CN109055583B

CN109055583B - Method for screening probiotics capable of achieving anti-depression function through 5-hydroxytryptophan related approach based on cell level

Info

Publication number: CN109055583B
Application number: CN201810963195.8A
Authority: CN
Inventors: 王刚; 陈卫; 田培郡; 赵建新; 张灏
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-08-22
Filing date: 2018-08-22
Publication date: 2022-02-01
Anticipated expiration: 2038-08-22
Also published as: CN109055583A

Abstract

The invention discloses a method for screening probiotics for realizing an anti-depression function through a 5-hydroxytryptophan related way based on a cell level. The RIN14B cells are used as a screening model, and the secretion amount of 5-hydroxytryptophan in cell supernatant and the mRNA level of tryptophan hydroxylase 1 in cells are detected through the co-incubation of probiotics and the cells, so as to determine the probiotics capable of exerting the antidepressant effect through the 5-hydroxytryptophan. The effective strain screened by the invention can relieve depression-like behaviors of depressed mice, improve the levels of neurotransmitter and neurotrophic factor in brain, and relieve the hyperthyroidism of hypothalamus-pituitary-adrenal axis caused by pressure, and the cell model screening result has obvious correlation with animal experiment results. The method can be used for measuring the anti-depression potentials of different bacterial strains in batches in a short time, and provides a faster and more efficient method for screening the anti-depression function probiotics.

Description

Method for screening probiotics capable of achieving anti-depression function through 5-hydroxytryptophan related approach based on cell level

Technical Field

The invention relates to the technical field of probiotic screening, in particular to a method for screening probiotics with an anti-depression function through a 5-hydroxytryptophan related way based on a cell level.

Background

Depression is also known as depressive disorder and is characterized clinically by marked and persistent mood depression. More than 3.5 million people worldwide currently suffer from depression, which is predicted to be the 1 st in global disease burden by 2030. Numerous studies have shown that the occurrence of depression is associated with a disturbance of monoamine neurotransmitters in the brain, mainly manifested by a decrease in the 5-hydroxytryptamine (5-HT) concentration in the brain's neurosynaptic space. Current treatment modalities for depression focus primarily on increasing the level of 5-HT in the brain, for example, first-line clinical antidepressants, selective 5-hydroxytryptamine reuptake inhibitors (SSRIs). In addition, 5 hydroxytryptophan (5-HTP), a precursor of 5-HT, also has some antidepressant effect, but cannot be used as a clinical drug because of its short half-life in humans. Current treatment modalities for depression focus primarily on increasing the level of 5-HT in the brain.

The "brain-gut axis" is a new concept proposed in recent years, which regulates the function and behavior of the brain mainly through the neural, endocrine and immune pathways as a two-way communication system between intestinal bacteria and the brain. Research shows that intestinal flora of depression patients can change, and animal experiments show that depression symptoms of sterile mice can be relieved, which proves that the intestinal flora is connected with the occurrence and development of depression. The probiotic bacteria can be used for regulating intestinal flora, so that the novel way for relieving depression can be realized.

The probiotics with relieving effect on depression reported at present are proved by animal experiments. However, the adoption of animals as a screening model of the anti-depression probiotics is long in time consumption and high in cost, and a specific mechanism for exerting the effect of the animals cannot be explained. The cell model has the advantages of short time consumption, low cost, high flux and the like, but no cell screening model of the anti-depression probiotics exists at present. Therefore, the development of a method for screening the probiotics with the anti-depression function based on the cell level measurement has very important scientific and practical significance.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

In order to solve the technical problems, the invention provides the following technical scheme: a method for screening probiotics for realizing an antidepressant function through a 5-hydroxytryptophan-related pathway based on a cell level, which comprises,

cell culture: the cell is capable of expressing tryptophan hydroxylase 1 and secreting 5-hydroxytryptophan;

and (3) incubating the cells with probiotics after the cells are treated by bovine serum albumin and fluoxetine, and screening the probiotics with the anti-depression function.

As a preferred embodiment of the method for screening probiotics for realizing antidepressant function through a 5-hydroxytryptophan related pathway based on the cell level, the method comprises the following steps: the cells include rat insulinoma cell RIN 14B.

