Stress marker and method for evaluating social stress using the same

Abstract

To provide an evaluation method capable of objectively evaluating social stress.SOLUTION: The present invention provides a method for evaluating social stress using, as an index, the content of at least one gene included in the Nan gene cluster or a by-product thereof in a fecal sample of a subject.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a stress marker and a method for assessing social stress using the same. [Background technology]

[0002] Stress is a physical and mental reaction (stress response) caused by external stimuli (stressors). Stressors include physical stressors such as cold and noise, chemical stressors such as drugs, and social stressors such as interpersonal relationships. Stress responses can occur in the form of psychological reactions such as anxiety, anger, tension, loneliness, and lethargy, as well as physical reactions such as palpitations, headaches, stomachaches, autonomic nervous system imbalance, and elevated blood pressure. If stress persists for a long period of time, or if the stress is severe enough to threaten life or body, there is a risk of developing various stress-related illnesses such as high blood pressure, heart disease, depression, and anxiety disorders.

[0003] Various social stimuli and responses to them during development are known to affect subsequent brain function and the development of the nervous system. For example, neglect and other abuse in childhood, coupled with exposure to social isolation stress due to isolation from social interactions with others, are risk factors for psychiatric disorders such as depression, schizophrenia, and attention-deficit hyperactivity disorder (ADHD) (Non-Patent Document 1).

[0004] When considering treatment and prevention methods for stress-related diseases, it is important to establish a method for evaluating stress. Conventionally, methods using salivary cortisol concentration as an indicator have been known as a method for evaluating stress. However, while cortisol increases in response to acute psychological and physical stress, as well as chronic stress due to work or unemployment (Patent Document 1), it has been reported that cortisol concentration decreases in response to traumatic stress (post-traumatic stress disorder) such as neglect (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2013-150558 [Non-patent literature]

[0006] [Non-Patent Document 1] Sara R et al., Annu. Rev. Psychol., 2017 [Non-patent document 2] Shuhei Izawa et al., Stress Assessment Using Saliva, Journal of the Japanese Society of Complementary and Alternative Medicine, Vol. 4, No. 3, pp. 91-101, 2007 [Non-patent document 3] Kosuge A, Kunisawa K, Arai S, Sugawara Y, Shinohara K, Iida T, Wulaer B, Kawai T, Fujigaki H, Yamamoto Y, Saito K, Nabeshima T, Mouri A. Heat-sterilized Bifidobacterium breve prevents depression-like behavior and interleukin-1β expression in mice exposed to chronic social defeat stress. Brain Behav Immun. 96:200-211, 2021 [Non-patent document 4] Ibi D, Takuma K, Koike H, Mizoguchi H, Tsuritani K, Kuwahara Y, Kamei H, Nagai T, Yoneda Y, Nabeshima T, Yamada K. Social isolation rearing-induced impairment of the hippocampal neurogenesis is associated with deficits in spatial memory and emotion-related behaviors in juvenile mice. J Neurochem. 105(3):921-32, 2008.

non-patent document5

non-patent document6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

[0007] An object of the present invention is to provide a biomarker that can objectively evaluate the degree of social stress load, etc., and an evaluation method using the same. [Means for solving the problem]

[0008] As a result of extensive research to solve the above-mentioned problems, the inventors discovered that when social stress is applied, the content of genes that make up the Nan gene cluster in fecal samples increases, and further that the occupancy rate of specific bacterial groups in the intestinal bacterial flora fluctuates, leading to the completion of the present invention.

[0009] Specifically, the present invention provides a method for assessing social stress and a diagnostic aid for PTSD or depression, using the content of one or more genes contained in the Nan gene cluster or their gene byproducts in a fecal sample from a subject as an index.

[0010] The present invention also relates to the following (1) to (4) in intestinal bacterial flora: (1) Verrucomicrobiota (2) Genus Akkermansia, Genus Alloprevotella, Genus Tidjanibacter, Genus Schaedlerella, Genus Ligilactobacillus, Genus Roseburia or Family Lacnospiraceae (3)Akkermansia muciniphila, Ruminococcus callidus, Parabacteroides distasonis or Bacteroides acidfaciens (4) Phocaeicola_A_858004, Alistipes_A_871400, Alistipes_A_871404, Parabacteroides_B_862066, Bacteroides_H, Muribaculaceae_UBA 3263, Peptostreptococcaceae_256921, Muribaculaceae_UBA7173, Muribaculaceae_CAG485, Lacnospiraceae_14-2 or Ruminococcus_C_58660 The present invention provides a method for assessing social stress and a diagnostic aid for PTSD or depression, using one or more types of bacteria occupancy rate as an index.

