{"paper_id":"17b42d0f-9138-4dd1-908a-015c2bb4dddb","body_text":"Probiotic strain released-carbamoyl phosphates modify corticosterone metabolism on colonic epithelial cells by elevating 11-beta-hydroxysteroid dehydrogenase isozyme 2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Probiotic strain released-carbamoyl phosphates modify corticosterone metabolism on colonic epithelial cells by elevating 11-beta-hydroxysteroid dehydrogenase isozyme 2 Sohei Arase, Kosuke Oana, Takashi Kurakawa, Tetsuji Hori, Satoshi Matsumoto This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7258444/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chronic psychological stress contributes to functional disorder development, including irritable bowel syndrome (IBS). Although probiotics have shown potential in ameliorating these disorders, the precise mechanisms remain incompletely understood. The aim of this study was to elucidate the mechanism underlying the effect of Lacticaseibacillus paracasei strain Shirota (LcS) on the host anti-stress response in a colonic epithelial cell line. The expression of the stress hormone-degrading enzyme, 11 β-hydroxysteroid dehydrogenase type 2 ( Hsd11b2 ), was suppressed by corticosterone and restored by LcS treatment. Fractionation of the LcS culture supernatant revealed a derivative of carbamoyl phosphate as the key factor responsible for inducing Hsd11b2 . Moreover, activation of acetylcholine receptor and inhibition of NF-κB p50 homodimer nuclear translocation were required to induce Hsd11b2 in colonic epithelial cells. These findings reveal a novel probiotic mechanism whereby an LcS metabolite triggers anti-stress responses, including Hsd11b2 induction, by modulating the acetylcholine and NF-κB pathways. This new mechanism by which probiotics can stimulate anti-stress effects in the colonic mucosa may contribute to IBS treatment. Biological sciences/Biochemistry Biological sciences/Cell biology Biological sciences/Microbiology Biological sciences/Molecular biology Biological sciences/Physiology psychological stress corticosterone colonic epithelial cells HSD11B2 acetylcholine NF-κB Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Psychological stress ranks second among global health concerns, with a global survey reporting that 63% of respondents experienced psychological stress in the past year, negatively impacting their daily lives 1 . Psychological stress responses occur within the central nervous system and peripheral tissues 2 . In the neuroendocrine system, glucocorticoids are released from the adrenal cortex by activating the hypothalamic–pituitary–adrenal (HPA) axis 3 . Chronic HPA axis activation or prolonged exposure to elevated glucocorticoid levels can cause stress hormone-driven gastrointestinal dysfunction 4 , 5 , 6 , 7 , 8 . These functional disorders are closely associated with chronic intestinal diseases, such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). In patients with IBS, psychological stress intensifies colonic contractile and motor activities 9 ; whereas, in IBD, it exacerbates disease activity 10 . Several studies in humans and animals suggest that psychological stress also impacts the gut environment, including alterations in the microbiota 11 , 12 . Indeed, the composition of the gut microbiota differs between patients with IBS and healthy adults 13 . In patients with IBD, compositional changes in gut microbiota exacerbate intestinal inflammation 14 . Thus, effective strategies to prevent and mitigate physiological stress responses are needed. Probiotics exert a range of physiological effects by improving the intestinal microbiota composition or promoting host tolerance to specific microbial strains or their metabolites 15 , 16 . Accordingly, oral administration of probiotics is often recommended in treatment guidelines for IBS 17 , 18 . Lacticaseibacillus paracasei strain Shirota (LcS, previously known as Lactobacillus casei strain Shirota) is among the most widely used probiotic strains globally. LcS has demonstrated myriad beneficial effects on the host’s gut microbiota composition, intestinal function, and immune responses. For example, LcS supplementation can modify defecation frequency and stool quality in healthy adults with soft stools 19 . Additionally, LcS can elicit immunomodulatory effects by enhancing natural killer (NK) cell activity in healthy individuals 20 , and alleviating IBD and colitis-associated cancer in murine models 21 . LcS also modulates psychological homeostasis via the gut–brain axis. For example, LcS has been shown to ameliorate sleep disorders in healthy adults experiencing academic examination stress 22 and enhance daytime cognitive performance in healthy office workers 23 . Moreover, in our previous study using murine model, we demonstrated that LcS suppressed increases in fecal water content and disruption of the mucosal microbiota under chronic psychological stress 24 . It also suppressed the increase in stress hormone levels. This led to the hypothesis that LcS may directly regulates stress-mediated dysfunction in intestinal epithelial cells. However, the specific LcS-derived components or metabolites responsible for these anti-stress responses remain unknown. The primary objective of the current study was to identify the key LcS-derived component(s) capable of suppressing stress hormone production in the host. To this end, an in vitro monolayer intestinal epithelial culture system was employed. Moreover, the molecular pathway(s) underlying the effect of LcS on the host anti-stress response was investigated. The findings of this investigation will inform the development of effective treatments for functional stress-related disorders such as IBS. Results Proteome analysis identifies HSD11B2 as a candidate anti-stress factor In our previous study 24 , colonic epithelial tissues were isolated from mice in the following groups: (a) placebo under no stress (Control + Placebo group), (b) placebo under chronic psychological stress (Stress + Placebo group), (c) LcS under chronic psychological stress (Stress + LcS group). To identify the molecular mechanism underlying the protective effect of long-term LcS administration on chronic stress in the gut, quantitative proteomic analysis of the murine colonic epithelial cells was performed. Overall, 14,367 distinct peptides were detected across all three groups and assigned to 1,092 proteins via a homology search (Table 1 ). Among these 1,092 proteins, 11 were downregulated and five were upregulated in the Stress + Placebo group by more than 1.5-fold compared with levels in the Control + Placebo group (Table 1 ). However, these alterations were reversed in the Stress + LcS group. Among the 16 differentially expressed proteins, 11 β-hydroxysteroid dehydrogenase type 2 (HSD11B2) was selected for further evaluation, as it is an enzyme that degrades corticosterone, the representative stress hormone 25 . The proteomic level of HSD11B2 was decreased by psychological stress stimuli but recovered by LcS administration. This suggests that HSD11B2 is a key mediator of stress response regulation in colonic epithelial cells. Table 1 Proteins affected by psychological stress and LcS detected by LC-TOF/MS Relative expression § No. Expression change * Total score † Protein name Accession No. ‡ Stress placebo Stress LcS 1 Down 10.71 Corticosteroid 11-beta-dehydrogenase isozyme 2 sp|P51661|DHI2 0.64 1.08 2 Down 7.73 Calcium-activated chloride channel regulator 1 sp|Q9D7Z6|CLCA1 0.67 1.28 3 Down 6.26 Solute carrier family 22-member 18 sp|Q78KK3|S22AI 0.67 1.81 4 Down 4.6 Peptidyl-prolyl cis-trans isomerase FKBP4 sp|P30416|FKBP4 0.74 2.54 5 Down 4.07 Elongation factor 1-delta sp|P57776|EF1D 0.12 0.75 6 Down 2.97 Nucleolar protein 56 sp|Q9D6Z1|NOP56 0.22 0.83 7 Down 1.82 Choline transporter-like protein 4 sp|Q91VA1|CTL4 0.36 1.24 8 Down 1.12 4-aminobutyrate aminotransferase sp|P61922|GABT 0.48 1.12 9 Down 1.12 Ubiquitin-conjugating enzyme E2 D2B sp|Q6ZWY6|U2D2B 0.34 3.03 10 Down 2.86 Ras-related protein Rab-5A sp|Q9CQD1|RAB5A 0.59 0.90 11 Down 0.96 Aquaporin-8 sp|P56404|AQP8 0.56 0.99 12 Up 12.93 Annexin A11 sp|P97384|ANX11 5.54 3.49 13 Up 4.08 Peroxiredoxin-5 sp|P99029|PRDX5 6.73 3.73 14 Up 4.17 Bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase 1 sp|Q60967|PAPS1 2.22 1.10 15 Up 1.16 RNA-binding protein 47 sp|Q91WT8|RBM47 2.23 1.31 16 Up 0.2 Coactosin-like protein sp|Q9CQI6|COTL1 1.58 0.63 * Proteins expressed in the Stress + Placebo group are listed that differed more than 1.5-fold in their expression from that in the Control + Placebo group. No. 1–11 were downregulated (Down) and No.12–16 were upregulated (Up) relative to the Control + Placebo group and are listed in order of the total score calculated by ProteinPilot. All other proteins detected are shown in Supplementary File 1. † Total score was identified based on quality parameters given by ProteinPilot. ‡ Accession No. in Uniprot. § Data were calculated as relative expression values relative to the Control + Placebo group and are presented as means of n = 2. Stress + Placebo: mice loaded with stress and administered a placebo. Stress + LcS: mice loaded with stress and administered LcS. Details of the experimental conditions are available in the previous report. Establishment of a novel cell line with normal colonic epithelial cell characteristics Commercially available versatile colon epithelial cell lines (i.e., CMT-93 and Caco-2) are frequently used for in vitro investigations. However, these cells were originally derived from colorectal cancer tissues, which may not accurately represent the physiology of normal intestinal epithelial cells. Meanwhile, given that the aim of the current study was to evaluate the influence of stress hormones and LcS on normal epithelial cells, a cell line with an intact intercellular tight junction structure that expresses glycoproteins and forms mucin layers was required. Accordingly, a new cell line, known as M3C-1B9, was cloned from the healthy mucosa tissue of B6.129P2-TcraTrp53/Yit mice. M3C-1B9 cells highly express several glycoproteins or glycolipids, including asialo GM1, (GA1), Dolichos biflorus agglutinin (DBA), fucosylated asialo GM1 (FGA1), and Ulex europaeus agglutinin-1, (UEA-1) that are abundantly expressed by murine normal colonic epithelial cells 26 . In contrast, CMT-93 cells lacked the expression of these proteins or lipids (Fig. 1 A, B, Figure S1 A). The expression of glycolipid synthesis genes, including glutaric aciduria type 1 ( Ga1 ) and fucosyltransferase 2 ( Fut2 ), was also higher in the M3C-1B9 cells than in CMT-93 cells (Fig. 1 C). Additionally, M3C-1B9 cells exhibited elevated expression of Muc2 and distinct expression profiles of tight junction-associated genes, such as E-cadherin ( Cdh1 ) and Claudin-1/2 ( Cldn1/2 ) from CMT-93 cells (Figure S1 B, C). Genes associated with protecting the colon mucosa from bacterial infection, namely, Toll-like receptor 5 (Tlr5) 27 , NADPH oxidase 1 (Nox1 ) 28 , and defensin beta 1 (Defb1 ) 29 , were also highly expressed in M3C-1B9 cells (Figure S1 C). Furthermore, the trans-epithelial electrical resistance (TEER) in M3C-1B9 cells—an indicator of epithelial cell barrier function—was significantly higher than that in CMT-93 cells (Fig. 1 D), indicating that M3C-1B9 cells have robust intercellular tight junction structures. In the current study, M3C-1B9 cells were cultured on cell culture inserts to evaluate the effect of LcS on the physiological stress response of normal colon epithelial cells. Using this culture method, cells can be accessed from the upper and lower layers, mimicking the luminal and serosal sides of the intestinal epithelial environment (Fig. 1 E). Exposure to corticosterone in the lower layer of the M3C-1B9 model, resulted in downregulation of Hsd11b2 expression compared with that in non-treated cells (Fig. 1 F). Next, to analyze the effect of LcS on the stress-induced intestinal epithelial cells, either live LcS cells or their culture supernatant was added to the upper layer of the M3C-1B9 model with corticosterone in the lower layer. Both treatments significantly increased Hsd11b2 expression, suggesting a positive effect on mitigating corticosterone-induced stress (Fig. 1 F). Identification of the active compounds in LcS responsible for upregulating Hsd11b2 expression To identify the bioactive compound(s) responsible for the induction of Hsd11b2 expression, the LcS culture supernatant was fractionated using gel filtration chromatography according to molecular weight (MW): < 3000 (Cort-LcSsup_un3k) and > 3000 (Cort-LcSsup_up3k). Each fraction was added separately to the upper layer of the M3C-1B9 model with corticosterone in the lower layer. Hsd11b2 expression in M3C-1B9 cells was evaluated via RT-PCR. Cort-LcSsup_un3k induced Hsd11b2 expression, whereas Cort-LcSsup_up3k did not (Fig. 2 A). The Cort-LcSsup_un3k was further fractionated via gel filtration chromatography, yielding 125 fractions. Four major peaks were identified corresponding to fractions A (No. 17–31), B (No. 38–44), C (No. 80–89), and D (No. 90–100) (Fig. 2 B). These four fractions were separately added to the upper layer of the M3C-1B9 model, with the corticosterone in the lower layer. Fraction D restored Hsd11b2 expression to levels similar to those observed with the LcS culture supernatant before fractionation (Fig. 2 C). In contrast, the other three fractions (A, B, and C) did not induce Hsd11b2 expression, whereas the molecules responsible for inducing Hsd11b2 were found within fraction D. Fraction D was concentrated via centrifugation and subjected to LC-TOF/MS. The compounds were estimated based on the elution time and MS/MS spectral data in both negative and positive ion modes and listed in order of increasing detection intensity (Fig. 2 D, Table S1 ). Among them, methyl N-(3-methoxyphenyl) carbamate was detected in both ion modes (Fig. 2 D). Carbamate-deficient LcS does not induce Hsd11b2 expression The effect of methyl N-(3-methoxyphenyl) carbamate on Hsd11b2 expression was assessed in M3C-1B9 cells with corticosterone. Treatment with methyl N-(3-methoxyphenyl) carbamate increased Hsd11b2 expression to levels similar to those observed the LcS culture supernatant (Fig. 3 A) in a dose-dependent manner (Figure S2 ). Methyl N-(3-methoxyphenyl) carbamate possesses carbamoyl groups. Hence, based on the metabolic pathway of the L. paracasei type strain American Type Culture Collection (ATCC) 334, the carbamates were presumed to be derivatives of carbamoyl phosphate, such as monoamines and polyamines (Fig. 3 B). Similar to L. paracasei ATCC 334, LcS possesses the carbamoyl phosphate synthase gene ( Carb ) (DDBJ Accession ID: LC859584). Thus, by inhibiting the synthesis of carbamoyl phosphate, the production of carbamates was expected to be suppressed. To test this, a mutant LcS strain lacking Carb was created. The culture supernatant of the mutant strain was added to the upper layer of M3C-1B9 cells with corticosterone in the lower layer. The mutant strain failed to induce Hsd11b2 expression in the M3C-1B9 with corticosterone, unlike the wild-type strain (Fig. 3 C). Thus, the carbamoyl phosphate or its derivatives were presumed to be major compounds responsible for upregulating Hsd11b2 expression in colonic epithelial cells. The non-neuronal acetylcholine pathway contributes to Hsd11b2 upregulation Several carbamate compounds inhibit acetylcholinesterase in the nervous system 30 . Considering that M3C-1B9 cells also express acetylcholine receptors (Figure S3 ), acetylcholine was expected to influence the physiological effects on non-neuronal cells. Notably, expression of muscarinic acetylcholine receptor subtypes Chrm3 and Chrm4 was upregulated in M3C-1B9 cells following corticosterone exposure (Figure S3 ). To evaluate the involvement of the acetylcholine cascade in Hsd11b2 induction, M3C-1B9 cells were pretreated with atropine—an acetylcholine receptor antagonist—in both upper and lower layers before adding LcS culture supernatants or methyl N-(3-methoxyphenyl) carbamate to the upper layer (Fig. 4 A, B). The upregulation of Hsd11b2 expression mediated by LcS or methyl N-(3-methoxyphenyl) carbamate was abolished by atropine pretreatment, indicating