CN118222463B - Lactobacillus acidophilus JYLA-722 for preventing and/or treating generalized anxiety disorder and its bacterial preparation and application - Google Patents

Abstract

The invention relates to the technical field of microbial application, in particular to lactobacillus acidophilus JYLA-722 for preventing and/or treating generalized anxiety disorder, and a microbial inoculum and application thereof. Lactobacillus acidophilus (Lactobacillus acidophilus) JYLA-722 is preserved in 2023, 10 and 25 days to China general microbiological culture Collection center with the preservation number of CGMCC No.28765 and the preservation address of North Star Xili No.1 and 3 in the Chaoyang district of Beijing. Lactobacillus acidophilus JYLA-722 can inhibit the levels of serum adrenocorticotropic hormone (ACTH) and Corticosterone (CORT), reduce the sensitivity of Hippocampus to aversive stimulus, and lower the expression level of Hippocampus indole 2, 3-dioxygenase-1 (IDO 1), thereby achieving the effect of relieving generalized anxiety disorder.

Description

Lactobacillus acidophilus JYLA-722 for preventing and/or treating generalized anxiety disorder and its bacterial preparation and application

Technical Field

The present invention relates to the technical field of microbial application, and in particular to Lactobacillus acidophilus JYLA-722 for preventing and/or treating generalized anxiety disorder, a bacterial agent and an application thereof.

Background Art

Generalized anxiety disorder (GAD) is a chronic anxiety disorder characterized by persistent and significant nervousness, accompanied by autonomic nervous system excitement and excessive vigilance. Patients with GAD often have characteristic appearances, such as distorted facial muscles, furrowed brows, tense postures, restlessness, and even trembling, pale skin, and sweaty palms, soles, and armpits. Patients tend to cry easily, which is a reflection of a generalized anxiety state, not a sign of depression.

At present, the treatment methods for generalized anxiety disorder mainly include: Chinese medicine treatment, physical therapy and drug-psychotherapy. Chinese medicine treatment can effectively relieve symptoms such as insomnia, easy fright, restlessness, muscle tension, palpitation and shortness of breath caused by generalized anxiety disorder, but it generally cannot achieve the effect of cure, and the single use of Chinese medicine for conditioning is slow to take effect. Physical therapy is a non-drug treatment method, such as transcranial magnetic stimulation, which is a painless and non-invasive electrophysiological technology that stimulates the cerebral cortex and reduces cortical excitability, thereby relieving the symptoms of generalized anxiety disorder. However, physical therapy alone can only have a slight relief effect on mild generalized anxiety disorder, and the effect on severe generalized anxiety disorder is not obvious. Drug-psychotherapy refers to the use of drug therapy combined with psychotherapy. Drug therapy is reliable, but there are disadvantages such as drug dependence, large side effects, and high recurrence rate; psychotherapy requires a high degree of cooperation from patients and is expensive, so the overall treatment effect is not stable enough.

Summary of the invention

In view of the technical problems that existing treatment methods for generalized anxiety disorder have insignificant effects or large side effects, the present invention provides a Lactobacillus acidophilus JYLA-722 for preventing and/or treating generalized anxiety disorder, a bacterial agent and an application thereof.

In the first aspect, the present invention provides a Lactobacillus acidophilus JYLA-722 for preventing and/or treating generalized anxiety disorder. Lactobacillus acidophilus JYLA-722 was deposited in the General Microbiology Center of the China Culture Collection Administration on October 25, 2023, with the deposit number CGMCC No. 28765, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

In a second aspect, the present invention provides a Lactobacillus acidophilus JYLA-722 bacterial agent, which is prepared by mixing the bacterial powder of the above-mentioned Lactobacillus acidophilus JYLA-722 with maltooligosaccharide.

Furthermore, the bacterial content of the Lactobacillus acidophilus JYLA-722 bacterial agent is 5.0×10 ⁹ ~2×10 ¹⁰ cfu/g.

Furthermore, the preparation method of the bacterial powder comprises the following steps:

(1) Prepare MRS liquid medium and MRS plate medium;

(2) Activating Lactobacillus acidophilus JYLA-722 on an MRS plate medium, inoculating the activated Lactobacillus acidophilus JYLA-722 into an MRS liquid medium, and culturing to obtain a bacterial liquid;

(3) After centrifuging the bacterial solution, the bacterial cells were collected, washed with sterile physiological saline, and resuspended in reconstituted skim milk to obtain a suspension; the concentration of the suspension was adjusted to obtain a bacterial suspension, and the bacterial suspension was freeze-dried to obtain bacterial powder of Lactobacillus acidophilus JYLA-722.