[0011] The present invention also provides a social stress assessment kit comprising a social stress marker consisting of one or more genes contained in the Nan gene cluster or their gene by-products in a fecal sample from a subject, and a reagent for measuring the stress marker.

[0012] The present invention also provides a method for evaluating a social stress-relieving agent, which comprises measuring one or more genes contained in the Nan gene cluster or their gene by-products in a fecal sample from a subject to which a test substance has been administered.

[0013] The present invention also provides the following (1) to (4) in the intestinal bacterial flora of a subject administered with a test substance: (1) Verrucomicrobiota (2) Genus Akkermansia, Genus Alloprevotella, Genus Tidjanibacter, Genus Schaedlerella, Genus Ligilactobacillus, Genus Roseburia or Family Lacnospiraceae (3)Akkermansia muciniphila, Ruminococcus callidus, Parabacteroides distasonis or Bacteroides acidfaciens (4) Phocaeicola_A_858004, Alistipes_A_871400, Alistipes_A_871404, Parabacteroides_B_862066, Bacteroides_H, Muribaculaceae_UBA 3263, Peptostreptococcaceae_256921, Muribaculaceae_UBA7173, Muribaculaceae_CAG485, Lacnospiraceae_14-2 or Ruminococcus_C_58660 The present invention relates to a method for evaluating a social stress-relieving agent, which comprises measuring the occupancy rate of one or more types of bacteria belonging to any one of the above. [Effects of the Invention]

[0014] According to the method of the present invention, it is possible to objectively evaluate stress conditions such as the presence or absence and degree of social stress. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a graph showing the content of the Nan gene in the social defeat stress group (CSDS) and the control group (Ctrl) in Example 1. [Figure 2] 1 is a graph showing the content of the Nan gene in the social isolation stress group (Isolation) and the control group (Ctrl) in Example 2. [Figure 3]1 is a graph showing the occupancy rate of bacterial groups that was significantly lower in the social isolation stress group (Isolation) than in the control group (Ctrl) in Example 2. [Figure 4] 1 is a graph showing the occupancy rate of bacterial groups that was significantly lower in the social isolation stress group (Isolation) than in the control group (Group) in Example 2. [Figure 5] 1 is a graph showing the occupancy rate of bacterial groups that was significantly lower in the social isolation stress group (Isolation) than in the control group (Control) in Example 2. [Figure 6] 1 is a graph showing the occupancy rate of bacterial groups that was significantly higher in the social isolation stress group (Isolation) than in the control group (Control) in Example 2. [Figure 7] 1 is a graph showing the occupancy rate of bacterial groups that was significantly higher in the social isolation stress group (Isolation) than in the control group (Control) in Example 2. [Figure 8] FIG. 1 shows the consensus sequence of nanA gene homologues. DETAILED DESCRIPTION OF THE INVENTION

[0016] The stress marker for social stress of the present invention comprises one or more genes contained in the Nan gene cluster or their gene by-products.

[0017] Bacterial enzymes involved in sialic acid metabolism include N-acetylneuraminate lyase (NanA), N-acetylmannosamine-6-P epimerase (NanE), N-acetylmannosamine kinase (NanK), N-acetylglucosamine-6-phosphate deacetylase (NagA), and glucosamine-6-phosphate deaminase (NagB) (Nan cluster). In the present invention, the content of one or more genes or their gene byproducts contained in the Nan gene cluster that encode these enzymes in a fecal sample from a subject is used as an indicator. Examples of bacteria having a Nan gene cluster include bacteria belonging to the genera Ruminococcus, Blautia, Clostridium, Eubacterium, Mediterraneeibacter, Lachnospiraceae, Faecalicatena, Butyricicoccus, Peptostreptococcus, Extibacter, Dorea, Robinsoniella, Schaedlerella, Latilactobacillus, Angelakisella, Dubosiella, Streptococcus, Akkermansia, Bacteroides, and Parabacteroides. By-products of one or more genes contained in the Nan gene cluster include mRNA, which is the transcription product of the gene, and proteins, which are the translation products.

