Application of lactobacillus reuteri in preparation of product for preventing and / or treating depression and anxiety

Abstract

The invention discloses application of lactobacillus reuteri in preparation of products for preventing and / or treating depression and anxiety, the lactobacillus reuteri comprises a lactobacillus reuteri SINE003 strain, the lactobacillus reuteri is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on September 7, 2022, and the preservation number is CGMCC NO.25664. The lactobacillus reuteri can be used for preventing and / or treating depression and anxiety. The lactobacillus reuteri SINE003 strain is deeply analyzed and studied, and it is found that the lactobacillus reuteri SINE003 strain shows the improvement effect on anxiety and depression symptoms in different behavioral tests, meanwhile, the lactobacillus reuteri SINE003 strain has the effects of regulating serum inflammatory factors and / or brain-derived neurotrophic factors, regulating intestinal short-chain fatty acid, regulating intestinal flora and the like, and the lactobacillus reuteri SINE003 strain has wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and relates to the application of Lactobacillus reuteri in the preparation of drugs for preventing and / or treating depression and anxiety. Background Art

[0002] With the development of society and economy, the pace has accelerated and the competition has become fierce. Under the influence of psychological factors, social factors, and the bad behaviors and lifestyles of humans themselves, the incidence of various psychosomatic diseases has been increasing among people of different ages. Depression is an expression of enhanced negative emotions. Patients feel depressed, worried all day long, underestimate their own intelligence and ability, and overestimate the difficulties around them. Mild patients show a lack of interest, listlessness, insufficient mental and physical strength and unwillingness to move. Severe patients have outbursts of sad emotions, and even more have despairing emotions and tendencies of self-harm and self-abuse.

[0003] Currently, the means of treating depression still mainly rely on taking drugs. Clinically used antidepressant drugs are mainly divided into three categories: monoamine oxidase inhibitors (MAOIs), tricyclics (TCAs), and selective serotonin (5-HT) reuptake inhibitors. And with the development of the pharmaceutical industry, new drugs for fighting depression emerge in an endless stream, such as venlafaxine, nefazodone, etc. Although the research and development of drugs for treating depression have developed rapidly, problems such as low drug efficiency, serious adverse reactions, and drug tolerance continue to trouble depression patients.

[0004] In recent years, the role of the gut microbiota in neuropsychiatric diseases has been gradually recognized by everyone. A large number of studies have found that there is a gut microbiota-gut-brain axis between the gut and the brain. Gut bacteria regulate the brain function and behavior of the host through this axis, and changes in the gut microbiota will also affect the development and function of the host's central nervous system through this. A number of studies have proved that after changing the gut microbiota of the host by means such as oral antibiotics, probiotics, or fecal microbiota transplantation, changes will occur in the mental state and behavior of the host. A large number of experiments have confirmed the important role of the gut microbiota in neuropsychiatric diseases. Therefore, improving the gut microbiota is an important auxiliary measure for preventing and treating neuropsychiatric diseases.

[0005] In summary, it is of great significance to explore gut microorganisms that can be used to prevent or treat depression and anxiety, and to provide new means and new ideas for preventing or treating depression and anxiety. Summary of the Invention

[0006] In view of the deficiencies of the prior art and the actual needs, the present invention provides the application of Lactobacillus reuteri in the preparation of products for preventing and / or treating depression and anxiety, providing new means and new ideas for preventing or treating depression and anxiety.

[0007] To achieve this purpose, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides the use of Lactobacillus reuteri in the preparation of a product for preventing and / or treating depression and anxiety; the Lactobacillus reuteri includes Lactobacillus reuteri SINE003 strain, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 7, 2022, with the deposit number CGMCC NO. 25664.

[0009] In the present invention, based on the mouse CUMS depression model, the ability of Lactobacillus reuteri SINE003 to prevent and relieve depression and anxiety was studied and verified. Through a number of behavioral experiments, the symptom changes of CUMS-depressed mice were monitored under the intervention of SINE003. The experiments included forced swimming test, tail suspension test, open field test, elevated plus maze, black and white box test, step-down test, and sucrose preference test. It was found that the behavioral changes of mice in the forced swimming test, black and white box test, and elevated plus maze test proved that SINE003 had a significant ability to improve depression; and in the tail suspension test, open field test, step-down test, and sucrose preference test, SINE003 showed potential anti-anxiety and anti-depression abilities; that is, SINE003 showed an improvement effect on anxiety and depression symptoms in different behavioral tests.

[0010] Preferably, the product includes drugs, health products, and so on.

[0011] In a second aspect, the present invention provides the use of Lactobacillus reuteri SINE003 described in the first aspect in the preparation of a product for regulating serum inflammatory factors and / or brain-derived neurotrophic factor.

[0012] Preferably, the regulation of serum inflammatory factors includes reducing the contents of IL-1β and LPS and increasing the content of IL-6.

[0013] Preferably, the regulation of brain-derived neurotrophic factor (BDNF) includes increasing the content of brain-derived neurotrophic factor.

[0014] In the present invention, it was found that Lactobacillus reuteri SINE003 can regulate mouse serum inflammatory factors (IL-1β, LPS, IL-6, IL-10, and TNF-α) and brain-derived neurotrophic factor, including reducing the contents of IL-1β and LPS, increasing the content of IL-6, and increasing the content of brain-derived neurotrophic factor, etc. It can be applied to the research of serum inflammatory factor- and brain-derived neurotrophic factor-related diseases, including the development of related drugs, as well as basic mechanism research and in vitro model construction for non-disease diagnosis and / or treatment purposes, and so on.

[0015] Preferably, the product includes drugs, inhibitors (such as IL-1β and LPS content inhibitors), or enhancers (such as brain-derived neurotrophic factor content enhancers), etc.

[0016] In a third aspect, the present invention provides the use of Lactobacillus reuteri SINE003 described in the first aspect in the preparation of a product for regulating short-chain fatty acids in the intestine.

[0017] Preferably, the product for regulating short-chain fatty acids in the intestine includes products that increase the contents of acetic acid, butyric acid, valeric acid, hexanoic acid, and propionic acid.

[0018] In the present invention, it is found that Lactobacillus reuteri SINE003 can regulate short-chain fatty acids (SCFAs) in mouse feces, including increasing the contents of acetic acid, butyric acid, valeric acid, hexanoic acid, and propionic acid, etc., indicating that SINE003 has a positive effect on the regulation of these SCFAs during the treatment process, and can also be applied to the research of short-chain fatty acid-related diseases, including the development of related drugs, as well as the construction of basic mechanism research and experimental models (cells / animals, etc.) for non-disease diagnosis and / or treatment purposes, etc.

[0019] Preferably, the product includes drugs, enhancers (such as short-chain fatty acid content enhancers), etc.

[0020] In a fourth aspect, the present invention provides the use of Lactobacillus reuteri SINE003 described in the first aspect in the preparation of a product for regulating the intestinal flora.

[0021] Preferably, the product for regulating the intestinal flora includes products that enhance the α-diversity and β-diversity of the intestinal flora.

[0022] Preferably, the α-diversity includes the ACE, Chao1, and Simpson indices of the intestinal flora.

[0023] Preferably, the product for regulating the intestinal flora also includes products that reduce the abundance of Bacteroidota.

[0024] Preferably, the product for regulating the intestinal flora also includes products that increase the abundances of Lactobacillus, ClostridiaUCG-014, Bifidobacterium, Ruminococcus, Blautia, Parabacteroides, Alloprevotella, Akkermansia, Prevotellaceae_UGG01, Butyricicoccus, Prevotellaceae_NK3B31_group, and Lachnospiraceae_NK4A136 group.