Furthermore, in step (1), the preparation method of MRS liquid culture medium is as follows: weigh 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of potassium hydrogen phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, and 1000mL of distilled water; mix the above raw materials, adjust the pH to natural, stir the bacterial solution, and sterilize it at 121°C and 0.1MPa for 20min; MRS plate culture medium is prepared by adding 15g of agar to the MRS liquid culture medium, and then pouring the sterilized culture medium into a plate and letting it cool for use.

Furthermore, in step (2), the inoculation amount of the activated Lactobacillus acidophilus JYLA-722 strain is 1%, the culture temperature in the MRS liquid culture medium is 37° C., and the culture time is 24 h.

Furthermore, in step (3), the concentration of the reconstituted skim milk is 15 wt %, and the concentration of the suspension is adjusted to 1.0×10 ¹⁰ -2.0×10 ¹⁰ cfu/mL.

In a third aspect, the present invention also provides a use of the above-mentioned Lactobacillus acidophilus JYLA-722 in the preparation of a drug for preventing and/or treating generalized anxiety disorder.

Furthermore, the drug relieves or treats generalized anxiety disorder by reducing serum ACTH and cortisol concentrations.

Furthermore, the drug alleviates or treats generalized anxiety disorder by downregulating hippocampal IDO1 protein expression.

The beneficial effects of the present invention are:

The Lactobacillus acidophilus JYLA-722 provided by the present invention has been verified by animal experiments to be able to inhibit the levels of serum adrenocorticotropic hormone (ACTH) and corticosterone (CORT), reduce the sensitivity of the hippocampus to aversive stimuli, and downregulate the expression level of hippocampal indole 2,3-dioxygenase-1 (IDO1), thereby achieving the effect of relieving generalized anxiety disorder. The bacterial agent prepared by Lactobacillus acidophilus JYLA-722 is used to prevent or treat generalized anxiety disorder, avoiding the negative effects caused by the use of Western medicine, without any side effects, and is not easy to relapse after taking it, and the effect of relieving generalized anxiety disorder can be maintained for a long time.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

FIG. 1 is a diagram showing the experimental results of horizontal cross-wires in Example 3.

FIG. 2 is a graph showing the results of the Vogel drinking water conflict experiment in Example 3.

FIG. 3 is a development diagram of the IDO1 band and the internal reference β-actin band on the PVDF membrane in Example 6.

FIG. 4 is a graph showing the grayscale value ratio of the IDO1 band and the internal reference β-actin band in Example 6.

Among them, * indicates significant difference (P＜0.05), and NS indicates no significant difference.

DETAILED DESCRIPTION

In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of 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 should fall within the scope of protection of the present invention.

Example 1 Isolation, screening and identification of bacterial strains

1. Strain isolation and purification

(1) Source of the strain: Yogurt was collected in Moyu County, Hotan Prefecture, Xinjiang Uygur Autonomous Region in June 2022, stored in sterile test tubes, and transported back to the laboratory through cold chain for use.

(2) Sample preparation:

① Place sterilized saline (0.85%) in a sterile conical flask, then add 1 g of the fermented yogurt from step (1), shake, and set aside;

② Dilute the solution in step ① to prepare samples with different concentration gradients, namely 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 , numbered 1#, 2#, 3#, 4#, 5#, 6#, 7#, respectively, for standby use.

(3) Preparation of MRS plate culture medium:

Weigh 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of potassium hydrogen phosphate heptahydrate, 1 mL of Tween 80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, 15 g of agar, and 1000 mL of distilled water; mix the above raw materials, adjust the pH to 6.8, heat and stir evenly, sterilize at 121°C and 0.1 MPa for 20 min, pour the sterilized culture medium into a plate, and let it cool for use.

(4) Cultivation of strains: Use a spreader to apply solutions 1#, 2#, 3#, 4#, 5#, 6#, and 7# onto MRS plate culture medium, and culture at 37°C under anaerobic conditions for 48 h.