[0018] Genes contained in the Nan gene cluster and the amino acid sequences encoded by these genes can be obtained from publicly known databases such as GeneBank. For example, the amino acid sequence encoded by the nanA gene of the Ruminococcus gnavus group is the amino acid sequence of SEQ ID NO: 1, registered in GeneBank under accession number EDN77078.1. Genes contained in the Nan gene cluster include, for example, genes encoding polypeptides consisting of the amino acid sequence of SEQ ID NO: 1 and amino acid sequences in which one or several amino acids have been deleted, substituted, or added. As used herein, "several amino acids" generally refers to 2 to 9, preferably 2 to 5, and more preferably 2 to 3 amino acids. Genes also include genes encoding polypeptides consisting of amino acid sequences that share 70% or more, preferably 80% or more, and more preferably 90% or more identity with the amino acid sequence of SEQ ID NO: 1. Examples include the R. gnavus nanA homologs listed in Table 1 (SEQ ID NOs: 1 to 26).

[0019] [Table 1]

[0020] The method for assessing social stress of the present invention uses the content of one or more genes included in the Nan gene cluster, which is a stress marker, or their gene byproducts in a fecal sample from a subject as an index. The subject to be assessed by the method of the present invention is a mammal, including humans and non-human mammals, and preferably a human.

[0021] The content of one or more genes contained in the Nan gene cluster or their gene byproducts in a fecal sample from the subject is measured. For measuring the content of one or more genes contained in the Nan gene cluster, for example, feces can be used as is, or a DNA extraction sample can be prepared by pretreating the feces as needed using a known method. The gene content in a fecal sample can be measured using known methods, such as a method using primers that can specifically amplify genes contained in the Nan gene cluster or a probe that specifically hybridizes with the gene.

[0022] When primers are used, primers capable of specifically amplifying genes contained in the Nan gene cluster can be designed based on the nucleotide sequences of the genes contained in the Nan gene cluster, and examples of suitable primers include the following: Using such a primer set, a diffusion amplification reaction is performed on a fecal sample by quantitative PCR or the like, and the amount of amplified fragments is measured, thereby measuring the content of genes contained in the Nan gene cluster in the fecal sample. (Primer set for amplifying the nanA gene of R. gnavus) 5'-ATTCCGGCATTTTATGC-3' (SEQ ID NO: 27) 5'-CCGTTTACATAGACACCTTTTAC-3' (SEQ ID NO: 28) (Primer set for amplifying the nanA gene of R. gnavus and Blautia) 5'-ATYCCGGCATTTTATGC-3' (SEQ ID NO: 29) 5'-CCRTTTACRTAGACACCYTTTAC-3' (SEQ ID NO: 30) (Primer set for amplifying the nanA gene of Drea) 5'-ATYCCDGCRTTYTATGC-3' (SEQ ID NO: 31) 5'-CCRTTHACATAYACACCTTTBAC-3' (SEQ ID NO: 32)

[0023] When a probe is used, the probe that specifically hybridizes to a gene contained in the Nan gene cluster can be designed based on the nucleotide sequence of the gene. The content of the gene contained in the Nan gene cluster can be measured by performing a hybridization reaction with a fecal sample using the designed probe and quantifying the amount of the hybridized probe by a known method such as Southern hybridization or DNA microarray.

[0024] Examples of by-products of one or more genes contained in the Nan gene cluster include mRNA, which is the transcription product of the gene, and proteins, which are the translation products. Measurement of mRNA can be performed according to known methods, such as quantitative PCR, which amplifies the mRNA from a fecal sample using primers capable of specifically amplifying the mRNA, or Northern hybridization, cDNA microarray, or other methods in which a specifically hybridizing probe is used to quantify the amount of hybridized probe. Quantification of proteins, which are transcription products, can be performed using antibodies that specifically bind to the protein. For example, antibodies can be produced by known methods such as the hybridoma method, and the amount of antibody binding in a fecal sample can be measured by ELISA or other methods.

[0025] The presence or absence, and degree, of social stress in a subject can be assessed by comparing the content of genes contained in the Nan gene cluster or their gene byproducts in a subject's fecal sample measured as described above with a reference value. The reference value is not particularly limited, but can be appropriately set based on statistical values ​​such as the mean, median, or third quartile of the content of genes contained in the Nan gene cluster or their gene byproducts in a fecal sample measured in a healthy group. A subject's measured value higher than the reference value can be evaluated as experiencing high social stress, while a value lower than the reference value can be evaluated as experiencing low social stress. Furthermore, as described below, in a social isolation stress model, which is a model of childhood neglect, and a social defeat stress model, which is a model of depression, the content of genes contained in the Nan gene cluster in a subject's fecal sample was lower than the reference value. Therefore, the genes or gene byproducts contained in the Nan gene cluster in a subject's fecal sample can be used as an indicator to similarly assist in the diagnosis of PTSD or depression.

