Inhibition of peripheral serotonin synthesis by a gut microbe-derived TPH1 inhibitor, hyodeoxycholic acid, alleviates irritable bowel syndrome

Inhibition of peripheral serotonin synthesis by a gut microbe-derived TPH1 inhibitor, hyodeoxycholic acid, alleviates irritable bowel syndrome | 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 Biological Sciences - Article Inhibition of peripheral serotonin synthesis by a gut microbe-derived TPH1 inhibitor, hyodeoxycholic acid, alleviates irritable bowel syndrome Lixiang Zhai, Gengyu Bao, Junfang Lyu, Mingchun Wang, Shujun Xu, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6769911/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 Gut dysbiosis significantly contributes to the pathogenesis of diarrhea-predominant irritable bowel syndrome (IBS-D) by enhancing serotonin (5-HT) biosynthesis, which exacerbates diarrhea symptoms. Current treatments aimed at peripheral 5-HT signaling in IBS-D are often limited by efficacy and side effects. This study presents hyodeoxycholic acid (HDCA), a gut-microbial metabolite, as a novel therapeutic strategy that directly inhibits peripheral 5-HT synthesis via tryptophan hydroxylase 1 (TPH1). Our research indicates that HDCA levels are notably reduced in IBS-D patients and show a negative correlation with both diarrhea severity and peripheral 5-HT levels. Furthermore, we demonstrate that HDCA and PULVIS FELLIS SUIS, a traditional Chinese medicine abundant in HDCA, effectively alleviate diarrhea symptoms and inhibit peripheral 5-HT production without impacting central 5-HT levels or mood behaviors in mouse models of IBS. Mechanistically, HDCA directly binds to and inhibits TPH1, thereby suppressing peripheral 5-HT biosynthesis, a critical pathological factor in IBS-D. These findings suggest that HDCA is a promising candidate for microbiota-driven therapeutic interventions and gut-brain axis regulation. This study demonstrates the use of a microbiota-derived metabolite and traditional medicine specifically targeting peripheral 5-HT biosynthesis for treating gastrointestinal disorders. Our results pave the way for new IBS treatment strategies and other conditions requiring TPH1 inhibition, offering novel insights and potential clinical applications. ClinicalTrials.gov no: NCT02822677 and NCT03457324 Health sciences/Diseases/Gastrointestinal diseases/Functional gastrointestinal disorders/Irritable bowel syndrome Health sciences/Biomarkers/Diagnostic markers Health sciences/Health care/Therapeutics/Drug therapy/Molecularly targeted therapy Hyodeoxycholic acid PULVIS FELLIS SUIS Irritable bowel syndrome TPH1 Serotonin Diarrhea Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Diarrhea-predominant irritable bowel syndrome (IBS-D) is a complicated gastrointestinal disorder affecting people globally. The persistent occurrence of diarrhea combined with abdominal discomfort profoundly affects the quality of life of patients. Due to its chronic gastrointestinal (GI) symptoms, IBS-D can also lead to exhaustion and emotional strain, deteriorating life quality. Given the diverse symptoms of IBS-D and the absence of a universally effective solution, devising treatment strategies for IBS-D remains intricate due to our limited understanding of its pathogenesis 1 . Serotonin (5-hydroxytryptamine, 5-HT) is implicated in the pathophysiology of IBS-D. 5-HT is an important factor in controlling various GI processes, including movement, secretion and sensitivity. Approximately 90% of peripheral 5-HT is synthesized by enterochromaffin (EC) cells located in the GI tract via the rate-limiting enzyme tryptophan hydroxylase (TPH1). In IBS-D, abnormalities of peripheral 5-HT biosynthesis and reuptake, along with EC cell hyperplasia, result in elevated peripheral 5-HT levels in the gut to aggravate diarrhea 2,3 . Research and drug development have concentrated on manipulating 5-HT signaling as a therapeutic approach for IBS-D. For example, medications impacting 5-HT receptors or 5-HT biosynthesis regulate bowel movement 4 . However, current FDA-approved drugs targeting 5-HT signaling require long-term treatment and with side effects such as constipation, ischemic colitis and cardiovascular risks 5 . Consequently, there is a need for new strategies that specifically fine-tune 5-HT signaling in the gut to provide more targeted and effective solutions for IBS-D. Recent research indicates a causal link between gut microbiota, gut-microbial metabolites and peripheral 5-HT production. These gut bacteria communicate with EC cells via gut-microbial metabolites such as short-chain fatty acids, deoxycholic acid and tyramine to affect the biosynthesis, release, and reuptake of 5-HT 6 . Our previous findings also showed gut microbes-derived aromatic trace amines contribute to diarrhea in IBS-D 3,7 . These intricate interactions between the gut microbiota and 5-HT signaling highlight the potential for developing gut-microbial metabolites as novel therapeutics in modulating peripheral 5-HT levels, thereby affecting gut movement to alleviate symptoms of IBS-D. However, whether there are microbial metabolites that improve diarrhea by inhibiting peripheral 5-HT in IBS-D remains unknown. In this study, we aimed to identify microbial metabolites that regulate 5-HT abnormalities for managing diarrhea in IBS-D. Through a targeted metabolomics approach and in vitro experiments, we found that a microbial metabolite hyodeoxycholic acid (HDCA, 3α,6α-dihydroxy-5β-cholanic acid), negatively correlates with the severity of diarrhea symptoms and peripheral 5-HT levels, suggesting HDCA is potentially beneficial for managing IBS-D and regulating 5-HT abnormalities. Through fecal microbiota transplantation, we further confirmed that HDCA-enriched fecal microbiota plays a crucial role in regulating 5-HT production and GI motility. In addition, we showed HDCA inhibits 5-HT production in ex vivo mice colonic tissues. Therefore, we investigated the action and underlying molecular mechanisms of HDCA and HDCA-enriched traditional medicine PULVIS FELLIS SUIS (PFS) on GI symptoms in a mouse model of IBS-D. Notably, we showed HDCA and PFS alleviated diarrhea symptoms and inhibited 5-HT production in mice with experimental IBS by directly inhibiting the TPH1 activity, a rate-limiting enzyme for 5-HT biosynthesis. Our findings suggest that HDCA is a promising microbe-derived therapeutic agent for IBS-D through inhibiting peripheral 5-HT biosynthesis. Results HDCA was negatively associated with serum 5-HT in patients with IBS-D To identify metabolites that correlated with 5-HT production in IBS-D patients, we first conducted sub-group analysis in IBS-D patients using a 75% cut-off value of serum 5-HT we determined in healthy controls (HC). Accordingly, IBS-D patients were grouped as IBS-D 5-HT + (>75% cut-off value of 5-HT in HC) group and IBS-D 5-HT control group ( < 75% cut-off value of 5-HT in HC) and we showed serum levels of 5-HT in IBS-D 5-HT + patients were significantly higher compared with subjects from IBS-D 5-HT control group and HC group (p<0.001 in all cases, Figure.1A). We then determined changes in the fecal metabolome of IBS-D 5-HT + patients in comparison with HC group and IBS-D 5-HT control group to further investigate the correlation between gut-microbial metabolites in feces, serum 5-HT and GI symptoms. As shown in Figure.1B, significant differences in metabolomic profiles were found in IBS-D 5-HT + group compared with IBS-D 5-HT control group and HC group. Notably, significant changes in fecal bile acid profiles were found in IBS-D 5-HT + patients as shown by reduction of hyocholic