(5) Select colonies according to the following colony characteristics: colonies are white, round, moist, opaque, and have neat edges.

(6) Separation and purification

According to the colony characteristics of step (5), 5 single colonies were picked and inoculated onto MRS plate culture medium by streak method. The culture was carried out at 37°C under anaerobic conditions for 48 h. A single colony was picked and placed in a glycerol tube for storage at -70°C.

2. Identification and preservation

The single colony isolated and purified in step (6) was sent for identification. The identification unit was Jinan Tianyi Biotechnology Co., Ltd. During the identification process, the primers used were as follows:

Primer sequences:

27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

1492R：5'-CTACGGCTACCTTGTTACGA-3'

The gene sequence of the strain obtained during the identification process is:

AGGTTAGGCCACCGGCTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTAAGAGATTCGCTTGCCTTCGCAGGCTTGCTCCTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCAT CCCCACCTTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATGACAAGGGTTGCGCTCGTTGCGG GACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGTGTCCCCGAAGGGAACTCCGTATCTCTACGGATTGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGCGGGGTGCTTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAA CACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCA GACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACC GTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGGCGGCGTTGCTC CATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTAGGCCGTTACCCTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTATAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAG ACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCGTTCCCAACGTCATCACCGCAAGTGAATCTGTTGGTTCAGCTCGCTCG.

The strain was identified as Lactobacillus acidophilus, so the strain was named Lactobacillus acidophilus JYLA-722. Lactobacillus acidophilus JYLA-722 was sent to the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, and the deposit information is as follows;

Classification name: Lactobacillus acidophilus , preservation date: October 25, 2023, preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Preservation number: CGMCC No.28765.

Example 2 Preparation of Lactobacillus acidophilus JYLA-722 bacterial agent

(1) Preparation of MRS liquid medium and MRS plate medium

The preparation method of MRS liquid culture medium is as follows: weigh 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of potassium hydrogen phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, and 1000mL of distilled water; mix the above raw materials, adjust the pH to natural, stir the bacterial solution, and sterilize at 121°C and 0.1MPa for 20min;

MRS plate culture medium is prepared by adding 15 g of agar to MRS liquid culture medium, then pouring the sterilized culture medium into the plate and letting it cool for later use.

(2) Activate Lactobacillus acidophilus JYLA-722 on an MRS plate medium, inoculate the activated Lactobacillus acidophilus JYLA-722 into an MRS liquid medium at an inoculation rate of 1%, and culture at 37°C for 24 hours to obtain a bacterial solution;

(3) After centrifuging the bacterial solution, the bacterial cells were collected, washed with sterile physiological saline, and resuspended in 15 wt% reconstituted skim milk to obtain a suspension; the concentration of the suspension was adjusted to 1.0×10 ¹⁰ ~2.0×10 ¹⁰ cfu/mL to obtain a bacterial suspension, and the bacterial suspension was freeze-dried to obtain bacterial powder of Lactobacillus acidophilus JYLA-722;

(4) The powder of Lactobacillus acidophilus JYLA-722 was mixed with isomaltooligosaccharide (purchased from Baolingbao Biotechnology Co., Ltd.) to prepare a Lactobacillus acidophilus JYLA-722 bacterial agent.

In this embodiment, by adjusting the ratio of Lactobacillus acidophilus JYLA-722 powder and isomaltooligosaccharide, Lactobacillus acidophilus JYLA-722 bacterial agents with bacterial contents of 5 billion/g, 10 billion/g, and 20 billion/g were obtained respectively.

Example 3 Effect of Lactobacillus acidophilus JYLA-722 on preventing generalized anxiety disorder in mice

1. Construction of a mouse model of generalized anxiety disorder

40 6-8 week old SPF grade C57BL/6J healthy male mice were purchased from Jackson Laboratory (JAX) and fed for one week under the following conditions: sufficient food and water, temperature of 25℃, 12h light and 12h dark.

After one week of adaptation, 40 male mice were randomly divided into 4 groups, 10 in each group, namely model group, JYLA-722 group, blank group, and prevention group. Before modeling, the mice in the prevention group were gavaged with Lactobacillus acidophilus JYLA-722 dissolved in saline (5 million cfu/day per mouse); the mice in the blank group, JYLA-722 group, and model group were gavaged with the same amount of saline for 14 consecutive days.