that activation of the acetylcholine receptor is required for Hsd11b2 upregulation in colonic epithelial cells. Additionally, acetylcholine production and release by M3C-1B9 cells were analyzed upon LcS addition. LcS increased the intracellular concentration of acetylcholine in M3C-1B9 cells (Fig. 4 C). Meanwhile, the extracellular concentration of acetylcholine was significantly increased in the lower layer of the M3C-1B9 cell culture model, not in the upper layer (Fig. 4 D). Moreover, the expression of choline acetyltransferase ( Chat ) and carnitine acetyltransferase ( Crat ), which induce acetylcholine production, was also increased in the M3C-1B9 cells following LcS addition (Fig. 4 E, F). These results suggested that LcS induces the release of acetylcholine from the basolateral layer of intestinal epithelial cells and stimulates acetylcholine signaling under stress conditions. NF-κB signaling induced by glucocorticoid receptor nuclear translocation impacts the anti-stress response Glucocorticoid receptor (GR) complexes are generally formed upon binding glucocorticoids, such as corticosterone. Subsequently, GR undergoes nuclear translocation, regulates the transcription of various genes and acts as a potent repressor of NF-κB signaling 31 . Addition of corticosterone to M3C-1B9 cells induced the nuclear translocation of the GR complex (Figure S4 A, B). Conversely, treating M3C-1B9 cells with LcS suppressed both GR nuclear translocation and the expression of nuclear receptor subfamily 3, group C, member 1 ( Nr3c1) , the gene encoding GR (Figure S4 C). Next, the effects of corticosterone and LcS on NF-κB signaling were evaluated. M3C-1B9 cells supplemented with corticosterone in the lower layer with or without LcS in the upper layer were periodically sampled. Fluctuations in the protein levels of NF-κB subunits in the nucleus and cytoplasm were analyzed via ELISA. Exposing M3C-1B9 cells to corticosterone significantly increased p50 expression in the nucleus after culturing for 2 and 4 h regardless of LcS addition; no change was observed in the cytoplasm (Fig. 5 A, B). Meanwhile, the persistent increase in nuclear p50 expression by corticosterone after 24 hours culture was significantly suppressed by LcS treatment (Fig. 5 A). LcS also increased IκB expression, suppressing NF-κB activation (Figure S5 ). However, additional LcS had no significant effect on the nuclear or cytoplasmic levels of the p65 subunit—another NF-κB subunit (Fig. 5 C, D). Moreover, blocking the acetylcholine cascade with atropine suppressed the effect of LcS on nuclear p50 expression (Fig. 5 E, F). Thus, it was suggested that the p50 homodimers, rather than p65, were translocated to the nucleus by corticosterone, whereaa LcS inhibited this process. Based on the collective results, a hypothesis was generated to describe how corticosterone and LcS mediate Hsd11b2 expression (Fig. 6 ). Under stress conditions, corticosterone binds to GR, facilitating its nuclear translocation and promoting p50 transcription. Concurrently, the p50 homodimers accumulate in the cytoplasm and translocate to the nucleus, repressing Hsd11b2 transcription. However, in the presence of viable LcS, carbamates produced by LcS promote acetylcholine synthesis, activating the acetylcholine receptor. The acetylcholine receptor cascade upregulates IκB expression while downregulating that of GR, thereby inhibiting the formation of p50 homodimers. Consequently, NF-κB signaling is suppressed, and Hsd11b2 transcription is ultimately repressed. Discussion In this study, HSD11B2 was identified as a key factor associated with host stress responses. HSD11B2 promotes the degradation of stress hormones in the colonic epithelium, potentially acting as an important suppressor of stress hormone-mediated stress responses. To clarify the bioactive components of LcS and the molecular mechanisms underlying stress hormone degradation in colonic epithelial cells, we fractionated the active components of LcS and evaluated their effect on Hsd11b2 expression in vitro . We also investigated the intracellular signaling pathway regulating Hsd11b2 expression. This is the first study to demonstrate corticosterone-induced downregulation of Hsd11b2 expression in the intestinal tract, implicating it as a key factor in host anti-stress effects. Other proteins (Table 1 ) also exhibited potential effects on the anti-stress response. For example, peptidyl-prolyl cis-trans isomerase (FKBP4), which participates in GR inactivation, was downregulated and may be involved in stress-induced GR activation 32 . Moreover, Aquaporin-8, which is involved in intestinal water regulation, was also decreased following LcS ingestion, suggesting a role in regulating intestinal stress responses associated with stool consistency 33 . In addition, choline transporter-like protein 4 may be involved in choline uptake, an acetylcholine substrate 34 Further investigation into the roles of these proteins in the LcS-associated anti-stress response is warranted. The current study required a normal colonic epithelial cell line to elucidate the molecular mechanism of colonic psychological stress response in vitro . Although primary culture systems and immortalized cells (e.g., those in which SV40 large T antigen or immortalization genes, such as telomerase reverse transcriptase [ TERT] ) are frequently used as intestinal epithelial cell lines in in vitro studies 35 – 37 , these systems have limitations. Primary culture systems are limited by their inability to culture cells long-term and the associated lot-to-lot variability of the cultured cells. Immortalized cells transfected with SV40 or viral vectors carrying immortalization genes may lack normal physiological characteristics and are not commercially available. To overcome these limitations, an immortalized normal colonic epithelial cell line, M3C-1B9, was established in this study. M3C-1B9 cells exhibited superior characteristics of normal colonic epithelial cells than the commonly used rectal cancer-derived CMT-93 cell line. Hence, M3C-1B9 cells are predicted to be a suitable model for evaluating the effects of intestinal microbiota, including probiotics, on normal colonic epithelial cells. We identified the carbamates as active compounds of LcS for increased expression of Hsd11b2. Carbamate is a class of compounds in which an amino group reacts with a hydroxyl group via a carbonyl moiety, forming a covalent bond between the nitrogen of the amine and the carbon of the carbonyl 38 . Several carbamate derivatives have been reported to increase the amount of acetylcholine in neurons by inhibiting acetylcholinesterase (AChE), an acetylcholine-degrading enzyme 30 However, no previous study has reported that intestinal bacteria produce carbamates. Polyamines (e.g., putrescine, agmatine, spermidine, and spermine) and GABA are derivatives of carbamoyl phosphate. Various bacteria, including Lactobacilli can metabolize polyamines 39 , 40 . Among these derivatives, putrescine has been reported to promote intestinal epithelial cell differentiation 39 . Moreover, exosomes released from intestinal epithelial cells in response to GABA contribute to neuronal cell activation and anti-stress responses, including decreasing corticosterone levels and improving anxiety behavior 41 . The results of the current study suggest that the LcS-derived carbamate increases Hsd11b2 expression in intestinal epithelial cells. Given that several carbamates function as AChE inhibitors, the acetylcholine cascade was selected as a potential major pathway driven by LcS. In the alimentary tract, acetylcholine functions primarily as a parasympathetic neurotransmitter that enhances intestinal motility 42 . However, acetylcholine is also released by non-neuronal cells and participates in intestinal epithelial cell differentiation and T-cell activation 43 . In the current study, acetylcholine was synthesized in intestinal epithelial cells and secreted in response to LcS treatment. Moreover, muscarinic acetylcholine receptors were expressed on intestinal epithelial cells and downstream intracellular cascade was activated upon acetylcholine binding. Considering the extremely short half-life of the released acetylcholine and the action of the degrading enzyme esterase, it is likely that the released acetylcholine exerts its effects on nearby cells, including immune cells in the mucosal intrinsic layer, intestinal endogenous neurons extending into the epithelia, and the neighboring epithelial cells. In this study, LcS suppressed NF-κB signaling via inhibiting the formation of p50 homodimers, and repressing Hsd11b2 transcription. Several studies have reported an association between psychological stress and the activation of NF-κB signaling pathways 44 , 45 . In the GR cascade, the activated GR complex binds to the GR element (GRE) sequence in the promoter regions of target genes, including Nfkb1 (p50) and Rela (p65). Hence, the regulation of these target genes is modulated by corticosterone levels or the presence of co-stimulators such as lipopolysaccharide 31 , 46 . Generally, the NF-κB complex is formed as a p50/p65 heterodimer or a p50/p50 homodimer and influences the transcription of target genes in the canonical NF-κB pathway 47 . Sustained activation of NF-κB signaling results in the NF-κB complex binding to the Hsd11b2 promoter, stimulating the transition from a p65/p50 heterodimer to a p50/p50 homodimer 48 . Consequently, Hsd11b2 transcription is repressed via p50/p50 homodimer binding. This aligns with the results of the current study: exposure to corticosterone induced formation of the p50/p50 homodimer, resulting in repression of Hsd11b2 transcription. Moreover, following its nuclear translocation, GR promoted the expression of p50, but not p65. Consistent with these findings, a recent study reported elevated p50 expression and altered localization in the intestines of patients with IBS compared to healthy controls 49 . Hence, increased p50 expression may be associated with an early inflammatory event and IBS pathogenesis. In the present study, LcS treatment with the M3C-1B9 cell model suppressed the stress-induced expression of p50 and its nuclear translocation. Interestingly, a carbamate analog has been shown to exert NF-κB inhibitory activity 50 , supporting our hypothesis. Two mechanisms underlying this NF-κB inhibitory effect were characterized in this study. First, LcS suppressed the expression and nuclear translocation of GR, preventing the transcriptional activation of Nfkb1 . Second, the expression of Ikb subunits ( Nfkbia , Nfkbib , and Nfkbiz ) was upregulated by LcS. Each IκB subunit negatively regulates NF-κB signaling through different mechanisms based on their cellular localization 51 . Specifically, the current study results suggest that increased IκBa and IκBb expression suppressed nuclear translocation of the p50/p50 homodimer, whereas IκBz likely inhibited the binding of the p50/p50 homodimer to the Hsd11b2 promoter, presumably alleviating Hsd11b2 downregulation. Despite the valuable insights provided by this study, additional analyses with clinical specimens are required, as the current study was performed exclusively using an in vitro model. Further investigations using clinical specimens is necessary to confirm the relevance of HSD11B2 in human colonic epithelium and determine how its expression is altered by intestinal disorders such as IBS. The metabolites produced by LcS involved in upregulating Hsd11b2 are not limited to the carbamate, methyl N-(3-methoxyphenyl) carbamate), identified in this study, but may also include other carbamoyl phosphate derivatives (e.g., polyamines). Further investigation is required to determine whether other carbamates or polyamines exert similar effects. In conclusion, it is hypothesized that metabolites produced by LcS, including derivatives of carbamoyl phosphate, promote corticosterone degradation in intestinal epithelial cells by activating the acetylcholine cascade and suppressing NF-κB signaling. Hence, the probiotic LcS was postulated to suppress corticosterone-driven stress responses in colonic epithelial cells, and this novel mechanism may contribute to the therapeutic strategies for IBS. Materials and Methods Cell Lines The M3C-1B9 cell line was previously established from the crypt epithelial cells of healthy mucosal tissue, as described in the Patent Publication No. 2023-146683. The CMT-93 rectal carcinoma cell line was obtained from the ATCC (CCL-223). Bacterial strain LcS was obtained from laboratory stock (strain YIT 9029). Proteomic analysis Colonic epithelial tissues were collected from 20-week-old female C57BL/6 mice in a previous study 24 and stored at − 80°C until proteomic analysis. The experimental protocols in the previous animal study were reviewed by the Institutional Animal Care and Use Committee of Yakult Central Institute and approved by the Director of Yakult Central Institute (Approval Numbers: 13–0091, 14–0130). All procedures involving animals were conducted in compliance with the Japanese Law for the Humane Treatment and Management of Animals (Law No. 105, issued on October 1, 1973). The study is reported in accordance to ARRIVE guidelines. Mice were assigned into the following three groups: (a) Control + Placebo group (ingested placebo for 12 weeks under no stress), (b) Stress + Placebo group (ingested placebo for 12 weeks under chronic psychological stress by loading 1 h water avoidance stress per day), and (c) Stress + LcS group (ingested LcS for 12 weeks under chronic psychological stress by loading 1 h water avoidance stress per day). Colonic epithelial cells from each mouse group were dissociated using EDTA treatment, as previously described 24 . The isolated cells were mixed with 10 mM formic acid (Sigma-Aldrich) and 20 mg alumina (a-Alumina 1–2 mm, Fujifilm Wako), then disrupted by sonication for 5 min using the Picoruptor 2 sonicator (Diagenode). The protein concentrations in the supernatant were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific). The solution containing 200 µg of total protein was centrifuged at 15,000 × g for 20 min, and the supernatant was discarded. Next, 50 mM triethylammonium bicarbonate solution containing 0.5% RapiGest (Waters) was added to the precipitate. The proteins were denatured by sonication for 5 min and incubated at 50°C for 30 min to hydrolyze RapiGest. The iTRAQ reagent kit (8-plex, SCIEX) was used for stable isotope labeling of proteins. The protein solutions were incubated with a reducing disulfide bonds agent at 60°C for 1 h, followed by alkylation with a cysteine-blocking agent at room temperature for 10 min. For enzymatic digestion, 10 mg of Trypsin (Pierce) was added to the protein samples and incubated at 37°C for 16 h. Eight different iTRAQ reagents (isotopic molecular weights: 113, 114, 115, 116, 117, 118, 119, 121) dissolved in dimethylformamide were mixed with each sample and incubated at room temperature for 2 h to label peptides. Next, formic acid was added to a final concentration of 5% and incubated at room temperature for 2 h to inactivate RapiGest. All eight samples were combined and applied to a solid-phase extraction column (Bond Elute Plexa, Agilent), washed with 60% acetonitrile, centrifuged, and concentrated using an evaporator, and redissolved in 5% acetonitrile. The redissolved sample was subjected to LC-TOF/MS (Triple TOF 6600, SCIEX). Each detected peptide peak was matched against the UniProt-Swiss-Prot database using ProteinPilot software (SCIEX) for protein identification. A quantitative comparison of iTRAQ peak intensities between samples was performed with ProteinPilot software. Preparation of LcS and culture supernatant LcS was cultured in 5 mL of De Man–Rogosa–Sharpe (MRS) broth at 37°C for 16 h under aerobic conditions. For LcS collection, the bacterial culture was centrifuged at 8,000 × g for 5 min at 4°C. The supernatant was collected and stored for further analysis. The pellet was washed with PBS and resuspended in 5 mL of 10% fetal calf serum (FCS)-supplemented Advanced DMEM/F12 (aDMEM/F12). Generation of the mutated LcS strain The Carb -deficient LcS strain was generated using a stepwise double-crossover method, as previously described 52 . The estimated 