Application of probiotic metabolite MEs in preparation of antidepressant drugs

Abstract

The invention belongs to the technical field of biology and new medicine, and particularly relates to application of probiotic metabolite MEs in preparation of antidepressant drugs. The probiotic metabolite MEs is separated from a fermentation solution of the bacillus subtilis BS-Z15. The probiotic metabolite MEs provided by the invention can improve the mental state, activeness and cognitive level of normal mice. In addition, after the probiotic metabolite MEs is used for treating a lipopolysaccharide-induced depression mouse model, the depression-like behavior of the mouse can be remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biology and new medicine technology, and specifically relates to the application of probiotic metabolites MEs in the preparation of antidepressant drugs. Background Art

[0002] Depression is manifested by persistent low mood, lack of energy, loss of interest or pleasure, insomnia, and even inability to enjoy life. It affects people's mental, emotional, and physical health. It is the leading cause of disability worldwide and the main cause of the global disease burden.

[0003] Scientists have conducted a lot of in-depth research on depression, but still cannot find its exact pathogenesis and pathophysiology. At present, the antidepressants widely used in clinical practice are mainly developed based on the "monoamine neurotransmitter hypothesis" and the theory of microglia-mediated neuroinflammation. The mechanism of action of antidepressants is mainly to inhibit the transporters of serotonin and dopamine, prevent the reuptake of these monoamine neurotransmitters, thereby increasing their concentration in the synaptic cleft between neurons, thereby alleviating depressive symptoms. Among them, the English name of serotonin is 5-HT. The English name of dopamine is DA.

[0004] The use of intestinal probiotics in depression is a relatively new area of ​​research. The connection between the intestinal flora and the brain, known as the "gut-brain axis," is a two-way communication system that includes the nervous system, immune system, and endocrine system. Studies have shown that an imbalance in the intestinal flora may be associated with mental health problems such as depression. Probiotics can help improve the balance of the intestinal flora by providing beneficial bacteria that can inhibit the growth of harmful bacteria and may have a positive impact on mental health. Depression patients are often accompanied by elevated levels of inflammation. Probiotics may help relieve depressive symptoms by regulating immune responses and reducing inflammation. They can also promote the production of neurotransmitters and affect brain function and mood. Although research in this area is promising, there is currently a lack of large-scale, high-quality randomized controlled trials to confirm the exact effect of probiotics in the treatment of depression. Therefore, although some small studies and preliminary evidence support that probiotics may have a positive effect on mental health, they cannot yet be used as a standard treatment for depression. Therefore, new research strategies are urgently needed to explore the pathological mechanisms of depression and the development of new antidepressant drugs.

[0005] At present, most antidepressants used in clinical practice are chemically synthesized drugs, and some are extracted from traditional Chinese medicines, but there have been no reports on extraction from probiotics.

[0006] A study reported that a probiotic, Bacillus subtilis, has attracted much attention due to its excellent ability to effectively secrete biological activity. The metabolites secreted by this probiotic can relieve obesity, promote skin wound healing, and have significant effects in anti-infection and anti-cancer. However, the antidepressant effect of the metabolites secreted by this probiotic has not yet been found. Summary of the invention

[0007] In order to solve the problem that the antidepressant effect of the metabolites secreted by Bacillus subtilis has not yet been found in the prior art, the present invention aims to develop a probiotic: the metabolites of Bacillus subtilis have the medicinal potential for treating depression, and provide an application of probiotic metabolites MEs in the preparation of antidepressant drugs. To achieve the above purpose, the present invention adopts the following technical scheme:

[0008] The first object of the present invention is to provide an application of probiotic metabolites MEs in the preparation of antidepressant drugs, wherein the probiotic metabolites MEs are separated from the fermentation broth of Bacillus subtilis BS-Z15. The present invention provides an application of probiotic metabolites MEs separated from Bacillus subtilis BS-Z15 in the preparation of antidepressant drugs, which solves the problem that the antidepressant effect of metabolites secreted by Bacillus subtilis has not yet been found.

[0009] Furthermore, the present invention has found that the metabolites MEs secreted by Bacillus subtilis BS-Z15 have an antidepressant effect, and the antidepressant efficacy component is different from the anti-infective component previously disclosed by the probiotics. Therefore, the present invention refers to the metabolites MEs secreted by Bacillus subtilis BS-Z15 as probiotic metabolites MEs. Based on this, the present invention provides the use of the probiotic metabolites MEs in the preparation of antidepressant drugs.