[0025] In the present invention, it is found that Lactobacillus reuteri SINE003 can regulate the intestinal flora of mice, including enhancing the α-diversity of the intestinal flora, with a more concentrated sample distribution and a relatively consistent and stable intestinal flora structure; increasing the abundances of probiotics such as Lactobacillus, Clostridia UCG-014, Bifidobacterium, Ruminococcus, Blautia, Parabacteroides, Alloprevotella, Akkermansia, Prevotellaceae_UGG01, Butyricicoccus, Prevotellaceae_NK3B31_group, and Lachnospiraceae_NK4A136 group, which helps regulate the intestinal pH value, enhance the intestinal barrier function, and promote overall intestinal health, and has significant anti-inflammatory and antioxidant effects, etc. It can be applied to the development of drugs for promoting intestinal health, etc., and can also be used for the development of specific bacterial enhancers in the intestine, etc., as well as research or model construction for non-disease treatment purposes, etc.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] In the present invention, in-depth analysis and research are carried out on the Lactobacillus reuteri SINE003 strain, and it is found that it shows an improvement effect on anxiety and depressive symptoms in different behavioral tests, and at the same time has effects such as regulating serum inflammatory factors and / or brain-derived neurotrophic factors, regulating intestinal short-chain fatty acids, and regulating intestinal flora, etc., and has broad application prospects. Brief Description of the Drawings

[0028] Figure 1 It is a graph showing the measurement results of the contents of IL-1β, IL-6, IL-10, TNF-α, LPS, and BDNF in the serum of mice in the SINE003 intervention group, where Figure A is the result of IL-1β, Figure B is the result of IL-6, Figure C is the result of IL-10, Figure D is the result of LPS, Figure E is the result of TNF-α, and Figure F is the result of BDNF;

[0029] Figure 2 It is a graph showing the measurement results of the contents of IL-1β, IL-6, IL-10, TNF-α, LPS, and BDNF in the serum of mice in the SINE003 treatment group, where Figure A is the result of IL-1β, Figure B is the result of IL-6, Figure C is the result of IL-10, Figure D is the result of LPS, Figure E is the result of TNF-α, and Figure F is the result of BDNF;

[0030] Figure 3 It is a graph showing the results of the effect of SINE003 intervention on the α-diversity of the intestinal flora of depressive mice;

[0031] Figure 4 The figure shows the results of the impact of SINE003 intervention on the beta diversity of the gut microbiota in depressive mice;

[0032] Figure 5A The figure shows the results of the impact of SINE003 intervention on the abundance of the gut microbiota in depressive mice (stacked plot at the phylum level);

[0033] Figure 5B The figure shows the results of the impact of SINE003 intervention on the abundance of the gut microbiota in depressive mice (changes in abundance at the phylum level);

[0034] Figure 6 The figure shows the results of the LEfSe analysis of the characteristics of the gut microbiota in depressive mice after SINE003 intervention;

[0035] Figure 7 The figure shows the results of the correlation analysis between the gut microbiota and serum inflammatory factors and BDNF in depressive mice;

[0036] Figure 8 The figure shows the results of the impact of SINE003 treatment on the alpha diversity of the gut microbiota in depressive mice. Figure A shows the results of the ACE index, Figure B shows the results of the Chao1 index, Figure C shows the results of the Shannon index, and Figure D shows the results of the Simpson index;

[0037] Figure 9 The figure shows the results of the impact of SINE003 treatment on the beta diversity of the gut microbiota in depressive mice;

[0038] Figure 10A The figure shows the results of the impact of SINE003 treatment on the abundance of the gut microbiota in depressive mice (stacked plot at the phylum level);

[0039] Figure 10B The figure shows the results of the impact of SINE003 treatment on the abundance of the gut microbiota in depressive mice (changes in abundance at the phylum level);

[0040] Figure 11 The figure shows the results of the LEfSe analysis of the characteristics of the gut microbiota in depressive mice after SINE003 treatment;

[0041] Figure 12 The figure shows the results of the correlation analysis between the gut microbiota and serum inflammatory factors and BDNF in depressive mice. Specific implementation manners

[0042] To further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with embodiments and the accompanying drawings. It can be understood that the specific implementation manners described herein are only used to explain the present invention, rather than limiting the present invention.

[0043] For those technical details or conditions not specified in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through regular commercial channels.

[0044] The Lactobacillus reuteri involved in the following examples is named Lactobacillus reuteri SINE003 strain (hereinafter referred to as SINE003). The depositary institution is the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The deposit date is September 7, 2022, and the deposit number is CGMCC NO.25664. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0045] In addition, according to the "List of Strains Allowed to be Used in Foods" promulgated by the National Health Commission of the People's Republic of China in 2022, the original Lactobacillus reuteri is now named Limosilactobacillus reuteri.

[0046] Example 1

[0047] In this example, behavioral evaluation of SINE003 - intervened CUMS - induced depressive mice was carried out.

[0048] 1. Sample information

[0049] Experimental animal information: 6 - week - old male C57BL / 6 mice (a total of 60 mice, supplied by the Shanghai Laboratory Animal Center) were raised in Shanghai Chengxi Biotechnology Co., Ltd. These mice were placed in a controlled environment with the indoor temperature maintained at 22 ± 2°C, humidity at 55% ± 5%, and following a 12 - h light / dark cycle. Mice had free access to food and water.

[0050] Normal group: 6 - week - old C57BL / 6 mice were fed normally;

[0051] Model group: Anxiety and depression models were established using 6 - week - old C57BL / 6 mice;

[0052] SINE003 - intervention group: Anxiety and depression models were established using 6 - week - old C57BL / 6 mice while being intervened with SINE003. Mice were gavaged with 1 mL of SINE003 aqueous solution with a concentration of 1^9 CFU / mL every day, and it was prepared freshly every day.

[0053] 2. Detection items

[0054] Behavioral evaluation of SINE003 in preventing depression in CUMS mice, including forced swimming test, tail suspension test, open field test, elevated plus maze, black and white box test, step-down test and sucrose preference test.

[0055] 3. Experimental methods

[0056] 3.1 Modeling method

[0057] 3.1.1 Brief description of the modeling method

[0058] After 1 week of adaptive breeding of mice, from the 2nd to the 6th week, a chronic stress program was carried out daily as described below. Mice were randomly exposed to 1-2 mild stressors daily; these stressors included forced swimming (10 min), restraint (3 h), water deprivation (24 h), isolation (24 h), food deprivation (24 h), wet bedding (24 h), no bedding (24 h), tilted cage (24 h), tail clamping (9 min), crowded space (24 h) and continuous lighting (24 h). Each stressor was used no more than three times. The stressor stimulation of mice started at different times every day to minimize its predictability (from 8:00 am to 8:00 pm).

[0059] 3.1.2 Chronic unpredictable mild stress (CUMS)

[0060] In this experiment, CUMS designed a random schedule of 1-2 stressors per day. Each stressor was used no more than three times in total. CUMS lasted for 5 weeks. The application of stress started at different times every day to minimize its predictability (from 8 am to 8 pm).

[0061] 3.1.2.1 Forced swimming

[0062] (1) Fill a glass jar 22 cm deep with water at 23 ± 2 °C;

[0063] (2) Place the mouse in the water in the glass tank and force it to swim for 10 min. When the mouse is swimming, it may try to get out of the water tank, so in order to prevent escape, it may be necessary to carefully cover it with a heavy lid;

[0064] (3) Return the animal to a clean, dry cage and line it with fresh bedding to avoid getting chilled and catching a cold.

[0065] 3.1.2.2 Restraint

[0066] (1) Place the mouse in a 50-ml plastic tube and adjust it outside with plastic tape so that the mouse cannot move. There must be a hole at the distal end of the tube for normal breathing. To put the mouse into the plastic tube, the animal's head must be placed near the entrance, and then it should enter by itself;

[0067] Although the plastic tube should be sufficient to prevent the mouse from getting out, it is best to leave the holder in the cage.