1,000 bp fragments of 5′- and 3′-terminal ends of Carb were amplified using the primer pairs (CarB_A_F and CarB_A_R for the 5’ region, and CarB_B_F and CarB_B_R for the 3’ region) listed in Table 2 . The pYSSE3.1 vector was also amplified to obtain a linear strand vector using the pYSSE3.1F and pYSSE3.1_R primer pair. After agarose electrophoresis of each amplified product, the bands corresponding to the insert and vector were excised, and the DNA fragments were purified using the QIAquick Gel Extraction Kit (Qiagen). The plasmid vector was prepared by ligating the two inserted DNA fragments with the vector via an In-Fusion cloning reaction. The plasmid vector was introduced into competent cells (XL10) via heat shock transformation. The transformants were selected on Lysogeny broth (LB) agar medium containing 500 µg/mL erythromycin and incubated aerobically at 37°C for 18 h. PCR was performed to select colonies carrying the inserted fragments using the 31F and 31R primer pair. Plasmid DNA of the selected colonies was purified using the GenElute Plasmid Miniprep Kit (Sigma-Aldrich). Table 2 List of primers used for generating the gene-disrupted strain of LcS Primer name Sequence (5′–3′) Application pYSSE3.1_F AGAGGATCCCCGGGTACCGAGCT Amplified pYSSE3.1 for in-fusion cloning of upstream and downstream regions of Carb pYSSE3.1_R AGAGTCGACCTGCAGGCATGCAA Carb_A_F ACCCGGGGATCCTCTAGATTTCCCTGGCGTCGGTTTCG Amplified upstream region of Carb Carb_A_R GGCAAGGCGCGATTATCCTTTCTGCGTGCG Carb_B_F AAGGATAATCGCGCCTTGCCATTCAAAGTG Amplified downstream region of Carb Carb_B_R CTGCAGGTCGACTCTAGACCAGTGCGATAATAGGCGTC 31F AGTTGGGTAACGCCAGG Amplified insert fragments 31R GGATAACAATTTCACAC CarB_Sc_F ATTCTTGAAGACGGCAGCGT Amplified flanking fragments upstream and downstream of Carb for in-fusion cloning CarB_Sc_R CAAGAGTTGTGGTCTTATCC CarB_Dc_F TTGGCGAGTGCGAATAGTTG Amplified flanking fragments of Carb CarB_Dc_R ACCTCCTTATGTGTAGGCTG Gene-disrupting plasmid was introduced into LcS by electroporation using Gene Pulser II (Bio-Rad). Plasmid-transfected LcS was plated on MRS agar containing 20 µg/mL erythromycin and incubated aerobically at 37°C for 72 h. Colony PCR was performed to check single crossovers using the CarB_Sc_F and CarB_Sc_R primer pair. Colonies with single crossovers were incubated in MRS liquid medium containing 20 µg/mL erythromycin in aerobic conditions at 37°C for 18 h and smeared on MRS agar medium containing 1 µg/mL vancomycin. As the plasmid vector contains a vancomycin ddl sequence, the success or failure of the double crossover was determined by the presence or absence of colony formation. Final colony PCR was performed using the CarB_Dc_F and CarB_Dc_R primers. Single colony isolation was performed to obtain carB-disrupted strains. Molecular weight fractionation of the LcS culture supernatant The LcS culture supernatant was subjected to molecular weight fractionation using an ultrafiltration spin column (VIVASPIN, Sartorius) with a 3 kDa fractional molecular weight cutoff, and centrifuged at 12,000 × g for 60 min at 4°C. The eluate fraction was collected and supplemented with FCS to a final concentration of 10% for subsequent testing. The components (> 3 kDa), retained on the column filter were resuspended in 10% FCS aDMEM/F12 medium. As a negative control, 10% FCS aDMEM/F12 medium without LcS supernatant was subjected to the same procedure for molecular weight fractionation. Gel filtration chromatography Gel filtration chromatography was performed on a gel carrier (Bio-Gel P2, Bio-Rad) with a molecular weight separation range of 100–1,800. The eluate fraction from the ultrafiltration spin column was concentrated and added to the Gel column, and Milli-Q water was used as the elution solvent. A total of 125 fractions (12 mL/fraction) were obtained. The absorbance of each fraction was measured at 220 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific), and four major peaks were identified. The fractions corresponding to each peak were lyophilized to remove the aqueous solvents, and the lyophilized powder was mixed and resuspended in 10% FCS aDMEM/F12 medium for functional assays. Cell culture insert assay The M3C-1B9 cells were seeded at a density of 5 × 10 4 cells/well onto cell culture inserts (Corning) mounted in 24-well plates (Corning) and cultured in 10% FCS aDMEM/F12 medium) at 37°C under 5% CO 2 . The culture medium was replaced every two days and cells were used for each assay on day 10 of culture. CMT-93 cells were cultured under the same conditions as M3C-1B9 cells. LcS was prepared at a final concentration of 1 × 10 7 CFU/well (1.4 × 10 7 CFU/mL) and added to the upper layer of the cell culture insert. The culture supernatant derived from 1 × 10 7 CFU of LcS was prepared and added to the upper layer of the cell culture insert. To identify the molecules responsible for inducing Hsd11b2 expression, 700 µL of the eluate was added to the upper layer of the insert. The equivalent volume of 10% FCS aDMEM/F12 medium was added as a negative control. Methyl N-(3-methoxyphenyl) carbamate (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO) and added to the upper layer of the insert in the concentration range of 0.001–100 µM. The culture supernatant of CarB -deficient LcS was prepared from a 1 × 10 7 CFU CarB -deficient LcS culture and added to the upper layer of the insert. Atropine (Wako) was dissolved in 10% FCS aDMEM/F12 medium and added to the upper and lower inserts at a final concentration of 10 µM 1 h before LcS addition. Corticosterone (Wako) was diluted in 10% FCS aDMEM/F12 to a final concentration of 30 ng/mL and added to the lower layer of the insert. The concentration was determined based on the plasma corticosterone concentration data from our previously established mice model of psychological stress 24 . RNA extraction and RT-PCR Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription was performed using 1 µg of RNA using 200 U of Superscript II Reverse Transcriptase (Invitrogen). The cDNA was amplified by PCR. Gene expression levels were quantified by RT-PCR. The data were calculated as relative expression, with glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) as the housekeeping gene. The primer pairs used for analysis are listed in Table 3 . Table 3 List of primers used for quantification of gene expression using RT-PCR Gene name Primer (Forward) Primer (Reverse) Reference Gapdh Not available Gene Globe ID: QT01658692 (QIAGEN) Hsd11b2 Not available Gene Globe ID: QT00252609 (QIAGEN) Chat Not available Gene Globe ID: QT00135212 (QIAGEN) Cdh1 Not available Gene Globe ID: QT00121163 (QIAGEN) Cldn1 Not available Gene Globe ID: QT00159278 (QIAGEN Cldn2 Not available Gene Globe ID: QT00261905 (QIAGEN Muc2 Not available Gene Globe ID: QT01060773 (QIAGEN Tlr5 Not available Gene Globe ID: QT00262549 (QIAGEN Nox1 Not available Gene Globe ID: QT00140091 (QIAGEN Defb1 Not available Gene Globe ID: QT00103271 (QIAGEN) Crat AGCTGGCATACTACAGGATCTATGG AGGTGAAACATGCGCAGAGA This study Chrm1 GCAGCAGCTCAGAGAGGTCACAG GATGAAGGCCAGCAGGATGG This study Chrm2 GCGGATCCTGTGGCCAACCAAGAC CGAATTCACGATTTTGCGGGCTA This study Chrm3 AAGGCACGAAACGGTCATCT GCAAACCTCTTAGCCAGCGT This study Chrm4 AGCCGCAGCCGTGTTCACAA TGGGTTGAGGGTTCGTGGCT This study Chrm5 GTCTCCGTCATGACCATACTCTA CCCGTTGTTGAGGTGCTTCTAC This study Nr3c1 ATGAGACTGCCGATTCCTCTGC TGCTTGGAATCTGCCTGAGA This study Nfkbia GCCAGGAATTGCTGAGGCACTT GTCTGCGTCAAGACTGCTACAC This study Nfkbib TCAGCATGAGCCCTTCCTGGAT CAAGGATGGCTGCTAGATGCAG This study Nfkbiz ATCCAGAAGGGAGCTGTGAGGA ATGAGACTGCCGATTCCTCTGC This study LC-TOF/MS analysis Fractions of LcS culture supernatant separated via gel filtration chromatography were lyophilized, resuspended in 100 µL of 10 mM ammonium formate solution (pH 6.5, Sigma-Aldrich), and subjected to LC-TOF/MS (Triple TOF 6600, SCIEX) using a Scherzo SW-C18 (Imtakt) column with a 10 mM ammonium formate solution and acetonitrile (5–65% gradient; Sigma-Aldrich) mobile phase. The LC-TOF/MS data were analyzed using Masterview software (SCIEX). The fractions that significantly increased Hsd11b2 expression in M3C-1B9 cells were compared with inactive fractions, and components with a ≥ 2-fold difference in relative abundance were screened. METLIN ( https://metlin.scripps.edu ) was used to estimate the chemical formula and structure of the detected components. Acetylcholine measurement M3C-1B9 cells were dissociated using TrypLE Express enzyme (Thermo Fisher Scientific) at 37°C for 20 min and collected by centrifugation (500 × g , 5 min). The choline and acetylcholine levels in the cell extracts and the culture supernatant were measured using a Choline/Acetylcholine Assay Kit (Abcam) according to the manufacturer’s instructions. The collected cells were suspended in the assay buffer and sonicated for 5 min to disrupt the cells. Cell extracts were collected after centrifugation (20,000 × g , 5 min) and analyzed for acetylcholine quantification. Extraction of cellular proteins and NF-κB measurement The ProteoExtract Subcellular Proteome Extraction Kit (Merck) was used to fractionate cytoplasmic and nuclear proteins from M3C-1B9 cells. The amounts of p50 and p65 proteins in the cytoplasmic and nuclear fractions were determined using the NF-κB p50 Transcription Factor Assay Kit (Cayman) and the NF-κB p65 Transcription Factor Assay Kit (Cayman), respectively. Quantification and statistical analysis For graphs, data are presented as mean ± SD. Statistical significance was defined as p < 0.05. Dunnett’s test was used to analyze significant differences in gene and protein expression affected by corticosterone, LcS, or its culture supernatant; cells were treated using corticosterone-only group as the reference. Analysis of variance (ANOVA) was performed to analyze differences in NF-κB expression. Graphs were constructed, and statistical analyses were performed using GraphPad Prism. Declarations Competing Interests The authors have declared that no conflict of interest exists. Funding Declaration This work was funded by Yakult Honsha Co., Ltd. The funder provided support in the form of salaries for all authors but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contribution S.A., K.O., T.H., and S.M. designed the study. S.A. and S.M. performed the experiments. S.A. and T.K. wrote the manuscript. Acknowledgement We greatly appreciate Ms. Hiromi Setoyama for assistance with the cell cultures and Dr. Haruo Ikemura for assistance with the proteomic analysis. This work was funded by Yakult Honsha Co., Ltd. The funder provided financial support in the form of salaries for all authors but did not influence the study design, data collection, analysis, preparation of the manuscript, or decision to publish. Data Availability Raw experimental data reported in this paper will be made available by the corresponding author upon reasonable request. This paper analyzes existing, publicly available data. The *Carb* coding sequence of LcS has been deposited in the DNA Data Bank of Japan under Accession No. LC859584. Any additional information required to reanalyze the data reported in this paper is available from the corresponding author upon request. References Ipsos IPSOS Global Health Service Monitor 2022 , (2022). Pereira, V. H., Campos, I. & Sousa, N. The role of autonomic nervous system in susceptibility and resilience to stress. Curr. Opin. Behav. Sci. 14 , 102–107 (2017). Gjerstad, J. K., Lightman, S. L. & Spiga, F. Role of glucocorticoid negative feedback in the regulation of HPA axis pulsatility. Stress 21 , 403–416 (2018). La Torre, D., Van Oudenhove, L., Vanuytsel, T. & Verbeke, K. Psychosocial stress-induced intestinal permeability in healthy humans: What is the evidence? Neurobiol. 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Microbiol. 74 , 4746–4755 (2008). Additional Declarations No competing interests reported. Supplementary Files Fig.S1page0001.jpg Fig.S2page0001.jpg Fig.S3page0001.jpg Fig.S4page0001.jpg Fig.S5page0001.jpg SupplementFigurelegend.docx TableS1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7258444\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":518151723,\"identity\":\"14a705bb-87ac-41de-bd91-80ce6997dad5\",\"order_by\":0,\"name\":\"Sohei 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\\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eHsd11b2\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003eexpression.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A, B) Immunostaining with antibodies against: (A) GA1 (Red) and DBA1 (Green), and (B) FGA1 (Red) with nuclei stained with DAPI (Blue).\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Relative gene expression of \\u003cem\\u003eGa1\\u003c/em\\u003e and \\u003cem\\u003eFut2\\u003c/em\\u003e normalized to \\u003cem\\u003eGapdh \\u003c/em\\u003eas the internal control gene. Data are presented as mean ± SD, \\u003cem\\u003en\\u003c/em\\u003e = 3/group. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 (vs. corticosterone (+) LcS (-)).\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Trans-epithelial electrical resistance (TEER) of the M3C-1B9 and CMT-93 cells periodically measured for 16 days after cell seeding.\\u003c/p\\u003e\\n\\u003cp\\u003e(E) Schematic diagram of the cell culture insert model. M3C-1B9 cells were cultured on the membrane of the insert.\\u003c/p\\u003e\\n\\u003cp\\u003e(F) Relative expression of \\u003cem\\u003eHsd11b2\\u003c/em\\u003e; data normalized to \\u003cem\\u003eGapdh\\u003c/em\\u003e and presented as mean ± SD; \\u003cem\\u003en\\u003c/em\\u003e = 6/group. Corticosterone (+): corticosterone added to the lower medium layer; LcS (+): LcS cells added to the upper medium layer; LcS (Supernatant) (+): LcS culture supernatants added to the upper medium layer. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 (vs. corticosterone (+) LcS (-)).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.1page0001.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/960840d9f1111c1c5956e47f.jpg\"},{\"id\":92203259,\"identity\":\"3d242b04-f04f-42a1-8703-07134b839b50\",\"added_by\":\"auto\",\"created_at\":\"2025-09-25 17:44:43\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":262420,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMolecular weight fractionation of LcS culture supernatants and comprehensive LC-TOF/MS component analysis.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) \\u003cem\\u003eHsd11b2 \\u003c/em\\u003eexpression following treatment with LcS culture supernatant fractions with an MW above or below 3,000 Da added to the upper layer of the M3C-1B9 model with corticosterone. Data are presented as mean ± SD, \\u003cem\\u003en\\u003c/em\\u003e = 3/group; *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 (vs. corticosterone (+) LcS (-)).\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Absorbance at 220 nm of fractions separated from the LcS culture supernatant with MW \\u0026lt; 3,000 Da by gel filtration chromatography. A: fractions No. 17–31, B: 38–44, C: 80–89, D: 90–100.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) \\u003cem\\u003eHsd11b2 \\u003c/em\\u003eexpression following the addition of fractions A–D separately to the upper layer of the M3C model. \\u003cem\\u003eGapdh\\u003c/em\\u003e served as the internal control. Data are presented as mean ± SD, \\u003cem\\u003en\\u003c/em\\u003e = 3/group. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 (vs. corticosterone (+) LcS (-)).\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Comprehensive component analysis performed by LC-TOF/MS with negative and positive ion mode measurements. The top five compounds with the highest intensity in each measurement are listed.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.2page0001.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/e492ed2963a557c2181f8d89.jpg\"},{\"id\":92203261,\"identity\":\"9d18a205-bef6-4466-bf95-4d7680b4cacd\",\"added_by\":\"auto\",\"created_at\":\"2025-09-25 17:44:43\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":166601,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eEffects of carbamate and carbamoyl phosphate on \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eHsd11b2\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003eexpression.