KR20230061882A - Preventive Effects of Lacticaseibacillus rhamnosus 4B15 on stress-related diseases - Google Patents

Abstract

The present invention relates to a composition that can be usefully used as food or feed to prevent or alleviate stress-related diseases by showing an effect of reducing stress-induced symptoms such as serum corticosterone levels, anxiety behavior, neurodegeneration, neuroinflammation, a brain-blood barrier-related gene and protein expression in an animal model in which Lactobacillus rhamnosus 4B15 strains are induced by stress.

Description

Composition for preventing or improving stress-related diseases of Lactobacillus rhamnosus 4B15 strain {Preventive Effects of Lacticaseibacillus rhamnosus 4B15 on stress-related diseases}

The present invention relates to a food composition or feed composition for preventing or improving stress-related diseases using a Lactobacillus rhamnosus 4B15 strain.

Many diseases and pre-existing physical ailments are caused by stress or are exacerbated by the effects of stress both large and small. Tension headache, migraine, coronary artery disease, bronchial asthma, hyperventilation syndrome, gastric/duodenal ulcer, irritable bowel syndrome, rheumatoid arthritis, and various mental disorders have been introduced as diseases that are generally known to be related to stress. External chronic stress is a risk factor for mental disorders, especially in vulnerable groups. Psychiatric disorders such as anxiety disorders and depression caused by chronic stress are complex and have heterogeneous symptoms, so the causes and mechanisms of the diseases are largely unknown. Chronic stress promotes decreased sucrose consumption and increased serum glucocorticoid levels, severe neuronal damage, immune and neuroendocrine system damage, anxiety-like behaviors and neurocognitive deficits. Stress is also known to be a major risk factor for irritable bowel syndrome (IBS), the most common gastrointestinal disorder that reflects pathologically altered colon-brain axis homeostasis.

According to recent research results, probiotics are very closely related to the intestinal microbiome and are recognized as beneficial microorganisms for our health. More specifically, it is known to have various biological effects such as inhibiting pathogenic bacteria, activating beneficial bacteria, preventing diarrhea and constipation, and enhancing immune activity. In a clinical trial testing the effects of probiotics Lactobacillus and Bifidobacteria on IBS and other lower gastrointestinal disorders, they reduced symptoms such as anxiety and depression, abdominal pain, and bloating, and reduced neuro-immune-endocrine pathways. It has been confirmed that it increases the composition of the intestinal microflora, which plays an important role in

On the other hand, studies on Lactobacillus rhamnosus have reported that the Lactobacillus rhamnosus LDTM7511 strain can be usefully used for preventing, improving or treating inflammatory bowel disease. There is no report that Lacticaseibacillus rhamnosus has an effect of preventing or improving stress-related diseases such as anxiety-like behavior, increased levels of neurotransmitters in the brain, or neuroinflammation. In particular, there has been no study on the efficacy of the Lactobacillus rhamnosus 4B15 strain on stress-related diseases.

Accordingly, while the present inventors were diligently trying to develop probiotics involved in preventing or improving stress-related diseases, the Lactobacillus rhamnosus 4B15 strain was mixed with milk, administered to mice, and then exposed to stress. As a result, serum corticosteroids The present invention was completed by confirming that stress-induced symptoms such as Theron level, anxious behavior, neurodegeneration, neuroinflammation, and expression of genes and proteins related to the blood-brain barrier were reduced.

Korean Patent Registration No. 10-2091175 Korean Patent Registration No. 10-2297271

An object of the present invention is to provide a food composition or feed composition for preventing or improving stress-related diseases by using a strain having an effect of preventing or improving stress diseases among probiotics strains beneficial to the human body.

In order to achieve the above object, the present invention is a group consisting of Lactobacillus rhamnosus 4B15 ( Lacticaseibacillus rhamnosus 4B15, accession number KCCM11983P) strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, and an extract of the culture It is characterized by providing a food composition for preventing or improving stress-related diseases, including at least one selected as an active ingredient.