acid (HCA) species including hyodeoxycholic acid (HDCA), taurohyocholic acid (THCA), taurohyodeoxycholic acid (THDCA), as well as increment of chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and lithocholic acid (LCA) (Figure.1C). Accordingly, we determined the correlation between fecal bile acid profiles, serum 5-HT and diarrhea symptoms indexes. Among all bile acid species, we showed that only fecal HCA species were negatively correlated with peripheral 5-HT and diarrhea indexes (r=-0.3, -0.27 and -0.36, p<0.05 in all cases, Figure.1D). Among all HCA species, we showed that only HDCA was significantly reduced in both serum and fecal samples from IBS-D 5-HT + patients compared with HC group among all HCA species (p<0.001 in both cases, Figure.1E-F). Correlation analysis revealed fecal and serum HDCA was negatively associated with serum 5-HT levels and diarrhea indexes in IBS-D patients with the lowest r-value and p-value (r=-0.41, -0.46, -0.23, -0.31, -0.37, and -0.27, p<0.05 in all cases, Figure.1G). Accordingly, we also validated the negative correlation between HDCA, diarrhea severity and 5-HT levels in another IBS-D cohort (r<-0.3, p<0.01 in all cases in 5-HT + IBS-D subgroup, Figure.2). These results established the clinical relevance of HDCA on 5-HT abnormalities in IBS-D and demonstrated that targeting HDCA is a potential therapeutic strategy for diarrhea management in IBS-D through regulating 5-HT. HDCA-associated fecal microbiota from IBS-D patients regulated peripheral 5-HT and GI motility in antibiotics-treated mice To understand whether endogenous HDCA is modulated by gut microbiota, we determined changes in HCA species in both germ-free mice and antibiotics-treated mice (Figure.3A). Notably, we showed both HCA and HDCA and their glycine and taurine-conjugated forms GHCA, THCA, GHDCA and THDCA are significantly reduced in both serum and fecal samples from both germ-free mice and antibiotics-treated mice compared with mice raised in an SPF environment (p<0.01 in both cases, Figure.3B-M), revealing endogenous HDCA is manipulated by gut microbiota. Based on this finding, we performed HDCA-associated fecal microbiota transplantation (FMT) from IBS-D patients in recipient mice after treatment with antibiotics. To determine the causal effects of HDCA-associated fecal microbiota on serum 5-HT and GI motility, fecal microbiota from IBS-D patients with high and low HDCA levels based on a top/bottom 25% cut-off value (labeled as HDCA + and HDCA - ) was orally administered to recipient antibiotic-treated mice (Figure.4A). Fecal HDCA levels from donor samples in HDCA + FMT group was significantly higher compared with HDCA - FMT group (p<0.001, Figure.4B). Notably, we showed GI motility was also regulated by HDCA-enriched FMT as evidenced by prolonged GI transit time and decreased defecation frequency, although no significant changes in fecal moisture content (p<0.05 in both cases, Figure.4C-D, n.s., Figure.4E). Moreover, peripheral 5-HT was also found significantly reduced in HDCA + FMT group compared with HDCA - FMT group as shown by 5-HT levels in serum, ileum and proximal colonic tissues (p<0.001 in both cases, Figure.4F-H). In alignment with fecal HCA species profiles from donor samples in HDCA + FMT group and HDCA - FMT group, we showed HDCA significantly increased in serum and ileum samples from recipient mice in HDCA + FMT group compared with HDCA - FMT group (p<0.001 in both cases, Figure.4I-J). These data suggested that HDCA-associated fecal microbiota regulate GI motility and peripheral 5-HT levels, highlighting the therapeutic potential of HDCA for the treatment of diarrhea in IBS-D. HDCA and Refined PULVIS FELLIS SUIS (RPFS) alleviated diarrhea and inhibited 5-HT production in a mouse model of IBS We then employed in vitro models, including QGP-1 cells along with mice intestinal segment cultures, to study the effects of HDCA on 5-HT production. Notably, we showed HDCA significantly inhibited 5-HT production in these models in a dose-dependent manner and time-dependent manner in QGP-1 cells (p<0.05, Figure.5A) and intestinal segment cultures from mice (p<0.05 in both cases, Figure.5B), suggesting HDCA is a potential candidate for the management of diarrhea through inhibiting 5-HT production. Subsequently, we evaluated the effects of Refined PULVIS FELLIS SUIS (RPFS), a traditional Chinese medicine enriched with HCA species, on 5-HT production. Our data showed RPFS extract containing 38.67% of total bile acids and 12.46% of total bile acids are HCA species (Figure.5C). We then showed RPFS significantly inhibited 5-HT production in mice intestinal segment cultures in a dose-dependent manner (p<0.05 in both cases, Figure.5D), revealing the therapeutic potential of RPFS, a commonly used non-toxic traditional medicine, on IBS-D via inhibiting 5-HT. Although RPFS also contains a high amount of UDCA and CDCA species, we did not find UDCA or CDCA affect 5-HT levels in QGP-1 cells at different dosages in the physiological range (n.s., Figure.5E-F), suggesting only HCA species in RPFS exhibited inhibitory effects on 5-HT production. To further evaluate the therapeutic effects of HDCA and RPFS on IBS-D, we employed a mouse model of IBS induced by TNBS, which is characterized by diarrhea and elevated 5-HT levels (Figure.6A). Notably, both HDCA and RPFS effectively alleviated diarrhea in TNBS-treated mice, as shown by prolonged GI transit time, reduced defecation frequency, and fecal water content (p<0.01 in both cases, Figure.6B-D). HDCA and RPFS significantly reduced 5-HT levels in both ileum and proximal colon in TNBS-treated mice, as demonstrated by LC-MS analysis and immunofluorescence (IF) staining of 5-HT (p<0.01 in both cases, Figure.6E-H). Moreover, 5-HT level in brain tissues is found significantly reduced in TNBS-treated mice (p<0.01, Figure.6J), but no significant changes in HDCA or 5-HT levels were found in brain tissues in the treatment of HDCA or RPFS in TNBS-treated mice, indicating HDCA is not distributed to brain tissues and affected brain 5-HT levels after oral administration (n.s., Figure.6I-J). Considering 5-HT inhibition is associated with alterations of mental disorder 8 , we evaluated general locomotor activity in TNBS-treated mice in the treatment of HDCA and we showed that both HDCA in high dose and RPFS treatment did not result in depression or anxiety symptoms in TNBS-treated mice (n.s., Figure.7A-D), revealing safety evidence of HDCA and RPFS treatment for IBS-D. Collectively, our findings demonstrated that HDCA and RPFS improved diarrhea phenotypes and inhibited peripheral 5-HT production in TNBS-treated mice. We then determined levels of HCA species in both ileum and proximal colon from TNBS-treated mice after the treatment of HDCA in various dosages and RPFS. Notably, we showed HDCA, THDCA and GHDCA but not HCA, THCA and GHCA were significantly increased in ileum and proximal colon tissues in the treatment of HDCA in a dose-dependent manner (p<0.05 in all cases, Figure.8A-L). Similarly, HDCA, GHDCA and THDCA were found upregulated in ileum and proximal colon tissues in the treatment of RPFS (p<0.05 in all cases, Figure.8A-L). These results revealed that treatment of HDCA and RPFS at indicated dosages resulted in around 50-300µM HDCA levels in the colonic tissues of TNBS-treated mice, while HDCA can be transformed into both taurine and glycine-conjugated HDCA (20-200µM) in vivo , suggesting peripheral 5-HT inhibition in the treatment of HDCA and RPFS can be mediated by HDCA, THDCA and GHDCA. HDCA suppressed 5-HT biosynthesis by inhibiting TPH1 activity TPH1 is a rate-limiting