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) \\u003cem\\u003eHsd11b2 \\u003c/em\\u003eexpression following methyl N-(3-methoxyphenyl) carbamate or LcS culture supernatant addition to M3C-1B9 cells with corticosterone compared with LcS culture supernatant. \\u003cem\\u003eGapdh\\u003c/em\\u003e served as the internal control. Data are presented as mean ± SD; \\u003cem\\u003en\\u003c/em\\u003e = 3/group. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 (vs. corticosterone (+) LcS (-)).\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Metabolic pathways of carbamoyl phosphate and its derivatives.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) \\u003cem\\u003eHsd11b2 \\u003c/em\\u003eexpression following treatment of M3C-1B9 cells with culture supernatant of LcS or the carbamoyl phosphate synthase gene-deficient mutant with corticosterone. \\u003cem\\u003eGapdh\\u003c/em\\u003e served as the internal control. Data are presented as mean ± SD; \\u003cem\\u003en\\u003c/em\\u003e = 3/group. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 (vs. corticosterone (+) LcS (-)).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.3page0001.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/a0c627915df652ce6c6587e9.jpg\"},{\"id\":92204283,\"identity\":\"25081590-040f-4fe3-b347-aa3dbf88c012\",\"added_by\":\"auto\",\"created_at\":\"2025-09-25 18:00:43\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":141302,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eEffects of LcS on the acetylcholine cascade in M3C-1B9 cells exposed to corticosterone.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A, B) \\u003cem\\u003eHsd11b2 \\u003c/em\\u003eexpression in M3C-1B9 cells pretreated with an acetylcholine receptor antagonist (atropine) followed by treatment with LcS or the carbamate (methyl N-(3-methoxyphenyl) carbamate) and corticosterone. \\u003cem\\u003eGapdh\\u003c/em\\u003e served as the internal control. Data are presented as mean ± SD, \\u003cem\\u003en\\u003c/em\\u003e = 3/group. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 (vs. corticosterone (+) LcS (-) atropine (-)). (C) Intracellular concentrations of acetylcholine in M3C-1B9 cells with or without LcS.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Acetylcholine concentrations in the upper and lower layer of the M3C-1B9 cell culture model with or without LcS. Data are presented as mean ± SD; \\u003cem\\u003en\\u003c/em\\u003e = 5 per group. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 (vs. LcS (-) group).\\u003c/p\\u003e\\n\\u003cp\\u003e(E, F) Relative gene expression of choline acetyltransferase (\\u003cem\\u003eChat\\u003c/em\\u003e) and carnitine acetyltransferase (\\u003cem\\u003eCrat\\u003c/em\\u003enormalized to \\u003cem\\u003eGapdh\\u003c/em\\u003e. Data are presented as mean ± SD, \\u003cem\\u003en\\u003c/em\\u003e = 3/group. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 (vs. LcS (-) group).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.4page0001.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/be24eebc5c7ab10b6f6853de.jpg\"},{\"id\":92203269,\"identity\":\"99b88c6e-21a3-46dc-866e-0da9b7dcc7e0\",\"added_by\":\"auto\",\"created_at\":\"2025-09-25 17:44:43\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":193865,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eEffects of LcS on the nuclear translocation of NF-κB subunits in M3C-1B9 cells.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A–D) Periodical changes in NF-κB subunit proteins: (A) p50 protein in the nucleus, (B) cytoplasmic p50 protein, (C) p65 protein in the nucleus, and (D) cytoplasmic p65 protein. Data are presented as mean ± SD; \\u003cem\\u003en\\u003c/em\\u003e = 4/group. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 (vs. 0 h before adding corticosterone or LcS); \\u003csup\\u003e#\\u003c/sup\\u003e\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 (LcS (+) group vs. LcS (-) group at each time point). LcS (-): corticosterone added to the lower medium layer, LcS (+): corticosterone added to the lower medium layer and LcS cells added to the upper medium layer.\\u003c/p\\u003e\\n\\u003cp\\u003e(E, F) p50 protein levels at 24 h in the nucleus (E) and cytoplasm (F) after adding corticosterone, LcS, or atropine. Data are presented as mean ± SD; \\u003cem\\u003en\\u003c/em\\u003e = 3 per group. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 (vs. corticosterone (-) LcS (-) atropine (-)).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.5page0001.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/890e853a6e908178c4a94937.jpg\"},{\"id\":92202722,\"identity\":\"35086e46-6553-40a7-b8c3-13f52b18a252\",\"added_by\":\"auto\",\"created_at\":\"2025-09-25 17:36:43\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":242560,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSchematic of glucocorticoid-mediated downregulation and LcS-mediated upregulation of \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eHsd11b2 \\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003ein colonic epithelial cells.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(Left) Corticosterone effect: Under stress conditions, corticosterone-bound GR complexes translocate to the nucleus and induce p50 (\\u003cem\\u003eNfkb1\\u003c/em\\u003e) homodimer transcription. The p50 homodimers repress the transcription of \\u003cem\\u003eHsd11b2\\u003c/em\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e(Right) LcS effect: Metabolites of LcS promote acetylcholine production and release in colon epithelial cells. Acetylcholine is accepted by colon epithelial cells, suppressing GR expression, nuclear translocation, and subsequent NF-κB activation by increasing IκB expression. Overall, probiotic treatment induces \\u003cem\\u003eHsd11b2\\u003c/em\\u003etranscription.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.6page0001.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/9d75c39ca691e02530e8aa4d.jpg\"},{\"id\":100363318,\"identity\":\"048ab5c8-97c5-4739-8853-beaa7ca50958\",\"added_by\":\"auto\",\"created_at\":\"2026-01-16 07:49:25\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3046072,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/14443ffb-454c-436f-914b-94ec97436b4e.pdf\"},{\"id\":92202715,\"identity\":\"33abfdd8-1b79-4ba2-bcec-35ac5fe8db37\",\"added_by\":\"auto\",\"created_at\":\"2025-09-25 17:36:43\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":347673,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Fig.S1page0001.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/7ae32bbeac56b178a14e1174.jpg\"},{\"id\":92202713,\"identity\":\"464c8e50-1a83-4386-9033-f9df10f67839\",\"added_by\":\"auto\",\"created_at\":\"2025-09-25 17:36:43\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":31720,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Fig.S2page0001.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/6426342391dd55d121d4b5b6.jpg\"},{\"id\":92203557,\"identity\":\"65adbd46-9025-487f-80fd-d00dd21c26a0\",\"added_by\":\"auto\",\"created_at\":\"2025-09-25 17:52:43\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":52698,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Fig.S3page0001.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/bfa3c9238adcd3985d977792.jpg\"},{\"id\":92203266,\"identity\":\"1dbabd11-d038-4cfb-9984-20bbbd97868f\",\"added_by\":\"auto\",\"created_at\":\"2025-09-25 17:44:43\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":73193,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Fig.S4page0001.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/3f5230295e7c6de09d13bc37.jpg\"},{\"id\":92203267,\"identity\":\"54212b1e-d9ae-4fc0-a0f7-cd4f3c8440b7\",\"added_by\":\"auto\",\"created_at\":\"2025-09-25 17:44:43\",\"extension\":\"jpg\",\"order_by\":7,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":54782,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Fig.S5page0001.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/1834e801cda024f880277622.jpg\"},{\"id\":92203559,\"identity\":\"fa8bec8c-30e3-4794-846b-51cf243aa02d\",\"added_by\":\"auto\",\"created_at\":\"2025-09-25 17:52:43\",\"extension\":\"docx\",\"order_by\":8,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":18262,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementFigurelegend.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/c7923a0d3da1f911fedbdd1e.docx\"},{\"id\":92202725,\"identity\":\"92cb645c-f33b-4b26-9909-8e4c4852537a\",\"added_by\":\"auto\",\"created_at\":\"2025-09-25 17:36:43\",\"extension\":\"docx\",\"order_by\":9,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":24255,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"TableS1.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7258444/v1/d0435cbb8e3e6635e4f6f0ed.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Probiotic strain released-carbamoyl phosphates modify corticosterone metabolism on colonic epithelial cells by elevating 11-beta-hydroxysteroid dehydrogenase isozyme 2\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003ePsychological stress ranks second among global health concerns, with a global survey reporting that 63% of respondents experienced psychological stress in the past year, negatively impacting their daily lives\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. Psychological stress responses occur within the central nervous system and peripheral tissues\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e. In the neuroendocrine system, glucocorticoids are released from the adrenal cortex by activating the hypothalamic\\u0026ndash;pituitary\\u0026ndash;adrenal (HPA) axis\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e. Chronic HPA axis activation or prolonged exposure to elevated glucocorticoid levels can cause stress hormone-driven gastrointestinal dysfunction\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e. These functional disorders are closely associated with chronic intestinal diseases, such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). In patients with IBS, psychological stress intensifies colonic contractile and motor activities\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e; whereas, in IBD, it exacerbates disease activity\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e. Several studies in humans and animals suggest that psychological stress also impacts the gut environment, including alterations in the microbiota\\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e. Indeed, the composition of the gut microbiota differs between patients with IBS and healthy adults\\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e. In patients with IBD, compositional changes in gut microbiota exacerbate intestinal inflammation\\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e. Thus, effective strategies to prevent and mitigate physiological stress responses are needed.\\u003c/p\\u003e\\u003cp\\u003eProbiotics exert a range of physiological effects by improving the intestinal microbiota composition or promoting host tolerance to specific microbial strains or their metabolites\\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e. Accordingly, oral administration of probiotics is often recommended in treatment guidelines for IBS\\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e. \\u003cem\\u003eLacticaseibacillus paracasei\\u003c/em\\u003e strain Shirota (LcS, previously known as \\u003cem\\u003eLactobacillus casei\\u003c/em\\u003e strain Shirota) is among the most widely used probiotic strains globally. LcS has demonstrated myriad beneficial effects on the host\\u0026rsquo;s gut microbiota composition, intestinal function, and immune responses. For example, LcS supplementation can modify defecation frequency and stool quality in healthy adults with soft stools\\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e. Additionally, LcS can elicit immunomodulatory effects by enhancing natural killer (NK) cell activity in healthy individuals\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e, and alleviating IBD and colitis-associated cancer in murine models\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. LcS also modulates psychological homeostasis via the gut\\u0026ndash;brain axis. For example, LcS has been shown to ameliorate sleep disorders in healthy adults experiencing academic examination stress\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u003c/sup\\u003e and enhance daytime cognitive performance in healthy office workers\\u003csup\\u003e\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e. Moreover, in our previous study using murine model, we demonstrated that LcS suppressed increases in fecal water content and disruption of the mucosal microbiota under chronic psychological stress\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e. It also suppressed the increase in stress hormone levels. This led to the hypothesis that LcS may directly regulates stress-mediated dysfunction in intestinal epithelial cells. However, the specific LcS-derived components or metabolites responsible for these anti-stress responses remain unknown.\\u003c/p\\u003e\\u003cp\\u003eThe primary objective of the current study was to identify the key LcS-derived component(s) capable of suppressing stress hormone production in the host. To this end, an \\u003cem\\u003ein vitro\\u003c/em\\u003e monolayer intestinal epithelial culture system was employed. Moreover, the molecular pathway(s) underlying the effect of LcS on the host anti-stress response was investigated. The findings of this investigation will inform the development of effective treatments for functional stress-related disorders such as IBS.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eProteome analysis identifies HSD11B2 as a candidate anti-stress factor\\u003c/h2\\u003e\\u003cp\\u003eIn our previous study\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e, colonic epithelial tissues were isolated from mice in the following groups: (a) placebo under no stress (Control\\u0026thinsp;+\\u0026thinsp;Placebo group), (b) placebo under chronic psychological stress (Stress\\u0026thinsp;+\\u0026thinsp;Placebo group), (c) LcS under chronic psychological stress (Stress\\u0026thinsp;+\\u0026thinsp;LcS group). To identify the molecular mechanism underlying the protective effect of long-term LcS administration on chronic stress in the gut, quantitative proteomic analysis of the murine colonic epithelial cells was performed. Overall, 14,367 distinct peptides were detected across all three groups and assigned to 1,092 proteins via a homology search (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Among these 1,092 proteins, 11 were downregulated and five were upregulated in the Stress\\u0026thinsp;+\\u0026thinsp;Placebo group by more than 1.5-fold compared with levels in the Control\\u0026thinsp;+\\u0026thinsp;Placebo group (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). However, these alterations were reversed in the Stress\\u0026thinsp;+\\u0026thinsp;LcS group. Among the 16 differentially expressed proteins, 11 β-hydroxysteroid dehydrogenase type 2 (HSD11B2) was selected for further evaluation, as it is an enzyme that degrades corticosterone, the representative stress hormone\\u003csup\\u003e\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e. The proteomic level of HSD11B2 was decreased by psychological stress stimuli but recovered by LcS administration. This suggests that HSD11B2 is a key mediator of stress response regulation in colonic epithelial cells.\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eProteins affected by psychological stress and LcS detected by LC-TOF/MS\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"9\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c9\\\" colnum=\\\"9\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c7\\\" namest=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c9\\\" namest=\\\"c8\\\"\\u003e\\u003cp\\u003eRelative expression \\u0026sect;\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eNo.