[0026] The social stress assessment kit of the present invention includes a reagent for measuring the genes contained in the Nan gene cluster, which is a stress marker, or their gene by-products in a fecal sample. Examples of such reagents include primers, probes, antibodies, etc. for the genes contained in the Nan gene cluster and their by-products, such as mRNA and proteins.

[0027] The method for evaluating social stress relief agents of the present invention involves measuring one or more genes contained in the Nan gene cluster or their gene by-products in a fecal sample from a subject to which a test substance has been administered. As described above, the amount of genes contained in the Nan gene cluster or their gene by-products in a fecal sample increases due to the load of social stress. Therefore, the amount of these genes before and after administration of the test substance is measured, and a test substance that reduces the amount can be evaluated as a social stress relief agent.

[0028] The present invention also relates to a method for detecting a bacterial flora of a subject, comprising: (1) Verrucomicrobiota (2) Genus Akkermansia, Genus Alloprevotella, Genus Tidjanibacter, Genus Schaedlerella, Genus Ligilactobacillus, Genus Roseburia or Family Lacnospiraceae (3)Akkermansia muciniphila, Ruminococcus callidus, Parabacteroides distasonis or Bacteroides acidfaciens (4) Phocaeicola_A_858004, Alistipes_A_871400, Alistipes_A_871404, Parabacteroides_B_862066, Bacteroides_H, Muribaculaceae_UBA 3263, Peptostreptococcaceae_256921, Muribaculaceae_UBA7173, Muribaculaceae_CAG485, Lacnospiraceae_14-2 or Ruminococcus_C_58660 This is a method for evaluating social stress using the occupancy rate of any one of the bacterial groups listed above as an index.

[0029] Analysis of a subject's intestinal microbiota can be performed using known intestinal microbiota analysis methods, using gastrointestinal contents such as feces and intestinal fluids as samples. For example, bacterial species present in the intestinal microbiota can be classified and identified based on the nucleotide sequence of the 16S rRNA gene. Specifically, when analyzing the 16S rRNA gene of intestinal bacteria in feces, collected feces is lysed according to known methods, genomic DNA is extracted from the resulting sample, and the 16S rRNA gene in the extracted genomic DNA is amplified by PCR to detect sequences specific to each bacterial species, thereby identifying intestinal bacteria and calculating their occupancy rate. PCR amplification of the 16S rRNA gene and detection of bacterial species-specific sequences can be performed using, for example, quantitative PCR, metagenomic analysis, DGGE / TGGE, FISH, clone library analysis, T-RFLP, and the like. Quantitative PCR involves performing PCR using primers specific to the target bacterial genus or species, and then detecting the amount of the amplified product using a real-time PCR device. Identification of bacterial species from sequences can be performed by homology searches in databases such as SILVA and Green genes. The above (1) to (4) are classifications based on the SILVA database.

[0030] When social stress is applied, the proportion of specific bacterial groups in the intestinal microbiota changes, and this can be used as an indicator to evaluate social stress. Specifically, social stress exposure can cause (1) a decrease in the proportion of bacterial groups belonging to the phylum Verrucomicrobiota, (2) a decrease in the proportion of bacterial groups belonging to the genera Akkermansia, Alloprevotella, Roseburia, or Tidjanibacter, and (3) a decrease in the proportion of bacterial groups belonging to Akkermansia muciniphila, Ruminococcus callidus, Parabacteroides distasonis, or Bacteroides (4) A decrease in the proportion of bacteria belonging to Phocaeicola_A_858004, Alistipes_A_871400, Alistipes_A_871404, Parabacteroides_B_862066, Bacteroides_H, Muribaculaceae_UBA3263, Peptostreptococcaceae_256921, or Ruminococcus_C_58660 occurs. Therefore, one or more of these bacterial groups in the subject's intestinal microbiota are measured and compared with a reference value. If the measured value of the subject is high, for example, it can be evaluated as low stress in terms of social stress, and if it is lower than the reference value, it can be evaluated as high stress. The reference value can be set appropriately based on statistical values ​​such as the mean, median, and third quartile of these bacterial groups in the intestinal bacteria measured in a healthy group.

[0031] On the other hand, due to the load of social stress, the proportion of (2) bacteria belonging to the genus Schaedlerella, the genus Ligilactobacillus, or the family Lacnospiraceae in the intestinal flora, and the proportion of bacteria belonging to the genus Muribaculaceae_UBA7173, Muribaculaceae_CAG485, or Lacnospiraceae_14-2 in the intestinal flora increases. Therefore, if the measured value of a subject is higher than the standard value, it can be evaluated as, for example, high social stress, and if it is lower than the standard value, it can be evaluated as low social stress.