enzyme responsible for the 5-HT production by catalyzing the conversion of tryptophan into 5-hydroxytryptophan (5-HTP), the precursor of 5-HT 9 . Through a TPH1 inhibitor screening assay, we preliminarily showed HDCA inhibited TPH1 activity in a dose-dependent manner (Figure.9A). To understand the underlying mechanisms of HDCA action on 5-HT production, we determined the molecular interactions between human TPH1 recombinant protein and HDCA species including HDCA, THDCA and GHDCA using surface plasmon resonance (SPR). It is noteworthy that HDCA exhibited rapid binding to the TPH1 with an equilibrium dissociation constant (K D ) value of 97µM, indicating a direct binding between human TPH1 activity with HDCA (Figure.9B-C). In contrast, both THDCA and GHDCA exhibited no binding to TPH1 with K D values of 6510µM and 8670µM (Figure.9D-E). We further showed inhibitory effects of HDCA on TPH1 in HEK 293 cells transfected with wildtype rhTPH1 with an IC 50 value of 265.7µM for HDCA (p<0.001, Figure.9F), while THDCA and GHDCA failed to inhibit 5-HTP synthesis within physiological dosage (n.s., Figure.9F). Herein, we revealed HDCA suppressed 5-HT biosynthesis by inhibiting TPH1 activity, thus alleviating diarrhea symptoms in patients with IBS-D. To address the binding sites of HDCA toward the TPH1 protein, we performed molecular docking and showed HDCA binds to the TPH1 protein at the following amino acid residues: Cys364, Pro268, Pro238, Leu236, Tyr312, Phe313 and Phe241 (Figure.10A) with a binding free energy of -45.40 kcal/mol (Table.S6). Accordingly, we constructed a HEK 293 cell line transfected with TPH1 plasmids containing mutations at those key residues involved in HDCA to determine the molecular mechanisms of HDCA binding to TPH1 (Figure.10B). Notably, HDCA displays a markedly suppressed binding affinity to the mutant forms of rhTPH1 Cys364 in comparison to the wild-type protein TPH1 (n.s., Figure.10C-J), suggesting Cys364 is a key binding site of HDCA for its inhibitory effects on TPH1. We also evaluated whether other receptors including FXR 10 , LXR 11 , PXR, VDR 12 , GPR119, TGR5 13 and PPARa 14 , are possibly involved in the HDCA action on 5-HT production, but we did not find agonism or antagonism of these receptors by small molecules affected inhibitory effects of HDCA on 5-HT production in QGP-1 cells (n.s., Figure.11A-L). Therefore, we demonstrated that HDCA and RPFS, as potent gut microbe-derived TPH1 inhibitors, effectively alleviated diarrhea in mice with experimental IBS. Discussion Peripheral 5-HT has been found significantly increased in IBS-D, which could be attributed to increased 5-HT biosynthesis, reduced 5-HT reuptake and EC cell hyperplasia. Currently, treatment strategies for IBS-D approved by the U.S. FDA that focus on modulating 5-HT levels or its activity at various receptors are limited 15 . To date, the U.S. FDA has approved only telotristat ethyl (LX-1032), an inhibitor of both TPH1 and TPH2, for the management of carcinoid syndrome-related diarrhea 16 . Alosetron (5-HT3 receptor antagonist) has been used to treat IBS-D in women, but severe adverse effects including increased abdominal pain, constipation and ischemic colitis have been reported 17 . Drugs that inhibit 5-HT synthesis such as LX-1031 are being explored for their potential to reduce 5-HT production in the gut, potentially alleviating symptoms of IBS-D. However, reducing peripheral 5-HT can lead to side effects such as constipation, depression, or mood changes. Achieving selective inhibition of peripheral 5-HT without affecting central nervous system 5-HT levels can be challenging. Therefore, there is a significant medical need for the development of inhibitors of 5-HT biosynthesis without substantial side effects in the treatment of IBS-D. Notably, we demonstrated that HDCA exclusively inhibits peripheral 5-HT level without affecting mental behaviors, which offers an advantage in treating IBS-D, particularly when co-morbid with anxiety or depression. In the present study, we delineated the therapeutic potential of HDCA in the treatment of IBS-D through inhibiting 5-HT biosynthesis. Our results showed that serum and fecal HDCA levels are negatively correlated with serum 5-HT and the severity of GI symptoms in IBS-D. We employed FMT studies to demonstrate that HDCA-associated microbiota is a key regulator of 5-HT production in recipient mice. The reduction of HDCA in IBS-D patients compared with HC and the regulatory role of HDCA-associated fecal microbiota on defecation frequency and fecal moisture emphasize the role of HDCA as not only a biomarker for IBS-D but also a potential therapeutic agent. Based on these observations, our preclinical studies in the TNBS-induced IBS mouse model further revealed the therapeutic roles of HDCA and RPFS in alleviating GI symptoms by inhibiting TPH1, the key enzyme in peripheral 5-HT biosynthesis. Taken together, our findings showed that HDCA is a promising drug candidate for IBS-D and other 5-HT-targeted diseases via inhibition of 5-HT biosynthesis. Clinical trials for TPH1 inhibitors have focused on conditions including carcinoid syndrome, IBS-D, pulmonary arterial hypertension (PAH), fibromyalgia, obesity, and metabolic disorders 18 . Herein, both HDCA and RPFS as potent TPH1 inhibitors can be further evaluated for their efficacy and safety profiles in these conditions. PFS, as a natural source of HDCA, has a long-standing history in traditional medicine for the treatment of diarrhea 18 . Considering that the levels of HCA and HDCA species in humans are much lower than those in pigs, the supplementation of exogenous PFS enriched with HDCA presents a promising strategy for managing IBS-D. In addition to their effects on IBS-D, HDCA and PFS have been discovered to bring numerous advantages in treating metabolic disorders such as obesity, non-alcoholic fatty liver disease (NAFLD), metabolic disorders and diabetes 12,14 . Given that inhibiting the 5-HT synthesis in the periphery has been demonstrated to improve obesity and metabolic dysfunction 19 , our research also offers a mechanism of action that explains the advantageous effects of HDCA on metabolic disorders. Considering the heterogeneous pathogenesis of IBS-D 20 and following results from this research, we are actively examining whether other pharmacological mechanisms are involved in the ameliorating effects of HDCA on GI symptoms in mouse models of IBS-D. For example, we will examine whether HDCA modulates gut-microbial metabolites (bile acids, short-chain fatty acids, amino acid-derived metabolites), neurotransmitters (histamine, glutamine, GABA, norepinephrine, dopamine, acetylcholine), neuroendocrine factors (GLP-1, PYY, NGF) and proteases to improve diarrhea and visceral hypersensitivity in different mouse models of IBS-D in the future. In conclusion, our investigation elucidates a pivotal molecular mechanism for IBS-D in which HDCA reduction is positively associated with diarrhea severity and 5-HT overproduction in IBS-D. We have revealed the therapeutic role of HDCA and RPFS, as TPH1 inhibitors, in modulating 5-HT biosynthesis, thus providing effective relief of GI symptoms in IBS-D. Furthermore, we highlight that a gut microbiota-HDCA-5HT axis is an important regulator for the management of IBS-D. These findings present a promising candidate for 5-HT-focused therapies in IBS-D and related GI disorders. Resource availability Lead contact Further information and requests for resources and reagents can be directed to and will be fulfilled by the lead contact Lixiang Zhai ( [email protected] ). Materials and data availability