\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eExpression change *\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eTotal\\u003c/p\\u003e\\u003cp\\u003escore \\u0026dagger;\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eProtein name\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003eAccession No. \\u0026Dagger;\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003eStress placebo\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003eStress LcS\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDown\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e10.71\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eCorticosteroid 11-beta-dehydrogenase isozyme 2\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|P51661|DHI2\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e0.64\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e1.08\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e2\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDown\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e7.73\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eCalcium-activated chloride channel regulator 1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|Q9D7Z6|CLCA1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e0.67\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e1.28\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e3\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDown\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e6.26\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eSolute carrier family 22-member 18\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|Q78KK3|S22AI\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e0.67\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e1.81\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e4\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDown\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e4.6\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003ePeptidyl-prolyl cis-trans isomerase FKBP4\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|P30416|FKBP4\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e0.74\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e2.54\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e5\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDown\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e4.07\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eElongation factor 1-delta\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|P57776|EF1D\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e0.12\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e0.75\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e6\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDown\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e2.97\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eNucleolar protein 56\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|Q9D6Z1|NOP56\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e0.22\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e0.83\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e7\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDown\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1.82\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eCholine transporter-like protein 4\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|Q91VA1|CTL4\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e0.36\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e1.24\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e8\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDown\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1.12\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003e4-aminobutyrate aminotransferase\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|P61922|GABT\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e0.48\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e1.12\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e9\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDown\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1.12\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eUbiquitin-conjugating enzyme E2 D2B\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|Q6ZWY6|U2D2B\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e0.34\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e3.03\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e10\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDown\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e2.86\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eRas-related protein Rab-5A\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|Q9CQD1|RAB5A\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e0.59\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e0.90\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e11\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDown\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0.96\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eAquaporin-8\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|P56404|AQP8\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e0.56\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e0.99\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e12\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eUp\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e12.93\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eAnnexin A11\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|P97384|ANX11\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e5.54\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e3.49\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e13\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eUp\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e4.08\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003ePeroxiredoxin-5\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|P99029|PRDX5\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e6.73\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e3.73\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e14\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eUp\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e4.17\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eBifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase 1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|Q60967|PAPS1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e2.22\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e1.10\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e15\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eUp\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1.16\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eRNA-binding protein 47\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|Q91WT8|RBM47\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e2.23\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e1.31\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e16\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eUp\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0.2\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e\\u003cp\\u003eCoactosin-like protein\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003esp|Q9CQI6|COTL1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e\\u003cp\\u003e1.58\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e0.63\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003ctfoot\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"9\\\"\\u003e* Proteins expressed in the Stress\\u0026thinsp;+\\u0026thinsp;Placebo group are listed that differed more than 1.5-fold in their expression from that in the Control\\u0026thinsp;+\\u0026thinsp;Placebo group. No. 1\\u0026ndash;11 were downregulated (Down) and No.12\\u0026ndash;16 were upregulated (Up) relative to the Control\\u0026thinsp;+\\u0026thinsp;Placebo group and are listed in order of the total score calculated by ProteinPilot. All other proteins detected are shown in Supplementary File 1.\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"9\\\"\\u003e\\u0026dagger; Total score was identified based on quality parameters given by ProteinPilot.\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"9\\\"\\u003e\\u0026Dagger; Accession No. in Uniprot.\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"9\\\"\\u003e\\u0026sect; Data were calculated as relative expression values relative to the Control\\u0026thinsp;+\\u0026thinsp;Placebo group and are presented as means of \\u003cem\\u003en\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;2. Stress\\u0026thinsp;+\\u0026thinsp;Placebo: mice loaded with stress and administered a placebo. Stress\\u0026thinsp;+\\u0026thinsp;LcS: mice loaded with stress and administered LcS.\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"9\\\"\\u003eDetails of the experimental conditions are available in the previous report.\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tfoot\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eEstablishment of a novel cell line with normal colonic epithelial cell characteristics\\u003c/h3\\u003e\\n\\u003cp\\u003eCommercially available versatile colon epithelial cell lines (i.e., CMT-93 and Caco-2) are frequently used for \\u003cem\\u003ein vitro\\u003c/em\\u003e investigations. However, these cells were originally derived from colorectal cancer tissues, which may not accurately represent the physiology of normal intestinal epithelial cells. Meanwhile, given that the aim of the current study was to evaluate the influence of stress hormones and LcS on normal epithelial cells, a cell line with an intact intercellular tight junction structure that expresses glycoproteins and forms mucin layers was required. Accordingly, a new cell line, known as M3C-1B9, was cloned from the healthy mucosa tissue of B6.129P2-TcraTrp53/Yit mice.\\u003c/p\\u003e\\u003cp\\u003eM3C-1B9 cells highly express several glycoproteins or glycolipids, including asialo GM1, (GA1), \\u003cem\\u003eDolichos biflorus\\u003c/em\\u003e agglutinin (DBA), fucosylated asialo GM1 (FGA1), and \\u003cem\\u003eUlex europaeus\\u003c/em\\u003e agglutinin-1, (UEA-1) that are abundantly expressed by murine normal colonic epithelial cells\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e. In contrast, CMT-93 cells lacked the expression of these proteins or lipids (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA, B, Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eA). The expression of glycolipid synthesis genes, including glutaric aciduria type 1 (\\u003cem\\u003eGa1\\u003c/em\\u003e) and fucosyltransferase 2 (\\u003cem\\u003eFut2\\u003c/em\\u003e), was also higher in the M3C-1B9 cells than in CMT-93 cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC). Additionally, M3C-1B9 cells exhibited elevated expression of \\u003cem\\u003eMuc2\\u003c/em\\u003e and distinct expression profiles of tight junction-associated genes, such as E-cadherin (\\u003cem\\u003eCdh1\\u003c/em\\u003e) and Claudin-1/2 (\\u003cem\\u003eCldn1/2\\u003c/em\\u003e) from CMT-93 cells (Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eB, C). Genes associated with protecting the colon mucosa from bacterial infection, namely, \\u003cem\\u003eToll-like receptor 5 (Tlr5)\\u003c/em\\u003e\\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eNADPH oxidase 1 (Nox1\\u003c/em\\u003e)\\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e, and \\u003cem\\u003edefensin beta 1 (Defb1\\u003c/em\\u003e)\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e, were also highly expressed in M3C-1B9 cells (Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eC). Furthermore, the trans-epithelial electrical resistance (TEER) in M3C-1B9 cells\\u0026mdash;an indicator of epithelial cell barrier function\\u0026mdash;was significantly higher than that in CMT-93 cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD), indicating that M3C-1B9 cells have robust intercellular tight junction structures.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eIn the current study, M3C-1B9 cells were cultured on cell culture inserts to evaluate the effect of LcS on the physiological stress response of normal colon epithelial cells. Using this culture method, cells can be accessed from the upper and lower layers, mimicking the luminal and serosal sides of the intestinal epithelial environment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE).\\u003c/p\\u003e\\u003cp\\u003eExposure to corticosterone in the lower layer of the M3C-1B9 model, resulted in downregulation of \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression compared with that in non-treated cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eF). Next, to analyze the effect of LcS on the stress-induced intestinal epithelial cells, either live LcS cells or their culture supernatant was added to the upper layer of the M3C-1B9 model with corticosterone in the lower layer. Both treatments significantly increased \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression, suggesting a positive effect on mitigating corticosterone-induced stress (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eF).\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eIdentification of the active compounds in LcS responsible for upregulating\\u003c/b\\u003e \\u003cb\\u003eHsd11b2\\u003c/b\\u003e \\u003cb\\u003eexpression\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003eTo identify the bioactive compound(s) responsible for the induction of \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression, the LcS culture supernatant was fractionated using gel filtration chromatography according to molecular weight (MW): \\u0026lt; 3000 (Cort-LcSsup_un3k) and \\u0026gt;\\u0026thinsp;3000 (Cort-LcSsup_up3k). Each fraction was added separately to the upper layer of the M3C-1B9 model with corticosterone in the lower layer. \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression in M3C-1B9 cells was evaluated via RT-PCR. Cort-LcSsup_un3k induced \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression, whereas Cort-LcSsup_up3k did not (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe Cort-LcSsup_un3k was further fractionated via gel filtration chromatography, yielding 125 fractions. Four major peaks were identified corresponding to fractions A (No. 17\\u0026ndash;31), B (No. 38\\u0026ndash;44), C (No. 80\\u0026ndash;89), and D (No. 90\\u0026ndash;100) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). These four fractions were separately added to the upper layer of the M3C-1B9 model, with the corticosterone in the lower layer. Fraction D restored \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression to levels similar to those observed with the LcS culture supernatant before fractionation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). In contrast, the other three fractions (A, B, and C) did not induce \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression, whereas the molecules responsible for inducing \\u003cem\\u003eHsd11b2\\u003c/em\\u003e were found within fraction D.\\u003c/p\\u003e\\u003cp\\u003eFraction D was concentrated via centrifugation and subjected to LC-TOF/MS. The compounds were estimated based on the elution time and MS/MS spectral data in both negative and positive ion modes and listed in order of increasing detection intensity (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD, Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e). Among them, methyl N-(3-methoxyphenyl) carbamate was detected in both ion modes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD).\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eCarbamate-deficient LcS does not induce\\u003c/b\\u003e \\u003cb\\u003eHsd11b2\\u003c/b\\u003e \\u003cb\\u003eexpression\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe effect of methyl N-(3-methoxyphenyl) carbamate on \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression was assessed in M3C-1B9 cells with corticosterone. Treatment with methyl N-(3-methoxyphenyl) carbamate increased \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression to levels similar to those observed the LcS culture supernatant (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA) in a dose-dependent manner (Figure \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eMethyl N-(3-methoxyphenyl) carbamate possesses carbamoyl groups. Hence, based on the metabolic pathway of the \\u003cem\\u003eL. paracasei\\u003c/em\\u003e type strain American Type Culture Collection (ATCC) 334, the carbamates were presumed to be derivatives of carbamoyl phosphate, such as monoamines and polyamines (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB). Similar to \\u003cem\\u003eL. paracasei\\u003c/em\\u003e ATCC 334, LcS possesses the carbamoyl phosphate synthase gene (\\u003cem\\u003eCarb\\u003c/em\\u003e) (DDBJ Accession ID: LC859584). Thus, by inhibiting the synthesis of carbamoyl phosphate, the production of carbamates was expected to be suppressed. To test this, a mutant LcS strain lacking \\u003cem\\u003eCarb\\u003c/em\\u003e was created. The culture supernatant of the mutant strain was added to the upper layer of M3C-1B9 cells with corticosterone in the lower layer. The mutant strain failed to induce \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression in the M3C-1B9 with corticosterone, unlike the wild-type strain (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC). Thus, the carbamoyl phosphate or its derivatives were presumed to be major compounds responsible for upregulating \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression in colonic epithelial cells.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eThe non-neuronal acetylcholine pathway contributes to\\u003c/b\\u003e \\u003cb\\u003eHsd11b2\\u003c/b\\u003e \\u003cb\\u003eupregulation\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003eSeveral carbamate compounds inhibit acetylcholinesterase in the nervous system\\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e. Considering that M3C-1B9 cells also express acetylcholine receptors (Figure \\u003cspan refid=\\\"MOESM3\\\" class=\\\"InternalRef\\\"\\u003eS3\\u003c/span\\u003e), acetylcholine was expected to influence the physiological effects on non-neuronal cells. Notably, expression of muscarinic acetylcholine receptor subtypes \\u003cem\\u003eChrm3\\u003c/em\\u003e and \\u003cem\\u003eChrm4\\u003c/em\\u003e was upregulated in M3C-1B9 cells following corticosterone exposure (Figure \\u003cspan refid=\\\"MOESM3\\\" class=\\\"InternalRef\\\"\\u003eS3\\u003c/span\\u003e). To evaluate the involvement of the acetylcholine cascade in \\u003cem\\u003eHsd11b2\\u003c/em\\u003e induction, M3C-1B9 cells were pretreated with atropine\\u0026mdash;an acetylcholine receptor antagonist\\u0026mdash;in both upper and lower layers before adding LcS culture supernatants or methyl N-(3-methoxyphenyl) carbamate to the upper layer (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA, B). The upregulation of \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression mediated by LcS or methyl N-(3-methoxyphenyl) carbamate was abolished by atropine pretreatment, indicating that activation of the acetylcholine receptor is required for \\u003cem\\u003eHsd11b2\\u003c/em\\u003e upregulation in colonic epithelial cells.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eAdditionally, acetylcholine production and release by M3C-1B9 cells were analyzed upon LcS addition. LcS increased the intracellular concentration of acetylcholine in M3C-1B9 cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC). Meanwhile, the extracellular concentration of acetylcholine was significantly increased in the lower layer of the M3C-1B9 cell culture model, not in the upper layer (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD). Moreover, the expression of choline acetyltransferase (\\u003cem\\u003eChat\\u003c/em\\u003e) and carnitine acetyltransferase (\\u003cem\\u003eCrat\\u003c/em\\u003e), which induce acetylcholine production, was also increased in the M3C-1B9 cells following LcS addition (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eE, F). These results suggested that LcS induces the release of acetylcholine from the basolateral layer of intestinal epithelial cells and stimulates acetylcholine signaling under stress conditions.\\u003c/p\\u003e\\n\\u003ch3\\u003eNF-κB signaling induced by glucocorticoid receptor nuclear translocation impacts the anti-stress response\\u003c/h3\\u003e\\n\\u003cp\\u003eGlucocorticoid receptor (GR) complexes are generally formed upon binding glucocorticoids, such as corticosterone. Subsequently, GR undergoes nuclear translocation, regulates the transcription of various genes and acts as a potent repressor of NF-κB signaling\\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e. Addition of corticosterone to M3C-1B9 cells induced the nuclear translocation of the GR complex (Figure \\u003cspan refid=\\\"MOESM4\\\" class=\\\"InternalRef\\\"\\u003eS4\\u003c/span\\u003eA, B). Conversely, treating M3C-1B9 cells with LcS suppressed both GR nuclear translocation and the expression of nuclear receptor subfamily 3, group C, member 1 (\\u003cem\\u003eNr3c1)\\u003c/em\\u003e, the gene encoding GR (Figure \\u003cspan refid=\\\"MOESM4\\\" class=\\\"InternalRef\\\"\\u003eS4\\u003c/span\\u003eC).\\u003c/p\\u003e\\u003cp\\u003eNext, the effects of corticosterone and LcS on NF-κB signaling were evaluated. M3C-1B9 cells supplemented with corticosterone in the lower layer with or without LcS in the upper layer were periodically sampled. Fluctuations in the protein levels of NF-κB subunits in the nucleus and cytoplasm were analyzed via ELISA. Exposing M3C-1B9 cells to corticosterone significantly increased p50 expression in the nucleus after culturing for 2 and 4 h regardless of LcS addition; no change was observed in the cytoplasm (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA, B). Meanwhile, the persistent increase in nuclear p50 expression by corticosterone after 24 hours culture was significantly suppressed by LcS treatment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA). LcS also increased \\u003cem\\u003eIκB\\u003c/em\\u003e expression, suppressing NF-κB activation (Figure \\u003cspan refid=\\\"MOESM5\\\" class=\\\"InternalRef\\\"\\u003eS5\\u003c/span\\u003e). However, additional LcS had no significant effect on the nuclear or cytoplasmic levels of the p65 subunit\\u0026mdash;another NF-κB subunit (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC, D). Moreover, blocking the acetylcholine cascade with atropine suppressed the effect of LcS on nuclear p50 expression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE, F). Thus, it was suggested that the p50 homodimers, rather than p65, were translocated to the nucleus by corticosterone, whereaa LcS inhibited this process.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eBased on the collective results, a hypothesis was generated to describe how corticosterone and LcS mediate \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e). Under stress conditions, corticosterone binds to GR, facilitating its nuclear translocation and promoting p50 transcription. Concurrently, the p50 homodimers accumulate in the cytoplasm and translocate to the nucleus, repressing \\u003cem\\u003eHsd11b2\\u003c/em\\u003e transcription. However, in the presence of viable LcS, carbamates produced by LcS promote acetylcholine synthesis, activating the acetylcholine receptor. The acetylcholine receptor cascade upregulates IκB expression while downregulating that of GR, thereby inhibiting the formation of p50 homodimers. Consequently, NF-κB signaling is suppressed, and \\u003cem\\u003eHsd11b2\\u003c/em\\u003e transcription is ultimately repressed.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eIn this study, HSD11B2 was identified as a key factor associated with host stress responses. HSD11B2 promotes the degradation of stress hormones in the colonic epithelium, potentially acting as an important suppressor of stress hormone-mediated stress responses. To clarify the bioactive components of LcS and the molecular mechanisms underlying stress hormone degradation in colonic epithelial cells, we fractionated the active components of LcS and evaluated their effect on \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression \\u003cem\\u003ein vitro\\u003c/em\\u003e. We also investigated the intracellular signaling pathway regulating \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression. This is the first study to demonstrate corticosterone-induced downregulation of \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression in the intestinal tract, implicating it as a key factor in host anti-stress effects. Other proteins (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e) also exhibited potential effects on the anti-stress response. For example, peptidyl-prolyl cis-trans isomerase (FKBP4), which participates in GR inactivation, was downregulated and may be involved in stress-induced GR activation\\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e. Moreover, Aquaporin-8, which is involved in intestinal water regulation, was also decreased following LcS ingestion, suggesting a role in regulating intestinal stress responses associated with stool consistency\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e. In addition, choline transporter-like protein 4 may be involved in choline uptake, an acetylcholine substrate\\u003csup\\u003e\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e Further investigation into the roles of these proteins in the LcS-associated anti-stress response is warranted.\\u003c/p\\u003e\\u003cp\\u003eThe current study required a normal colonic epithelial cell line to elucidate the molecular mechanism of colonic psychological stress response \\u003cem\\u003ein vitro\\u003c/em\\u003e. Although primary culture systems and immortalized cells (e.g., those in which SV40 large T antigen or immortalization genes, such as telomerase reverse transcriptase [\\u003cem\\u003eTERT]\\u003c/em\\u003e) are frequently used as intestinal epithelial cell lines in \\u003cem\\u003ein vitro\\u003c/em\\u003e studies\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR36\\\" citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u003c/sup\\u003e, these systems have limitations. Primary culture systems are limited by their inability to culture cells long-term and the associated lot-to-lot variability of the cultured cells. Immortalized cells transfected with SV40 or viral vectors carrying immortalization genes may lack normal physiological characteristics and are not commercially available. To overcome these limitations, an immortalized normal colonic epithelial cell line, M3C-1B9, was established in this study. M3C-1B9 cells exhibited superior characteristics of normal colonic epithelial cells than the commonly used rectal cancer-derived CMT-93 cell line. Hence, M3C-1B9 cells are predicted to be a suitable model for evaluating the effects of intestinal microbiota, including probiotics, on normal colonic epithelial cells.\\u003c/p\\u003e\\u003cp\\u003eWe identified the carbamates as active compounds of LcS for increased expression of \\u003cem\\u003eHsd11b2.\\u003c/em\\u003e Carbamate is a class of compounds in which an amino group reacts with a hydroxyl group via a carbonyl moiety, forming a covalent bond between the nitrogen of the amine and the carbon of the carbonyl\\u003csup\\u003e\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e. Several carbamate derivatives have been reported to increase the amount of acetylcholine in neurons by inhibiting acetylcholinesterase (AChE), an acetylcholine-degrading enzyme\\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e However, no previous study has reported that intestinal bacteria produce carbamates.\\u003c/p\\u003e\\u003cp\\u003ePolyamines (e.g., putrescine, agmatine, spermidine, and spermine) and GABA are derivatives of carbamoyl phosphate. Various bacteria, including \\u003cem\\u003eLactobacilli\\u003c/em\\u003e can metabolize polyamines\\u003csup\\u003e\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e\\u003c/sup\\u003e. Among these derivatives, putrescine has been reported to promote intestinal epithelial cell differentiation\\u003csup\\u003e\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u003c/sup\\u003e. Moreover, exosomes released from intestinal epithelial cells in response to GABA contribute to neuronal cell activation and anti-stress responses, including decreasing corticosterone levels and improving anxiety behavior\\u003csup\\u003e\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e. The results of the current study suggest that the LcS-derived carbamate increases \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression in intestinal epithelial cells.\\u003c/p\\u003e\\u003cp\\u003eGiven that several carbamates function as AChE inhibitors, the acetylcholine cascade was selected as a potential major pathway driven by LcS. In the alimentary tract, acetylcholine functions primarily as a parasympathetic neurotransmitter that enhances intestinal motility\\u003csup\\u003e\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e\\u003c/sup\\u003e. However, acetylcholine is also released by non-neuronal cells and participates in intestinal epithelial cell differentiation and T-cell activation\\u003csup\\u003e\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e\\u003c/sup\\u003e. In the current study, acetylcholine was synthesized in intestinal epithelial cells and secreted in response to LcS treatment. Moreover, muscarinic acetylcholine receptors were expressed on intestinal epithelial cells and downstream intracellular cascade was activated upon acetylcholine binding. Considering the extremely short half-life of the released acetylcholine and the action of the degrading enzyme esterase, it is likely that the released acetylcholine exerts its effects on nearby cells, including immune cells in the mucosal intrinsic layer, intestinal endogenous neurons extending into the epithelia, and the neighboring epithelial cells.\\u003c/p\\u003e\\u003cp\\u003eIn this study, LcS suppressed NF-κB signaling via inhibiting the formation of p50 homodimers, and repressing \\u003cem\\u003eHsd11b2\\u003c/em\\u003e transcription. Several studies have reported an association between psychological stress and the activation of NF-κB signaling pathways\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e\\u003c/sup\\u003e. In the GR cascade, the activated GR complex binds to the GR element (GRE) sequence in the promoter regions of target genes, including \\u003cem\\u003eNfkb1\\u003c/em\\u003e (p50) and \\u003cem\\u003eRela\\u003c/em\\u003e (p65). Hence, the regulation of these target genes is modulated by corticosterone levels or the presence of co-stimulators such as lipopolysaccharide\\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e\\u003c/sup\\u003e. Generally, the NF-κB complex is formed as a p50/p65 heterodimer or a p50/p50 homodimer and influences the transcription of target genes in the canonical NF-κB pathway\\u003csup\\u003e\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e\\u003c/sup\\u003e. Sustained activation of NF-κB signaling results in the NF-κB complex binding to the \\u003cem\\u003eHsd11b2\\u003c/em\\u003e promoter, stimulating the transition from a p65/p50 heterodimer to a p50/p50 homodimer\\u003csup\\u003e\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e\\u003c/sup\\u003e. Consequently, \\u003cem\\u003eHsd11b2\\u003c/em\\u003e transcription is repressed via p50/p50 homodimer binding. This aligns with the results of the current study: exposure to corticosterone induced formation of the p50/p50 homodimer, resulting in repression of \\u003cem\\u003eHsd11b2\\u003c/em\\u003e transcription. Moreover, following its nuclear translocation, GR promoted the expression of p50, but not p65. Consistent with these findings, a recent study reported elevated p50 expression and altered localization in the intestines of patients with IBS compared to healthy controls\\u003csup\\u003e\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e\\u003c/sup\\u003e. Hence, increased p50 expression may be associated with an early inflammatory event and IBS pathogenesis.