After 14 days, the mice in the model group, JYLA-722 group, and prevention group were raised with one mouse per cage, and a generalized anxiety disorder mouse model was established by referring to the uncertain empty bottle stimulation method and restraint method within 28 days. The specific method is as follows:

Seven days before the start of the experiment, mice were given regular water feeding training. They were given water bottles with water for 10 minutes at 9:00-9:10 in the morning and 21:00-21:10 in the evening. The water bottles were taken away at other times to keep the mice in a thirsty state. When the mice were accustomed to drinking water at two time points every day, from the 8th day to the 28th day, a water bottle with water was randomly selected for 10 minutes at 9:00-9:10 in the morning and 21:00-21:10 in the evening every day. In another time period, the mice were stimulated with empty water bottles to observe the mice's attack cage behavior, looking around the water bottle, and grooming behavior; at the same time, the mice were fixed for 6 hours continuously from 9:30 in the morning to 15:30 in the afternoon using a mouse roller experimental fixture (purchased from Henan Zhike Hongrun Environmental Protection Technology Co., Ltd.) for 28 days, and the changes in the mice's food intake were observed every week.

2. Modeling evaluation and effect evaluation

2.1 Observe external manifestations

After 28 days of modeling, the mice in the model group and JYLA-722 group gradually reduced their food intake, showed mental restlessness, irritability, more grooming behaviors, and were prone to attacking water bottles. Their fur color was dark yellow and lusterless, and hair loss was obvious. Their skin and mucous membranes were dark purple, and their nails lacked luster, indicating that the generalized anxiety disorder mouse model was successfully established. The mice in the prevention group had normal food intake, moist fur, ruddy nails, vigorous energy, and were lively and active, indicating that Lactobacillus acidophilus JYLA-722 has a certain effect on preventing generalized anxiety disorder in mice.

2.2 Behavioral assessment

2.2.1 Pentobarbital-induced sleep experiment

The mice in the blank group, model group, JYLA-722 group, and prevention group were placed in a light-proof environment, and the room temperature was maintained at about 25°C. They were fasted but not watered for 12 hours before the experiment. Each group of mice was intraperitoneally injected with 46 mg/kg of sodium pentobarbital to induce sleep in the mice. The timing was started after the injection, and the time from intraperitoneal injection to the disappearance of the righting reflex of each mouse was observed and recorded, that is, the sleep latency; after the righting reflex disappeared, the mouse was placed on a black foam pad with its abdomen facing up, and the time from the disappearance of the righting reflex to the recovery of the mouse was observed and recorded, that is, the sleep time. The results are shown in Table 1.

The experimental data obtained were expressed as "mean ± standard deviation" and analyzed using SPSS26.0 statistical software. When the comparison between multiple groups satisfied the normal distribution and homogeneity of variance, one-way ANOVA analysis was used, and P < 0.05 was considered to be statistically significant.

Table 1 Sleeping conditions of mice

Note: * indicates significant difference compared with the blank group (P＜0.05); # indicates significant difference compared with the prevention group (P＜0.05).

As shown in Table 1, compared with the blank group, the sleep latency in the model group and JYLA-722 group was significantly increased (P < 0.05), and the sleep time was significantly reduced (P < 0.05), indicating that the generalized anxiety disorder mouse model was successfully established. Compared with the prevention group, the sleep latency in the model group and JYLA-722 group was significantly increased (P < 0.05), and the sleep time was significantly reduced (P < 0.05); the sleep latency and sleep time in the prevention group were not significantly different from those in the blank group, indicating that Lactobacillus acidophilus JYLA-722 has a certain effect on preventing generalized anxiety disorder in mice.

2.2.2 Horizontal cross-wire experiment

The mice in the blank group, model group, JYLA-722 group, and prevention group were placed at room temperature of 25°C and fasted but not watered for 12 hours before the experiment. The mice were grabbed by their tails and their forelimbs were made to grab a horizontally stretched line 15 cm long and 20 cm above the horizontal plane, and then released. The number of mice in each group that did not grab the line or actively grabbed the line with at least one hind limb within 5 seconds was recorded. If the above situation occurred within 5 seconds, the mouse was considered to have muscle relaxation. The line-grasping rate of each group of mice was calculated according to the following formula, and the results are shown in Figure 1.