[0032] The method for evaluating a social stress-relieving agent of the present invention is to evaluate the intestinal bacterial flora of a subject to which a test substance is administered. (1) Verrucomicrobiota (2) Genus Akkermansia, Genus Alloprevotella, Genus Tidjanibacter, Genus Schaedlerella, Genus Ligilactobacillus, Genus Roseburia or Family Lacnospiraceae (3)Akkermansia muciniphila, Ruminococcus callidus, Parabacteroides distasonis or Bacteroides acidfaciens (4) Phocaeicola_A_858004, Alistipes_A_871400, Alistipes_A_871404, Parabacteroides_B_862066, Bacteroides_H, Muribaculaceae_UBA 3263, Peptostreptococcaceae_256921, Muribaculaceae_UBA7173, Muribaculaceae_CAG485, Lacnospiraceae_14-2 or Ruminococcus_C_58660 As mentioned above, the intestinal bacterial flora is affected by social stress. (1) Verrucomicrobiota (2) Akkermansia, Alloprevotella, Roseburia, or Tidjanibacter (3)Akkermansia muciniphila, Ruminococcus callidus, Parabacteroides distasonis or Bacteroides acidfaciens (4) Phocaeicola_A_858004, Alistipes_A_871400, Alistipes_A_871404, Parabacteroides_B_862066, Bacteroides_H, Muribaculaceae_UBA3263, Peptostreptococcaceae_256921 or Ruminococcus_C_58660 Therefore, for example, by measuring these occupancies before and after administration of a test substance, a test substance that increases the occupancy can be evaluated as a social stress reliever. In contrast, social stress can alter the gut microbiota (2) Schaedlerella genus, Ligilactobacillus genus or Lacnospiraceae family (4) Muribaculaceae_UBA7173, Muribaculaceae_CAG485 or Lacnospiraceae_14-2 Since the occupancy rate of one of the bacterial groups increases, these occupancies can be measured before and after administration of the test substance, and a test substance that reduces the occupancy rate can be evaluated as a social stress reliever. [Example]

[0033] The present invention will be described in detail below by way of examples, but the present invention is not limited to these examples in any way.

[0034] Example 1 Social Defeat Stress Model (CSDS): Seven-week-old male C57BL / 6J mice were used. Mice were housed 4–5 per cage in plastic cages under a 12-hour light / dark cycle (lights on at 8:00 A.M.) and provided with food and water ad libitum. Chronic social defeat stress was administered according to a published method (Non-Patent Document 3). Seven-week-old C57BL / 6J mice (test mice) were individually placed in an away cage containing one aggressive ICR male mouse and exposed to the stress for 10 minutes per day. The ICR male mouse attacked the test mouse in the away cage, and the test mouse was returned to its home cage after 10 minutes. This procedure was repeated for 10 consecutive days to induce chronic social defeat stress (CSDS). A control group was exposed to the stress for 10 minutes per day in an away cage containing one deeply anesthetized ICR male mouse. Ten days of CSDS exposure has been shown to result in decreased social skills and motivation, and increased anxiety. (Measurement of Nan gene content) We created primer sets to amplify nanA in R. gnavus and Blautia, and examined whether there was a difference in the amount of Nan gene between the social defeat stress group (CSDS) and the control group (Ctrl). Note that Y represents C and T, R represents A and G, D represents G, A, and T, and H represents A, T, and C. [Primer set for amplifying nanA from R. gnavus and Blautia] nanA_R.gnavus+Blautia_113F: 5'-ATYCCGGCATTTTATGC-3' (SEQ ID NO: 29) nanA_R.gnavus+Blautia_113R: 5'-CCRTTTACRTAGACACCYTTTAC-3' (SEQ ID NO: 30) Quantitative PCR was performed on fecal samples from the control and social defeat stress groups using these primer sets. Specifically, amplification was performed using PowerTrack SYBR Green Master Mix (Thermo Fisher Scientific) with 40 cycles of 95°C for 120 seconds, 95°C for 10 seconds, 50°C for 15 seconds, and 72°C for 15 seconds, followed by a final extension reaction at 72°C for 1 minute. Amplification and detection were performed using QuantStudio7 (ThermoFisher). A PCR fragment of the nanA gene from R. gnavus was used for the qPCR standard curve. Quantitative PCR of the 16S rRNA gene of all bacteria was also performed using the primer set (F_Bact1369: 5'-CGGTGAATACGTTCCCGG-3' (SEQ ID NO: 33) and R_Prok1492: 5'-TACGGCTACCTTGTTACGAC-3' (SEQ ID NO: 34)) according to Non-Patent Document 7, and normalization was performed by dividing by this value.