As a preferred embodiment of the method for screening probiotics for realizing antidepressant function through a 5-hydroxytryptophan related pathway based on the cell level, the method comprises the following steps: the cell culture, wherein the cell culture medium comprises RPMI1640 medium.

As a preferred embodiment of the method for screening probiotics for realizing antidepressant function through a 5-hydroxytryptophan related pathway based on the cell level, the method comprises the following steps: the probiotic bacteria include Bifidobacterium, Lactobacillus, Streptococcus, lactococcus, Leuconostoc, Propionibacterium, Pediococcus, and Bacillus.

As a preferred embodiment of the method for screening probiotics for realizing antidepressant function through a 5-hydroxytryptophan related pathway based on the cell level, the method comprises the following steps: the screening of the probiotics with the function of realizing the antidepressant function through the 5-hydroxytryptophan related pathway comprises that passage cells are divided into 10 parts⁵～10⁶The cells were inoculated in a 24-well plate, washed with Hank's balanced salt solution containing 0.1% bovine serum albumin and 1-4. mu. mol/L fluoxetine, and HBSS suspensions containing different probiotics were added, the concentration of the probiotics was adjusted to 10⁷-10⁹cfu/mL, incubating at 37 ℃ for 20min, collecting suspension, centrifuging for 5-10min at 5000-6000 g to remove precipitates, and taking supernatant to be tested.

As a preferred embodiment of the method for screening probiotics for realizing antidepressant function through a 5-hydroxytryptophan related pathway based on the cell level, the method comprises the following steps: the screening of the probiotics with the function of realizing the antidepressant function through a 5-hydroxytryptophan related pathway comprises the detection of 5-hydroxytryptophan secreted by cells and the mRNA level of tryptophan hydroxylase 1 in the cells after the cells are incubated with the probiotics.

As a preferred embodiment of the method for screening probiotics for realizing antidepressant function through a 5-hydroxytryptophan related pathway based on the cell level, the method comprises the following steps: and taking the probiotic strain with the cell secretion of 5-hydroxytryptophan increased by more than 10 times as an effective strain for screening.

As a preferred embodiment of the method for screening probiotics for realizing antidepressant function through a 5-hydroxytryptophan related pathway based on the cell level, the method comprises the following steps: and the secretion amount of the 5-hydroxytryptophan is detected and analyzed by adopting a fluorescence high performance liquid chromatography.

As a preferred embodiment of the method for screening probiotics for realizing antidepressant function through a 5-hydroxytryptophan related pathway based on the cell level, the method comprises the following steps: the probiotic strain in which the mRNA of tryptophan hydroxylase 1 is increased by more than 50% is used as an effective strain.

As a preferred embodiment of the method for screening probiotics for realizing antidepressant function through a 5-hydroxytryptophan related pathway based on the cell level, the method comprises the following steps: the tryptophan hydroxylase 1mRNA level was detected by qPCR.

The invention has the beneficial effects that: according to the invention, RIN14B cells are used as a screening model, after fluoxetine treatment, probiotics are added for co-incubation, cell supernatant is taken to detect the content of 5-HTP, and the cells are cracked to detect the expression level of the mRNA of Tryptophan hydroxylase 1(TPH1) so as to determine whether the probiotics can promote intestinal cells to generate neurotransmitter precursor 5-hydroxytryptophan (5-hydroxytryptophan, 5-HTP) and further exert the anti-depression effect.

The invention adopts experiments to prove that: the probiotics can stimulate continuous and slow secretion of 5-HTP by pheochromocyte in the intestinal tract through colonization of the intestinal tract, thereby providing precursor substances for synthesis of 5-HT in the brain and realizing the anti-depression effect. Specifically, the invention utilizes 'simulated enterochromaffin cells' (namely RIN14B cells) to detect the secretion amount of 5-HTP and the expression amount of TPH1mRNA through probiotic stimulation, so as to screen probiotics with an antidepressant function, and the effectiveness of a screening model is verified in an animal test. The invention provides a method for rapidly and efficiently screening anti-depression functional probiotic strains based on a 5-HTP (high-temperature high-performance protein) approach.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 is a schematic of chromatographic retention times and standard curves for 5-HTP. (A) Chromatographic retention times for 5-HTP and 5-HT; (B)5-HTP standard curve.