As shown in Figure 1, compared with the blank group, the thread-grabbing rate of the model group and JYLA-722 group was significantly reduced (P < 0.05), indicating that the generalized anxiety disorder mouse model was successfully established. Compared with the model group, the thread-grabbing rate of the prevention group was significantly increased (P < 0.05), and the thread-grabbing rate of the prevention group was not significantly different from that of the blank group, indicating that Lactobacillus acidophilus JYLA-722 has a certain effect on preventing generalized anxiety disorder in mice.

2.2.3 Vogel drinking water conflict experiment

The drinking water conflict test box for rats and mice was purchased from Wuhan Yihong Technology Co., Ltd. The mice in the blank group, model group, JYLA-722 group, and prevention group were placed under fluorescent light and conducted in two stages. In the first stage, non-punishment drinking water training: each group of mice was deprived of water for 24 hours and then placed in the experimental box individually to allow them to fully explore until they found the bottle mouth and began to lick the water. The number of licking times of the mice in 3 minutes was recorded, and the mice that licked the water less than 300 times were eliminated. In the second stage, the punishment experiment: the above mice that were not eliminated continued to be deprived of water for 24 hours (a total of 48 hours) and were placed in the operation box. The mice could quickly find the bottle mouth and start licking the water. After licking 20 times, the instrument automatically started timing and gave an electric shock (the ratio of licking water to electric shock was 20:1). The electric shock intensity was generally 0.2-0.5mA (punishment period) and lasted for 2 seconds. The number of licking times of the mice in 3 minutes was recorded. The results are shown in Figure 2.

As shown in Figure 2, compared with the blank group, the model group and JYLA-722 group licked water significantly less (P < 0.05), indicating that the generalized anxiety disorder mouse model was successfully established. Compared with the model group, the prevention group licked water significantly more (P < 0.05), and the prevention group licked water no significantly different from the blank group, indicating that Lactobacillus acidophilus JYLA-722 has a certain effect on preventing generalized anxiety disorder in mice.

Example 4 Effect of Lactobacillus acidophilus JYLA-722 on body weight in mice with generalized anxiety disorder

According to Example 3, the model group, JYLA-722 group mice and blank group mice were weighed after successful modeling, and the JYLA-722 group was gavaged with Lactobacillus acidophilus JYLA-722 bacterial agent dissolved in physiological saline (the dosage was 5 million cfu/day per mouse). The blank group and model group mice were gavaged with the same amount of physiological saline and fed continuously for 24 days. The weight of each group of mice was weighed every 6 days during the period. The results are shown in Table 2.

The experimental data obtained were expressed as "mean ± standard deviation" and were analyzed using statistical software (SPSS 26.0). When the comparisons among multiple groups satisfied the normal distribution and homogeneity of variance, one-way ANOVA analysis was used, and P < 0.05 was considered to be statistically significant.

Table 2 Changes in mouse body weight

Note: * indicates significant difference compared with the blank group (P < 0.05); # indicates significant difference compared with the model group (P < 0.05).

As shown in Table 2, after successful modeling, the weight of mice in the model group at 0d/g was not significantly different from that of mice in the JYLA-722 group, but was significantly different from that of mice in the blank group (P < 0.05). As the number of days of oral administration of probiotics to mice in the JYLA-722 group increased, the weight of mice in the JYLA-722 group gradually increased, while the weight of the model group gradually decreased. From the 6th day, there was a significant difference in the weight of mice in the model group and the JYLA-722 group (P < 0.05). It can be seen that JYLA-722 can restore the weight of mice with generalized anxiety disorder, and has a certain alleviating and therapeutic effect on generalized anxiety disorder in mice.

Example 5 Effects of Lactobacillus acidophilus JYLA-722 on serum ACTH and CORT in mice with generalized anxiety disorder

Studies have shown that the hypothalamus-pituitary-adrenal (HPA) axis is one of the important mechanisms of stress response in the brain and is the key to the pathogenesis of generalized anxiety disorder. Adrenocorticotropic hormone (ACTH) and cortisol (CORT) are important hormones of the HPA axis, which regulate human metabolism, cognitive function and emotions, especially for generalized anxiety disorder. When the human body is stimulated by exogenous stimuli, the activity of the HPA axis is enhanced, resulting in the release of a large amount of adrenocorticotropic hormone (ACTH), and serum cortisol (CORT) is always at a high level, causing the endocrine and immune system functions of the human body to be disturbed, seriously affecting the normal function of the body, thereby stimulating generalized anxiety disorder. Generalized anxiety disorder can be improved by reducing the levels of adrenocorticotropic hormone (ACTH) and cortisol (CORT).