[0068] (2) Return the mouse to the cage. The best way to extract the mouse is to make a sudden downward movement and drop it into the cage, trying to avoid pulling from the tail.

[0069] 3.1.2.3 Water resource shortage

[0070] (1) Remove the water bottle from the cage within 24 hours to deprive the mouse of water for 24 hours.

[0071] (2) Replace the water bottle after the time point.

[0072] 3.1.2.4 Isolation

[0073] (1) Keep each mouse separately in a cage for 24 hours.

[0074] (2) Return the mouse to the original cage for group housing.

[0075] 3.1.2.5 Food shortage

[0076] (1) Remove the food from the cage for 24 hours to deprive the mouse of food for 24 hours.

[0077] (2) Supply food after the time point.

[0078] 3.1.2.6 Wet bedding

[0079] (1) Keep each mouse separately in a cage. Add 200 ml of water to every 100 g of bedding and continuously use the wet bedding to raise the mouse for 24 hours.

[0080] (2) Return the mouse to the original cage for group housing.

[0081] 3.1.2.7 No bedding

[0082] (1) Keep each mouse separately in a new cage without bedding for 24 hours.

[0083] (2) Return the mouse to the original cage for group housing.

[0084] 3.1.2.8 Inclined cage

[0085] (1) Place the cage for raising the mouse at a 45° inclination for 24 hours.

[0086] (1) Restore the cage to the normal state.

[0087] 3.1.2.9 Tail clamping

[0088] (1) Clamp the plastic forceps 2 cm from the end of the mouse's tail, lasting for 3 minutes each time, for a total of 3 times a day.

[0089] (2) Return the mice to the original cage for group housing.

[0090] 3.1.2.10 Crowded space

[0091] (1) Place a plastic partition in the breeding cage to reduce the activity area of the mice in the whole cage by half.

[0092] (2) After 24 hours, remove the partition to restore the mice's movement space.

[0093] 3.2 Behavioral testing methods

[0094] The experimental groups include (1) normal mouse group: healthy mice are fed normally; (2) model group: fed under the same conditions as the anxiety and depression models constructed with healthy mice; (3) SINE003 intervention group: fed under the same conditions as the anxiety and depression models constructed with healthy mice, and at the same time, SINE003 is given for intervention every day. There are 12 mice in each group.

[0095] 3.2.1 Forced swimming test

[0096] On the first day, force all mice to swim for 15 minutes for training; on the second day, place the mice in the same environment for 10 minutes. Use a video tracking system (EthoVision pro, Noldus Inc., Leesburg, VA) to monitor the motor activity of the mice, and use EthoVision software to calculate the immobility time of each mouse during the test. Immobility is defined as no movement except for the floating movements required to keep the head above the water surface.

[0097] 3.2.2 Tail suspension test

[0098] Use adhesive tape to attach the tip of the mouse's tail (2 cm from the tail tip) to the suspension rod (30 cm above the ground), and monitor its motor activity through a video tracking system for 6 minutes. Score the duration of immobility. Immobility is defined as no voluntary or evasive movement.

[0099] 3.2.3 Black and white box test

[0100] The device consists of a box (50×21×25 cm), divided into a dark chamber and a light chamber (each occupying half). The mice are placed in the dark chamber, initially facing the door of the light chamber. Record the time the mice spend in the light chamber and the number of times they enter the light chamber.

[0101] 3.2.4 Elevated plus maze

[0102] Mice were placed in the central area of the maze, facing the open area arms, allowed to move freely for 6 min, and their behaviors were recorded by a video tracking system. The entries into the light and dark arms and the time spent in each area were scored.

[0103] 3.2.5 Open-field test

[0104] The open-field experiment included a square arena (50 cm × 50 cm) enclosed by continuous opaque walls made of plexiglass. Mice were placed in the middle of the area facing the wall, and their activity behaviors were monitored by a video tracking system for 10 min. The time spent by the mice in the center and the distance walked in the center were recorded, and the numerical ratio was calculated.

[0105] 3.2.6 Step-down test

[0106] The experiment was divided into an electroshock training stage and a memory reproduction stage. An experiment was set up in a narrow enclosed space with an electric grid (energized at 0.2 mA) at the bottom. A platform was placed on the electric grid for the mice to stand on to avoid the electric grid. When the mice jumped off the platform, they received an electroshock stimulus, thus generating a pain memory. In the electroshock training stage, the mice were placed on the platform, the power switch of the electric grid was turned on, and the time when the mice jumped off the platform was recorded. The training time was 3 min. After the training, all the mice were returned to the original breeding cages. 24 h after the training was the memory reproduction stage. At this stage, the power of the electric grid was turned off, the mice were placed on the platform, and a 3-min test was carried out to record the latency time for the mice to jump off the platform.

[0107] 3.3 Data analysis method

[0108] The experimental data were expressed as mean ± data range. Statistical analysis was completed using SPSS 27.0 software. The Shapiro-Wilk method was used to test the normality of the data. For data conforming to a normal distribution, unpaired t-tests, one-way analysis of variance (ANOVA), and Dunnett's post hoc test were used to analyze the significance of differences; for non-normally distributed data, the Mann-Whitney U test non-parametric test and Dunnett's post hoc test were used to analyze the significance; the significance criterion was set as p < 0.05; the P values for post hoc multiple tests were all corrected according to the Familywise Error Rate and 95% confidence interval.

[0109] 4. Test results

[0110] 4.1 Forced swimming experiment

[0111] In the forced swimming test, the depressive level of mice was evaluated by counting the immobile time and the number of movements of each group of mice. The longer the immobile time and the fewer the number of movements, the more severe the depressive symptoms of the mice. The results are shown in Table 1. Compared with the normal group (CTL), the immobile time of the mice in the model group (Depression) increased significantly, and the number of movements decreased substantially, indicating that the mice were in a state of anxiety and depression, proving that the depression and anxiety model was successfully established. In the SINE003 intervention group (SINE003), it was found that the frequency and duration of the mice's movement increased significantly, proving that taking SINE003 could effectively prevent the tendency of the mice to have movement stagnation, indicating that SINE003 effectively prevented the mice from developing anxiety and depression.

[0112] Table 1 Results of the forced swimming experiment of mice

[0113] Normal group Model group SINE003 intervention group Number of activities 305.58±97.05 274.92±78.42 302.25±90.41 Static time (s) 66.6±32.91 123.82±62.89 62.26±30.8

[0114] 4.2 Tail suspension test

[0115] In the tail suspension test, the depressive degree of mice was evaluated by analyzing the immobile time and the number of movements of each group of mice. The longer the immobile time and the fewer the number of movements of the mice, the more severe the anxiety and depressive symptoms. The results are shown in Table 2. Compared with the normal group, the immobile time of the mice in the model group increased after the tail was suspended, and the number of movements decreased significantly. The frequency and duration of the movements of the mice in the SINE003 group were significantly higher than those of the mice in the model group, indicating that the probiotic SINE003 has the potential to prevent the occurrence of anxiety and depression.

[0116] Table 2 Results of the tail suspension experiment of mice

[0117] Normal group Model group SINE003 intervention group Number of activities 123.67±56.3 93.75±20.7 106.08±38.56 Static time (s) 140.34±34.92 168.62±48.01 149.74±41.18

[0118] 4.3 Light-dark box test

[0119] Mice usually tend to explore in bright and relatively safe environments and feel uneasy or disgusted with dark rooms. Therefore, the light-dark box test can effectively reflect the emotional state of mice and provide a reliable means for evaluating the anxiety level of mice. The number of entries and the immobile time of the mice in each area were recorded separately. The results are shown in Table 3. Compared with the normal group, the time of the mice in the model group in the dark area increased, the immobile time in the white area decreased significantly, and the number of entries also decreased, proving that the whole group of mice was in a state of depression and anxiety; the immobile time of the mice in the SINE003 intervention group in the white area increased, the number of entries in the dark area decreased significantly, and the immobile time in the dark area also decreased. The experimental results proved that SINE003 could prevent the mice from developing anxiety and depressive symptoms.