[0010] At the same time, the good antidepressant function of the metabolite MEs of Bacillus subtilis BS-Z15 provided by the present invention is the first discovery. That is, although the probiotic metabolites have been reported to have antifungal and anticancer activities, their antidepressant function has never been reported. Therefore, the application of a probiotic metabolite MEs in the preparation of antidepressant drugs provided by this application belongs to a new application direction of the probiotic metabolite MEs, which can provide a new research and development idea for the preparation of antidepressant drugs.

[0011] Among them, the metabolites MEs of Bacillus subtilis BS-Z15 can also be called probiotic metabolites MEs.

[0012] Preferably, the method for preparing the probiotic metabolites MEs comprises the following steps:

[0013] The Bacillus subtilis BS-Z15 is inoculated into a beef extract peptone liquid culture medium and cultured for 12 hours to 22 hours to obtain a fermentation liquid of the Bacillus subtilis BS-Z15.

[0014] The fermentation liquid of Bacillus subtilis BS-Z15 is first centrifuged at 3°C ​​to 5°C; after the first centrifugation, the supernatant is collected and the pH of the solution is adjusted to 2 to 3, and the solution is first allowed to stand at 38°C to 4.2°C for 11.5h to 12.5h to obtain the probiotic acid fermentation liquid.

[0015] The probiotic acid fermentation liquid is subjected to a second low-temperature centrifugation at 3°C ​​to 5°C; after the second low-temperature centrifugation, the precipitate is collected, the precipitate is dissolved with sterile water, and the pH of the solution is adjusted to 6.5 to 7.5 to obtain a probiotic acid precipitate crude extract.

[0016] Precooled acetone is added to the crude extract of probiotic acid precipitation, and the extract is allowed to stand for a second time at 3°C ​​to 5°C for 2.5h to 3.5h, and then centrifuged for a third time to collect the supernatant; after removing acetone from the supernatant, the supernatant is first frozen into a solid, and then freeze-vacuum dried to obtain the probiotic metabolites MEs.

[0017] Wherein, the volume of acetone added to the crude extract of probiotic acid precipitation is 3 to 5 times the volume of the crude extract of probiotic acid precipitation.

[0018] Preferably, the volume of acetone added to the crude extract of probiotic acid precipitation is 4 times the volume of the crude extract of probiotic acid precipitation. The purpose of the 4 times acetone is to dilute more impurities in the crude extract while saving acetone. 2.5h to 3.5h is enough time for diluting protein. After removing acetone, the supernatant is ultra-low temperature solidified, which is the prerequisite for freeze drying.

[0019] Preferably, the conditions of the first low-temperature centrifugation, the second low-temperature centrifugation and the third centrifugation are: 3.5°C to 4.5°C, 11500 r / min to 12500 r / min. 3.5°C to 4.5°C is to keep the MEs stable during centrifugation, and the speed of 11500 r / min to 12500 r / min is adapted to the centrifuge to achieve the best centrifugal effect.

[0020] Preferably, the culture conditions of the Bacillus subtilis BS-Z15 are: temperature 36° C. to 38° C., and culture time 15 h to 18 h, so that the probiotics can ferment to obtain the best metabolites.

[0021] Preferably, the temperature when freezing into a solid is -75°C to -85°C; the freeze vacuum drying conditions are -40°C to -50°C, 15h to 20h. The ultra-low temperature solidification of the supernatant is a prerequisite for freeze drying, and -40°C to -50°C, 15h to 20h meets the best conditions for freeze drying of solid into powder.

[0022] Preferably, the temperature during low temperature standing is 3.5° C. to 4.5° C., in order to keep the effect of MEs stable.

[0023] Preferably, the drug is prepared with the probiotic metabolites MEs as active ingredients and supplemented with pharmaceutically acceptable excipients.

[0024] Preferably, the effective content of the probiotic metabolites MEs in the medicine is 80 wt% to 90 wt%.

[0025] Preferably, the auxiliary materials include any one or more of starch, sodium chloride, glucose, lactose and mannitol.

[0026] Preferably, the medicament comprises an oral dosage form.