5.1 Serum sample preparation

The mice weighed in Example 4 were fasted but not watered for 12 h, anesthetized with 0.4% pentobarbital at a dose of 1 mL/g by intraperitoneal injection, and then placed in a supine position on an ice tray. The head and limbs were fixed with transparent medical tape, the eyeballs of the mice were removed with tweezers, and then the mice were killed and about 1 mL of peripheral blood was collected with an EP tube. The blood was allowed to stand at room temperature for 20 to 30 min until coagulation, and then centrifuged at low temperature for 10 min (4°C, 3000 rpm), and the supernatant was aspirated with a pipette and placed in a -80°C refrigerator for testing.

5.2Detection and methods of ACTH and CORT

The ELISA kit purchased from Shanghai Ke Ai Bo Biotechnology Co., Ltd. was used to detect the concentrations of ACTH and CORT in the serum of each group of mice using the double antibody sandwich method. The specific steps are as follows:

(1) Equilibrate all reagents in the ELISA kit to room temperature (i.e., leave them at room temperature for 15 to 30 minutes).

(2) Sample addition: Set up several blank wells, standard wells of various concentrations, and sample wells on the ELISA plate. Add 50 μL of standard diluent, standard of various concentrations, and serum sample to the wells (add the sample to the bottom of the well without touching the well wall). Immediately add 50 μL of the prepared working solution (prepared within 15 minutes before use), shake gently, cover with film, and incubate at 37°C for 45 minutes.

(3) Pour out the liquid in the ELISA wells and rinse the ELISA plate three times with the diluted plate washing solution. The specific rinsing steps are as follows: add an appropriate amount of plate washing solution and soak for 2 minutes, then pour out the liquid and pat dry on absorbent paper.

(4) Add 100 μL of the working solution prepared within 15 min to each well, cover with a film and incubate at 37°C for 30 min.

(5) Pour out the working solution in the ELISA wells and wash the plate 5 times, following the same procedure as above.

(6) Add 50 μL of colorimetric reagent A and B to each well, cover with film, and incubate at 37°C in the dark for about 15 min.

(7) Add 50 μL of stop solution to each well according to the order of sample addition to terminate the reaction.

(8) Set the wavelength of the Multlskan Mk3 microplate reader to 450 nm and read the optical density (OD) value of each microplate well.

(9) The sample concentration was calculated based on the measured sample OD value. The results are shown in Table 3.

The experimental data obtained were expressed as "mean ± standard deviation" and were analyzed using statistical software (SPSS 26.0). When the comparisons among multiple groups satisfied the normal distribution and homogeneity of variance, one-way ANOVA analysis was used, and P < 0.05 was considered to be statistically significant.

Table 3 Serum ACTH and CORT concentrations in mice

Note: * indicates significant difference compared with the blank group (P < 0.05); # indicates significant difference compared with the model group (P < 0.05).

As shown in Table 3, compared with the blank group, the serum ACTH and CORT concentrations of mice in the model group were significantly increased (P < 0.05). Compared with the model group, the serum ACTH and CORT concentrations of mice in the JYLA-722 group were significantly decreased (P < 0.05), and the serum ACTH and CORT concentrations of mice in the JYLA-722 group were not significantly different from those in the blank group, indicating that JYLA-722 can reduce the serum ACTH and CORT concentrations of mice with generalized anxiety disorder, and has a certain alleviating and therapeutic effect on generalized anxiety disorder in mice.

Example 6 Effect of Lactobacillus acidophilus JYLA-722 on IDO1 protein expression in the hippocampus of mice with generalized anxiety disorder

Indole 2,3-dioxygenase-1 (IDO1) is not only a "metabolic" enzyme, but also an important immune mediator. The increase in indole 2,3-dioxygenase-1 (IDO1) activity is positively correlated with the severity of generalized anxiety disorder scores. Inhibiting hippocampal indole 2,3-dioxygenase-1 (IDO1) expression can improve generalized anxiety disorder.