FIG. 2 is a graph showing the content of 5-HTP secreted by the cells RIN14B stimulated by probiotics; wherein P <0.05, P <0.01, P <0.0001(vs control).

FIG. 3 is a graph showing TPH1mRNA levels in RIN14B cells following probiotic stimulation; wherein P <0.05, P <0.0001(vs control).

Fig. 4 is a schematic representation of the behavioural changes of the groups of mice six weeks after probiotic intervention in depressed mice. (A) Forced swimming experiment; (B) a syrup preference experiment; (C) performing a diving platform experiment; wherein P <0.05, P <0.01, P <0.0001(vs model group).

FIG. 5 is a graphical representation of the change in levels of 5-HT (A) and 5-HTP (B) in hippocampal tissue of groups of mice six weeks after probiotic intervention in depressed mice; wherein P <0.05, P <0.01(vs model group).

FIG. 6 is a graph showing the change in BDNF levels in the prefrontal cortex of various groups of mice six weeks after probiotic intervention in depressed mice; wherein P <0.05, P <0.01, P <0.001(vs model group).

FIG. 7 is a graph showing the variation of ACTH and corticosterone levels in the serum of mice six weeks after the present strain intervenes in depressed mice; wherein P <0.05(vs model group)

FIG. 8 is a graph showing the correlation between the results of the 5-HTP and TPH1mRNA measurements and the animal experimental indices; where blue represents a negative correlation and red represents a positive correlation.

Fig. 9 is a schematic diagram of an index with significant correlation between cell models and animal experiments.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Example 1: screening of probiotic bacteria with potential for antidepressant realization through 5-hydroxytryptophan-related pathway by RIN14B cells

Cell culture:

RIN14B (ATCC CRL-2059) cells belong to the rat insulinoma cell line in RPMI-1640 complete medium (or DMEM medium) supplemented with 1% diabody (100U/mL penicillin and 100. mu.g/mL streptomycin) and 10% Fetal Bovine Serum (FBS). The cells contained 5% CO at 37 deg.C₂The culture medium is replaced every 2-3d according to the growth state of the cells. At passage and frozen storage, cells were digested with 0.25% pancreatin digest. The digestion time is 1-2 min. The number of cell generations used in this example was 10-20.

And (3) probiotic culture:

the strains used are shown in table 1. Activating the strain, inoculating to MRS liquid culture medium at 1%, standing at 37 deg.C for 20 hr, centrifuging at 3000rpm for 10min to collect thallus, washing with Hank's Balanced Salt Solution (HBSS) for 3 times, suspending in HBSS, adjusting viable bacteria concentration to 2 × 10⁸CFU/mL。

TABLE 1 Strain name and numbering

Probiotics stimulate RIN14 cells to secrete 5-HTP:

RIN14B cells at 4X 10⁵Density seed/mL in 24 well plates, incubate for 72 h. Discarding the culture medium, washing cells with HBSS (1mL) containing 0.1% Bovine Serum Albumin (BSA) and 2. mu.M fluoxetine, adding 1mL HBSS suspension containing different probiotics (control group is HBSS without thallus), incubating at 37 ℃ for 20min, collecting the supernatant, centrifuging 6000g for 5min to remove the precipitate, and taking the supernatant to freeze at-80 ℃ for testing. HPLC-FLD detection of 5-HTP in cell supernatants:

liquid phase conditions: detection analysis of 5-HTP was performed on a Waters 2695Alliance HPLC device equipped with a fluorescence detector (Waters 2695). The column was Inert sustatin C18(150 mm. times.4.6 mm, 5 um). Mobile phase a was acetate buffer (0.1M NaAC and 0.1mM EDTA-2Na, pH 5.1) and mobile phase B was methanol. Mobile phase A: b-85: 15(v/v), a flow rate of 1mL/min, a column temperature of 25 ℃, a fluorescence detector excitation wavelength of 290nm, and an emission wavelength of 330 nm. The amount of sample was 20. mu.L.