Reagents and resource details are provided in Table.S1. The clinical data used in this study were from the accession number PRJNA1139229 in NCBI, and are available for public access. Methods Human study We determined the changes in bile acid (BA) profiles and metabolic metabolites in 5-HT signaling in IBS-D patients from a clinical study of healthy controls and IBS-D patients which was approved by the Research Ethics Committee of Hong Kong Baptist University (HASC/15-16/0300 and HASC/16-17/0027) (ClinicalTrials.gov Identifier: NCT02822677 and NCT03457324). Briefly, IBS-D patients (n=345) meeting Rome IV diagnostic criteria and age and gender-matched healthy volunteers (n=91) as controls were recruited. Written consent was obtained from each subject prior to sample collection and analysis. Biological samples, including serum and feces of all participants, were collected and transported to the laboratory in dry ice and stored at -80 until further LC-MS analysis as previously described 3,12 . Mouse study The mouse study was approved by the Research Ethics Committee of Hong Kong Baptist University at Hong Kong Baptist University (REC/23-24/0346). 6-8 weeks old C57BL/6J mice were purchased from the Jicui Yaokang Co., Ltd. (Jiangsu Province, China) and raised in Animal Unit of Centre for Chinese Medicine Drug Development Limited. Mice were housed with a 12-h light/dark cycle at a controlled temperature of around 25℃ and a controlled humidity of 60% with free access to standard rodent diet and water. Animal experiment 1: Measurement of HCA species in germ-free mice To determine the changes of HCA species in germ-free mice and normal mice, germ-free mice were purchased from Nanjing GemPharmatech Co., Ltd. (Jiangsu Province, China) and were housed in sterile plastic isolators with fully blocked exposure to microorganisms, with the intent of keeping them free of detectable bacteria, viruses, and eukaryotic microbes at the Laboratory Animal Facility of Nanjing GemPharmatech Co., Ltd. Animal experiment 2: Fecal microbiota transplantation from IBS-D patients with either high or low fecal HDCA levels in an antibiotic-treated mouse model 6-8 weeks male wild-type mice were gavaged with 10 mL/kg antibiotics mixture containing 50 mg/kg vancomycin, 100 mg/kg neomycin, 100 mg/kg metronidazole, 100 mg/kg ampicillin, 50 mg/kg streptomycin for 10 consecutive days to establish an antibiotics-treated mouse model following a previous study 2 . The gut microbiota deletion was induced by the administration of the antibiotic mixture and confirmed by DNA gel electrophoresis of fecal samples. The antibiotics-treated mice were randomly divided into 4 groups: HDCA-enriched (HDCA + ) and HDCA-reduced (HDCA - ) fecal microbiota from IBS-D patients (n=5/group). For fecal samples of humans, about 2 g of fresh fecal samples were added with 5 times sterilized phosphate-buffered saline (PBS, m/v) and homogenized as fecal microbiota suspension. The oral gavage of fecal microbiota suspension (equal to 6mg for each mouse in 200μL PBS) was conducted for 10 days daily after finishing the antibiotics treatment. The serum and fecal levels of HDCA were measured on day 5 and 10 to determine whether the fecal microbiota transplantation affects HDCA levels in recipient mice. Defecation frequency, GI transit, fecal moisture content and visceral hypersensitivity were measured on day 10 and day 11 to determine whether fecal microbiota transplantation affects diarrhea-related phenotypes in recipient antibiotics-treated mice. Animal experiment 3: Evaluation of HDCA/Refined PULVIS FELLIS SUIS treatment on a mouse model of IBS induced by TNBS 6-8 weeks old C57BL/6J mice were fasted overnight and provided with 5% glucose in drinking water. After weighing, mice were anesthetized using 2% isoflurane. A 100 μL TNBS solution in 30% ethanol was then slowly administered into the colon using a 1 mL syringe and 3.5F catheter. The catheter was then gently removed, and the mice were positioned head-down for 5 min. Subsequently, the mice were maintained in a head-down vertical position for an additional 5 min. The control group received 100 μL saline. Mice were weighed on day 1 to 7, 14, and 21 following TNBS administration. Diarrhea-related phenotypes including defecation frequency, gastrointestinal (GI) transit and fecal moisture content were measured on day 14 to examine the establishment of the IBS model. On day 28, mice model of IBS induced by TNBS were divided into 4 groups: (1) TNBS group (n=5): mice were gavaged with vehicle control (0.5% CMC-Na); (2) 25 mg/kg HDCA (n=5): mice were administered with HDCA by oral gavage at a dose of 25 mg/kg/day; (3) 50 mg/kg HDCA (n=5): mice were administered with HDCA by oral gavage at a dose of 50 mg/kg/day; (4) 75 mg/kg HDCA (n=5): mice were administered with HDCA by oral gavage at a dose of 75 mg/kg/day; (5) Refined PULVIS FELLIS SUIS (n=5): mice were administered with Refined PULVIS FELLIS SUIS by oral gavage at a dose of 100 mg/kg/day; (6) Alosetron: mice were administered with alosetron by oral gavage at a dose of 3 mg/kg/day. Both diarrhea-related phenotypes were measured as previously described 2 after 14 days of HDCA and Refined PULVIS FELLIS SUIS treatment. In vitro study QGP-1 cells QGP-1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). QGP-1 cells were incubated in a humidified atmosphere with 95% air and 5% CO 2 at 37 ℃. When reached 80% confluence, cells were seeded to a 96-well plate at a density of 5x10 4 cells/well. After 24 hours, cells were incubated with different concentrations of HDCA, GHDCA, and THDCA for 24 hours for the determination of 5-HT production in the culture medium. Intestinal segments The intestinal tissues from 6-8 week-old mice were collected and cut into small pieces (0.5 cm) and placed in a 12-well cell culture plate. Intestinal segments were incubated at 37 ℃ with 5% CO 2 and 95% air, and cultivated with RPMI-1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin, followed by treatment with HDCA and Refined PULVIS FELLIS SUIS with various concentrations as indicated. Metabolomics analysis For fecal and colonic samples, about 50 mg of feces or colonic tissues were extracted with 10-fold 95% methanol (m/v) containing 125 ng/mL L-Tryptophan-d 5 , 50 ng/mL cholic acid-d 4 and 50 ng/mL deoxycholic acid-d 4 as internal standards. The samples were then homogenized in TissueLyser II for 2 min and stored in 4 ℃ fridges overnight. After centrifugation at 15, 000 rpm for 15 min at 4 ℃ on the second day, the supernatant was collected for LC-MS/MS analysis. For serum samples, about 80 µL serum samples were extracted with methanol containing internal standards (125 ng/mL L-Tryptophan-d 5 , 50 ng/mL cholic acid-d 4 and 50 ng/mL deoxycholic acid-d 4 ) in a ratio of 1:4 (v/v) and stored overnight at -20 ℃. After centrifugation at 15, 000 rpm for 15 min at 4 ℃, the supernatant was collected for LC-MS/MS analysis. For brain samples, about 100 mg of brain tissues were extracted with 2-fold 95% methanol (m/v) containing internal standards, and then homogenized in TissueLyser II for 2 min and stored at -20 ℃ overnight. After centrifugation at 15, 000 rpm for 15 min at 4 ℃, 100 µL supernatant was transferred was used for LC-MS/MS analysis. An Agilent 1290 Infinity II UPLC system coupled with a triple quadrupole (QQQ) 6470 mass spectrometer was used for targeted metabolomics analysis. An Agilent ECLIPSE PLUS C18 column (2.1 × 5 mm, 1.8 μm) with a pre-column was used. For the LC-MS-QQQ analysis of 5-HT, the mobile phases were solution A (water containing 0.1% formic acid) and solution B (acetonitrile containing 