In addition, in the food composition for preventing or improving stress-related diseases of the present invention, the stress-related diseases are characterized by neurodegeneration, neuroinflammation, weakening of the blood-brain barrier and anxiety behavior.

In addition, in the food composition for preventing or improving stress-related diseases of the present invention, the food composition is a patient nutritional food, meat, grains, caffeinated beverages, general beverages, dairy products, chocolate, breads, snacks, confectionery, pizza, jellies, noodles, gums , It is characterized in that at least one selected from ice creams, alcoholic beverages, alcohol, vitamin complexes, and other health supplements.

In addition, in the food composition for preventing or improving stress-related diseases of the present invention, the prevention or improvement of stress-related diseases is the concentration of corticosterone in serum, CRFR1, CRFR2, BDNF, Bax/Bcl-2, Caspase- Regulates expression of at least one gene and protein selected from the group consisting of 3, or modulates expression of at least one gene and protein selected from the group consisting of Zo-1, Occludin and Claudin, or NF-κB, COX-2, It is characterized by regulating the expression of at least one protein selected from the group consisting of IL-6, IL-1β, TNF-α, IL-10, and TGF-β.

In addition, in the food composition for preventing or improving stress-related diseases of the present invention, the prevention or improvement of stress-related diseases is characterized in that anxiety and depression-like behaviors are suppressed.

In addition, the present invention is Lactobacillus rhamnosus 4B15 (Lacticaseibacillus rhamnosus 4B15, accession number KCCM11983P) strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and at least one selected from the group consisting of extracts of the culture is effective It is characterized by providing a feed composition for preventing or improving stress-related diseases, including as components.

In addition, in the feed composition for preventing or improving stress-related diseases of the present invention, the stress-related diseases are characterized by neurodegeneration, neuroinflammation, weakening of the blood-brain barrier and anxiety behavior.

In addition, in the feed composition for preventing or improving stress-related diseases of the present invention, the prevention or improvement of stress-related diseases is the concentration of corticosterone in serum, CRFR1, CRFR2, BDNF, Bax/Bcl-2, Caspase- Regulates expression of at least one gene and protein selected from the group consisting of 3, or modulates expression of at least one gene and protein selected from the group consisting of Zo-1, Occludin and Claudin, or NF-κB, COX-2, It is characterized by regulating the expression of at least one protein selected from the group consisting of IL-6, IL-1β, TNF-α, IL-10, and TGF-β.

In addition, in the feed composition for preventing or improving stress-related diseases of the present invention, the prevention or improvement of stress-related diseases is characterized in that behaviors similar to anxiety and depression are suppressed.

The Lactobacillus rhamnosus 4B15 strain of the present invention induces stress such as serum corticosterone levels, anxiety behavior, neurodegeneration, neuroinflammation and brain-vascular barrier related gene and protein expression in stress-induced animal models Since it shows the effect of reducing symptoms, it can be usefully used as a food or feed for preventing or improving stress-related diseases, and is very useful in related industries.

1 is a schematic diagram of an in vivo experiment for verifying the effects of Lactobacillus rhamnosus 4B15 on stress-induced neurological diseases and anxiety behavior prevention and improvement in chronic stress (unpredictable chronic mild stress, UCMS) and inflammation-induced mouse models and weight change in mice am.
Figure 2 shows the effect of ingestion of Lactobacillus rhamnosus 4B15 on anxiety behavioral characteristics in UCMS/DSS-exposed mice. (a) Open field test (b) Tail suspension test (c) Elevated plus maze test (d) Light and dark box test .
Figure 3 shows the effect of ingestion of Lactobacillus rhamnosus 4B15 in UCMS/DSS-exposed mice on the expression of genes and proteins related to the HPA axis and neurodegeneration in brain tissue ((a) concentration of corticosterone in blood Numerical values, (b) gene expression analysis result through real-time PCR, (c) protein expression analysis result through western blot Lane 1 = Control group; Lane 2 = UCMS/DSS treated group; Lane 3 = Lactobacillus rhamnosus 4B15 treatment group).
Figure 4 shows the effect of ingestion of Lactobacillus rhamnosus 4B15 in UCMS/DSS-exposed mice on the expression of genes and proteins related to blood-brain barrier function in brain tissue ((a) through real-time PCR Gene expression analysis results, (b) protein expression analysis results through Western blot, lane 1 = control group; lane 2 = UCMS/DSS treatment group; lane 3 = Lactobacillus rhamnosus 4B15 treatment group).
Figure 5 shows the effect of ingestion of Lactobacillus rhamnosus 4B15 in UCMS/DSS-exposed mice on neuroinflammation-related gene and protein expression in brain tissue ((a) gene expression analysis result through real-time PCR , (b) Results of protein expression analysis by Western blot (lane 1 = control group; lane 2 = UCMS/DSS treatment group; lane 3 = Lactobacillus rhamnosus 4B15 treatment group).