Preparing 5-HTP and 5-HT mixed standard solution: 5-HTP and 5-HT standards (Sigma-Aldrich, Chemicals, USA) were dissolved in 2.5% perchloric acid solution to prepare 1g/L of 5-HTP standard stock solution. By adopting a gradient dilution method, 200 mug/L, 100 mug/L, 50 mug/L, 25 mug/L, 12.5 mug/L and 6.25 mug/L standard solutions are respectively prepared, filtered by a 0.22 mu m filter membrane and transferred to a liquid phase small bottle for determination.

Qualitative and quantitative: the peak-out retention time is used for qualitative determination, and the external standard method is used for measuring the peak area for quantitative determination, as shown in figure 1A, the retention time of 5-HTP is about 4.0min, the retention time of 5-HT is about 5.8min, the chromatographic peak type is stable, the base line is stable, no other impurity interference peak appears, the separation effect of the two is good, the 5-HTP can be accurately determined, the detection time is short, and the determination can be completed within 8 minutes. Standard curves were prepared for the concentrations of peak area values measured in the series of standard solutions of 200. mu.g/L, 100. mu.g/L, 50. mu.g/L, 25. mu.g/L, 12.5. mu.g/L and 6.25. mu.g/L (FIG. 1B). And substituting the peak area of the sample to be detected into a formula, and multiplying the formula by the dilution times to obtain the content of the 5-HTP in the sample.

The 5-HTP content in the supernatant after stimulation of RIN14B cells by the different strains is shown in FIG. 2. Bi1, Bi3, BB1, BB2, BB4, BB5 and LH1 can obviously improve the secretion of 5-HTP in RIN14B cells (figure 2). 5-HTP, which is a precursor of the neurotransmitter 5-HT, is able to enter the blood circulation and cross the blood brain barrier and is involved in the synthesis of 5-HT in brain tissue. Therefore, the strain capable of stimulating RIN14B cells to secrete 5-HTP has potential antidepressant function.

The probiotics stimulate the gene expression of the tryptophan hydroxylase 1(TPH1) of the RIN14B cells:

extraction of cellular RNA: and (3) taking the cells in the step (3), washing adherent cells in the pore plate three times by HBSS after probiotic stimulation, adding 1mL of Trizol, placing on ice for incubation for 5-10min, blowing to make the cells fall off, and transferring the lysate to an enzyme-free EP tube. Adding 200 μ L chloroform, shaking to emulsify thoroughly, standing at room temperature for 15min, and centrifuging at 12000g at 4 deg.C for 15 min. Absorbing 300-400 mu L of supernatant, transferring the supernatant to a new enzyme-free EP tube, adding isopropanol with the same volume, gently inverting and mixing the mixture, standing the mixture at room temperature for 10min, and then centrifuging the mixture at 12000g for 10min at 4 ℃. Carefully discard the supernatant, slowly add 1mL of 75% glacial ethanol along the tube wall, mix well, centrifuge at 8000g for 10min at 4 ℃. The supernatant was carefully discarded, centrifuged briefly and the residual liquid was aspirated off, dried in a clean bench for 5min to evaporate the ethanol. Finally, 20. mu.L of DEPC water was added and the pellet was dissolved by incubation at 50 ℃ for 10 min. Taking 1 μ L RNA sample, and detecting its concentration and purity (A) with ultramicro spectrophotometer (NanoDrop 2000C)₂₆₀/A₂₈₀) And the rest samples are stored at-80 ℃ for later use.

Synthesis of cellular cDNA: synthesis of cDNA according to the instructions of the reverse transcription kitTo (PrimeScript RT reagent Kit gDNAeraser, Takara). The concentration and purity of the synthesized cDNA sample were determined by ultramicro-spectrophotometer (NanoDrop 2000C) (A)₂₆₀/A₂₈₀) And storing at-80 ℃ for later use.