6.1 Protein extraction from hippocampal tissue of mice in each group

(1) Obtaining mouse hippocampal tissue: After blood was collected from the eyeballs in Example 5, the mouse skulls were cut open to expose the skull caps, and the skull caps were pried open with forceps. After the cortex was seen, the cortex was gently pushed aside until the crescent-shaped brain tissue was found under the cortex. The brain tissue was removed, the hippocampus was separated, and the left and right hippocampi were separated with a blade. The hippocampus was stored in a cryopreservation tube, temporarily frozen in a liquid nitrogen tank, and after the sampling was completed, it was transferred to a -80°C refrigerator for storage.

(2) Lysis and homogenization: Accurately weigh 100 mg of hippocampal tissue from each group of mice frozen at -80°C, add 0.5 mL of lysis buffer, and use an electric homogenizer to crush and homogenize the tissue.

(3) Centrifugation and denaturation: Centrifuge the sample homogenate for 10 min (4°C, 15,000 rpm), take the supernatant and add it to an EP tube, add protein loading buffer, place in a metal bath (100°C, 10 min), and then cool for use.

6.2 Protein concentration determination

(1) Prepare the working solution: Prepare the standard sample at a nominal concentration of 0.5 mg/mL.

(2) Setting up a concentration gradient: Take 8 enzyme-free EP tubes and label them as A, B, C, D, E, F, G, and H, extract 20 μL of the standard with a concentration of 0.5 mg/mL, and dilute it serially to obtain standard with concentrations of 0.50 mg/mL, 0.40 mg/mL, 0.30 mg/mL, 0.20 mg/mL, 0.15 mg/mL, 0.10 mg/mL, 0.05 mg/mL, and 0.00 mg/mL, respectively, and add 20 μL to a 96-well plate.

(3) Determine the protein concentration of the sample: add 20 μL of BCA working solution to the ELISA well, cover the ELISA plate with sealing film (37°C, 30 min), calibrate the ELISA reader to A562nm, and calculate the protein concentration.

6.3 Gel preparation, electrophoresis, transfer, and antibody incubation

(1) Preparation: Clean the BIO-RAD glass plate with distilled water and mount it on the gel holder.

(2) Prepare 10% separation gel: Mix 30% gel preparation solution (3.3 mL), 1.5 M Tris-HCl (2.5 mL), 10% SDS (100 μL), 10% PAGE gel solid (100 μL), PAGE gel coagulant (10 μL), and ddH ₂ O (4.0 mL), and pour into a glass plate.

(3) Prepare 5% concentrated gel: Mix 30% gel preparation solution (0.83 mL), 1.0 M Tris-HCl (0.625 mL), 10% SDS (50 μL), 10% PAGE gel solid (75 μL), PAGE gel coagulant (7.5 μL), and ddH ₂ O (3.42 mL), and pour it onto the separation gel. At the same time, insert a 10-well loading comb into the concentrated gel and let it stand for 10 min.

(4) Gel transfer and sample loading: Open the gel holder, fix the glass plate in the electrophoresis tank, add electrophoresis buffer until it exceeds the top edge of the glass plate, vertically remove the sample loading comb, first add 3 μL of protein molecular weight standard (Marker), add 3.5 μL of IDO1 to the sample loading well, and finally add 3 μL of Marker to calibrate the protein position.

(5) Electrophoresis: First set the constant voltage electrophoresis at 80V. When the band moves into the concentrated gel, change the voltage to 120V for electrophoresis. Stop the electrophoresis when the molecular weight of the target protein is roughly equivalent to that of the marker.

(6) Transfer: After activating the PVDF membrane with formaldehyde, wrap the double-layer sponge, filter paper, and PVDF membrane on the gel in sequence, add the transfer solution for electrotransfer, and cut the PVDF membrane based on the molecular weight of the target protein and the mark on the marker.

(7) Blocking and incubating with antibodies: Place the PVDF membrane in an incubation box, add 20 mL of milk blocking solution, block for 2.5 h, place the blocked PVDF membrane in an antibody incubation box, add 20 mL each of GAPDH Anti-body (concentration 1:2000) and IDO1 (concentration 1:1000), and incubate overnight at 37°C on a constant temperature water bath shaker.