[0027] According to the actual treatment purpose and treatment plan, the probiotic metabolites MEs can be prepared into different types of drugs to achieve the anti-depressant effect.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention provides an application of probiotic metabolites MEs in the preparation of antidepressant drugs. The metabolites MEs provided by the present invention can improve the mental state, activity and cognitive level of normal mice. Moreover, the metabolites MEs can significantly improve the depressive behavior of mice after treating the lipopolysaccharide-induced depression mouse model. Compared with the model group, the expression levels of 5-hydroxytryptamine and dopamine in the MEs group mice were significantly increased. The metabolites MEs have an inhibitory effect on the expression of Iba-1, an activation marker of hippocampal microglia in lipopolysaccharide-induced depression-like mice, and the metabolites MEs can significantly increase the density of hippocampal neurons in depressed mice and restore their morphology, reduce the intercellular gap, make them arranged in a regular manner, improve extracellular edema, increase the number of nissoloids in the cytoplasm, and effectively repair damaged neurons. Regulate the expression of cyclic adenosine monophosphate, cAMP response element binding protein, phosphatidylinositol kinase / protein kinase B and nuclear factor κB pathway-related proteins in the hippocampus of lipopolysaccharide-induced depression-like mice.

[0030] Among them, the English name of cyclic adenosine monophosphate is cAMP. The English name of cAMP response element binding protein is CREB. The English name of phosphatidylinositol kinase / protein kinase B is PI3K / Akt. The English name of nuclear factor κB is NF-κB. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the number of single grasping struggles of normal mice treated with the bacterial solution of Bacillus subtilis BS-Z15 and the probiotic metabolites MEs by oral administration in the present invention.

[0032] Figure 2 The open field test (OFT), forced swimming test and tail suspension test were performed on normal mice gavaged with the probiotic metabolites MEs in the present invention; wherein, Figure 2 Figure A shows the total distance that mice moved in the open field test within 5 minutes; Figure 2 Figure B shows the time that the mouse was forced to swim immobile within 4 minutes; Figure 2 Figure C shows the immobility time of mice in the tail suspension test within 5 minutes; where *: P < 0.05; **: P < 0.01; ***: P < 0.001;

[0033] Among them, the English name of the open field test is OFT; the English name of the forced swim test is FST; the English name of the tail suspension test is TST.

[0034] Figure 3 The present invention provides a lipopolysaccharide-induced depression mouse model establishment and a probiotic metabolite MEs and positive drug treatment experimental plan.

[0035] Figure 4 This is a diagram of the sugar water preference experiment in the present invention, wherein, #: P < 0.05; ##: P < 0.01; blank group and model group; *: P < 0.05; **: P < 0.01 metabolite group or positive drug group and model group.

[0036] Figure 5 The effect of the probiotic metabolites MEs in the present invention on the open field test of lipopolysaccharide-induced depression mice; wherein, Figure 5 Figure A shows the movement trajectory of mice in the open field test within 5 minutes; Figure 5 Figure B shows the total distance that mice moved in the open field test within 5 min; #: P < 0.05; ##: P < 0.01 blank group and model group; *: P < 0.05; **: P < 0.01 metabolite group or positive drug group and model group.

[0037] Figure 6 This is a diagram for analyzing the forced swimming and tail suspension experiments of the probiotic metabolites MEs in the present invention on lipopolysaccharide-induced depressed mice; wherein, Figure 6 Figure A shows the time that mice were forced to swim immobile within 4 min; Figure 6 Figure B shows the immobility time of mice in the tail suspension test within 5 minutes; where, #: P < 0.05; blank group and model group; *: P < 0.05; metabolite group or positive drug group and model group.

[0038] Figure 7 This is an analysis diagram of the effects of the probiotic metabolites MEs of the present invention on monoamine neurotransmitters 5-hydroxytryptamine and dopamine in lipopolysaccharide-induced depression mice; wherein, Figure 7Figure A shows the 5-HT content in the mouse hippocampus; Figure 7 Figure B shows the dopamine content in the hippocampal tissue of mice; among them, #: P < 0.05; ##: P < 0.01; ###: P < 0.001 blank group and model group; *: P < 0.05; **: P < 0.01 metabolite group or positive drug group and model group.