[0120] Table 3 Results of the light-dark box test of mice

[0121] Normal group Model group SINE003 intervention group Static time in white area (s) 4.3±0.04 4.19±0.04 4.42±0.04 Number of entries into white area 19.58±3.32 17.33±7.63 16.58±4.06 Static time in black area (s) 1.7±2.25 3.16±5.09 1.72±1.56 Number of entries into black area 17.58±6.99 19.92±3.23 16.58±4.21

[0122] 4.4 Elevated Maze Test

[0123] In the elevated maze test, the anxiety level of mice was evaluated by recording the percentage of the number of entries into the open arms and the percentage of the time spent in the open arms. The percentage of the number of entries into the open arms and the percentage of the time spent in the open arms were negatively correlated with the anxiety level of mice. The results are shown in Table 4. Compared with the normal group of mice, the percentage of the number of entries into the open arms and the percentage of the time spent in the open arms of the model group of mice both decreased. The number of entries of the SINE003 group of mice into the open arms increased significantly compared with that of the model group of mice, and the percentage of the time spent in the open arms also increased. The experiment proved that SINE003 could effectively prevent the occurrence of anxiety in mice.

[0124] Table 4 Results of the Elevated Maze Test for Mice

[0125] Normal group Model group SINE003 intervention group Percentage of entries into open arms 0.45±0.2 0.33±0.13 0.43±0.07 Percentage of time spent in open arms 0.61±0.31 0.5±0.32 0.66±0.21

[0126] 4.5 Open Field Test

[0127] In the open field test, the depression level / status of mice was evaluated by the immobile time of mice and the number of entries into the central area. The movement tendency of depressed mice decreased significantly, specifically manifested as a significant reduction in the horizontal movement distance of mice, mostly hiding in a corner of the open field without moving, and the number of entries into the central area and the residence time would also be significantly reduced. The results are shown in Table 5. Compared with the normal group, the total movement distance and immobile time of the model group of mice decreased, and the number of entries into the central area decreased significantly. Compared with the model group, the total movement distance, immobile time and the number of entries into the central area of mice in the probiotic SINE003 intervention group increased, indicating that the probiotic SINE003 has the effect of preventing depression in mice.

[0128] Table 5 Results of the Open Field Test for Mice

[0129] Total distance (cm) Number of entries Latency (s) Normal group 4032.11±1083.02 55.33±14.67 44.76±49.08 Model group 3520.81±604.54 43.08±11.07 18.53±12.83 SINE003 intervention group 3989.48±668.09 52.17±18.05 33.26±32.76

[0130] 4.6 Step-Down Test

[0131] In the step-down test, the depression level / status of mice was evaluated by the latency time of mice. The latency time was negatively correlated with the depression level / status. The results are shown in Table 6. Compared with the control group, the latency time of the model group of mice decreased, while in the probiotic SINE003 group, the latency time of mice was significantly prolonged, indicating that the probiotic SINE003 has the effect of preventing depression in mice.

[0132] Table 6 Results of the Step-Down Test

[0133] Normal group Model group SINE003 intervention group Latency (s) 5.98±5.42 1.05±1.8 3.76±3

[0134] In summary, behavioral tests were conducted on mice in each group through forced swimming, tail suspension, open field, elevated plus maze, black and white box, and step-down tests. The experimental results demonstrated that SINE003 could effectively improve depressive and anxiety symptoms in mice, specifically manifested as enhanced survival awareness, increased voluntary exploratory behavior, and elevated activity levels. Specifically, in the forced swimming, open field, black and white box, and elevated plus maze tests, SINE003 was shown to have a preventive effect on the occurrence of depression in mice, and in the tail suspension and step-down tests, SINE003 was verified to have a preventive effect on anxiety. The above results indicate that the probiotic SINE003 exhibits preventive effects on the occurrence and progression of anxiety and depressive symptoms in different behavioral tests.

[0135] Example 2

[0136] In this example, a behavioral evaluation of SINE003 in treating the CUMS depression model was performed.

[0137] The sample grouping, model construction, and testing methods refer to Example 1. The SINE003 treatment group included administering SINE003 to mice by gavage every day after the mouse model was constructed. In addition, a sucrose preference test was added. The sucrose test was used to monitor the preference degree of mice for sugar water. After the occurrence of depressive symptoms, the interest of mice in sugar water decreased significantly. Before the test, a randomly assigned sucrose bottle and a water bottle were placed on one side of the mouse cage to allow the mice to adapt for 72 h. After adaptation, the mice were fasted for 24 h, and then each mouse was housed individually in a cage, and 1% sucrose water and pure water were provided simultaneously. Bottles of the same type and volume were used. After two hours, the bottles were weighed, and the sucrose intake and water intake of the mice were recorded to calculate the sucrose preference degree of the mice. The calculation formula is as follows:

[0138] Sucrose preference = [intake of sucrose solution / (intake of sucrose solution + water)] × 100%.

[0139] Test results

[0140] 1 Forced swimming test

[0141] Normal mice will maintain regular and smooth swimming movements in water, but mice in a depressive state will not. The longer the immobile time of the mice, the more severe their depressive state. Therefore, the forced swimming test is one of the main research methods for evaluating the depressive degree and state of mice. The results are shown in Table 7. Compared with the mice in the normal control group, the immobile time of the mice in the model group in water increased significantly, and the number of activities tended to decrease. The immobile time of the mice in the SINE003 group decreased significantly compared with that of the mice in the model group, and the number of activities increased. In summary, the mice in the model group showed an obvious depressive state, while taking SINE003 could increase the activity tendency and survival desire of the mice and improve the depressive symptoms of the mice.

[0142] Table 7 Results of forced swimming test in mice

[0143] Normal group Model group SINE003 treatment group Number of activities 219.75±142.41 174.25±126.75 184.75±88.46 Static time 26.96±22.13 67.18±57.85 28.75±27.14

[0144] 2 Tail suspension test

[0145] When normal mice are suspended by their tails and cannot escape, they will struggle due to biological instinct. When the suspended mice stop struggling, their bodies remain vertically inverted and motionless, which can be regarded as the behavioral manifestation of the mice giving up struggling after developing learned helplessness. In the tail suspension test, the depressive degree and state of the mice are evaluated by analyzing the immobile time and the number of activities of the mice. The longer the immobile time of the mice in the tail suspension test, the more severe their depression. The results are shown in Table 8. Compared with the mice in the normal group, the mice in the model group show a tendency to give up struggling, marked by an increase in immobile time and a decrease in the number of activities. The number of activities and time of the mice in the SINE003 group increase, improving the depressive symptoms of the mice. This result suggests that SINE003 can improve the activity of the mice and relieve their depressive symptoms.

[0146] Table 8 Results of the tail suspension test

[0147] Normal group Model group SINE003 treatment group Number of activities 101.67±22.68 99.91±28.32 106.58±31.92 Static time 155.97±55.76 162.34±49.98 159.21±57.35

[0148] 3 Light-dark box test

[0149] The principle of the light-dark box test is as described in Example 1. The number of times the mice enter the area and the residence time are shown in Table 9. Compared with the normal group, the latency time of the mice in the model group increases, and the number of entries decreases significantly because the mice are in a state of anxiety. After treatment with the probiotic SINE003, the immobile time of the mice in the dark area decreases, and the number of entries increases, indicating obvious exploratory behavior. This reaction is considered to represent a better emotional state and stress adaptation ability in psychology.