\\u003c/p\\u003e\\u003cp\\u003eIn the present study, LcS treatment with the M3C-1B9 cell model suppressed the stress-induced expression of p50 and its nuclear translocation. Interestingly, a carbamate analog has been shown to exert NF-κB inhibitory activity\\u003csup\\u003e\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e\\u003c/sup\\u003e, supporting our hypothesis. Two mechanisms underlying this NF-κB inhibitory effect were characterized in this study. First, LcS suppressed the expression and nuclear translocation of GR, preventing the transcriptional activation of \\u003cem\\u003eNfkb1\\u003c/em\\u003e. Second, the expression of \\u003cem\\u003eIkb\\u003c/em\\u003e subunits (\\u003cem\\u003eNfkbia\\u003c/em\\u003e, \\u003cem\\u003eNfkbib\\u003c/em\\u003e, and \\u003cem\\u003eNfkbiz\\u003c/em\\u003e) was upregulated by LcS. Each IκB subunit negatively regulates NF-κB signaling through different mechanisms based on their cellular localization\\u003csup\\u003e\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e\\u003c/sup\\u003e. Specifically, the current study results suggest that increased IκBa and IκBb expression suppressed nuclear translocation of the p50/p50 homodimer, whereas IκBz likely inhibited the binding of the p50/p50 homodimer to the \\u003cem\\u003eHsd11b2\\u003c/em\\u003e promoter, presumably alleviating \\u003cem\\u003eHsd11b2\\u003c/em\\u003e downregulation.\\u003c/p\\u003e\\u003cp\\u003eDespite the valuable insights provided by this study, additional analyses with clinical specimens are required, as the current study was performed exclusively using an \\u003cem\\u003ein vitro\\u003c/em\\u003e model. Further investigations using clinical specimens is necessary to confirm the relevance of HSD11B2 in human colonic epithelium and determine how its expression is altered by intestinal disorders such as IBS. The metabolites produced by LcS involved in upregulating \\u003cem\\u003eHsd11b2\\u003c/em\\u003e are not limited to the carbamate, methyl N-(3-methoxyphenyl) carbamate), identified in this study, but may also include other carbamoyl phosphate derivatives (e.g., polyamines). Further investigation is required to determine whether other carbamates or polyamines exert similar effects.\\u003c/p\\u003e\\u003cp\\u003eIn conclusion, it is hypothesized that metabolites produced by LcS, including derivatives of carbamoyl phosphate, promote corticosterone degradation in intestinal epithelial cells by activating the acetylcholine cascade and suppressing NF-κB signaling. Hence, the probiotic LcS was postulated to suppress corticosterone-driven stress responses in colonic epithelial cells, and this novel mechanism may contribute to the therapeutic strategies for IBS.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eCell Lines\\u003c/h2\\u003e\\u003cp\\u003eThe M3C-1B9 cell line was previously established from the crypt epithelial cells of healthy mucosal tissue, as described in the Patent Publication No. 2023-146683. The CMT-93 rectal carcinoma cell line was obtained from the ATCC (CCL-223).\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eBacterial strain\\u003c/h3\\u003e\\n\\u003cp\\u003eLcS was obtained from laboratory stock (strain YIT 9029).\\u003c/p\\u003e\\n\\u003ch3\\u003eProteomic analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eColonic epithelial tissues were collected from 20-week-old female C57BL/6 mice in a previous study\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e and stored at \\u0026minus;\\u0026thinsp;80\\u0026deg;C until proteomic analysis. The experimental protocols in the previous animal study were reviewed by the Institutional Animal Care and Use Committee of Yakult Central Institute and approved by the Director of Yakult Central Institute (Approval Numbers: 13\\u0026ndash;0091, 14\\u0026ndash;0130). All procedures involving animals were conducted in compliance with the Japanese Law for the Humane Treatment and Management of Animals (Law No. 105, issued on October 1, 1973). The study is reported in accordance to ARRIVE guidelines. Mice were assigned into the following three groups: (a) Control\\u0026thinsp;+\\u0026thinsp;Placebo group (ingested placebo for 12 weeks under no stress), (b) Stress\\u0026thinsp;+\\u0026thinsp;Placebo group (ingested placebo for 12 weeks under chronic psychological stress by loading 1 h water avoidance stress per day), and (c) Stress\\u0026thinsp;+\\u0026thinsp;LcS group (ingested LcS for 12 weeks under chronic psychological stress by loading 1 h water avoidance stress per day).\\u003c/p\\u003e\\u003cp\\u003eColonic epithelial cells from each mouse group were dissociated using EDTA treatment, as previously described\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e. The isolated cells were mixed with 10 mM formic acid (Sigma-Aldrich) and 20 mg alumina (a-Alumina 1\\u0026ndash;2 mm, Fujifilm Wako), then disrupted by sonication for 5 min using the Picoruptor 2 sonicator (Diagenode). The protein concentrations in the supernatant were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific). The solution containing 200 \\u0026micro;g of total protein was centrifuged at 15,000 \\u0026times; \\u003cem\\u003eg\\u003c/em\\u003e for 20 min, and the supernatant was discarded. Next, 50 mM triethylammonium bicarbonate solution containing 0.5% RapiGest (Waters) was added to the precipitate. The proteins were denatured by sonication for 5 min and incubated at 50\\u0026deg;C for 30 min to hydrolyze RapiGest. The iTRAQ reagent kit (8-plex, SCIEX) was used for stable isotope labeling of proteins. The protein solutions were incubated with a reducing disulfide bonds agent at 60\\u0026deg;C for 1 h, followed by alkylation with a cysteine-blocking agent at room temperature for 10 min. For enzymatic digestion, 10 mg of Trypsin (Pierce) was added to the protein samples and incubated at 37\\u0026deg;C for 16 h. Eight different iTRAQ reagents (isotopic molecular weights: 113, 114, 115, 116, 117, 118, 119, 121) dissolved in dimethylformamide were mixed with each sample and incubated at room temperature for 2 h to label peptides. Next, formic acid was added to a final concentration of 5% and incubated at room temperature for 2 h to inactivate RapiGest. All eight samples were combined and applied to a solid-phase extraction column (Bond Elute Plexa, Agilent), washed with 60% acetonitrile, centrifuged, and concentrated using an evaporator, and redissolved in 5% acetonitrile. The redissolved sample was subjected to LC-TOF/MS (Triple TOF 6600, SCIEX). Each detected peptide peak was matched against the UniProt-Swiss-Prot database using ProteinPilot software (SCIEX) for protein identification. A quantitative comparison of iTRAQ peak intensities between samples was performed with ProteinPilot software.\\u003c/p\\u003e\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003ePreparation of LcS and culture supernatant\\u003c/h2\\u003e\\u003cp\\u003eLcS was cultured in 5 mL of De Man\\u0026ndash;Rogosa\\u0026ndash;Sharpe (MRS) broth at 37\\u0026deg;C for 16 h under aerobic conditions. For LcS collection, the bacterial culture was centrifuged at 8,000 \\u003cem\\u003e\\u0026times; g\\u003c/em\\u003e for 5 min at 4\\u0026deg;C. The supernatant was collected and stored for further analysis. The pellet was washed with PBS and resuspended in 5 mL of 10% fetal calf serum (FCS)-supplemented Advanced DMEM/F12 (aDMEM/F12).\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eGeneration of the mutated LcS strain\\u003c/h2\\u003e\\u003cp\\u003eThe \\u003cem\\u003eCarb\\u003c/em\\u003e-deficient LcS strain was generated using a stepwise double-crossover method, as previously described \\u003csup\\u003e\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e\\u003c/sup\\u003e. The estimated 1,000 bp fragments of 5\\u0026prime;- and 3\\u0026prime;-terminal ends of \\u003cem\\u003eCarb\\u003c/em\\u003e were amplified using the primer pairs (CarB_A_F and CarB_A_R for the 5\\u0026rsquo; region, and CarB_B_F and CarB_B_R for the 3\\u0026rsquo; region) listed in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e. The pYSSE3.1 vector was also amplified to obtain a linear strand vector using the pYSSE3.1F and pYSSE3.1_R primer pair. After agarose electrophoresis of each amplified product, the bands corresponding to the insert and vector were excised, and the DNA fragments were purified using the QIAquick Gel Extraction Kit (Qiagen). The plasmid vector was prepared by ligating the two inserted DNA fragments with the vector via an In-Fusion cloning reaction. The plasmid vector was introduced into competent cells (XL10) via heat shock transformation. The transformants were selected on Lysogeny broth (LB) agar medium containing 500 \\u0026micro;g/mL erythromycin and incubated aerobically at 37\\u0026deg;C for 18 h. PCR was performed to select colonies carrying the inserted fragments using the 31F and 31R primer pair. Plasmid DNA of the selected colonies was purified using the GenElute Plasmid Miniprep Kit (Sigma-Aldrich).\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eList of primers used for generating the gene-disrupted strain of LcS\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"3\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePrimer name\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eSequence (5\\u0026prime;\\u0026ndash;3\\u0026prime;)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eApplication\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003epYSSE3.1_F\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eAGAGGATCCCCGGGTACCGAGCT\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003eAmplified pYSSE3.1 for in-fusion cloning of upstream and downstream regions of \\u003cem\\u003eCarb\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003epYSSE3.1_R\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eAGAGTCGACCTGCAGGCATGCAA\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCarb_A_F\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eACCCGGGGATCCTCTAGATTTCCCTGGCGTCGGTTTCG\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003eAmplified upstream region of \\u003cem\\u003eCarb\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCarb_A_R\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eGGCAAGGCGCGATTATCCTTTCTGCGTGCG\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCarb_B_F\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eAAGGATAATCGCGCCTTGCCATTCAAAGTG\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003eAmplified downstream region of \\u003cem\\u003eCarb\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCarb_B_R\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eCTGCAGGTCGACTCTAGACCAGTGCGATAATAGGCGTC\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e31F\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eAGTTGGGTAACGCCAGG\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003eAmplified insert fragments\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e31R\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eGGATAACAATTTCACAC\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCarB_Sc_F\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eATTCTTGAAGACGGCAGCGT\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003eAmplified flanking fragments upstream and downstream of \\u003cem\\u003eCarb\\u003c/em\\u003e for in-fusion cloning\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCarB_Sc_R\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eCAAGAGTTGTGGTCTTATCC\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCarB_Dc_F\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eTTGGCGAGTGCGAATAGTTG\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003eAmplified flanking fragments of \\u003cem\\u003eCarb\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCarB_Dc_R\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eACCTCCTTATGTGTAGGCTG\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eGene-disrupting plasmid was introduced into LcS by electroporation using Gene Pulser II (Bio-Rad). Plasmid-transfected LcS was plated on MRS agar containing 20 \\u0026micro;g/mL erythromycin and incubated aerobically at 37\\u0026deg;C for 72 h. Colony PCR was performed to check single crossovers using the CarB_Sc_F and CarB_Sc_R primer pair. Colonies with single crossovers were incubated in MRS liquid medium containing 20 \\u0026micro;g/mL erythromycin in aerobic conditions at 37\\u0026deg;C for 18 h and smeared on MRS agar medium containing 1 \\u0026micro;g/mL vancomycin. As the plasmid vector contains a vancomycin \\u003cem\\u003eddl\\u003c/em\\u003e sequence, the success or failure of the double crossover was determined by the presence or absence of colony formation. Final colony PCR was performed using the CarB_Dc_F and CarB_Dc_R primers. Single colony isolation was performed to obtain carB-disrupted strains.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eMolecular weight fractionation of the LcS culture supernatant\\u003c/h2\\u003e\\u003cp\\u003eThe LcS culture supernatant was subjected to molecular weight fractionation using an ultrafiltration spin column (VIVASPIN, Sartorius) with a 3 kDa fractional molecular weight cutoff, and centrifuged at 12,000 \\u003cem\\u003e\\u0026times; g\\u003c/em\\u003e for 60 min at 4\\u0026deg;C. The eluate fraction was collected and supplemented with FCS to a final concentration of 10% for subsequent testing. The components (\\u0026gt;\\u0026thinsp;3 kDa), retained on the column filter were resuspended in 10% FCS aDMEM/F12 medium. As a negative control, 10% FCS aDMEM/F12 medium without LcS supernatant was subjected to the same procedure for molecular weight fractionation.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eGel filtration chromatography\\u003c/h2\\u003e\\u003cp\\u003eGel filtration chromatography was performed on a gel carrier (Bio-Gel P2, Bio-Rad) with a molecular weight separation range of 100\\u0026ndash;1,800. The eluate fraction from the ultrafiltration spin column was concentrated and added to the Gel column, and Milli-Q water was used as the elution solvent. A total of 125 fractions (12 mL/fraction) were obtained. The absorbance of each fraction was measured at 220 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific), and four major peaks were identified. The fractions corresponding to each peak were lyophilized to remove the aqueous solvents, and the lyophilized powder was mixed and resuspended in 10% FCS aDMEM/F12 medium for functional assays.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eCell culture insert assay\\u003c/h2\\u003e\\u003cp\\u003eThe M3C-1B9 cells were seeded at a density of 5 \\u0026times; 10\\u003csup\\u003e4\\u003c/sup\\u003e cells/well onto cell culture inserts (Corning) mounted in 24-well plates (Corning) and cultured in 10% FCS aDMEM/F12 medium) at 37\\u0026deg;C under 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. The culture medium was replaced every two days and cells were used for each assay on day 10 of culture. CMT-93 cells were cultured under the same conditions as M3C-1B9 cells.\\u003c/p\\u003e\\u003cp\\u003eLcS was prepared at a final concentration of 1 \\u0026times; 10\\u003csup\\u003e7\\u003c/sup\\u003e CFU/well (1.4 \\u0026times; 10\\u003csup\\u003e7\\u003c/sup\\u003e CFU/mL) and added to the upper layer of the cell culture insert. The culture supernatant derived from 1 \\u0026times; 10\\u003csup\\u003e7\\u003c/sup\\u003e CFU of LcS was prepared and added to the upper layer of the cell culture insert. To identify the molecules responsible for inducing \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression, 700 \\u0026micro;L of the eluate was added to the upper layer of the insert. The equivalent volume of 10% FCS aDMEM/F12 medium was added as a negative control.\\u003c/p\\u003e\\u003cp\\u003eMethyl N-(3-methoxyphenyl) carbamate (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO) and added to the upper layer of the insert in the concentration range of 0.001\\u0026ndash;100 \\u0026micro;M. The culture supernatant of \\u003cem\\u003eCarB\\u003c/em\\u003e-deficient LcS was prepared from a 1 \\u0026times; 10\\u003csup\\u003e7\\u003c/sup\\u003e CFU \\u003cem\\u003eCarB\\u003c/em\\u003e-deficient LcS culture and added to the upper layer of the insert. Atropine (Wako) was dissolved in 10% FCS aDMEM/F12 medium and added to the upper and lower inserts at a final concentration of 10 \\u0026micro;M 1 h before LcS addition.