The present invention is further specifically illustrated by the following examples. However, the following examples are only for helping understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

Technical terms and scientific terms used in the present invention represent meanings commonly understood by those of ordinary skill in the art to which this invention belongs, unless otherwise defined.

(Example 1) Effects of Lactobacillus rhamnosus 4B15 on preventing and improving chronic stress-induced anxiety and depressive behavior

(1) Production of powdered milk rich in probiotics and melatonin

Lactobacillus rhamnosus 4B15 strain, which was isolated from newborn feces as an experimental strain and deposited with the Korea Microorganism Conservation Center on February 28, 2017 under accession number KCCM11983P, was prepared in powder form and added to mouse feed.

(2) Chronic stress treatment in animal models

Thirty-six C57BL/6 male mice (Central Lab. Animal Inc., Seoul, South Korea) were obtained at 5 weeks of age and acclimated for one week. Mice were housed in a 22 cm wide x 28 cm long x 13 cm high polycarbonate cage covered with a filter top with a stainless steel lid under standard conditions (12-h light/dark cycle, 23 ± 2 °C, 50 ± 10% relative humidity). Mice were housed 4 per cage and sorted into 3 groups (n = 12 in each group). The three groups were ① a group that consumed regular feed without treatment with chronic stress and dextran sulfate sodium (UCMS/DSS) (normal control group, hereinafter referred to as 'Con'), ② a group that consumed regular feed while being treated with UCMS/DSS (UCMS/DSS control group, hereinafter referred to as 'SD'), ③ UCMS/DSS treatment while adding Lactobacillus rhamnosus 4B15 to normal feed and ingested group (10 ⁹ CFU/kg/day, hereinafter referred to as '4B15' )am. The 4B15 diet has previously been reported (Bachmanov, AA; Reed, DR; Beauchamp, GK; Tordoff, MG Food intake, water intake, and drinking spout side preference of 28 mouse strains. Behav Genet 2002, 32, 435-443, doi:10.1023/a:1020884312053.). The UCMS/DSS treatment group was treated with dextran sulfate sodium (DSS) and then randomly exposed to various stressors, such as daily sleep cycle changes, wet bedding, tilted cage, lighting, lack of water, confinement, and cold baths for 6 weeks. (see Table 1), and their body weight was checked every week during the experiment period.