[0150] Table 9 Results of the light-dark box test

[0151] Normal group Model group SINE003 treatment group Number of entries 18.42±8.46 11.25±3.72 14.42±4.48 Latency 0.22±0.34 1.35±2.45 1.04±1.27

[0152] 4 Results of the elevated plus-maze test

[0153] In the elevated plus-maze test, the depressive degree and state of the mice are evaluated by the percentage of the number of times the mice enter the open arms and the percentage of the time they stay in the open arms. The results are shown in Table 10. Compared with the normal group, the percentage of the number of times the mice in the model group enter the open arms shows a downward trend, and the percentage of the time they stay in the open arms decreases significantly, indicating that the mice are in a state of anxiety and depression. Compared with the performance of the depressive mice in the model group, the number of times the mice in the SINE003 group enter the open arms and the percentage of the time they stay increase. The experimental results prove that the probiotic SINE003 can effectively treat and relieve the anxiety and depressive degree and state of the mice.

[0154] Table 10 Results of the elevated plus-maze test

[0155] Normal group Model group SINE003 treatment group Percentage of entries 0.3±0.1 0.29±0.1 0.31±0.09 Percentage of time spent 0.81±0.16 0.64±0.19 0.74±0.28

[0156] 5 Open-field test

[0157] In the open-field test, the depressive degree and state of mice were evaluated by the immobile time of mice, the number of entries into the central area, and the number of rearing. It was found in the experiment that the immobile time of depressive mice increased in the open field and the number of entries into the central area decreased, and the results are shown in Table 11. By comparing the behavioral data of the model group and the normal group of mice, it was found that the latency time of the model group of mice increased, and the number of entries into the central area and the number of rearing decreased, proving that the model group of mice was already in a depressive state. Compared with the model group, the number of entries of the SINE003 group of mice into the central area increased significantly. The experimental results prove that SINE003 can relieve the depressive degree and state of mice.

[0158] Table 11 Open-field test of mice

[0159] Normal group Model group SINE003 treatment group Number of entries 54.5±16.8 39.33±14.04 45.5±15.96 Latency 48.93±59.06 17.93±26.02 21.23±27.92

[0160] 6 Step-down test

[0161] In the step-down test, the depressive degree and state of mice were evaluated by recording the number of step-downs and the latency time of mice, and the results are shown in Table 12. By comparing the behavioral data of the model group and the normal group of mice, it was found that the number of times the mice jumped off the platform increased, while the SINE003 group of mice showed a trend opposite to that of the model group. The number of times the mice jumped off the platform decreased and approached that of normal mice. The experimental results prove that SINE003 can relieve the depressive degree and state of mice.

[0162] Table 12 Results of step-down test of mice

[0163] Normal group Model group SINE003 treatment group Number of entries 4.33±5.61 5.33±3.85 4.08±5.42

[0164] 7 Sucrose preference test

[0165] In the sucrose preference test, the depressive degree and state of mice were evaluated by observing and recording the preference degree of mice for sucrose, and the results are shown in Table 13. By observing the normal group, the model group and the SINEFREE group of mice, it was found that there was no obvious difference in the preference of the three groups of mice for sucrose.

[0166] Table 13 Results of sucrose preference test

[0167] Normal group Model group SINE003 treatment group Before treatment 47.81±1.02 47.34±0.38 47.85±1.44 After treatment 47.54±0.94 45.97±1.6 47.74±1.32

[0168] In summary, through seven behavioral experiments including forced swimming, tail suspension, open field, elevated plus maze, black and white box, step-down test, and sucrose preference test, behavioral tests and evaluations were conducted on normal mice, model mice (anxiety and depression models), and mice intervened with SINE003. The experimental results showed that SINE003 exhibited the ability to improve the depressive degree and state of mice. SINE003 showed significant antidepressant ability in forced swimming, black and white box, and elevated plus maze experiments; it showed potential anxiolytic and antidepressant effects in tail suspension, open field, step-down test, and sucrose preference experiments. The above results indicate that the probiotic SINE003 has the effect of improving anxiety and depression symptoms.

[0169] Example 3

[0170] This example verified the effect of SINE003 on regulating the levels of serum inflammatory factors (IL-1β, LPS, IL-6, IL-10, and TNF-α) and brain-derived neurotrophic factor (BDNF) in mice.

[0171] 1 Sample information

[0172] 1.1 SINE003 prevents CUMS depressive model mice and obtains serum samples

[0173] The method of model establishment and administration refers to Example 1, and drugs were administered simultaneously during the process of mouse model establishment.

[0174] Method for collecting blood from mice: After anesthetizing the mice with an animal anesthesia machine, gently compress both sides of the mouse's neck with the left hand to make the orbital venous sinus congested and the eyeballs protrude fully. Hold a capillary tube with the right hand, insert its tip between the inner corner of the eye and the eyeball, and gently pierce it towards the larynx direction. Rotate the blood collection tube to cut the venous sinus, and the blood will flow into the blood collection and centrifugation tube. Place the blood in the centrifugation tube in a 37 °C incubator or water bath for 1 h; then place it in a 4 °C refrigerator for 3 - 4 h; after serum separation, centrifuge at 4000 rpm for 10 min. Take the supernatant into a clean centrifugation tube, store it at -20 °C, and place it in a 4 °C refrigerator overnight for standby.

[0175] 1.2 SINE003 treats CUMS depressive model mice and obtains serum samples

[0176] The method of model establishment and administration refers to Example 1, and the mice in the treatment group were administered drugs after model establishment.

[0177] The method for collecting blood from mice refers to 1.1 SINE003 prevents CUMS depressive model mice and obtains serum samples.

[0178] 2. Detection items

[0179] Serum inflammatory factors (IL-1β, LPS, IL-6, IL-10, and TNF-α) and brain-derived neurotrophic factor (BDNF) in mice.

[0180] 3. Experimental methods

[0181] The concentrations of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), lipopolysaccharide (LPS), and brain-derived neurotrophic factor (BDNF) in the serum of mice were detected using an ELISA kit.

[0182] The double-antibody one-step sandwich enzyme-linked immunosorbent assay was adopted:

[0183] 1. Take out the required strips from the aluminum foil bag after equilibration at 25 °C for 20 min, and seal the remaining strips with a self-sealing bag and return them to 4 °C;

[0184] 2. Set up standard wells and sample wells, and add 50 μL of standards with different concentrations to each standard well;

[0185] 3. First add 10 μL of the sample to be tested to the sample wells, then add 40 μL of sample diluent, and do not add anything to the blank wells;

[0186] 4. Except for the blank wells, add 100 μL of the detection antibody labeled with horseradish peroxidase (HRP) to each well in the standard wells and sample wells, seal the reaction wells with a sealing film, and incubate in a 37 °C water bath or incubator for 60 min;

[0187] 5. Discard the liquid, pat dry on the absorbent paper, fill each well with washing solution, let stand for 1 min, discard the washing solution, pat dry on the absorbent paper, and repeat the washing process 5 times (the plate can also be washed with a plate washer);

[0188] 6. Add 50 μL of substrate A and B to each well, and incubate at 37 °C in the dark for 15 min;

[0189] 7. Add 50 μL of stop solution to each well, and within 15 min, measure the OD value of each well at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader;

[0190] 8. Plot the standard curve: In the Excel worksheet, use the standard concentration as the abscissa and the corresponding OD value as the ordinate to plot the standard linear regression curve, and calculate the concentration values of each sample according to the curve equation.