\\u003c/p\\u003e\\u003cp\\u003eCorticosterone (Wako) was diluted in 10% FCS aDMEM/F12 to a final concentration of 30 ng/mL and added to the lower layer of the insert. The concentration was determined based on the plasma corticosterone concentration data from our previously established mice model of psychological stress\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eRNA extraction and RT-PCR\\u003c/h2\\u003e\\u003cp\\u003eTotal RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer\\u0026rsquo;s instructions. Reverse transcription was performed using 1 \\u0026micro;g of RNA using 200 U of Superscript II Reverse Transcriptase (Invitrogen). The cDNA was amplified by PCR. Gene expression levels were quantified by RT-PCR. The data were calculated as relative expression, with glyceraldehyde-3-phosphate dehydrogenase (\\u003cem\\u003eGapdh\\u003c/em\\u003e) as the housekeeping gene. The primer pairs used for analysis are listed in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e.\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eList of primers used for quantification of gene expression using RT-PCR\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"4\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eGene name\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003ePrimer (Forward)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003ePrimer (Reverse)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eReference\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eGapdh\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e\\u003cp\\u003eNot available\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eGene Globe ID: QT01658692 (QIAGEN)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eHsd11b2\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e\\u003cp\\u003eNot available\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eGene Globe ID: QT00252609 (QIAGEN)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eChat\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e\\u003cp\\u003eNot available\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eGene Globe ID: QT00135212 (QIAGEN)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eCdh1\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e\\u003cp\\u003eNot available\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eGene Globe ID: QT00121163 (QIAGEN)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eCldn1\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e\\u003cp\\u003eNot available\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eGene Globe ID: QT00159278 (QIAGEN\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eCldn2\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e\\u003cp\\u003eNot available\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eGene Globe ID: QT00261905 (QIAGEN\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eMuc2\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e\\u003cp\\u003eNot available\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eGene Globe ID: QT01060773 (QIAGEN\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eTlr5\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e\\u003cp\\u003eNot available\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eGene Globe ID: QT00262549 (QIAGEN\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eNox1\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e\\u003cp\\u003eNot available\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eGene Globe ID: QT00140091 (QIAGEN\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eDefb1\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e\\u003cp\\u003eNot available\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eGene Globe ID: QT00103271 (QIAGEN)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eCrat\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eAGCTGGCATACTACAGGATCTATGG\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eAGGTGAAACATGCGCAGAGA\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eThis study\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eChrm1\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eGCAGCAGCTCAGAGAGGTCACAG\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eGATGAAGGCCAGCAGGATGG\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eThis study\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eChrm2\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eGCGGATCCTGTGGCCAACCAAGAC\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eCGAATTCACGATTTTGCGGGCTA\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eThis study\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eChrm3\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eAAGGCACGAAACGGTCATCT\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eGCAAACCTCTTAGCCAGCGT\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eThis study\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eChrm4\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eAGCCGCAGCCGTGTTCACAA\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eTGGGTTGAGGGTTCGTGGCT\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eThis study\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eChrm5\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eGTCTCCGTCATGACCATACTCTA\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eCCCGTTGTTGAGGTGCTTCTAC\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eThis study\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eNr3c1\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eATGAGACTGCCGATTCCTCTGC\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eTGCTTGGAATCTGCCTGAGA\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eThis study\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eNfkbia\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eGCCAGGAATTGCTGAGGCACTT\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eGTCTGCGTCAAGACTGCTACAC\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eThis study\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eNfkbib\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eTCAGCATGAGCCCTTCCTGGAT\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eCAAGGATGGCTGCTAGATGCAG\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eThis study\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cem\\u003eNfkbiz\\u003c/em\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eATCCAGAAGGGAGCTGTGAGGA\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eATGAGACTGCCGATTCCTCTGC\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eThis study\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eLC-TOF/MS analysis\\u003c/h2\\u003e\\u003cp\\u003eFractions of LcS culture supernatant separated via gel filtration chromatography were lyophilized, resuspended in 100 \\u0026micro;L of 10 mM ammonium formate solution (pH 6.5, Sigma-Aldrich), and subjected to LC-TOF/MS (Triple TOF 6600, SCIEX) using a Scherzo SW-C18 (Imtakt) column with a 10 mM ammonium formate solution and acetonitrile (5\\u0026ndash;65% gradient; Sigma-Aldrich) mobile phase. The LC-TOF/MS data were analyzed using Masterview software (SCIEX). The fractions that significantly increased \\u003cem\\u003eHsd11b2\\u003c/em\\u003e expression in M3C-1B9 cells were compared with inactive fractions, and components with a\\u0026thinsp;\\u0026ge;\\u0026thinsp;2-fold difference in relative abundance were screened. METLIN (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://metlin.scripps.edu\\u003c/span\\u003e\\u003cspan address=\\\"https://metlin.scripps.edu\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) was used to estimate the chemical formula and structure of the detected components.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eAcetylcholine measurement\\u003c/h2\\u003e\\u003cp\\u003eM3C-1B9 cells were dissociated using TrypLE Express enzyme (Thermo Fisher Scientific) at 37\\u0026deg;C for 20 min and collected by centrifugation (500 \\u0026times; \\u003cem\\u003eg\\u003c/em\\u003e, 5 min). The choline and acetylcholine levels in the cell extracts and the culture supernatant were measured using a Choline/Acetylcholine Assay Kit (Abcam) according to the manufacturer\\u0026rsquo;s instructions. The collected cells were suspended in the assay buffer and sonicated for 5 min to disrupt the cells. Cell extracts were collected after centrifugation (20,000 \\u0026times; \\u003cem\\u003eg\\u003c/em\\u003e, 5 min) and analyzed for acetylcholine quantification.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eExtraction of cellular proteins and NF-κB measurement\\u003c/h2\\u003e\\u003cp\\u003eThe ProteoExtract Subcellular Proteome Extraction Kit (Merck) was used to fractionate cytoplasmic and nuclear proteins from M3C-1B9 cells. The amounts of p50 and p65 proteins in the cytoplasmic and nuclear fractions were determined using the NF-κB p50 Transcription Factor Assay Kit (Cayman) and the NF-κB p65 Transcription Factor Assay Kit (Cayman), respectively.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eQuantification and statistical analysis\\u003c/h2\\u003e\\u003cp\\u003eFor graphs, data are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD. Statistical significance was defined as \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05. Dunnett\\u0026rsquo;s test was used to analyze significant differences in gene and protein expression affected by corticosterone, LcS, or its culture supernatant; cells were treated using corticosterone-only group as the reference. Analysis of variance (ANOVA) was performed to analyze differences in NF-κB expression. Graphs were constructed, and statistical analyses were performed using GraphPad Prism.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003ch2\\u003eCompeting Interests\\u003c/h2\\u003e\\u003cp\\u003eThe authors have declared that no conflict of interest exists.\\u003c/p\\u003e\\u003c/p\\u003e\\u003ch2\\u003eFunding Declaration\\u003c/h2\\u003e\\u003cp\\u003eThis work was funded by Yakult Honsha Co., Ltd. The funder provided support in the form of salaries for all authors but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eS.A., K.O., T.H., and S.M. designed the study. S.A. and S.M. performed the experiments. S.A. and T.K. wrote the manuscript.\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgement\\u003c/h2\\u003e\\u003cp\\u003eWe greatly appreciate Ms. Hiromi Setoyama for assistance with the cell cultures and Dr. Haruo Ikemura for assistance with the proteomic analysis. This work was funded by Yakult Honsha Co., Ltd. The funder provided financial support in the form of salaries for all authors but did not influence the study design, data collection, analysis, preparation of the manuscript, or decision to publish.\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eRaw experimental data reported in this paper will be made available by the corresponding author upon reasonable request. This paper analyzes existing, publicly available data. The *Carb* coding sequence of LcS has been deposited in the DNA Data Bank of Japan under Accession No. LC859584. Any additional information required to reanalyze the data reported in this paper is available from the corresponding author upon request.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eIpsos \\u003cem\\u003eIPSOS Global Health Service Monitor 2022\\u003c/em\\u003e, (2022).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003ePereira, V. H., Campos, I. \\u0026amp; Sousa, N. The role of autonomic nervous system in susceptibility and resilience to stress. \\u003cem\\u003eCurr. Opin. Behav. Sci.\\u003c/em\\u003e \\u003cb\\u003e14\\u003c/b\\u003e, 102\\u0026ndash;107 (2017).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eGjerstad, J. K., Lightman, S. L. \\u0026amp; Spiga, F. Role of glucocorticoid negative feedback in the regulation of HPA axis pulsatility. \\u003cem\\u003eStress\\u003c/em\\u003e \\u003cb\\u003e21\\u003c/b\\u003e, 403\\u0026ndash;416 (2018).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLa Torre, D., Van Oudenhove, L., Vanuytsel, T. \\u0026amp; Verbeke, K. Psychosocial stress-induced intestinal permeability in healthy humans: What is the evidence? \\u003cem\\u003eNeurobiol. Stress\\u003c/em\\u003e. \\u003cb\\u003e27\\u003c/b\\u003e, 100579 (2023).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eTurner, J. R. Intestinal mucosal barrier function in health and disease. \\u003cem\\u003eNat. Rev. Immunol.\\u003c/em\\u003e \\u003cb\\u003e9\\u003c/b\\u003e, 799\\u0026ndash;809 (2009).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eShaler, C. R. et al. Psychological stress impairs IL22-driven protective gut mucosal immunity against colonising pathobionts. \\u003cem\\u003eNat. 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Med.\\u003c/em\\u003e \\u003cb\\u003e175\\u003c/b\\u003e, 1181\\u0026ndash;1194 (1992).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSethi, G. \\u0026amp; Tergaonkar, V. Potential pharmacological control of the NF-κB pathway. \\u003cem\\u003eTrends Pharmacol. Sci.\\u003c/em\\u003e \\u003cb\\u003e30\\u003c/b\\u003e, 313\\u0026ndash;321 (2009).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eYasuda, E., Serata, M. \\u0026amp; Sako, T. Suppressive effect on activation of macrophages by \\u003cem\\u003eLactobacillus casei\\u003c/em\\u003e strain Shirota genes determining the synthesis of cell wall-associated polysaccharides. \\u003cem\\u003eAppl. Environ. Microbiol.\\u003c/em\\u003e \\u003cb\\u003e74\\u003c/b\\u003e, 4746\\u0026ndash;4755 (2008).\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"psychological stress, corticosterone, colonic epithelial cells, HSD11B2, acetylcholine, NF-κB\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7258444/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7258444/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eChronic psychological stress contributes to functional disorder development, including irritable bowel syndrome (IBS). Although probiotics have shown potential in ameliorating these disorders, the precise mechanisms remain incompletely understood. The aim of this study was to elucidate the mechanism underlying the effect of \\u003cem\\u003eLacticaseibacillus paracasei\\u003c/em\\u003e strain Shirota (LcS) on the host anti-stress response in a colonic epithelial cell line. The expression of the stress hormone-degrading enzyme, 11 β-hydroxysteroid dehydrogenase type 2 (\\u003cem\\u003eHsd11b2\\u003c/em\\u003e), was suppressed by corticosterone and restored by LcS treatment. Fractionation of the LcS culture supernatant revealed a derivative of carbamoyl phosphate as the key factor responsible for inducing \\u003cem\\u003eHsd11b2\\u003c/em\\u003e. Moreover, activation of acetylcholine receptor and inhibition of NF-κB p50 homodimer nuclear translocation were required to induce \\u003cem\\u003eHsd11b2\\u003c/em\\u003e in colonic epithelial cells. These findings reveal a novel probiotic mechanism whereby an LcS metabolite triggers anti-stress responses, including \\u003cem\\u003eHsd11b2\\u003c/em\\u003e induction, by modulating the acetylcholine and NF-κB pathways. This new mechanism by which probiotics can stimulate anti-stress effects in the colonic mucosa may contribute to IBS treatment.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Probiotic strain released-carbamoyl phosphates modify corticosterone metabolism on colonic epithelial cells by elevating 11-beta-hydroxysteroid dehydrogenase isozyme 2\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-09-25 17:36:38\",\"doi\":\"10.21203/rs.3.rs-7258444/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"3f0eb471-fa69-4f61-bcef-8460d2ce0902\",\"owner\":[],\"postedDate\":\"September 25th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":55063192,\"name\":\"Biological sciences/Biochemistry\"},{\"id\":55063193,\"name\":\"Biological sciences/Cell biology\"},{\"id\":55063194,\"name\":\"Biological sciences/Microbiology\"},{\"id\":55063195,\"name\":\"Biological sciences/Molecular biology\"},{\"id\":55063196,\"name\":\"Biological sciences/Physiology\"}],\"tags\":[],\"updatedAt\":\"2026-01-12T09:55:07+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-09-25 17:36:38\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7258444\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7258444\",\"identity\":\"rs-7258444\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}