level of memory-related proteins in the hippocampus. Among the five probiotic intervention groups (HAP-A1, HAP-A2, HAP-B1, HAP-B2 and HAP-AB), the effect of the HAP-AB group was significantly better than that of the other four groups.

[0092] Effects of different treatment groups on the antioxidant capacity of hippocampus:

[0093] The results of the determination of the antioxidant capacity of the hippocampus are as follows Figure 11-Figure 14 As shown:

[0094] In terms of T-AOC, MDA and GSH-Px activity levels, the HA group was significantly lower than the control group, and there was no significant difference between the four probiotic single bacteria groups (HAP-A1, HAP-B1, HAP-A2, HAP-B2) and the HA group; the HAP-AB group was significantly higher than the four probiotic single bacteria groups (HAP-A1, HAP-B1, HAP-A2, HAP-B2), and close to the control group. In terms of MDA activity level, the HA group was significantly higher than the control group, and there was no significant difference between the four probiotic single bacteria groups (HAP-A1, HAP-B1, HAP-A2, HAP-B2) and the HA group; the HAP-AB group was significantly lower than the four probiotic single bacteria groups (HAP-A1, HAP-B1, HAP-A2, HAP-B2), and close to the control group. T-AOC (total antioxidant capacity), GSH-Px (glutathione peroxidase) and SOD (superoxide dismutase) can directly reflect the antioxidant capacity of the body or tissue. The higher the value, the stronger the antioxidant capacity of the tissue and the stronger the ability to cope with oxidative stress damage. MDA (malondialdehyde) is the final product of lipid peroxidation reaction under the action of free radicals in the body. The higher the MDA content, the higher the degree of oxidative damage to the body's tissues. In summary, Figure 11-Figure 14 The results showed that exposure to high-altitude hypoxic environment could reduce the antioxidant capacity of the hippocampus in the mouse brain and cause oxidative damage. Among the five probiotic intervention groups (HAP-A1, HAP-A2, HAP-B1, HAP-B2 and HAP-AB), the effect of the HAP-AB group was significantly better than that of the other four groups.

[0095] In summary, the present invention discloses the use of a Lactobacillus johnsonii composite bacterial agent in the preparation of a drug for preventing and treating memory impairment caused by plateau hypoxia, and belongs to the field of microorganisms. The composite bacterial agent of Lactobacillus johnsonii YH1136 and Bacillus subtilis BS01 provided by the present invention can effectively prevent and treat brain memory dysfunction caused by exposure to a plateau hypoxic environment. The composition can restore spatial memory ability, significantly reduce the escape latency, significantly increase the expression level of hippocampal memory-related proteins, significantly increase the antioxidant capacity of the hippocampus in the brain, and alleviate oxidative damage to the hippocampus. The composite bacterial agent of Lactobacillus johnsonii YH1136 and Bacillus subtilis BS01 provided by the present invention can be used to prepare a drug for preventing and treating brain memory dysfunction caused by exposure to a plateau hypoxic environment and a composition thereof, and can also be used as an auxiliary product of existing therapeutic drugs and treatment methods, with very broad application prospects.

[0096] At this point, those skilled in the art recognize that, although the embodiments of the present invention have been shown and described in detail herein, many other variations or modifications that conform to the principles of the present invention can still be directly determined or derived based on the contents disclosed in the present invention without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention should be understood and recognized as covering all such other variations or modifications.

Claims

1. The application of a Lactobacillus johnsonii composite bacterial agent in the preparation of a medicine for preventing and treating memory impairment caused by plateau hypoxia, characterized in that: The Lactobacillus johnsonii composite bacterial agent is formed by combining the Lactobacillus johnsonii YH1136 strain and the Bacillus subtilis BS01 strain; The deposit number of the Lactobacillus johnsonii YH1136 strain is: CCTCC NO: M 20221116; The deposit number of the Bacillus subtilis BS01 strain is: CCTCC NO: M 2012485; The number of Lactobacillus johnsonii YH1136 strains is 1-2×10 9 CFU / mL; The number of the Bacillus subtilis BS01 strain is 1-2×10 9 CFU / mL; The ratio of the number of the Lactobacillus johnsonii YH1136 strain to the number of the Bacillus subtilis BS01 strain is 1-2:1; The method for preventing and treating brain memory impairment caused by plateau hypoxia includes at least one of (a) to (c): (a) Restore the memory ability of mammals and significantly reduce the escape latency of mammals; (b) Significantly increased the expression level of mammalian hippocampal memory-related proteins; (c) Significantly improve the antioxidant capacity of the hippocampus in the mammalian brain and alleviate oxidative damage in the hippocampus.

2. The use as claimed in claim 1, characterized in that The composite bacterial agent of the Lactobacillus johnsonii YH1136 strain and the Bacillus subtilis BS01 strain is in the form of powder or aqueous solution.

3. The use as claimed in claim 1, characterized in that The drug also contains a pharmaceutically acceptable carrier.

4. The use as claimed in claim 1, characterized in that The application method is to administer the medicine or medicine composition prepared by intragastric administration or oral administration of the composite bacterial agent.

5. The use as claimed in claim 1, characterized in that The mammals include humans.

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