[0035] The nanA levels of R. gnavus and Blautia amplified using primers SEQ ID NOs: 29 and 30 were significantly higher in the CSDS group compared to the Ctrl group (Fig. 1), indicating that the Nan gene cluster correlates with social defeat stress.

[0036] Example 2 Social isolation stress model: (Animals used) Three-week-old (P21) male C57BL / 6J mice were used in this experiment. Mice were housed in plastic cages under a 12-hour light-dark cycle (lights on at 8:00 AM) and provided with food and water ad libitum. Social isolation stress, which mimics stress experienced in juveniles, was administered according to a published method (Non-Patent Document 4). Three-week-old (P21) mice were randomly divided into two groups: isolation and group. Mice in the isolation group were housed individually in cages, while the group group was maintained under standard mouse housing conditions (5 mice per cage). After four weeks of social isolation, the following behavioral analyses were performed. All behavioral analyses were performed between 10:00 AM and 6:00 PM. Mice were allowed to acclimate to the laboratory for at least two hours before behavioral analysis. Social isolation stress was also imposed during the behavioral analysis period.

[0037] (Social Behavior Test) The social behavior test (SIT) was performed to evaluate sociality according to a known method (Non-Patent Document 5). The social behavior test was performed in an open box (30 × 30 × 35 cm) under 10 lux illumination. Each mouse was habituated to the box for 10 minutes each for two days prior to the social behavior test. On the day of the test, the test mouse and a C57BL / 6J mouse of the same age and sex were placed in the box as a partner mouse for 10 minutes, and the duration of social interactions (sniffing, grooming, chasing, mounting, crawling, etc.) was recorded. A social behavior test was performed after social isolation stress, and a significant decrease in sociality was observed in the isolation-housed group compared to the group-housed group (n = 10-12 each; **p < 0.01).

[0038] (Forced swimming test) The forced swimming test (FST) was conducted as a test to evaluate motivation, following a known method (Non-Patent Document 5). Each mouse was placed in a plastic cylinder (20 × 15 cm) filled with water under 100 lux of illumination and forced to swim for 6 minutes. Swimming time was measured using a SCANET-40 device (Melquest), and the immobility time (total time - swimming time) was calculated from 1 minute after the start of swimming. A forced swimming test was conducted after social isolation stress. The results showed that the immobility time was significantly increased in the isolated group compared to the group-housed group (n=10-12 each; **p<0.01), suggesting a decrease in motivation.

[0039] (Object location recognition test) The object location recognition test (NOLT) was conducted to evaluate spatial cognitive function according to a known method (Non-Patent Document 6). For the object location recognition test, mice were first acclimatized to an open box (30 × 30 × 35 cm) for 3 days. On the fourth day, a training session was conducted in which two identical objects were placed 7 cm from the rear wall of the box, and contact time to each object was measured for 10 minutes. On the following day (Day 5), a test session was conducted in which the position of one of the two objects was changed, and contact time to the object (F) placed in the same position as in the training session and the relocated object (N) was measured for 10 minutes. The proportion of contact with the novel object (N) was calculated as the spatial discrimination index [DI (%) = 100 × (contact time to object N - contact time to object F) / (contact time to object N + contact time to object F)], and the DI was evaluated as an index of spatial cognition. After the social isolation stress test, an object location recognition test was conducted. Although no differences were observed between the two groups in the training session, a significant decrease in the spatial discrimination index was observed in the isolated group compared to the group-reared group in the test session (n=10-12 each; **p<0.01), suggesting that spatial cognitive function may be impaired.