mRNA assay for TPH 1: the samples were mixed with the fluorescent dye SYBR Green super mix (Qiagen, Germany) and the PCR system was 5. mu.L mix, 1. mu.L cDNA, 1. mu.L forward and reverse primers supplemented with dd water to a total volume of 10. mu.L. In real-time fluorescent quantitative gene amplification instrument CFX96^TMThe detection was performed on the Real-Time System (Bio-Rad, USA), 3 parallel wells were set for each sample, and housekeeping gene β -Actin was used as an internal reference, and the results were obtained using 2^-ΔΔCqThe method of (1) for analysis; the primer sequences used are shown in Table 2. The expression level of TPH1mRNA of RIN14B cells stimulated by different probiotics is shown in FIG. 3. The results show that probiotics Bi1 and BB4 can not only improve the secretion of 5-HTP, but also improve the mRNA level of TPH1 of RIN14B cells. The experimental result of TPH1 further narrows the range of effective bacteria. In the present example, probiotics capable of simultaneously and significantly improving the expression of TPH1mRNA and the secretion of 5-HTP of RIN14B cells are identified as strains with potential antidepressant functions, namely Bi1 and BB 4. Animal experimental verification is performed next.

TABLE 2 qPCR primer sequences

Example 2: animal experiment verification of effective strain for cell screening

1. Establishment and processing method of chronic stress depression mouse model

78 male C57BL/6J mice at 6 weeks of age were randomly divided into four groups according to body weight after acclimation for one week: a control group, a model group, a fluoxetine intervention group, and a probiotic intervention group (10 probiotics shown in table 1 were gavaged, respectively), each group containing 6 mice. Animal groups and treatment methods are shown in table 2.

TABLE 2 animal experiment grouping and processing method

Chronic unpredictable stress-depressed mouse model: the stimulation is randomly applied in 1-2 times every day, and the stimulation time is randomly determined, so that the circadian rhythm is avoided. Each method does not exceed three times, and takes five weeks. The stimulus factors include: (1) fasting for 24 h; (2) water is forbidden and the bottle is empty for 24 hours; (3) clamping tail for 3 min; (4) 24h for wet padding; (5) braking for 1-2 h; (6) inclining the cage box for 24h at 45 ℃; (7) continuously illuminating for 24 h; (8) no padding is used for 24 hours; (9) forced swimming for 15 minutes; (10) and performing isolated culture for 24 hours.

Probiotic intragastric administration agent: culturing activated 2 generation probiotic at 37 deg.C for 24 hr, centrifuging at 4 deg.C and 8000r/min for 3min to collect thallus, discarding supernatant, and re-suspending with 5% sterile defatted emulsion to reach probiotic concentration of 5 × 10⁹CFU/mL. The gavage volume was 0.2 mL/day.

2. Improvement effect of probiotics on depression behavior of mice

Starting in the fifth week, daily chronic unpredictable stress and drug and probiotic intervention was discontinued while all mice were behaviorally tested. Including forced swimming experiments, sugar water preference experiments and diving platform experiments. The specific implementation method and results are as follows:

(1) forced swimming experiment:

the forced swimming test is a behavior despair test method and is a classic test model for evaluating the antidepressant action of the medicine. The experimental bucket is filled with clear water with the height of about 20cm, the water temperature is about 24 +/-1 degrees, and each mouse is subjected to swimming training test for 15 minutes 24 hours before the formal experiment. For the official experiment, each mouse was tested for 6 minutes. The whole course of the experiment was videotaped using a camera. Immobility (floating) time, i.e. immobility of the limbs or only slight hind limb movements, was recorded. As shown in fig. 4A, the swimming time of the depressed mice in water was significantly reduced, indicating a behavior despair. The phenomenon can be obviously improved by taking Bi1 and BB4, and the antidepressant effect of Bi1 and BB4 is proved. In example 1, it has been demonstrated that Bi1 and BB4 can improve the expression of TPH1mRNA and the secretion of 5-HTP in RIN14B cells, and the results of the animal experiments are consistent with the results of the cell model screening.

(2) Sugar water preference experiment:

the carbohydrate preference test is a model for detecting anhedonia in depression. Before starting the test, two identical drinking bottles were placed in the cages and mice were acclimatized to drink for at least 3 days. After the adaptation was completed, one of the bottles of water was replaced with an aqueous solution containing 1% sucrose. The intake of water and sucrose solution was checked by weighing the bottle. The position of the two bottles is changed every day to reduce drinking preferences due to different water volumes. The sucrose preference calculation formula is: sucrose preference is V (sucrose solution)/[ V (sucrose solution) + V (water) ] x 100%, for a total of 3 days tested and averaged. The experimental results are shown in fig. 4B, the sugar water preference degree of depressed mice is significantly reduced, and the mice recover to the normal sugar water preference degree after taking Bi1, BB2, BB4 and LH 1. Wherein Bi1 and BB4 are consistent with the results of cell model screening.