0.1% formic acid). The gradient was set as follows: 2% B (0-0.5 min), 2%-30% B (0.5-4 min), 30%–100% B (4-6 min), 100% B (6-8 min), 100%-2% B (8-8.5 min), and maintained at 2% B (8.5-10 min). For the LC-MS-QQQ analysis of HDCA and HCA species, the mobile phases were solution A (water containing 0.1% formic acid) and solution B (acetonitrile containing 0.1% formic acid). The gradient was as follows: 15% B (0-2 min), 15%-25% B (2-4 min), 25%-35% B (4-20 min), 35%-50% B (20-30 min), and 50%-100% B (30-33 min). The column temperature was maintained at 40°C, the flow rate was set as 0.4 mL/min, and the sample injection volume was set as 2 μL. The TQ MS was operated in positive/negative ion mode with an electrospray ionization (ESI) source separately. The ESI capillary voltage was set to 4000 V and 3500 V respectively, and the nozzle voltage to 1000 V. The nitrogen drying gas flow rate was 10 L/min, heated to 350°C. The sheath gas temperature was 350°C, with a flow rate of 8 L/min, and the nebulizer pressure was 45 psi. The mass spectrometry was performed in multiple reaction monitoring (MRM) mode. MS data were collected and processed using Agilent software, with data acquisition information provided in Table.S2. Surface plasmon resonance analysis All experiments were performed on a Biacore 8k (GE Healthcare, Sweden). The target recombinant human protein TPH1 was diluted to 40 μg/mL using 10 mM acetate at pH 4.0 and covalently immobilized on parallel channels of CM5 sensor chip surface for 10 min. The CM5 chip surface was activated by 0.4 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.1 M N-hydroxysuccinimide for 7 min at a flow rate of 10 μL/min. The immobilized chip was blocked with 1 M ethanolamine for 7 min at a flow rate of 10 μL/min. The HDCA was diluted in running buffer containing phosphate buffered saline (PBS), 1mM dithiothreitol, 0.5% P20 and 5% dimethylsulfoxide (DMSO) at a series of concentrations 2.5, 5, 10, 25, 50, 100 μM, respectively. The solution of HDCA was injected for 3 min at 30 μL/min, and the dissociation was followed for 1.5 min. All data were analyzed using kinetic models with Biacore 8k Evaluation Software. A TPH1 inhibitor LX-1031 and tryptophan were used as positive controls for the binding experiment of HDCA. Molecular docking Docking analysis was performed by Schrödinger v.2023 software with default parameters. The protein structure of TPH1 (PDB ID: 5JD6) and HDCA structure (CAS: 83-49-8) were uploaded to the software. MOE was applied for the interaction analysis between TPH1 and HDCA. Cloning strategies The full-length human TPH1 gene (NM_004179.3) was from Pcr-bluntII-TOPO-TPH1 plasmid (Miaoling). TPH1 was recombined into PLV3-CMV-MCS-3xFLAG-Puro vector and one FLAG tag at the amino terminus through BmaHI and NheI. The plasmids were confirmed by Sanger sequencing by BGI. 50 μl PCR reactions were performed using Phusion ® high-fidelity DNA polymerase (New England Biolabs). The cycling protocol was 98°C, 30 sec, then 35 cycles of 98°C 15s, 55°C 30s, 72°C 1min. The final extension is 72°C, 10min. The resulting PCR products are digested using BmaHI and NheI (New England Biolabs). DNA ligation reactions were performed in a final volume of 10 μl using T4 DNA ligase following the manual from New England Biolabs. Site-directed mutagenesis by PCR Amplify the parental plasmid (PLV3-CMV-MCS-3xFLAG-Puro vector) containing TPH1. 50 μl PCR reactions were performed using Phusion® high-fidelity DNA polymerase (New England Biolabs). The cycling protocol was 98°C, 30 sec, then 35 cycles of 98°C 15s, 55°C 30s, 72°C 5min. The final extension is 72°C, 10min. And then PCR products are digested by DpnI at 37°C for 2h. The digested PCR products are transformed into DH5α. Establishment of TPH1 overexpression cell line The 293T cells were cultured with DMEM supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco) under the conditions of 37 °C, 5% CO2. Briefly, 6 x 10 6 293 T cells were seeded in a 10 cm dish in complete DMEM medium 24h prior to transfection with plasmid DNA (TPH1, point mutant TPH1 Leu236, Pro238, Phe241, Pro268, Ala 309, Tyr312 and Phe313) at a ratio of 4:3:1. Polyethylenimine (PEI) was used following the manufacturer’s instructions to produce lentivirus particles. Viral harvest was carried out at 48 post-transfection. The harvested medium was subsequently filtered to remove cell debris using a 0.45‐μm filter. Transfected cells were cultured under the same conditions for another 48h before puromycin selection. TPH1 activity assay TPH1 wildtype and TPH1 mutant cells are seeded in 24 well plates overnight. And then the cells are treated with HDCA and LX-1031 for 12h. A TPH1 inhibitor LX-1031 and tryptophan were used as positive controls for the binding experiment of HDCA. About 100 µL medium was extracted with methanol containing internal standards (125 ng/mL L-Tryptophan-d5) in a ratio of 1:4 (v/v) and stored overnight at -20 ℃. After centrifugation at 15, 000 rpm for 15 min at 4 ℃, the supernatant was collected for LC-MS/MS analysis. Statistical analysis Unless otherwise specified, data are presented as mean ± standard error of the mean (SEM) in figures and text. Normality tests were conducted, followed by either parametric or non-parametric tests as appropriate. Student’s t-tests (paired or unpaired) or one-way ANOVA were used in relevant scenarios as indicated. Data were analyzed using GraphPad Prism 10.0, and p -values less than 0.05 were considered statistically significant. Abbreviations 5-HIAA 5-hydroxyindoleacetic acid 5-HT Serotonin 5-HTP 5-hydroxytryptophan BA Bile acid EC Enterochromaffin FMT Fecal microbiota transplantation GHCA Glycohyocholic acid GHDCA Glycohyodeoxycholic acid GI Gastrointestinal HCA Hyocholic acid HC Healthy controls HDCA Hyodeoxycholic acid IBS Irritable bowel syndrome IBS-D Diarrhea-predominant irritable bowel syndrome K D Dissociation constant NAFLD Non-alcoholic fatty liver disease PCA Principle component analysis RPFS Refined PULVIS FELLIS SUIS SPR Surface plasmon resonance THCA Taurohyocholic acid THDCA Taurohyodeoxycholic acid TNBS 2,4,6-trinitrobenzene sulfonic acid TPH1 Tryptophan hydroxylase 1 TPH2 Tryptophan hydroxylase 2 Trp L-tryptophan Declarations Acknowledgments This study is supported by General Research Fund from the University Grants Committee (12100323 to Zhaoxiang Bian), Health@InnoHK Initiative Fund from the Hong Kong SAR Government (ITC RC/IHK/4/7 to Zhaoxiang Bian), the General Program from the Guangdong Basic and Applied Basic Research Foundation (2414050003325 to Lixiang Zhai) and Collaborative Research Fund from the University Grants Committee (C2004-23Y to Lixiang Zhai and Lu Zhang). The authors express their gratitude to all the participants in this study and to the Chinese medicine practitioners who will contribute to the study but are not listed as authors. The authors also declare that they have no competing interests. The authors also thank Vincent and Lily Woo Foundation for the funding support of this work and Wu Jieh Yee Institute of Translational Chinese Medicine Research, HKBU for the technical support. Authorship Contributions Z. B., L.Zhai and H.X. are the principal investigators and design this study. L.Zhai, G.B., J.L. and M.W. drafted the manuscript. G.B., S.X., J.L., B.L., Y. L., and M.Z. conducted the in vivo and in vitro studies including animal experiments and cell experiments. G.B. and S.X. conducted the metabolomics analysis. S.X., Y.Z., Z.N., B.L., Y.L., W.Y., and M.Z. manage the clinical specimens collected from all participants. L.Zhao, L.Zhang, C.L. and X.HL.W. provided important suggestions for the study