[0040] (Measurement of Nan gene content) We created a primer set to amplify nanA in R. gnavus and Blautia, and examined whether there was a difference in the amount of Nan gene between the social isolation stress group (Isolation) and the control group (Ctrl; group-reared group). Note that Y represents a mixture of C and T, R represents A and G, D represents G, A, and T, and H represents a mixture of A, T, and C. [Primer set for amplifying nanA of R. gnavus and Blautia (see Figure 8)] nanA_R.gnavus+Blautia_113F: 5'-ATYCCGGCATTTTATGC-3' (SEQ ID NO: 29) nanA_R.gnavus+Blautia_113R: 5'-CCRTTTACRTAGACACCYTTTAC-3' (SEQ ID NO: 30) Quantitative PCR was performed on fecal samples from the control and social isolation stress groups using these primer sets. Specifically, amplification was performed using PowerTrack SYBR Green Master Mix (Thermo Fisher Scientific) with 40 cycles of 95°C for 120 seconds, 95°C for 10 seconds, 50°C for 15 seconds, and 72°C for 15 seconds, followed by a final extension reaction at 72°C for 1 minute. Amplification and detection were performed using a QuantStudio7 (ThermoFisher). A PCR fragment of the nanA gene from R. gnavus was used for the qPCR standard curve. Quantitative PCR of the 16S rRNA gene of total bacteria was also performed using the primer set (F_Bact1369: 5'-CGGTGAATACGTTCCCGG-3' (SEQ ID NO: 33) and R_Prok1492: 5'-TACGGCTACCTTGTTACGAC-3' (SEQ ID NO: 34)) according to non-patent literature 7, and normalization was performed by dividing by this value.

[0041] The nanA levels of R. gnavus and Blautia amplified using primers SEQ ID NOs: 29 and 30 were significantly higher in the isolation group than in the control group (P = 0.0148, Figure 2), indicating that the Nan gene cluster correlates with social isolation stress.

[0042] (Analysis of intestinal flora) DNA extraction and 16S amplicon sequence analysis DNA extraction from each fecal sample was performed according to the method described in Non-Patent Document 8, and the crude DNA was purified using a GENE PREP STAR P I-480 (Kurashiki Boseki). PCR amplification was performed using a primer set (Pro341F-Pro805R, Non-Patent Document 8) that amplifies the 16S rRNA gene (V3-V4 region) of bacilli and archaea. The PCR products were subjected to paired-end sequencing (600 cycles) using a MiSeq system and MiSeq Reagent Kit version 3 (Illumina) with 2x301-bp cycles. Obtid sequences were removed from the paired-end sequences combined with the primer sequences using Cutadapt software (Non-Patent Document 9) and fastq-join software (Non-Patent Document 10). Reads containing 99% or more sequences with a quality value (QV) of 20 or higher were selected, and chimeric sequences were removed from these reads.

[0043] 16S rRNA gene analysis was performed using QIIME2 (ver. 2022.2, Non-Patent Document 11). Sequence data were quality filtered and denoised using the DADA2 (Non-Patent Document 12) pipeline. Filtered output sequences were assigned to taxa using the "qiime feature-classifier classify-sklearn" command (Non-Patent Documents 13, 14). Silva SSU Ref Nr 99 (ver. 138) was used as the reference database for taxonomic assignment (Non-Patent Document 15).

[0044] Significant differences were observed in certain bacterial groups between the Ctrl and Isolation groups. (1) The phylum Verrucomicrobiota; (2) the genera Akkermansia, Alloprevotella, Roseburia, or Tidjanibacter; (3) Akkermansia muciniphila, Ruminococcus callidus, Parabacteroides distasonis, or Bacteroides acidfaciens; and (4) Phocaeicola_A_858004, Alistipes_A_871400, Alistipes_A_871404, Parabacteroides_B_862066, Bacteroides_H, Muribaculaceae_UBA3263, Peptostreptococcaceae_256921, or Ruminococcus_C_58660. The occupancy rates of these bacterial groups were lower in the Isolation group than in the Ctrl group (Figures 3–5). In contrast, for bacterial groups belonging to (2) the genus Schaedlerella, the genus Ligilactobacillus, or the family Lacnospiraceae, and (4) Muribaculaceae_UBA7173, Muribaculaceae_CAG485, or Lacnospiraceae_14-2, the occupancy rate was higher in the Isolation group than in the Ctrl group (Figures 6-7). [Industrial Applicability]

[0045] The present invention is capable of objectively evaluating social stress and can therefore be used as a method for evaluating social stress, an evaluation kit, a method for evaluating agents for alleviating social stress, and the like.

Claims

1. A method for social stress assessment using as an index the content of one or more genes or gene by-products contained in the Nan gene cluster in a fecal sample of a subject.

2. The method for social stress assessment according to Claim 1, wherein the content of one or more genes or gene by-products contained in the Nan gene cluster in a fecal sample of a subject is measured, and when the content is equal to or higher than a reference value, the subject is evaluated as being under high stress.