[0039] Figure 8 This is an analysis diagram of the expression of the probiotic metabolites MEs in the present invention on the activation marker (Iba-1) of hippocampal microglia in lipopolysaccharide-induced depression-like mice; wherein, Figure 8 Figure A shows IBA-1-positive astrocytes in hippocampal CA1 by immunofluorescence. The nuclei (blue) are stained with DAPI. Figure 8 Figure B is a bar graph showing the average fluorescence intensity of astrocytes in mouse hippocampal CA1; among them, #: P < 0.05; ##: P < 0.01; ###: P < 0.001 blank group and model group; *: P < 0.05; **: P < 0.01 metabolite group or positive drug group and model group.

[0040] Fig. 9 This is an analysis diagram of the effect of the metabolites MEs in the present invention on the synapses of hippocampal microglia in lipopolysaccharide-induced depression-like mice; wherein, Fig. 9 Figure A shows the maximum intensity projection of the microglia image converted into a binary image and then skeletonized; Fig. 9 Figure B is a statistical diagram of microglial cell neural intersection Sholl analysis; among them, #: P < 0.05; ##: P < 0.01; ###: P < 0.001 blank group and model group; *: P < 0.05; **: P < 0.01 metabolite group or positive drug group and model group.

[0041] Fig.10 This is the effect of the metabolites MEs in the present invention on Nissl bodies in the hippocampus of lipopolysaccharide-induced depression-like mice.

[0042] Fig.11 This is a graph analyzing the expression of PI3K / Akt pathway-related proteins in the hippocampus of lipopolysaccharide-induced depression-like mice by the metabolites MEs of the present invention; wherein, Fig.11 Figure A shows the relative expression statistics of phosphorylated protein kinase B / protein kinase B; Fig.11 Figure B shows the relative expression statistics of phosphorylated phosphatidylinositol kinase / phosphatidylinositol kinase; Fig.11 Panel C in the figure shows immunoblots of phosphorylated protein kinase B, protein kinase B, and β-actin; Fig.11Figure D in the figure is the immunoblot of phosphorylated phosphatidylinositol kinase, phosphatidylinositol kinase and β-actin; among them, #: P < 0.05; ##: P < 0.01; ###: P < 0.001 blank group and model group; *: P < 0.05; **: P < 0.01 metabolite group or positive drug group and model group.

[0043] Fig.12 This is a graph analyzing the expression of NF-κB pathway-related proteins in the hippocampus of lipopolysaccharide-induced depression-like mice by the metabolites MEs of the present invention; wherein, Fig.12 Panel A in the figure shows immunoblots for tumor necrosis factor, interleukin-6, NOD-like receptor pyrin domain-associated protein 3, nuclear factor κB, and β-actin; Fig.12 Panel B in the figure shows the relative expression statistics of tumor necrosis factor; Fig.12 Figure C in the figure shows the relative expression statistics of interleukin-6; Fig.12 Figure D in the figure shows the relative expression statistics of NOD-like receptor pyrin domain-associated protein 3; Fig.12 Figure E shows the relative expression statistics of nuclear factor-κB.

[0044] Fig.13 This is an analysis diagram of the effect of the metabolites MEs in the present invention on the expression of cAMP pathway-related proteins in the hippocampus of lipopolysaccharide-induced depression-like mice; wherein, Fig.13 Panel A in the figure is immunoblot of protein kinase A, phosphorylated cAMP response element binding protein, cAMP response element binding protein, brain-derived neurotrophic factor and β-actin; Fig.13 Figure B in the figure shows the relative expression statistics of protein kinase A; Fig.13 Figure C shows the relative expression statistics of acidified cAMP response element binding protein / AMP response element binding protein; Fig.13 Figure D in the figure is the relative expression statistics of brain-derived neurotrophic factor; among them, #: P < 0.05; ##: P < 0.01; ###: P < 0.001 blank group and model group; *: P < 0.05; **: P < 0.01 metabolite group or positive drug group and model group.

[0045] Fig.14 The probiotic metabolites MEs in the present invention are shown to have antidepressant effects from three aspects.

[0046] Fig.15 This is a schematic diagram of the probiotic metabolites MEs in the present invention improving the depressive-like behavior of lipopolysaccharide mice. DETAILED DESCRIPTION

[0047] The following will be combined with the specific implementation of the present invention to clearly and completely describe the technical solution in the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] The methods described in the embodiments of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0049] The materials, reagents and experimental animals used in the examples are as follows:

[0050] 1. Materials and Reagents

[0051] Physiological saline was purchased from Tianyifeng Biotechnology Co., Ltd. with a volume percentage concentration of 0.9%.