(3) A jump bench experiment:

depression is usually accompanied by a degree of anxiety and memory impairment. The diving platform experiment can reflect the memory degree of the mouse to the passive injury and the anxiety degree of the mouse during the memory reproduction. The first phase is the shock training phase. The mouse is placed on the platform of the diving platform reaction box, the electric grid at the bottom of the reaction box is electrified (38V), and when the mouse jumps down from the platform, the mouse is subjected to one-time electric shock stimulation. Training time is 3min, if individual mouse does not jump down the platform within 3min, then it is manually driven down to ensure all animals are hurt by electric shock, resulting in nociceptive memory. And after training, returning to the original cage for feeding. The second phase (24 h after shock training) is the memory reproduction test phase. The mouse is placed on a reaction box platform, the test time is 3 minutes, the power is not supplied to the test, the time from the platform on the station to the 1 st jump (the time of staying on the platform) of the mouse is recorded, namely the jump platform latency, and if the mouse does not jump down the platform all the time, the calculation is carried out for 180 s. The artificial interference to the animal behavior is avoided as much as possible in the whole experimental process. The experimental result shows (figure 4C) that the diving platform latency of the depressed mice is obviously increased, which represents that the depression and anxiety degree are increased, and the diving platform latency of the depressed mice can be obviously reduced by taking Bi1, Bi3, BB2, BB4 and BB5, which shows that the strains can not only relieve depression symptoms, but also improve anxiety symptoms accompanied by depression. Wherein Bi1 and BB4 are consistent with the results of cell model screening.

3. Effect of Probiotics on neurotransmitters and neurotrophic factors in the brain of depressed mice

Mice in example 2 were euthanized at the end of the sixth week, and mouse brain tissue was taken and hippocampus and prefrontal cortex were isolated on ice. Respectively taking a certain mass of fresh hippocampal and prefrontal cortex tissues (the weight is not less than 50mg), adding 9 times of sterile PBS buffer solution (equivalent to 1g of tissues and 9ml of homogenate), homogenizing by using a tissue homogenizer, centrifuging the tissue solution for 3000g and 20min, taking supernatant, and detecting the content of 5-HT and BDNF by using an ELISA kit. Adding 5% perchloric acid precipitated protein with the same volume into the supernatant of the hippocampal tissue fluid, centrifuging for 10min at 10000g, sucking the supernatant, filtering by a 0.22 mu m water system filter membrane, and performing content determination of 5-HTP by adopting a high performance liquid chromatography-fluorescence detection method (HPLC-FLD). The experimental results are shown in figure 2, and the results show that the administration of Bi1 and LF3 can remarkably reverse the stress-caused reduction of 5-HT and 5-HTP levels in hippocampal tissues (figure 5). Wherein Bi1 is consistent with the results of cell experiments. In addition, administration of Bi1, BB4, and BB5 significantly increased the concentration of brain-derived neurotrophic factor in the mouse prefrontal cortex (fig. 6), where Bi1 and BB4 matched the results of the cell screening.

4. Alleviation of HPA hyperactivity by probiotics:

the mice in example 2 were euthanized at the end of the sixth week, and blood was collected from the mice and centrifuged at 1000g for 15min to obtain serum. The serum contents of adrenocorticotropic hormone (ACTH) and Corticosterone (Corticosterone) were measured using ELISA kits. The experimental results show (figure 7) that depressed mice show hypothalamus-pituitary-adrenal axis (HPA) hyperfunction due to continuous chronic stress, the levels of ACTH and corticosterone in serum (P <0.05) are increased, the concentrations of ACTH in serum can be obviously reduced by taking BB1 and BB2, and the levels of corticosterone in serum can be obviously reduced by taking Bi3, BB4 and LF 2. Wherein BB4 is consistent with the expected results of the cell experiment.