3. One or more of the following (1) to (4) in the gut microbiota of a subject; (1) Phylum Verrucomicrobiota (2) Genus Akkermansia, Genus Alloprevotella, Genus Tidjanibacter, Genus Schaedlerella, Genus Ligilactobacillus, Genus Roseburia, or Family Lacnospiraceae (3) Akkermansia muciniphila, Ruminococcus callidus, Parabacteroides distasonis, or Bacteroides acidfaciens (4) Phocaeicola_A_858004, Alistipes_A_871400, Alistipes_A_871404, Parabacteroides_B_862066, Bacteroides_H, Muribaculaceae_UBA3263, Peptostreptococcaceae_256921, Muribaculaceae_UBA7173, Muribaculaceae_CAG485, Lacnospiraceae_14-2, or Ruminococcus_C_58660 A method for social stress assessment using as an index one or more of the occupancy rates of any of the bacterial groups.

4. One or more of the following (1) to (4) in the gut microbiota of a subject; (1) Phylum Verrucomicrobiota (2) Genus Akkermansia, Genus Alloprevotella, Genus Roseburia, or Genus Tidjanibacter (3) Akkermansia muciniphila, Ruminococcus callidus, Parabacteroides distasonis, or Bacteroides acidfaciens (4) Phocaeicola_A_858004, Alistipes_A_871400, Alistipes_A_871404, Parabacteroides_B_862066, Bacteroides_H, Muribaculaceae_UBA3263, Peptostreptococcaceae_256921 or Ruminococcus_C_58660 The social stress evaluation method according to claim 3, wherein one or more of the occupancy rates of any of the bacterial flora are measured, and when the occupancy rate is less than or equal to a reference value, the subject is evaluated as being under high stress.

5. The following (2) and (4) in the gut microbiota of the subject; (2) The genus Schaedlerella, the genus Ligilactobacillus or the family Lacnospiraceae (4) Muribaculaceae_UBA7173, Muribaculaceae_CAG485 or Lacnospiraceae_14-2 The social stress evaluation method according to claim 3, wherein one or more of the occupancy rates of any of the bacterial flora are measured, and when the occupancy rate is greater than or equal to a reference value, the subject is evaluated as being under high stress.

6. A social stress marker comprising one or more genes contained in the Nan gene cluster or gene by-products thereof.

7. A social stress evaluation kit comprising a reagent for measuring one or more genes contained in the Nan gene cluster or gene by-products thereof in a fecal sample of a subject.

8. A method for evaluating a social stress reliever, comprising measuring one or more genes contained in the Nan gene cluster or gene by-products thereof in a fecal sample of a subject administered with a test substance.

9. The following (1) to (4) in the gut microbiota of a subject administered with a test substance; (1) The phylum Verrucomicrobiota (2) The genus Akkermansia, the genus Alloprevotella, the genus Tidjanibacter, the genus Schaedlerella, the genus Ligilactobacillus, the genus Roseburia or the family Lacnospiraceae (3) Akkermansia muciniphila, Ruminococcus callidus, Parabacteroides distasonis or Bacteroides acidfaciens (4) Phocaeicola_A_858004, Alistipes_A_871400, Alistipes_A_871404, Parabacteroides_B_862066, Bacteroides_H, Muribaculaceae_UBA3263, Peptostreptococcaceae_256921, Muribaculaceae_UBA7173, Muribaculaceae_CAG485, Lacnospiraceae_14-2 or Ruminococcus_C_58660 A method for evaluating a social stress reliever, comprising measuring one or more of the occupancy rates of any of the above bacterial flora

10. A method for assisting in the diagnosis of PTSD or depression, using as an index the content of one or more genes or gene by-products contained in the Nan gene cluster in a fecal sample of a subject.

11. The following (1) to (4) in the gut microbiota; (1) The phylum Verrucomicrobiota (2) The genus Akkermansia, Alloprevotella, Tidjanibacter, Schaedlerella, Ligilactobacillus, Roseburia or the family Lacnospiraceae (3) Akkermansia muciniphila, Ruminococcus callidus, Parabacteroides distasonis or Bacteroides acidfaciens (4) Phocaeicola_A_858004, Alistipes_A_871400, Alistipes_A_871404, Parabacteroides_B_862066, Bacteroides_H, Muribaculaceae_UBA3263, Peptostreptococcaceae_256921, Muribaculaceae_UBA7173, Muribaculaceae_CAG485, Lacnospiraceae_14-2 or Ruminococcus_C_58660 A method for assisting in the diagnosis of PTSD or depression, using as an index one or more of the occupancy rates of any of the above bacterial flora.

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