[0191] 4. Test results

[0192] 4.1 Effects of SINE003 on the levels of serum inflammatory factors and BDNF in CUMS-induced depressive model mice

[0193] In the animal experiment of probiotic SINE003 for preventing depression in the CUMS depression model, the effects of probiotic SINE003 on preventing depression and anxiety were evaluated by detecting and analyzing the contents of IL-1β, IL-6, IL-10, TNF-α, LPS and BDNF in the serum of mice. The results of the levels of inflammatory factors and BDNF in the serum of mice are as Figure 1 shown. Compared with the normal group (CTL), the concentrations of serum IL-1β and LPS in the model group (Depression) mice were significantly increased, and the concentration of IL-6 was significantly decreased, indicating that systemic inflammatory response and immune activation occurred in the body of depressed mice. This change may be related to the pathogenesis of depression. The concentrations of IL-1β and LPS in the mice pre-administered with probiotic SINE003 were significantly decreased and tended to be normal levels, indicating that SINE003 intervention could regulate the levels of inflammatory factors and inhibit the occurrence of inflammation.

[0194] In addition, compared with the CTL group, the concentration of BDNF in the serum of the depression model group mice was significantly decreased, which was closely related to the occurrence of depressive symptoms. The concentration of BDNF in the serum of the mice pre-administered with probiotic SINE003 was significantly increased, indicating that SINE003 has the ability to prevent the occurrence and progression of depression.

[0195] 4.2 Levels of serum inflammatory factors and BDNF in mice with CUMS depression model treated with SINE003

[0196] In the animal experiment of probiotic SINE003 for treating the CUMS depression model, the therapeutic effects of probiotic SINE003 were evaluated by detecting and analyzing the contents of IL-1β, IL-6, IL-10, TNF-α, LPS and BDNF in the serum of mice. The results are as Figure 2 shown. Compared with the normal control group, IL-1β in the serum of the depression model mice was decreased while IL-6 and IL-10 were significantly increased, suggesting that the inflammatory level in the mice was on the rise. The significant increase in IL-6 and LPS in the serum of the SINE003 treatment group implied the activation of inflammatory response in the organism, while the decrease in IL-10 and TNF-α indicated the remission of the inflammatory state, reflecting the multi-level and complex effects of SINE003 on immune regulation during the treatment of depression.

[0197] In addition, compared with the CTL group, the concentration of BDNF in the serum of the depression model group mice was significantly decreased, which was closely related to the occurrence of depressive symptoms. Under the intervention of probiotic SINE003, the concentration of BDNF in the serum of mice was significantly increased, indicating that SINE003 has the effect of improving and relieving the depressive state of mice.

[0198] In the intervention experiment: The detection results of serum inflammatory factors and BDNF in the depression model mice proved that probiotic SINE003 could effectively prevent the increasing trend of IL-1β and LPS concentrations in the organism caused by depressive and anxiety disorders, and at the same time regulate the increase of IL-6 and BDNF concentrations, gradually restoring them to normal levels. These results indicated that probiotic SINE003 could effectively prevent depressive and anxiety symptoms in mice and might play a role by regulating the levels of inflammatory factors and BDNF.

[0199] In the treatment experiment: In the depression model, the detection results of serum cytokines in the model group mice showed abnormal changes in inflammatory factors and BDNF. Compared with the control group, the concentration of IL-1β in the model group mice decreased significantly, while the concentrations of IL-6 and IL-10 increased, and the BDNF concentration decreased significantly. These results indicated the imbalance of the immune system and impaired neurodevelopment under the depressive state. After treatment with probiotic SINE003, the concentration of IL-1β in the mice serum showed a downward trend, and the BDNF concentration in the mice serum increased significantly, which further confirmed the therapeutic potential of SINE003 in relieving depressive symptoms.

[0200] Example 4

[0201] This example detected the effects of SINE003 on fecal fat and short-chain fatty acids (SCFAs) in mice.

[0202] The animal experiment groups included: (1) Normal group: 6-week-old C57BL / 6 male mice were fed normally; (2) Model group: An anxiety and depression model was constructed using 6-week-old C57BL / 6 male mice and they were fed under the same conditions; (3) SINE003 treatment group: The anxiety and depression model was treated with SINE003, and the depressive mice were given oral probiotic SINE003 every day. There were 12 mice in each group.

[0203] At the initial stage of the experiment, the baseline levels of SCFAs in the fecal samples of the three groups of mice were measured to evaluate the initial concentrations of fecal SCFAs in mice before the start of the experiment. As shown in Table 14, for the seven detected SCFAs (isobutyric acid, acetic acid, butyric acid, isovaleric acid, valeric acid, caproic acid, and propionic acid), no significant differences were observed among the CTL group, the model group, and the probiotic SINE003 treatment group (p>0.05).

[0204] Table 14 Sequencing of fecal SCFAs at baseline (μg / mL)

[0205]

[0206]

[0207] Note: The p value represents the p value between the model group and the normal feeding CTL group; the p value* represents the p value between the SINE003 treatment group and the CTL group, and the numerical range in [] represents the ±SD (standard deviation) value.

[0208] At the end of the experiment, quantitative analysis of SCFAs was performed on the fecal samples of the three groups of mice to evaluate the effect of probiotic SINE003 treatment on the fecal SCFAs level in mice. As shown in Table 15, compared with CTL, the concentrations of the 7 main SCFAs in the feces of the model group mice were significantly decreased (p<0.05), indicating that the depressive state had a significant negative impact on the SCFAs level. Compared with the model group, the contents of acetic acid, butyric acid, valeric acid, hexanoic acid and propionic acid in the fecal SCFAs of the mice treated with probiotic SINE003 were significantly increased (p<0.05).

[0209] Table 15 Detection of fecal SCFAs at the end of the experiment (μg / mL)

[0210] SCFAs Normal group Model group SINE003 treatment group p value p value* Isobutyric acid 2.31[0.47-3.13] 1.37[0.77-2.87] 1.90[2.74-3.94] 0.004 0.342 Acetic acid 202.45[87.12-290.87] 142.91[77.15-234.88] 196.23[16.41-162.05] 0.013 0.025 Butyric acid 82.26[14.69-151.36] 39.34[14.13-93.82] 84.67[16.41-162.05] 0.008 0.010 Isovaleric acid 1.33[0.24-1.80] 0.77[0.40-1.62] 1.21[0.45-2.61] 0.039 0.079 Valeric acid 3.13[1.01-6.02] 1.64[0.68-2.99] 3.11[0.48-6.15] 0.019 0.023 Hexanoic acid 0.04[0.02-0.08] 0.02[0.01-0.03] 0.03[0.01-0.04] 0.007 0.006 Propionic acid 37.40[19.77-53.24] 29.38[21.83-40.03] 37.73[21.44-53.74] 0.018 0.041

[0211] Note: The numerical range in [] represents the ±SD (standard deviation) value.

[0212] In summary, the detection results of fecal SCFAs at the baseline of each group of mice showed that there was no significant difference in the SCFAs level among the three groups of mice (p>0.05), proving that there was no significant difference among the groups of mice before the experiment and ensuring the stability and credibility of the experiment. The experimental results showed that the contents of the 7 short-chain fatty acids in the depressive group mice were significantly decreased, proving that depression had a significant impact on biological metabolites. Treatment with probiotic SINE003 could effectively reverse this phenomenon, promote the increase of the SCFAs level in mice and approach that of the normal group. The experimental results proved that probiotic SINE003 had a positive effect on the regulation of these SCFAs during the treatment process (p<0.05).

[0213] Example 5

[0214] This example verifies the effect of SINE003 on the intestinal flora of mice.