[0052] Lipopolysaccharide was purchased from Sigma.

[0053] Fluoxetine was purchased from Maclean.

[0054] PBS was purchased from Pronose, with a concentration of 0.01 M and a pH of 7.3.

[0055] ECL detection reagents were purchased from White Shark.

[0056] 2. Experimental Animals

[0057] KM mice were sourced from the Experimental Animal Center of a medical university.

[0058] Example 1: Preparation of probiotic metabolites MEs

[0059] 1. Source of probiotics: The probiotics used in this embodiment are Bacillus subtilis BS-Z15. The specific strain screening, separation, purification and identification methods of Bacillus subtilis BS-Z15 are detailed in "Isolation and Identification of Antagonistic Spore Strains of Verticillium dahliae and Study on Antibacterial Properties of Two Strains" published by Zhao Junjie et al. It is referred to as strain BS-Z15 below.

[0060] 2. Activation of probiotics: After the frozen strain BS-Z15 was quickly thawed at room temperature, it was inoculated into beef extract peptone liquid culture medium, and activated in a 37°C shaker for 12 hours. After that, an inoculation loop was used to dip the bacterial liquid and streak it on a beef extract peptone solid plate, and then it was placed in a 37°C incubator for 12 hours to observe the single colony.

[0061] 3. Preparation of probiotic metabolites MEs: Inoculate a single colony of the above-cultured strain BS-Z15 into 20 mL of beef extract peptone liquid medium and shake at 37°C and 180 r / min until the OD 600=0.9, and the expanded culture strain is obtained. According to the inoculation amount of 1% v / v, the expanded culture strain is inoculated into the new culture medium and continued to expand the culture for 18h. After the expansion culture is completed, the obtained fermentation liquid is subjected to the first low-temperature centrifugation at 4°C and 12000r / min for 15min, and the supernatant is taken. The obtained supernatant is adjusted to pH=2 and left to stand at 4°C overnight. After the overnight fermentation liquid is subjected to the second low-temperature centrifugation at 4°C and 12000r / min for 20min, the precipitate is taken and the precipitate is dissolved with sterile water and adjusted to pH=7 to obtain a crude extract of probiotic acid precipitate. After mixing 4 times the volume of pre-cooled acetone into the crude extract of probiotic acid precipitate, it is placed in a 4°C refrigerator for 3h, and then centrifuged for the third time at 4°C and 12000r / min. After the third centrifugation, the supernatant was subjected to rotary evaporation at 35°C to remove acetone, and the supernatant was frozen into a solid in a -80°C refrigerator and then dried in a freeze vacuum dryer at -40°C for 18 h to finally obtain the probiotic metabolites MEs.

[0062] Among them, overnight refers to a time ≥ 12h.

[0063] The reagent grade of acetone was analytical grade.

[0064] The probiotic metabolites MEs prepared in Example 1 of the present invention have shown antidepressant efficacy and improved depressive-like behavior of lipopolysaccharide mice from three aspects. Fig.14 and Fig.15 The specific research is as follows:

[0065] Example 2: Effects of probiotic metabolites MEs on normal mouse status

[0066] Experimental studies have found that probiotic metabolites MEs can improve the mental state, activity and cognitive level of normal mice. They have the potential to be used as antidepressant drugs. The specific experimental studies are as follows:

[0067] 1. Methods

[0068] Thirty-six KM mice were randomly divided into three groups, with 12 mice in each group. The probiotic metabolites MEs were dissolved in 0.9% saline. The mice were gavaged with 0.1 mL / 10 g of saline, 100 mg / kg of probiotic metabolites MEs, and 50 mg / kg of probiotic metabolites MEs, once a day for three days, and behavioral tests were performed.

[0069] Among them, the test group treated with normal saline gavage was the blank group; the test group using probiotic metabolites MEs at a dose of 100 mg / kg was the metabolite 100 mg / kg group; and the test group using probiotic metabolites MEs at a dose of 50 mg / kg was the metabolite 50 mg / kg group.