< Chronic stress conditions treated for the present invention > Mon Tue Wed Thu Fri Sat Sun Week 1 Change cage Water deprivation sleep cycle change Sleep cycle change Sleep cycle change
Empty cage Empty cage
tilted cage tilted cage
Illumination
Foreign cage Foreign cage
Water deprivation
Wet cage Week 2 Wet cage
Pause/Changed cage Restraint
tilted cage tilted cage
Wet cage Wet cage
Water deprivation
Empty cage Empty cage
Restraint
Foreign cage Foreign cage
Sleep cycle change Sleep cycle change Week 3 Sleep cycle change
Pause/Changed cage Empty cage
Restraint
tilted cage tilted cage
Cold water bath
Empty cage Empty cage
Water deprivation
Wet cage Wet cage
Restraint
Sleep cycle change Sleep cycle change Sleep cycle change
Cold water bath
tilted cage Week 4 tilted cage
Water deprivation
Foreign cage Foreign cage
Empty cage Empty cage
Restraint
tilted cage Empty + tilted cage
Foreign + wet cage Change cage
Cold water bath
Illumination Illumination
Water deprivation
Empty cage Empty cage
Restraint Week 5 Water deprivation
Sleep cycle change tilted cage
Wet cage Empty cage
Water deprivation
Wet cage Change cage
Cold water bath
Illumination tilted cage
Illumination
Foreign cage Sleep cycle change Sleep cycle change
Pause/Changed cage Week 6 Empty cage
Restraint Wet cage
Restraint
Sleep cycle change tilted cage
Cold water bath
Empty cage Foreign cage
Empty cage Change cage tilted cage
Illumination
Foreign cage Water deprivation
Sleep cycle change

As a result, the body weight increased from an average of about 24.2 g to 27.6 g during the experiment period, but the change did not show a significant difference between the groups (Fig. 1b).

(3) Conducting behavioral tests of animal models

To investigate the effects of Lactobacillus rhamnosus 4B15 or melatonin-enriched milk on mood-related behaviors induced by chronic stress (UCMS), behavioral tests were conducted from week 7 to week 10 using animals treated with chronic stress. .

(3-1) Open field test

The open-field behavioral test measured locomotive activity and anxiety-like behavior in the same way as previously reported (Oh, N.S.; Joung, J.Y.; Lee, J.Y.; Song, J.G.; Oh, S.; Kim, Y.; Kim H. W.; Mice were placed in an opaque acrylic arena (40 cm x 40 cm x 40 cm).

Animals were placed in one of the corner areas and allowed to explore the open field for 20 minutes. The exploratory activity of the mice was recorded and analyzed using ANY-maze software (Stoelting, Wood Dale, IL, USA). Between subjects, the boxes were thoroughly washed with 70% ethyl alcohol and allowed to dry completely.

(3-2) Tail suspension test

In order to measure depressive-like behavior, a non-destination test was performed (see J vis Exp 2012;(59):3769). Rats were allowed to hang by attaching their tails with adhesive tape at locations where they could not escape or cling to a nearby surface.

The test had a duration of 6 minutes, the time spent waiting without moving and the total time spent without moving were measured and recorded. Rats did not move when passively suspended and were considered immobile for at least 1 second. Between subjects, the instruments were thoroughly washed with 70% ethyl alcohol and dried thoroughly.

(3-3) Elevated plus maze test

To measure anxiety-like behavior, the high-ranking cross maze test was performed (Oh, N.S.; Joung, J.Y.; Lee, J.Y.; Song, J.G.; Oh, S.; Kim, Y.; Kim, H.W.; Kim, S.H. Glycated milk protein fermented with Lactobacillus rhamnosus ameliorates the cognitive health of mice under mild-stress condition. Gut Microbes 2020, 11, 1643-1661, doi:10.1080/19490976.2020.1756690).

The device consisted of two open arms and two closed arms arranged in the direction of the plus sign. Each arm is 5 cm wide × 30 cm long × 40 cm high, and the wall of the closed arm is 15 cm high. The rat was initially placed in the center of the high cross maze (EPM), facing the closed arm.

Rats were allowed to explore the maze for 5 min, and activity in the open and closed arms was recorded and analyzed using the ANYmaze system. Between subjects, the instruments were thoroughly washed with 70% ethyl alcohol and dried thoroughly.

(3-4) Light and dark box test

A dark-light transfer test was performed to measure the behavior-like anxiety of mice (Zou J; Storm DR,; Xia Z. Conditional deletion of ERK5 MAP kinase in the nervous system impairs pheromone information processing and pheromone-evoked behaviors. PLoS One 2013;8:e76901).

The apparatus used in the experiment consisted of a dark chamber and a light chamber (34x24x24cm). A small hole (8 × 8 cm) is created between the two chambers so that it can move freely between the two chambers.