[0215] The test mouse groups included (1) mouse normal group: healthy mice were fed normally; (2) model group: after constructing an anxiety and depression model with healthy mice, they were fed under the same conditions; (3) SINE003 intervention group: an anxiety and depression model was constructed with healthy mice and SINE003 intervention was given to the mice every day, (4) SINE003 treatment group: after modeling, SINE003 was given to the mice orally every day.

[0216] 1 16s rRNA sequencing method for intestinal flora

[0217] (1) Extraction and detection of total DNA:

[0218] Use a DNA extraction kit (MoBio, Carlsbad, CA, USA) to extract bacterial genomic DNA from mouse cecal contents. Use a NanoDrop 2000 micro-spectrophotometer (Thermo Scientific, Wilmington, DE, USA) to measure the purity (A260 / A280 ratio between 1.8 and 2.1) and concentration of the extracted genomic DNA. Verify the integrity of the DNA by 1% agarose gel electrophoresis to ensure clear, bright bands, no trailing, and no smeared bands.

[0219] (2) PCR amplification of the 16S rRNA gene:

[0220] Use the universal primers 338_F (5'-ACTCCTACGGGAGGCAGCA-3') and 806_R (5'-GGACTACHVGGGTWTCTAAT-3') to perform two rounds of PCR amplification on the V3-V4 region of the bacterial 16S rRNA gene. The first-round PCR amplification system is 50 μL, containing 10 μL Buffer, 0.2 μL Q5 High-Fidelity DNA polymerase, 10 μL HighGC Enhancer, 1 μL dNTP, 10 μM forward primer, 10 μM reverse primer, and 60 ng genomic DNA. The thermal cycling conditions are pre-denaturation at 95 °C for 5 min, followed by denaturation at 95 °C for 1 min, annealing at 50 °C for 1 min, extension at 72 °C for 1 min, for a total of 10 cycles, and finally extension at 72 °C for 7 min. After purification of the first-round PCR product with VAHTSTM DNA Clean Beads, perform the second-round PCR amplification. The second-round amplification system is 40 μL, including 20 μL 2×Phusion HF MM, 8 μL ddH2O, 10 μM forward primer, 10 μM reverse primer, and 10 μL of the first-round PCR product. The thermal cycling conditions are pre-denaturation at 98 °C for 10 min, denaturation at 98 °C for 10 s, annealing at 65 °C for 30 s, extension at 72 °C for 30 s, for a total of 10 cycles, and finally extension at 72 °C for 5 min. Use Quant-iT TM dsDNA HS reagent to perform fluorescence quantification on the second-round PCR amplification product and mix according to the sequencing amount requirements.

[0221] (3) Sequencing and bioinformatics analysis: The purified samples were subjected to 16S rRNA sequencing analysis using the Illumina Hiseq 2500 platform (2×250bp paired-end). In the data preprocessing stage, USEARCH11 was used for quality control and denoising to ensure data quality. USEARCH11 was used to cluster the preprocessed data into ASVs, and the relative abundances of different microbial ASVs in the samples were calculated. Principal Coordinates Analysis (PCoA) was performed using the QIIME software, and Bray-Curtis distance was used to analyze the differences in microbial community structures among samples. The LEfSe software was used to analyze biomarker species in each sample. Pearson correlation coefficient analysis was conducted using the R language (version 4.2.3) to explore the correlations among microbial populations.

[0222] 2 Statistical analysis methods

[0223] The t-test was used to analyze continuous variables that follow a normal distribution. When the data did not meet the normal distribution assumption and did not depend on the specific distribution form, the non-parametric test method, Wilcoxon rank-sum test, was used to analyze whether there were significant differences between two independent samples.

[0224] 3 16S rRNA sequencing results of gut microbiota

[0225] 3.1 SINE003 prevents gut microbiota in the CUMS depression model

[0226] 3.1.1 Analysis of α-diversity and β-diversity of gut microbiota

[0227] The effect of SINE003 prevention on the α-diversity of gut microbiota in depressive model mice was evaluated by ACE, Chao1, Shannon, and Simpson indices, and the results are as Figure 3 shown. Compared with the model group (Depression), the ACE, Chao1, and Simpson indices of gut microbiota in the SINE003 intervention group of mice were significantly increased (p<0.05), indicating that the species richness and evenness of gut microbiota were significantly improved, and the gut microbiota structure showed a tendency to recover to the normal physiological state.

[0228] The results of β-diversity analysis were presented by non-metric multidimensional scaling plots (NMDS), which compared the differences in gut microbiota structures among the three groups of mice. The NMDS plot ( Figure 4)Revealed the basic similarity in the gut microbiota structure among the three groups, manifested as partial overlap of the sample clustering regions. Nevertheless, the samples in the depression model group were more dispersed in the figure, indicating a greater variability in its gut microbiota structure. In contrast, the samples in the control group and the SINE003 prevention group were more concentrated, reflecting the relative consistency and stability of the gut microbiota structure in these two groups.

[0229] 3.1.2 Gut microbiota phylum-level analysis

[0230] Analysis of the mouse gut microbiota at the phylum level showed that the relative abundances of gut microorganisms varied among different groups, but the two major phyla, Firmicutes and Bacteroidota, always dominated the gut microbial communities of mice in each group before (Baseline) and after (End) the experiment ( Figure 5A ). There were no significant differences in the abundances of Actinobacteria and Patescibacteria among groups. Compared with the model (Depression) group, the abundance of Bacteroidota in the gut of mice intervened with SINE003 was significantly reduced ( Figure 5B ).

[0231] 3.1.3 Gut microbiota genus-level analysis

[0232] The differences in the genus levels of gut microbiota among CTL, Depression, and SINE003 groups of mice were compared through LEfSe analysis ( Figure 6 ), and the results showed that SINE003 intervention significantly changed the abundances of specific microbiota. Compared with the Depression group, SINE003 intervention significantly increased the abundances of beneficial genera such as Alloprevotella, Akkermansia, Prevotellaceae_UGG01, Butyricicocuss, Prevotellaceae_NK3B31_group, and Lachnospiraceae_NK4A136 group.

[0233] Both Prevotellaceae_UGG01 and Prevotellaceae_NK3B31_group belong to the family Prevotellaceae. These bacterial communities play a key role in carbohydrate metabolism, capable of decomposing plant-derived polysaccharides and producing SCFAs such as acetic acid, propionic acid, and butyric acid. These SCFAs not only provide an energy source for the host but also help regulate intestinal pH, enhance intestinal barrier function, and promote overall intestinal health. Meanwhile, Butyricicoccus belongs to the genus Butyricicoccus, and Lachnospiraceae_NK4A136 group belongs to the family Lachnospiraceae. These bacterial communities play an important role in the production of butyric acid. Butyric acid is not only the main energy source for intestinal epithelial cells but also has significant anti-inflammatory and antioxidant effects, which are crucial for maintaining the balance and health of the intestinal microecosystem. Members of the family Lachnospiraceae are closely related to the degradation of dietary fiber and the production of SCFAs, further emphasizing their key role in maintaining intestinal health.

[0234] 3.1.4 Correlation analysis of gut microbiota with serum inflammatory factors and BDNF levels in depressive mice

[0235] To deeply understand the relationship between gut microbiota and serum inflammatory factors in depressive mice, correlation analysis was performed and a heatmap was drawn ( Figure 7 ). After SINE003 intervention, the relative abundance of Alloprevotella increased significantly, showing a positive correlation with the elevation of anti-inflammatory cytokine IL-10 and BDNF levels in mouse serum. In addition, Muribaculaceae was significantly enriched in the model group, showing a negative correlation with mouse serum BDNF level and a positive correlation with serum pro-inflammatory factor IL-1β level.