[0070] The specific test experiments are as follows:

[0071] (1) Open field test, OFT: The spontaneous activity of mice in an unfamiliar open area was measured. Each test mouse was placed in a device consisting of a 50 cm × 50 cm black square, and a camera placed above the experimental box automatically tracked the entire process. Each mouse moved freely for 5 minutes, and the activity trajectory of the mouse was recorded by a video tracking system. The time spent in the middle area was analyzed using a video tracking system. At the end of each test, the instrument was wiped with 75% alcohol by volume to avoid the influence of the residual odor of the previous mouse on the test results.

[0072] (2) Forced swimming test, FST: The forced swimming test was conducted in a glass cylinder with a diameter of 10 cm and a height of 25 cm. The experiment lasted for 6 min, and the resting state of each mouse was recorded in the last 5 min.

[0073] (3) Tail suspension test, TST: The mouse was placed head down with its tail suspended from a fixed rod with tape. The distance from the ground to the mouse's head was controlled at 30 cm. The entire experimental process lasted for 6 minutes. During the 6-minute test, each mouse was allowed to adapt for 1 minute after the start of the suspension, and the time the animal remained relatively motionless was recorded in the remaining 5 minutes. The despair behavior of the mouse was measured by recording the immobility time of the mouse in TST.

[0074] 2. Results

[0075] The results are as follows Figure 1 and Figure 2 shown.

[0076] From the OFT results on the left, it can be seen that the total movement distance of mice treated with the metabolite 100 mg / kg group was significantly different from that of the blank group, while the total movement distance of mice treated with the metabolite 50 mg / kg group was not different from that of the blank group.

[0077] The above results indicate that the metabolite 100 mg / kg group has an effect on the exercise capacity of mice.

[0078] In addition, the results of the FST in the middle and the TST on the right show that the immobility time of mice treated with the 100 mg / kg metabolite group was significantly reduced compared with the blank group. The immobility time of mice treated with the 50 mg / kg metabolite group was not different from that of mice in the blank group in the FST experiment.

[0079] The above results all confirm that probiotic metabolites MEs have antidepressant effects.

[0080] Example 3: Establishment of LPS-induced acute depression model in mice

[0081] In order to further evaluate the ability of probiotic metabolites MEs as antidepressant drugs, the present invention uses lipopolysaccharide to establish an acute depression model. The following experimental studies were conducted:

[0082] 1. Methods

[0083] 60 mice were randomly divided into 4 groups: Group 1: gavage with normal saline, the amount of normal saline was 1mL / kg per day; Group 2: intraperitoneal injection of 1mg / kg lipopolysaccharide; Group 3: intraperitoneal injection of 1mg / kg lipopolysaccharide + gavage of 100mg / kg probiotic metabolites MEs; Group 4: intraperitoneal injection of 1mg / kg lipopolysaccharide + gavage of 8mg / kg fluoxetine.

[0084] Among them, lipopolysaccharide was dissolved in endotoxin-free physiological saline for use, and probiotic metabolites MEs and fluoxetine were dissolved in 0.9% by mass physiological saline for use.

[0085] In the first three days, the mice were pretreated with probiotic metabolites MEs and fluoxetine by gavage. From the fourth day, lipopolysaccharide was injected intraperitoneally and probiotic metabolites MEs and fluoxetine were gavaged. A sugar preference experiment was conducted to verify whether the model was successfully established. The reduction in sucrose intake can be used to evaluate the anhedonia of mice. The model refers to the lipopolysaccharide-induced mouse depression model.

[0086] First, mice were trained to ingest 1% by mass sucrose solution from two different bottles for 24 hours. Afterwards, one of the bottles was changed to pure water and soaked for 24 hours. To avoid bottle-side preference, the two bottles were replaced after 12 hours. After acclimation, mice were deprived of water for 12 hours, and each mouse was allowed to freely obtain 1% by mass sucrose and pure water from two different bottles. After 12 hours, the quantities in the two bottles were measured, and the sucrose preference was calculated according to the following formula:

[0087] Sucrose preference (%) = sucrose water consumption / (sucrose + purified water) consumption × 100%.

[0088] 2. Results

[0089] The results are as follows Figure 3 and Figure 4 shown.

[0090] The results showed that the preference for sugar water in the model group was significantly reduced. 24 hours after the last injection of lipopolysaccharide, behavioral tests were performed on the same group of mice.

[0091] Example 4: Treatment with probiotic metabolites MEs alleviates depressive-like behavior in mice

[0092] 1. Methods

[0093] After the lipopolysaccharide-induced mouse depression model was established, behavioral experiments were performed. The operating procedures of OFT, FST, and TST were the same as those in Example 2.