The chamber is made of opaque plexiglass in white and black. The dark chamber was covered with a black lid (dark chamber), while the light chamber was covered with a transparent lid and irradiated with 450 lux light using ambient room lighting.

During the test, subjects were placed in the center of a light box facing away from the hole connecting the two chambers and allowed to explore both chambers freely for 5 minutes. After each experiment, the box was washed with 70% ethanol and dried completely. All sessions were recorded and analyzed using the ANY-maze system (Stoelting, Wood Dale, IL).

As a result, mice fed Lactobacillus rhamnosus 4B15 under chronic stress conditions spent more time in the central area of the open field box and less time in the corner area of the box. The total distance traveled in the entire field of mice administered with Lactobacillus rhamnosus 4B15 was similar to that of mice fed a control diet without chronic stress (Fig. 2a).

In the higher cross maze (EPM), mice administered with Lactobacillus rhamnosus 4B15 spent significantly less time in the closed arm and more time in the open arm than did the UCMS-Con group (Fig. 2c). The total distance traveled during the EPM test was higher in mice administered with Lactobacillus rhamnosus 4B15 than in the UCMS-Con group (Fig. 2c).

In the light and dark box test, mice administered with Lactobacillus rhamnosus 4B15 spent more time in the light field and showed a greater number of conversions than mice in the UCMS-Con group (Fig. 2d).

Because mice instinctively prefer dark, enclosed areas, a greater willingness to explore the central area of the open box, the open arms and bright areas of the EPM indicates less anxious behavior, while spending more time in the corner areas, closed arms. and staying in the dark indicate increased anxiety.

In addition, to investigate the effect of Lactobacillus rhamnosus 4B15 on depression-like behavior, a tail suspension test was performed using the same group of rats. As a result, compared to the UCMS-Con group, the Lactobacillus rhamnosus 4B15 group had significantly less immobility time.

This means that there are fewer depressive behaviors (Fig. 2b). Overall, these experimental results indicate that Lactobacillus rhamnosus 4B15 attenuates or prevents anxiety and depression-like behaviors induced by chronic stress.

(Example 2) Effect of Lactobacillus rhamnosus 4B15 on chronic stress-induced stress response mechanism and prevention and improvement of neurodegeneration

(1) Serum analysis

Blood samples were collected on a BD Vacutainer® SST™ II Advance (Becton Dickinson, Franklin Lakes, NJ, USA). After centrifugation at 2,000 rpm for 15 minutes, corticosterone enzyme-linked immunosorbent assay kit (Abnova, Taipei, Taiwan) was used to measure corticosterone enzyme activity according to the manufacturer's instructions. .All measurements were performed in 3 repetitions.

(2) Gene expression analysis

To analyze the effect of ingestion of Lactobacillus rhamnosus 4B15 on the expression of genes and proteins related to the HPA axis and neurodegeneration in brain tissue, total RNA was extracted from frozen brain (whole brain) tissue (Oh, N.S.; Joung, J.Y.; Lee, J.Y.; Song, J.G.; Oh, S.; Kim, Y.; Kim, H.W.; Kim, S.H. Glycated milk protein fermented with Lactobacillus rhamnosus ameliorates the cognitive health of mice under mild-stress condition. 2020, 11, 1643-1661, doi:10.1080/19490976.2020.1756690).

cDNA was generated using a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). cDNA samples were stored at -20 °C until further use.

qRT-PCR (CFX Connect Real-Time PCR Detection System; Bio-Rad, Hercules, CA, USA) is KAPA SYBR FAST qPCR kit universal master mix (2×) (Kapa Biosystems, Boston, MA) to analyze the amount of mRNA expression , USA).

The performance conditions of qRT-PCR were denaturation at 95 °C for 10 minutes, annealing at 95 °C for 15 seconds, and extension at the primer melting temperature for 30 seconds.

The expression level of the related gene was calculated using a comparative threshold cycle method, and the value was quantified based on the expression level of the glyceraldehyde-3-phosphate dehydrogenase gene.