[0236] 3.2 SINE003 treatment of the gut microbiota in the CUMS depression model

[0237] 3.2.1 Analysis of gut microbiota α-diversity and β-diversity

[0238] The effect of SINE003 treatment on the gut microbiota α-diversity of depressive model mice was evaluated by ACE, Chao1, Shannon, and Simpson indices. The results are as Figure 8 shown. Compared with the control group, the Simpson index of mice in the depressive model group decreased significantly; compared with the model group, the Simpson index of the gut microbiota of mice increased significantly after SINE003 treatment (p<0.05) and approached the control group. However, there were no significant changes in ACE, Chao1, and Shannon indices among the three groups.

[0239] The results of β-diversity analysis, presented by non-metric multidimensional scaling plots, compared the differences in the gut microbiota structures of three groups of mice. The NMDS plots ( Figure 9 ) showed the similarities in the gut microbiota structures among the three groups of mice, with most of the sample clustering regions overlapping, indicating that the differences in the gut microbiota structures among the samples were not significant. In contrast, the samples in the SINE003 treatment group were more concentratedly distributed, and the gut microbiota structure was more consistent.

[0240] 3.2.2 Gut microbiota phylum-level analysis

[0241] Analysis of the gut microbiota of mice at the phylum level showed that the gut microorganisms of mice in different groups were diverse at the phylum level, but the two major phyla, Firmicutes and Bacteroidota, always dominated the gut microbial community of mice ( Figure 10A and Figure 10B ).

[0242] 3.2.3 Gut microbiota genus-level analysis

[0243] By LEfSe analysis, the differences in the enrichment of gut microbiota genera among CTL, Depression, and SINE003 groups of mice were compared ( Figure 11 ). The results showed that SINE003 treatment significantly changed the abundances of specific microbiota. Compared with the Depression group, the SINE003 treatment group significantly increased the abundances of beneficial genera such as Lactobacillus, ClostridiaUCG-014, Alloprevotella, Bifidobacterium, Ruminococcus, Blautia, Parabacteroides, and Akkermansia.

[0244] Lactobacillus and Bifidobacterium are probiotics in the gut and have multiple functions such as improving the gut environment, inhibiting the growth of harmful bacteria, and enhancing immunity. Their increase may help relieve depressive symptoms and improve gut microbial balance. ClostridiaUCG-014 and Ruminococcus belong to the Clostridiales order and are related to the degradation of dietary fiber and the production of short-chain fatty acids, which are of great significance for maintaining gut homeostasis. The increase in Alloprevotella may be related to carbohydrate metabolism and the production of short-chain fatty acids, while Blautia and Parabacteroides are closely related to gut inflammation and immune regulation. As a gut symbiotic bacterium, the increase in Akkermansia abundance may help improve gut permeability and reduce the production of inflammatory factors.

[0245] 3.2.4 Correlation analysis of gut microbiota with serum inflammatory factors and BDNF levels in depressive mice

[0246] To deeply understand the relationship between gut microbiota and serum inflammatory factors in depressive mice, correlation analysis was performed and a heatmap was drawn, as Figure 12 shown. The relative abundances of Lachnoclostridium and Bacteroides were significantly enriched in the SINE003 treatment group. Lachnoclostridium was negatively correlated with the serum LPS level and positively correlated with the BDNF level in depressive mice; Bacteroides was negatively correlated with the serum pro-inflammatory factor IL-6 level in depressive mice.

[0247] From the above results, it can be seen that SINE003 significantly increased the α-diversity of gut microbiota, as reflected by the increase in ACE, Chao1, and Simpson indices; β-diversity analysis showed that there were significant differences in the gut microbiota species composition between the Depression group and the SINE003 group; at the phylum level, after SINE003 intervention, the abundance of Bacteroidota decreased significantly, while the abundances of other phyla such as Actinobacteriot and Patescibacteria did not change significantly; at the genus level, after SINE003 treatment, the abundances of Alloprevotella, Akkermansia, Prevotellaceae_UGG01, Butyricicoccus, Prevotellaceae_NK3B31_group, and Lachnospiraceae_NK4A136 group and other microbiota increased significantly; according to the correlation analysis of gut microbiota with inflammatory factors and BDNF, SINE003 treatment significantly increased the relative abundance of Alloprevotella, which was positively correlated with the serum IL-10 and BDNF levels in mice; Muribaculaceae in the Depression group was significantly enriched, which was negatively correlated with the serum BDNF and positively correlated with the inflammatory factor IL-1 level in mice.

[0248] SINE003 treatment significantly improved the α-diversity of the gut microbiota, with a significant increase in the Simpson index; β-diversity analysis showed that the differences in the gut microbiota structure among samples were small; at the phylum level, after SINE003 treatment, there were no obvious differences in the abundances of Actinobacteriota, Bacteroidota, Campilobacterota, Desulfobacterota, Firmicutes, and Verrucomicrobiota; at the genus level, after SINE003 treatment, the abundances of beneficial genera such as Lactobacillus, ClostridiaUCG-014, Alloprevotella, Bifidobacterium, Ruminococcus, Blautia, Parabacteroides, and Akkermansia were significantly increased; the correlations between the gut microbiota and inflammatory factors and BDNF were analyzed, and the relative abundances of Lachnoclostridium and Bacteroides in the SINE003 treatment group were significantly enriched. Lachnoclostridium was negatively correlated with the serum LPS level and positively correlated with the BDNF level in depressive mice; Bacteroides was negatively correlated with the serum inflammatory factor IL-6 level in depressive mice.

[0249] In summary, through in-depth analysis and research on the Lactobacillus reuteri SINE003 strain of the present invention, it was found that it showed an improvement effect on anxiety and depressive symptoms in different behavioral tests, and at the same time had the effects of regulating serum inflammatory factors and / or brain-derived neurotrophic factor, regulating intestinal short-chain fatty acids, and regulating the gut microbiota, with broad application prospects.

[0250] The applicant declares that the present invention uses the above embodiments to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. Use of Lactobacillus reuteri in the preparation of products for preventing and / or treating depression and anxiety; The Lactobacillus reuteri includes the Lactobacillus reuteri SINE003 strain, which was deposited in the General Microbiology Center of the China Culture Collection Administration on September 7, 2022, with the deposit number CGMCCNO.25664.

2. Use of the Lactobacillus reuteri described in claim 1 in the preparation of products for regulating serum inflammatory factors and / or brain-derived neurotrophic factor.

3. The use according to claim 2, characterized in that: The regulation of serum inflammatory factors includes reducing the content of IL-1β and LPS and increasing the content of IL-6.

4. The use according to claim 2, characterized in that: The regulating brain-derived neurotrophic factor includes increasing the content of brain-derived neurotrophic factor.

5. Use of the Lactobacillus reuteri described in claim 1 in preparing products for regulating intestinal short-chain fatty acids.

6. The use according to claim 5, characterized in that: The products for regulating intestinal short-chain fatty acids include products for increasing the contents of acetic acid, butyric acid, valeric acid, caproic acid and propionic acid.

7. Use of the Lactobacillus reuteri described in claim 1 in preparing products for regulating intestinal flora.

8. The use according to claim 7, characterized in that: The products for regulating intestinal flora include products for improving α diversity and β diversity of intestinal flora; Preferably, the alpha diversity includes intestinal flora ACE, Chao1 and Simpson index.

9. The use according to claim 7 or 8, characterized in that: The products for regulating intestinal flora also include products for reducing the abundance of Bacteroidota.

10. The use according to any one of claims 7 to 9, characterized in that: The products for regulating intestinal flora also include products that increase the abundance of Lactobacillus, ClostridiaUCG-014, Bifidobacterium, Ruminococcus, Blautia, Parabacteroides, Alloprevotella, Akkermansia, Prevotellaceae_UGG01, Butyricicocuss, Prevotellaceae_NK3B31_group and Lachnospiraceae_NK4A136 group.

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