Example 3 correlation analysis of cell screening results and animal experiment results:

to further validate the cell screening model, the results of the cell experiments (5-HTP and TPH1) were analyzed for Pearson correlation with various measures of animal experiments. The results are shown in FIG. 8, where blue represents positive correlation and red represents negative correlation, and the size of the dot area reflects the size of the correlation coefficient. It can be seen that the expression level of TPH1mRNA and the secretion level of 5-HTP in RIN14B cells are positively correlated with the behavioral manifestations (alleviation of depressive symptoms) in animal experiments and the BDNF level in prefrontal cortex and negatively correlated with HPA hyperfunction. Wherein, the secretion of 5-HTP of RIN14B cells is in positive correlation with the glucose water preference of mice, the reduction of the diving platform latency and the BDNF level in brain. The expression level of TPH1mRNA in RIN14B cells was in positive correlation with the swimming time of mice and the BDNF level in brain (FIG. 9). The results show that the probiotics which are screened by the RIN14B cell model and have the potential of realizing antidepressant action through a 5-hydroxytryptophan related way can be verified for effectiveness in animal experiments, and the results of the animal experiments have obvious correlation with the results of the cell model screening. Therefore, the RIN14B cell is taken as a model, and based on the determination of 5-HTP related pathways (TPH1 and 5-HTP), the method can be a quick and effective screening method for realizing the antidepressant probiotics through the 5-hydroxytryptophan related pathway.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A method for screening probiotics for achieving an antidepressant function through a 5-hydroxytryptophan-related pathway based on a cellular level, characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

cell culture: the cell can express tryptophan hydroxylase 1 and secrete 5-hydroxytryptophan, wherein the cell comprises rat insulinoma cell RIN 14B;

and (2) the cells are treated by bovine serum albumin and fluoxetine and then incubated with probiotics, 5-hydroxytryptophan secreted by the cells and the mRNA level of tryptophan hydroxylase 1 in the cells after incubation with the probiotics are detected, and the probiotics with the anti-depression function are screened.

2. The method for cell-based level screening of probiotics for antidepressant function via 5-hydroxytryptophan-related pathway according to claim 1, characterized in that: the cell culture, wherein the cell culture medium comprises RPMI1640 medium.

3. The method for cell-based level screening of probiotics for antidepressant function via the 5-hydroxytryptophan-related pathway according to claim 1 or 2, characterized in that: the probiotic bacteria include Bifidobacterium, Lactobacillus, Streptococcus, lactococcus, Leuconostoc, Propionibacterium, Pediococcus, and Bacillus.

4. The method for cell-based level screening of probiotics for antidepressant function via the 5-hydroxytryptophan-related pathway according to claim 1 or 2, characterized in that: the screening of the probiotics with the function of realizing the antidepressant function through the 5-hydroxytryptophan related pathway comprises that passage cells are divided into 10 parts⁵～10⁶The cells were inoculated in a 24-well plate, washed with Hank's balanced salt solution containing 0.1% bovine serum albumin and 1-4. mu. mol/L fluoxetine, and HBSS suspensions containing different probiotics were added, the concentration of the probiotics was adjusted to 10⁷-10⁹cfu/mL, incubating at 37 ℃ for 20min, collecting suspension, centrifuging for 5-10min at 5000-6000 g to remove precipitates, and taking supernatant to be tested.

5. The method for cell-based level screening of probiotics for antidepressant function via 5-hydroxytryptophan-related pathway according to claim 1, characterized in that: and taking the probiotic strain with the cell secretion of 5-hydroxytryptophan increased by more than 10 times as an effective strain for screening.

6. The method for cell-based level screening of probiotics for antidepressant function via 5-hydroxytryptophan-related pathway according to claim 5, characterized in that: and the secretion amount of the 5-hydroxytryptophan is detected and analyzed by adopting a fluorescence high performance liquid chromatography.

7. The method for cell-based level screening of probiotics for antidepressant function via 5-hydroxytryptophan-related pathway according to claim 1, characterized in that: the probiotic strain in which the mRNA of tryptophan hydroxylase 1 is increased by more than 50% is used as an effective strain.

8. The method for cell-based level screening of probiotics for antidepressant function via 5-hydroxytryptophan-related pathway according to claim 7, characterized in that: the tryptophan hydroxylase 1mRNA level was detected by qPCR.