Primer sequences used to perform qRT-PCR are shown in Table 2. To confirm neurodegeneration, the expression levels of BDNF, bax, bcl-2, and caspase-3 genes were confirmed, and the expression levels of zo-1, occludin, and claudin-2 genes were confirmed to confirm the effect on tight junction structure. In order to confirm neuroinflammation, the expression levels of fIL-6, IL-1β, TNF-α, COX-2, IL-10 and TGF-β genes were confirmed. All experiments were performed in triplicate.

<Primer sequences used in the present invention> Gene Sequence (5’-3’) Annealing Temp. BDNF Forward 5’-TCATACTTCGGTTGCATGAAGG-3’ 61.3 °C Reverse 5'-AGACCTCTCGAACCTGCCC-3' Bax Forward 5'-AGACAGGGGCTTTTTGCTAC-3' 61.5 °C Reverse 5'-AATTCGCCGGAGACACTCG-3' Bcl-2 Forward 5'-GCTACCGTCGTGACTTCGC-3' 60.5 °C Reverse 5’-CCCCACCGAACTCAAAGAAGG-3’ Caspase-3 Forward 5'-TGGTGATGAAGGGGTCATTTATG-3' 59.5 °C Reverse 5'-TTCGGCTTTCCAGTCAGACTC-3' Zo-1 Forward 5'-AGGACACCAAAGCATGTGAG-3' 53.5 °C Reverse 5'-GGCATTCCTGCTGGTTACA-3' Occludin Forward 5'-TCTGCTCATCGCTTCCTTAG-3' 56.0 °C Reverse 5'-GTCGGGTTCACTCCCATTA-3' Claudin-2 Forward 5'-GCAAGGTGTATGAATCTGTGCT-3' 59.5 °C Reverse 5'-GTCAAGGTAACAAAGAGTGCCA-3' IL-6 Forward 5’-AAGTCGGAGGCTTAATTACACATGT-3’ 56.0 °C Reverse 5’-CCATTGCACAACTCTTTTCTCATTC-3’ TNF-α Forward 5’-CTGAACTTCGGGGTGATCGG-3’ 58.0 °C Reverse 5'-GGCTTGTCACTCGAATTTTTGAGA-3' COX-2 Forward 5'-GAAGTCTTTGGTTCTGGTGCCT-3' 58.5 °C Reverse 5'-GCTCCTGCTTGAGTATGTCG-3' GAPDH Forward 5'-GACGGCCGCATCTTCTTGT-3' 58.5 °C Reverse 5'-CAGTGCCAGCCTCGTCCCGTACAA-3'

(3) Protein expression analysis

Protein expression levels in brain and colon tissues were analyzed by Western blot analysis as previously reported (Oh, N.S.; Joung, J.Y.; Lee, J.Y.; Song, J.G.; Oh, S.; Kim, Y.; Kim, H. W.;

In detail, brain and colon tissues were homogenized and lysed in RIPA buffer (Thermo Scientific, Rockford, IL, USA) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). After centrifugation, the clear lysate was collected and 15 μg of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 12% gel (Bio-Rad, Hercules, CA, USA). It was performed in Yeongdong.

The gel was transferred to a polyvinylidene difluoride membrane and the membrane was incubated with the primary antibody. The blots were then incubated with an appropriate horseradish peroxidase-conjugated secondary antibody.

Protein bands were visualized using a Bio-Rad ChemiDoc MP imaging system (Bio-Rad) and protein expression was calculated with normalization to β-actin. Antibodies used are as shown in Table 3.