[0094] 2. Results

[0095] The results are as follows Figure 5 and Figure 6 shown.

[0096] The analysis results show that compared with the model group, treatment with probiotic metabolites MEs alleviated the depressive symptoms of lipopolysaccharide-induced depression model mice, increased the total movement distance in the OFT experiment, and reduced the immobility time in TST and FST.

[0097] From the above, we can see that treatment with probiotic metabolites MEs alleviates depressive-like behavior and has the potential to be a drug for the treatment of depression.

[0098] Example 5: Probiotic metabolites MEs increase lipopolysaccharide-induced neurotransmitter levels

[0099] 1. Methods

[0100] After the behavioral test, the mice were killed, blood was collected from the eyeballs, the brain tissue was peeled off to separate the hippocampus and cerebral cortex, and the expression levels of 5-HT and DA in the hippocampus tissue were detected using ELISA Kits, and the operation was strictly carried out according to the instructions of the mouse 5-HT and DA kits. Statistical comparison was also performed.

[0101] 3. Results

[0102] The results are as follows Figure 7 shown.

[0103] The results showed that probiotic metabolites MEs can increase the expression of serotonin (5-HT) and dopamine (DA) in the hippocampus of depressed mice.

[0104] From the above, we can see that probiotic metabolites MEs improve the expression of neurotransmitters in lipopolysaccharide-induced depressive mice and have the potential to be used as a drug for the treatment of depression.

[0105] Example 6: Probiotic metabolites MEs improve lipopolysaccharide-induced activation of microglia in hippocampal brain tissue

[0106] 1. Methods

[0107] After the behavioral experiment is completed, the experimental mice are dissected, brain samples are collected, and fixed in PBS containing 4% paraformaldehyde by volume. The paraffin-embedded slices are cut, dewaxed, and finally rinsed with water. After adding repair solution, serum blocking, primary antibody, secondary antibody and DAPI working solution are added, and finally anti-fluorescence mounting medium is added to the tissue and covered with a coverslip. The prepared fluorescent slides are stored at 4°C in the dark, and the slices are imaged under a light microscope. Statistical analysis is also performed.

[0108] 2. Results

[0109] The results are as follows Figure 8 and Fig. 9 shown.

[0110] From the above, we can see that probiotic metabolites MEs inhibit the activation of hippocampal microglia in lipopolysaccharide-induced depressive mice and reduce the differentiation of microglia, and have the potential to be used as a drug for the treatment of depression.

[0111] Example 7: Probiotic metabolites MEs improve lipopolysaccharide-induced pathological changes in hippocampal brain tissue 1. Methods

[0112] The sections were sectioned as described in Example 6, stained with hematoxylin and eosin, and imaged under a light microscope.

[0113] 2. Results

[0114] The results are as follows Fig.10 shown.

[0115] The results showed that the probiotic metabolites MEs significantly increased the density of hippocampal neurons in depressed mice and restored their morphology, reduced the intercellular gaps, made them arranged in a regular manner, improved extracellular edema, increased the number of Nissler bodies in the cytoplasm, and could effectively repair damaged neurons.

[0116] From the above, we can see that probiotic metabolites MEs can improve hippocampal Ni-bodies in lipopolysaccharide-induced depressive mice and have the potential to be used as a drug for the treatment of depression.

[0117] Example 8: Antidepressant effect of probiotic metabolites MEs via cAMP and PI3K / Akt-related signaling pathways

[0118] 1. Methods

[0119] After weighing the hippocampus, the tissue fragments were soaked in 1×RIPA buffer and ground with a homogenizer. The protein concentration was determined by the BCA method. Each sample was separated by 12% SDS-PAGE gel according to the molecular weight and transferred to a PVDF membrane. Then it was blocked with 5% skim milk powder for 2 hours at room temperature and washed three times with 1×TBST for 15 minutes each time. The membrane was incubated with the indicated primary antibody at 4°C overnight. It was washed three times with 1×TBST for 15 minutes each time and incubated with goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibodies for 1.5 hours. It was washed three times with 1×TBST for 15 minutes each time and the immunoreactive bands were detected with ECL detection reagent. The results showed that MEs could reduce the expression of inflammatory proteins related to PI3K / Akt and NF-κB pathways.