(8) Incubation with secondary antibody: Wash the PVDF membrane incubated with primary antibody with TBST solution for 4 times (10 min/time), place it in an incubation box, add 20 mL of antibody HRP*Goat Anti-Rabbit IgG (H+L) (concentration 1:10000), incubate at 37°C on a constant temperature shaker for 2 h, and wash the PVDF membrane with TBST solution for 4 times (10 min/time).

6.4 ECL color development and analysis results

According to the instructions for use of the developer, the prepared developer was evenly dropped on the PVDF membrane and developed using Bio-RADQuantity one imaging software (Figure 3). Using GAPDH as a reference, the grayscale values were compared using Image J software, and the ratio of the grayscale values of the IDO1 band to the internal reference β-actin band was used as the basis for relative quantitative analysis. The results are shown in Figure 4.

As shown in Figure 4, compared with the blank group, the gray value ratio of IDO1 to the internal reference β-actin in the model group was significantly increased (P < 0.05). Compared with the model group, the gray value ratio of IDO1 to the internal reference β-actin in the JYLA-722 group was significantly decreased (P < 0.05), and the gray value ratio of IDO1 to the internal reference β-actin in the JYLA-722 group was not significantly different from that in the blank group, indicating that JYLA-722 can downregulate the expression level of IDO1 protein in the hippocampus. It can be seen that JYLA-722 has a certain alleviating and therapeutic effect on generalized anxiety disorder in mice.

Although the present invention has been described in detail by combining the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims (8)

1. Lactobacillus acidophilus JYLA-722 for preventing and/or treating generalized anxiety disorder, wherein lactobacillus acidophilus (Lactobacillus acidophilus) JYLA-722 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.28765 and the preservation address of the American ginseng district North Star Xiyu No.1, 3 in 10-25 of 2023.

2. A lactobacillus acidophilus JYLA-722 microbial agent, which is prepared by mixing the bacterial powder of lactobacillus acidophilus JYLA-722 according to claim 1 with isomaltooligosaccharide.

3. The lactobacillus acidophilus JYLA-722 microbial agent of claim 2, wherein the lactobacillus acidophilus JYLA-722 microbial agent has a bacterial content of 5.0 x 10 ⁹~2×10¹⁰ cfu/g.

4. The lactobacillus acidophilus JYLA-722 microbial inoculum according to claim 2, wherein the preparation method of the microbial powder comprises the following steps:

(1) Preparing MRS liquid culture medium and MRS plate culture medium;

(2) Activating Lactobacillus acidophilus JYLA-722 on MRS plate culture medium, inoculating activated Lactobacillus acidophilus JYLA-722 into MRS liquid culture medium, and culturing to obtain bacterial liquid;

(3) Centrifuging the bacterial liquid, collecting bacterial cells, washing the bacterial cells with sterile physiological saline, and re-suspending the bacterial cells in reconstituted skim milk to obtain suspension; the concentration of the suspension is regulated to obtain bacterial suspension, and the bacterial suspension is freeze-dried to obtain bacterial powder of lactobacillus acidophilus JYLA-722.

5. The lactobacillus acidophilus JYLA-722 as claimed in claim 4, wherein in step (1), the preparation method of the MRS liquid medium is as follows: weighing 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate and 1000mL of distilled water, mixing, naturally adjusting the pH, stirring the bacterial liquid, and sterilizing for 20min at 121 ℃ and 0.1 MPa; the MRS flat-plate culture medium is prepared by adding 15g of agar into MRS liquid culture medium, pouring sterilized culture medium into a plate, and cooling for later use.

6. The lactobacillus acidophilus JYLA-722 as claimed in claim 4, wherein in the step (2), the inoculation amount of the activated lactobacillus acidophilus JYLA-722 strain is 1%, the culture temperature in the MRS liquid culture medium is 37 ℃ and the culture time is 24 hours.

7. The lactobacillus acidophilus JYLA-722 microbial agent as claimed in claim 4, wherein in step (3), the concentration of reconstituted skim milk is 15wt%, and the concentration of the suspension is adjusted to 1.0x10 ¹⁰~2.0×10¹⁰ cfu/mL.

8. Use of lactobacillus acidophilus JYLA-722 as claimed in claim 1 in the manufacture of a medicament for the prevention and/or treatment of generalized anxiety disorder by down regulating hippocampal IDO1 protein expression by lowering the concentration of corticotropin and cortisol in the serum.