<Antibodies and conditions used in the present invention> Antibody Clone Source Dilution CRF1 PC Goat 1:1000 CRF2 PC Rabbit 1:500 NF-κB PC Rabbit 1:500 COX-2 MC Rabbit 1:1000 ZO1 MC Mouse 1:500 Occludin MC Rabbit 1:5000 Claudin 5 PC Rabbit 1:500 BDNF MC Rabbit 1:1000 Bcl-2 MC Rabbit 1:1000 Bax PC Rabbit 1:1000 Caspase-3 PC Rabbit 1:500 β-actin MC Rabbit 1:1000 Rabbit IgG H&L (HRP) PC Goat 1:1000 Mouse IgGκ BP-HRP MC Mouse 1:500 Goat IgG H&L (HRP) PC Donkey 1:1000

(4) Statistical analysis

All data were expressed as mean ± standard deviation (SD). Statistical significance of differences between groups was analyzed by one-way ANOVA using SPSS (version 22.0; IBM, Armonk, NY, USA). Differences were considered significant at p < 0.05.

To analyze the effect of Lactobacillus rhamnosus 4B15 on the hypothalamic-pituitary-adrenal axis, which is a major component of the neuroendocrine system in the brain that responds to stress, and the expression of genes and proteins related to neurodegeneration After administering Lactobacillus rhamnosus 4B15 to DSS/UCMS-treated mice, the blood corticosterone concentration, a representative stress marker, was measured. As a result, the corticosterone concentration was significantly lower than that of the UCMS/DSS-only group This showed a value similar to that of the Con group.

In addition, the expression of CRFR1 and CRFR2, which are major regulators of the HPA axis in brain tissue, brain derived neuronal factor [BDNF], which is a representative marker of brain function, and BAX, BCL-2, and Caspase-3, which are related to neurodegeneration. was measured. CRFR1 and CRFR2 proteins were significantly increased in expression in the SD group, but were expressed at significantly lower concentrations in the Lactobacillus rhamnosus 4B15 intake group than in the SD group.

CRF plays an important role in mediating the hormonal response to stress. When a stressor is activated, the hypothalamus releases corticotropin-releasing factor (CRF) to send a message to the pituitary gland, and when CRF binds to a CRF receptor in the anterior pituitary gland, adrenocorticotropic hormone (ACTH) is released.

ACTH induces a stress response by stimulating the production of the stress hormone corticosterone in the adrenal glands. Recently, CRF receptor antagonists have been discussed as antidepressants and antianxiety agents in relation to the role of CRF and its receptors in mediating stress responses, and the possibility of preventive treatment in various neuropsychiatric diseases has been raised.

BDNF, a neurotrophin-based growth factor involved in neural development, showed a reduced expression level in the UCMS/DSS-treated group compared to the untreated group (Fig. 3). However, the decrease in BDNF expression by UCMS/DSS was significantly prevented in the group fed with Lactobacillus rhamnosus 4B15. In addition, in the 4B15 group, it was confirmed that UCMS/DSS suppressed the increase in Bax/Bcl-2 ratio and Caspase-3 expression involved in neurodegeneration (FIG. 3).

Parkinson's disease, Alzheimer's disease, and Huntington's disease are typical diseases that occur in the process of neurodegeneration, and neurodegeneration is a gradual loss of structure and function of neurons, and inhibiting this process is essential for preventing new dog diseases.

(Example 3) Effect of Lactobacillus rhamnosus 4B15 on blood-brain barrier function

Exposure to UCMS/DSS significantly reduced the expression of genes and proteins of Zo-1, Occludin, and Claudin-5 involved in tight junction structure (FIG. 4).

On the other hand, ingestion of Lactobacillus rhamnosus 4B15 significantly inhibited the decrease in expression of Zo-1, Occludin, and Claudin-5 caused by UCMS/DSS (FIG. 4).

The blood-brain barrier is a medium that protects the brain and maintains neurovascular homeostasis by selectively controlling the movement of pathogenic substances inside and outside the brain. When the integrity of the blood-brain barrier is compromised, glial activation and changes in the extracellular milieu may occur, resulting in neuroinflammation.

Therefore, strengthening of the tight junction involved in brain-vascular barrier function and permeability plays an important role in the barrier function of tissues in the body.

(Example 4) Effect of Lactobacillus rhamnosus 4B15 on chronic stress-induced neuroinflammation

In order to confirm the effect of Lactobacillus rhamnosus 4B15 on neuroinflammation induced by chronic stress (DSS/UCMS), the expression patterns of genes and proteins related to neuroinflammation were confirmed.