[0120] 2. Results

[0121] The results are as follows Fig.11 shown.

[0122] From the above, we can see that probiotic metabolites MEs improve the symptoms of lipopolysaccharide-induced depression-like mice by regulating the expression of proteins related to the PI3K / Akt and NF-κB pathways, and have the potential to be used as a drug for the treatment of depression.

[0123] Embodiment 9:

[0124] 1. Methods

[0125] The procedure was carried out as described in Example 8.

[0126] 2. Results

[0127] The results are as follows Fig.12 shown.

[0128] The results showed that probiotic metabolites MEs could increase the expression of nutrient-derived neurotrophic factor proteins related to the cAMP pathway.

[0129] From the above, we can see that probiotic metabolites MEs improve nutrient-derived neurotrophic factors in the brain of mice with lipopolysaccharide-induced depression by regulating the expression of proteins related to the cAMP pathway, and have the potential to be used as a drug for the treatment of depression.

[0130] In summary, the probiotic metabolites MEs provided by the present invention can alleviate depressive-like behaviors and can be used to prepare antidepressant drugs.

[0131] The present invention provides the use of probiotic metabolites MEs in the preparation of antidepressant drugs. The research results of the present invention show that probiotic metabolites MEs can alleviate depressive-like behavior, inhibit the activation of microglia in hippocampal tissue, reduce the expression of inflammatory factors, and reduce neuroinflammation, indicating that probiotic metabolites MEs have the potential to be developed into antidepressant drugs.

[0132] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0133] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of probiotic metabolites MEs in the preparation of antidepressant drugs, It is characterized in that The probiotic metabolites MEs are separated from the fermentation broth of Bacillus subtilis BS-Z15; The method for separating probiotic metabolites MEs comprises the following steps: The fermentation liquid of Bacillus subtilis BS-Z15 is centrifuged for the first time at 3°C ​​to 5°C; after the first centrifugation, the supernatant is collected and the solution is adjusted to an acidic solution, and the probiotic acid fermentation liquid is obtained after the first standing; The probiotic acid fermentation liquid is centrifuged for the second time at 3°C ​​to 5°C; after the second centrifugation, the precipitate is collected, the precipitate is dissolved and the solution is adjusted to a neutral solution to obtain a probiotic acid precipitate crude extract; Acetone is added to the crude extract of the probiotic acid precipitation, and the mixture is allowed to stand for 2.5 hours to 3.5 hours at 3° C. to 5° C. for a second time, and then centrifuged for a third time to collect the supernatant; after removing acetone from the supernatant, the supernatant is first frozen into a solid, and then freeze-dried in a vacuum to obtain the probiotic metabolites MEs; The volume of acetone is 3 to 5 times the volume of the probiotic acid precipitate crude extract.

2. The use according to claim 1, It is characterized in that The pH of the acidic solution is 2-3.

3. The use according to claim 1, It is characterized in that The pH of a neutral solution is 6.5 to 7.

5.

4. The use according to claim 1, It is characterized in that The first standing condition is: 3.8℃~4.2℃ for 11.5h~12.5h.

5. The use according to claim 1, It is characterized in that The preparation method of the fermentation liquid of the Bacillus subtilis BS-Z15 is as follows: the Bacillus subtilis BS-Z15 is inoculated into a liquid culture medium, and cultured for 12 hours to 22 hours to obtain the fermentation liquid of the Bacillus subtilis BS-Z15.

6. The use according to claim 5, It is characterized in that The culture conditions of the Bacillus subtilis BS-Z15 are: temperature 36° C. to 38° C., and culture time 15 h to 18 h.

7. The use according to claim 1, It is characterized in that The conditions of the first centrifugation, the second centrifugation and the third centrifugation are all: 3.5°C to 4.5°C, 11500r / min to 12500r / min.

8. The use according to claim 1, It is characterized in that The temperature when frozen into a solid is -75℃~-85℃; the conditions for freeze vacuum drying are -40℃~-50℃, 15h~20h.

9. The use according to claim 1, It is characterized in that The antidepressant drug is prepared by using the probiotic metabolites MEs as active ingredients and supplemented with pharmaceutically acceptable excipients.

10. The use according to claim 9, It is characterized in that The effective content of the probiotic metabolites MEs in the antidepressant drug is 80 wt% to 90 wt%.

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