As a result, the expression of pro-inflammatory cytokines IL-6, IL-1β, TNF-α and COX-2 genes, NF-κB and COX-2 proteins were significantly up-regulated by UCMS/DSS exposure. , IL-10, which is an anti-inflammatory cytokine, and TGF-β gene expression were significantly decreased by UCMS/DSS (FIG. 5).

However, in the group ingesting Lactobacillus rhamnosus 4B15, it was confirmed that changes in related inflammatory mediators were significantly inhibited despite the DSS/UCMS treatment (FIG. 5). NF-κB signaling is activated by stress or inflammatory responses, and the expression of inflammatory mediators is cascaded.

Sustained exposure to stress affects immune responses in the brain and colon due to interactions between the HPA axis, the colonic nervous system and the immune system.

According to the experimental results performed in the present invention, it was found that intestinal infectious microorganisms affect depression and anxiety-like behavior through the activation of the immune signal transduction pathway from the large intestine to the brain.

As described above, although the present invention has been described by limited examples and comparative examples, the present invention is not limited thereto, and the technical spirit of the present invention and the following Of course, various modifications and variations are possible within the scope of equality of the claims to be described in.

Korea Microbial Conservation Center (Korea) KFCC11983P 20170228

Claims (9)

Lactobacillus rhamnosus 4B15 ( Lacticaseibacillus rhamnosus 4B15, accession number KCCM11983P) strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, and an extract of the culture. Containing as an active ingredient at least one selected from the group consisting of A food composition for preventing or improving stress-related diseases.
The food composition according to claim 1, wherein the stressful disease is neurodegeneration, neuroinflammation, weakening of the blood-brain barrier and anxiety behavior.
The food composition according to claim 1, wherein the food composition is used for patient nutrition, meat, grains, caffeinated beverages, general beverages, dairy products, chocolate, breads, snacks, confectionery, pizza, jellies, noodles, chewing gum, ice creams, alcoholic beverages, alcohol, vitamins A food composition for preventing or improving stress-related diseases, characterized in that it is any one selected from complexes and other health supplements.
The method of claim 1, wherein the prevention or improvement of the stress-related disease is at least one selected from the group consisting of serum corticosterone concentration, CRFR1, CRFR2, BDNF, Bax/Bcl-2, and Caspase-3 Regulating gene and protein expression, or modulating at least one gene and protein expression selected from the group consisting of Zo-1, Occludin and Claudin, or NF-κB, COX-2, IL-6, IL-1β, TNF -α, IL-10, to regulate the expression of at least one protein selected from the group consisting of TGF-β, a food composition for preventing or improving stress-related diseases.
The food composition for preventing or improving stress-related diseases according to claim 1, wherein the stress-related diseases are prevented or improved by attenuating behaviors similar to anxiety and depression.
Lactobacillus rhamnosus 4B15 ( Lacticaseibacillus rhamnosus 4B15, accession number KCCM11983P) strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, and an extract of the culture. Containing as an active ingredient at least one selected from the group consisting of Feed composition for preventing or improving stress-related diseases.
The feed composition according to claim 6, wherein the stressful disease is neurodegeneration, neuroinflammation, brain-vascular barrier weakening, and anxiety behavior.
The method of claim 6, wherein the prevention or improvement of the stress-related disease is at least one selected from the group consisting of serum corticosterone concentration, CRFR1, CRFR2, BDNF, Bax/Bcl-2, and Caspase-3 Regulating gene and protein expression, or modulating at least one gene and protein expression selected from the group consisting of Zo-1, Occludin and Claudin, or NF-κB, COX-2, IL-6, IL-1β, TNF -α, IL-10, to regulate the expression of at least one protein selected from the group consisting of TGF-β, a stress-related disease prevention or improvement feed composition.
According to claim 6, wherein the prevention or improvement of the stress-related disease is to attenuate behaviors similar to anxiety and depression, stress-related disease prevention or improvement for feed composition.

Images