design and helped revise the manuscript. All authors have read and approved the manuscript. Declaration of interests The authors declare that they have no competing interests. The authors have filed patent based in part on the findings in this study. References Wechsler, E. V. & Shah, E. D. Diarrhea-Predominant and Constipation-Predominant Irritable Bowel Syndrome: Current Prescription Drug Treatment Options. Drugs 81 , 1953-1968 (2021). https://doi.org/10.1007/s40265-021-01634-7 Gao, J., Xiong, T. T., Grabauskas, G. & Owyang, C. Mucosal Serotonin Reuptake Transporter Expression in Irritable Bowel Syndrome Is Modulated by Gut Microbiota Via Mast Cell-Prostaglandin E2. Gastroenterology 162 , 1962-+ (2022). https://doi.org/10.1053/j.gastro.2022.02.016 Zhai, L. X. et al. Ruminococcus gnavus plays a pathogenic role in diarrhea-predominant irritable bowel syndrome by increasing serotonin biosynthesis. Cell Host & Microbe 31 , 33-+ (2023). https://doi.org/10.1016/j.chom.2022.11.006 Lembo, A. et al. AGA Clinical Practice Guideline on the Pharmacological Management of Irritable Bowel Syndrome With Diarrhea. Gastroenterology 163 , 137-153 (2022). https://doi.org/10.1053/j.gastro.2022.04.017 Binienda, A., Storr, M., Fichna, J. & Salaga, M. Efficacy and Safety of Serotonin Receptor Ligands in the Treatment of Irritable Bowel Syndrome: A Review. Current Drug Targets 19 , 1774-1781 (2018). https://doi.org/10.2174/1389450119666171227225408 Yano, J. M. et al. Indigenous Bacteria from the Gut Microbiota Regulate Host Serotonin Biosynthesis. Cell 161 , 264-276 (2015). https://doi.org/10.1016/j.cell.2015.02.047 Zhai, L. X. et al. Gut microbiota-derived tryptamine and phenethylamine impair insulin sensitivity in metabolic syndrome and irritable bowel syndrome. Nature Communications 14 (2023). https://doi.org/10.1038/s41467-023-40552-y Ligneul, R. & Mainen, Z. F. Serotonin. Curr. Biol. 33 , R1216-R1221 (2023). https://doi.org/10.1016/j.cub.2023.09.068 Nowak, E. C. et al. Tryptophan hydroxylase-1 regulates immune tolerance and inflammation. Journal of Experimental Medicine 209 , 2127-2135 (2012). https://doi.org/10.1084/jem.20120408 Song, M. et al. Hyodeoxycholic acid (HDCA) suppresses intestinal epithelial cell proliferation through FXR-PI3K/AKT pathway, accompanied by alteration of bile acids metabolism profiles induced by gut bacteria. Faseb Journal 34 , 7103-7117 (2020). https://doi.org/10.1096/fj.201903244R De Marino, S. et al. Hyodeoxycholic acid derivatives as liver X receptor α and G-protein-coupled bile acid receptor agonists. Scientific Reports 7 (2017). https://doi.org/10.1038/srep43290 Zheng, X. J. et al. Hyocholic acid species improve glucose homeostasis through a distinct TGR5 and FXR signaling mechanism. Cell Metabolism 33 , 791-+ (2021). https://doi.org/10.1016/j.cmet.2020.11.017 Zhu, H. et al. Hyodeoxycholic acid inhibits lipopolysaccharide-induced microglia inflammatory responses through regulating TGR5/AKT/NF-κB signaling pathway. Journal of Psychopharmacology 36 , 849-859 (2022). https://doi.org/10.1177/02698811221089041 Kuang, J. L. et al. Hyodeoxycholic acid alleviates non-alcoholic fatty liver disease through modulating the gut-liver axis. Cell Metabolism 35 , 1752-+ (2023). https://doi.org/10.1016/j.cmet.2023.07.011 Brenner, D. M. & Sayuk, G. S. Current US Food and Drug Administration-Approved Pharmacologic Therapies for the Treatment of Irritable Bowel Syndrome with Diarrhea. Advances in Therapy 37 , 83-96 (2020). https://doi.org/10.1007/s12325-019-01116-z Dillon, J. S. & Chandrasekharan, C. Telotristat ethyl: a novel agent for the therapy of carcinoid syndrome diarrhea. Future Oncology 14 , 1155-1164 (2018). https://doi.org/10.2217/fon-2017-0340 Alosetron approved for treatment of irritable bowel syndrome. American Journal of Health-System Pharmacy 57 , 519-519 (2000). Bader, M. Inhibition of serotonin synthesis: A novel therapeutic paradigm. Pharmacology & Therapeutics 205 (2020). https://doi.org/10.1016/j.pharmthera.2019.107423 Crane, J. D. et al. Inhibiting peripheral serotonin synthesis reduces obesity and metabolic dysfunction by promoting brown adipose tissue thermogenesis. Nature Medicine 21 , 166-172 (2015). https://doi.org/10.1038/nm.3766 Enck, P. et al. Irritable bowel syndrome. Nature Reviews Disease Primers 2 (2016). https://doi.org/10.1038/nrdp.2016.14 Additional Declarations There is NO Competing Interest. Supplementary Files 2025052803Supplementaldataset.xlsx Supplemental dataset 1-6 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. 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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-6769911","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":483189288,"identity":"a8f024c4-33f5-4d82-b80a-fd0bcc4c4895","order_by":0,"name":"Lixiang 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Hong Kong Baptist University","correspondingAuthor":false,"prefix":"","firstName":"Zhao-Xiang","middleName":"","lastName":"Bian","suffix":""}],"badges":[],"createdAt":"2025-05-28 16:25:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6769911/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6769911/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87053592,"identity":"1e0741b2-0ca1-4485-beed-67d807c3975c","added_by":"auto","created_at":"2025-07-18 15:13:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1302575,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFecal and serum HDCA were negatively associated with serum 5-HT and diarrhea symptoms in patients with IBS-D. (A) \u003c/strong\u003eSerum 5-HT level in IBS-D patients with normal 5-HT (5-HT\u003csup\u003econtrol\u003c/sup\u003e IBS-D group) and IBS-D patients with high 5-HT (5-HT\u003csup\u003e+\u003c/sup\u003e IBS-D group) based on a 75% cut-off value of serum 5-HT from healthy control (HC) group. \u003cstrong\u003e(B) \u003c/strong\u003ePrincipal component analysis\u003cstrong\u003e (\u003c/strong\u003ePCA) score plot of fecal metabolomics profiles in 5-HT\u003csup\u003econtrol\u003c/sup\u003e IBS-D group, 5-HT\u003csup\u003e+\u003c/sup\u003e IBS-D group and HC group. \u003cstrong\u003e(C) \u003c/strong\u003eVolcano plot of fecal metabolites in 5-HT\u003csup\u003e+\u003c/sup\u003e IBS-D group and HC group. \u003cstrong\u003e(D)\u003c/strong\u003e Nonparametric Spearman correlation analysis between fecal bile acid (BA) species with peripheral 5-HT, defecation frequency and Bristol stool scale in 5-HT\u003csup\u003econtrol\u003c/sup\u003e IBS-D group and 5-HT\u003csup\u003e+\u003c/sup\u003e IBS-D group. \u003csup\u003e*** \u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, \u003csup\u003e** \u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e\u0026lt;0.05. \u003cstrong\u003e(E-F) \u003c/strong\u003eFecal and serum HCA species levels in 5-HT\u003csup\u003econtrol\u003c/sup\u003e IBS-D group, 5-HT\u003csup\u003e+\u003c/sup\u003e IBS-D group and HC group. (\u003cstrong\u003eG\u003c/strong\u003e) Nonparametric Spearman correlation analysis between fecal and serum HCA species with peripheral 5-HT, defecation frequency and Bristol stool scale in 5-HT\u003csup\u003e+\u003c/sup\u003e IBS-D group.\u003csup\u003e *** \u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, \u003csup\u003e** \u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e\u0026lt;0.05. Data were determined by ordinary one-way ANOVA. Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6769911/v1/6ffc2ad0606c0435b58f2145.png"},{"id":87051768,"identity":"9c7183b4-64fa-4af3-b1fc-b2cf82b7dddc","added_by":"auto","created_at":"2025-07-18 15:05:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":215420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe negative association between fecal and serum HDCA with serum 5-HT and diarrhea symptoms in patients with IBS-D in a validation cohort. (A)\u003c/strong\u003e Nonparametric Spearman correlation analysis between fecal HDCA with peripheral 5-HT, defecation frequency and Bristol stool scale in 5-HT\u003csup\u003econtrol\u003c/sup\u003e IBS-D group and 5-HT\u003csup\u003e+\u003c/sup\u003e IBS-D group. \u003cstrong\u003e(B)\u003c/strong\u003e Nonparametric Spearman correlation analysis between serum HDCA with peripheral 5-HT, defecation frequency and Bristol stool scale in 5-HT\u003csup\u003econtrol\u003c/sup\u003e IBS-D group and 5-HT\u003csup\u003e+\u003c/sup\u003e IBS-D group.\u003csup\u003e *** \u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, \u003csup\u003e** \u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6769911/v1/f4f854a6dc801fd7b8013b9f.png"},{"id":87051767,"identity":"ed551d75-35e2-4c51-8883-a1e2648e2bbd","added_by":"auto","created_at":"2025-07-18 15:05:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":647546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHCA species were depleted in germ-free mice and antibiotics-treated mice. (A) \u003c/strong\u003eSchematic illustration of experimental design for evaluation of changes in HCA species in germ-free mice and antibiotics-treated mice compared with mice raised in an SPF environment. (\u003cstrong\u003eB-G\u003c/strong\u003e) Fecal levels of HCA, HDCA, GHCA, GHDCA, THCA, and THDCA in SPF mice, germ-free mice, and antibiotics-treated mice. \u003cstrong\u003e(H-M)\u003c/strong\u003e Serum levels of HCA, HDCA, GHCA, GHDCA, THCA and THDCA in SPF mice, germ-free mice, and antibiotics-treated mice.\u003cstrong\u003e \u003c/strong\u003eData were determined by ordinary one-way ANOVA. Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6769911/v1/37230fb492978ec6f86c4b58.png"},{"id":87051770,"identity":"079ffc3f-d2b3-42f9-9d3b-225f5aa1bbc2","added_by":"auto","created_at":"2025-07-18 15:05:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":612736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDCA-associated fecal microbiota from IBS-D patients regulated peripheral 5-HT and GI motility in antibiotics-treated mice. (A) \u003c/strong\u003eSchematic illustration of experimental design for FMT study in recipient mice with different levels of HDCA from IBS-D patients in HDCA-deficient microbiota (HDCA\u003csup\u003e-\u003c/sup\u003e FMT) group and HDCA-enriched microbiota (HDCA\u003csup\u003e+\u003c/sup\u003e FMT) group. \u003cstrong\u003e(B)\u003c/strong\u003e Fecal HCA profiles in IBS-D donors in HDCA\u003csup\u003e-\u003c/sup\u003e FMT group and HDCA\u003csup\u003e+\u003c/sup\u003e FMT group. \u003cstrong\u003e(C-E) \u003c/strong\u003eGI transit time, defecation frequency, and fecal moisture content in recipient mice transplanted with fecal microbiota from HDCA\u003csup\u003e-\u003c/sup\u003e FMT group and HDCA\u003csup\u003e+\u003c/sup\u003e FMT group. \u003cstrong\u003e(F-H) \u003c/strong\u003e5-HT levels in serum, ileum and proximal colon tissues of recipient mice transplanted with fecal microbiota from HDCA\u003csup\u003e-\u003c/sup\u003e FMT group and HDCA\u003csup\u003e+\u003c/sup\u003e FMT group. \u003cstrong\u003e(I-J)\u003c/strong\u003e HCA species profiles in serum and ileum tissues of recipient mice transplanted with fecal microbiota from HDCA\u003csup\u003e-\u003c/sup\u003e FMT group and HDCA\u003csup\u003e+\u003c/sup\u003e FMT group. Data were analyzed by student’s t-test (two-tailed Mann-Whitney test). Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6769911/v1/74c824b36bebe5cc7ab0da1a.png"},{"id":87053593,"identity":"bb3173ad-e277-4585-acee-4b710d170a52","added_by":"auto","created_at":"2025-07-18 15:13:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":403226,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDCA and RPFS inhibited 5-HT production \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (A-B) \u003c/strong\u003e5-HT levels in the culture medium of QGP-1 cells and mice intestinal tissues in the treatment of HDCA at indicated concentration. \u003cstrong\u003e(C) \u003c/strong\u003eTotal\u003cstrong\u003e \u003c/strong\u003eBA concentrations and BA profiles in RPFS. \u003cstrong\u003e(D)\u003c/strong\u003e 5-HT levels in the culture medium of mice intestinal tissues in the treatment of RPFS at indicated concentration. \u003cstrong\u003e(E-F)\u003c/strong\u003e5-HT levels in the culture medium of QGP-1 cells in the treatment of UDCA and CDCA at indicated concentration. Data were determined by ordinary one-way ANOVA. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6769911/v1/36fcd7a9fe55d507af95eb02.png"},{"id":87051769,"identity":"a9250997-baf5-4315-8bde-479e59d6f1a0","added_by":"auto","created_at":"2025-07-18 15:05:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1789650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDCA and RPFS alleviated diarrhea and inhibited 5-HT production in a mouse model of IBS. (A) \u003c/strong\u003eSchematic illustration of experimental design for evaluation of HDCA (25, 50, or 75 mg/kg) and RPFS (100mg/kg) in TNBS-induced IBS model in mice. Eluxadoline (15 mg/kg) was used as the positive control. \u003cstrong\u003e(B-D)\u003c/strong\u003eGI transit time, defecation frequency, and fecal moisture content in TNBS-treated mice in the treatment of HDCA, RPFS and eluxadoline. \u003cstrong\u003e(E-F) \u003c/strong\u003e5-HT levels in ileum and proximal colon tissues of TNBS-treated mice in the treatment of HDCA, RPFS and eluxadoline. \u003cstrong\u003e(G-H) \u003c/strong\u003eIF staining of chromogranin A (CgA) and 5-HT inproximal colon tissues of TNBS-treated mice in the treatment of HDCA and RPFS. \u003cstrong\u003e(I-J) \u003c/strong\u003eHDCA and 5-HT levels in brain tissues of TNBS-treated mice in the treatment of HDCA and RPFS. Data were analyzed using ordinary one-way ANOVA. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6769911/v1/e475c3285336442c83d7e980.png"},{"id":87051771,"identity":"ae7965e1-47f5-4677-a3ab-3791de71f758","added_by":"auto","created_at":"2025-07-18 15:05:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":752316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDCA and RPFS showed no detrimental effects on mental behaviors in a mouse model of IBS. (A-C) \u003c/strong\u003eTime spent, central zone entries and total distance in the open field experiments in TNBS-treated mice in the treatment of HDCA and RPFS. \u003cstrong\u003e(D)\u003c/strong\u003e Representative moving trace of TNBS-treated mice in the treatment of HDCA and RPFS in the open field test. Data were analyzed using ordinary one-way ANOVA. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6769911/v1/4e09ef41e44a7de1dae73bb2.png"},{"id":87053595,"identity":"803acbf5-a135-4939-9e73-90638533a227","added_by":"auto","created_at":"2025-07-18 15:13:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":954071,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDCA and RPFS increased HDCA, GHDCA and THDCA in a mouse model of IBS. (A-F) \u003c/strong\u003eHCA (HCA, GHCA and THCA) species and HDCA species (HDCA, GHDCA and THDCA) in ileum tissues of TNBS-treated mice in the treatment of HDCA and RPFS.\u003cstrong\u003e (G-L)\u003c/strong\u003e HCA species and HDCA species in proximal colon tissues of TNBS-treated mice in the treatment of HDCA and RPFS.\u003cstrong\u003e \u003c/strong\u003eData were analyzed using ordinary one-way ANOVA. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6769911/v1/2c907ff02c9a41b86635a729.png"},{"id":87051775,"identity":"b476d4e5-1232-4dcf-bff9-1fccfc8d5fd1","added_by":"auto","created_at":"2025-07-18 15:05:49","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":402659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDCA is a moderate TPH1 inhibitor. (A) \u003c/strong\u003eInhibitory activities of HDCA at the indicated concentration on TPH1 by \u003cem\u003ein vitro\u003c/em\u003e enzyme activity assay. \u003cstrong\u003e(B-C)\u003c/strong\u003eSPR binding studies of human TPH1 with tryptophan, TPH1 inhibitor (LX-1031) and HDCA at indicated concentration. \u003cstrong\u003e(D-E)\u003c/strong\u003e SPR binding studies of human TPH1 with GHDCA and THDCA at indicated concentration. \u003cstrong\u003e(F)\u003c/strong\u003e Inhibitory activities of HDCA, GHDCA and THDCA at indicated concentration on 5-HTP production in HEK 293 cells transfected with wildtype TPH1. Data were analyzed using ordinary one-way ANOVA. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-6769911/v1/9b56f236c83f5f11eae6a2e7.png"},{"id":87051777,"identity":"10acceb1-50c2-47b4-9680-71a5d97478a6","added_by":"auto","created_at":"2025-07-18 15:05:49","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1172923,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCys364 is a key binding site of HDCA for TPH1 inhibition. (A) \u003c/strong\u003eMolecular binding modes of HDCA with the key residues of TPH1 and 2D contour of the binding modes. \u003cstrong\u003e(B) \u003c/strong\u003eExpression of Flag-TPH1 with amino acids mutation of Leu236, Pro238, Phe241, Pro268, Tyr312, Phe313 and Cys364 in HEK 293 cells. \u003cstrong\u003e(C-J) \u003c/strong\u003eEffects of HDCA and LX-1031 on 5-HTP production in HEK 293 cells transfected with Flag-TPH1 with amino acids mutation. Data were analyzed using ordinary one-way ANOVA. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-6769911/v1/34b43529d13e899b51fc3cf8.png"},{"id":87051776,"identity":"3d8493c6-7c66-408c-97ef-e3c25e8137e2","added_by":"auto","created_at":"2025-07-18 15:05:49","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":775728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlockade and activation of bile acid receptors failed to affect the inhibitory effects of HDCA on 5-HT production in QGP-1 cells.\u003c/strong\u003e (A) Effects of pregnane X receptor (PXR) agonist forskolin and antagonist resveratrol on 5-HT production in QGP-1 cells. (B) Cell viability of forskolin and resveratrol in QGP-1 cells. (C) Effects of farnesoid X receptor (FXR) agonist GW4064, and antagonists NDB and tauro-β-muricholic acid on 5-HT production in QGP-1 cells. (D) Cell viability of GW4064, NDB and tauro-β-muricholic acid in QGP-1 cells. (E) Effects of the G protein-coupled receptor 119 (GPR119) agonist GSK1292263 and MBX-2982 on 5-HT production in QGP-1 cells. (F)Cell viability of GSK1292263 and MBX-2982 in QGP-1 cells. (G) Effects of vitamin D receptor (VDR) agonist calcitriol and antagonist ZK168281 on 5-HT production in QGP-1 cells. (H)Cell viability of calcitriol and ZK168281 in QGP-1 cells. (I) Effects of takeda G protein-coupled receptor 5 (TGR5) agonist INT777 and antagonist SBI-115 on 5-HT production in QGP-1 cells. (J)Cell viability of INT777 and SBI-115 in QGP-1 cells. (K) Effects of the liver X receptor (LXR) agonist GW3965 hydrochloride and antagonist GSK2033 on 5-HT production in QGP-1 cells. (L)Cell viability of GW3965 hydrochloride and GSK2033 in QGP-1 cells. (M) Effects of the peroxisome proliferator-activated receptor α (PPARα) agonist GW7647 on 5-HT synthesis. (N) Cell viability of GW7647 in QGP-1 cells. Data were analyzed using the Kruskal-Wallis test. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-6769911/v1/38841a4425cda430fe50d8c4.png"},{"id":88567509,"identity":"a215a720-a838-4e36-90c4-d261f2cea179","added_by":"auto","created_at":"2025-08-07 20:28:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8850755,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6769911/v1/ed7f6aeb-22d0-4fa7-951a-30935388c0e3.pdf"},{"id":87053594,"identity":"563800fa-2f78-4dc0-b5f1-de4b248a85c5","added_by":"auto","created_at":"2025-07-18 15:13:49","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28377,"visible":true,"origin":"","legend":"Supplemental dataset 1-6","description":"","filename":"2025052803Supplementaldataset.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6769911/v1/3d7dc8617f11283c5cc1c76a.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Inhibition of peripheral serotonin synthesis by a gut microbe-derived TPH1 inhibitor, hyodeoxycholic acid, alleviates irritable bowel syndrome","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiarrhea-predominant irritable bowel syndrome (IBS-D) is a complicated gastrointestinal disorder affecting people globally. The persistent occurrence of diarrhea combined with abdominal discomfort profoundly affects the quality of life of patients. Due to its chronic gastrointestinal (GI) symptoms, IBS-D can also lead to exhaustion and emotional strain, deteriorating life quality. Given the diverse symptoms of IBS-D and the absence of a universally effective solution, devising treatment strategies for IBS-D remains intricate due to our limited understanding of its pathogenesis\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSerotonin (5-hydroxytryptamine, 5-HT) is implicated in the pathophysiology of IBS-D. 5-HT is an important factor in controlling various GI processes, including movement, secretion and sensitivity. Approximately 90% of peripheral 5-HT is synthesized by enterochromaffin (EC) cells located in the GI tract via the rate-limiting enzyme tryptophan hydroxylase (TPH1). In IBS-D, abnormalities of peripheral 5-HT biosynthesis and reuptake, along with EC cell hyperplasia, result in elevated peripheral 5-HT levels in the gut to aggravate diarrhea\u003csup\u003e2,3\u003c/sup\u003e. Research and drug development have concentrated on manipulating 5-HT signaling as a therapeutic approach for IBS-D. For example, medications impacting 5-HT receptors or 5-HT biosynthesis regulate bowel movement\u003csup\u003e4\u003c/sup\u003e. However, current FDA-approved drugs targeting 5-HT signaling require long-term treatment and with side effects such as constipation, ischemic colitis and cardiovascular risks\u003csup\u003e5\u003c/sup\u003e. Consequently, there is a need for new strategies that specifically fine-tune 5-HT signaling in the gut to provide more targeted and effective solutions for IBS-D.\u003c/p\u003e\n\u003cp\u003eRecent research indicates a causal link between gut microbiota, gut-microbial metabolites and peripheral 5-HT production. These gut bacteria communicate with EC cells via gut-microbial metabolites such as short-chain fatty acids, deoxycholic acid and tyramine to affect the biosynthesis, release, and reuptake of 5-HT\u003csup\u003e6\u003c/sup\u003e. Our previous findings also showed gut microbes-derived aromatic trace amines contribute to diarrhea in IBS-D\u003csup\u003e3,7\u003c/sup\u003e. These intricate interactions between the gut microbiota and 5-HT signaling highlight the potential for developing gut-microbial metabolites as novel therapeutics in modulating peripheral 5-HT levels, thereby affecting gut movement to alleviate symptoms of IBS-D. However, whether there are microbial metabolites that improve diarrhea by inhibiting peripheral 5-HT in IBS-D remains unknown.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we aimed to identify microbial metabolites that regulate 5-HT abnormalities for managing diarrhea in IBS-D. Through a targeted metabolomics approach and \u003cem\u003ein vitro\u003c/em\u003e experiments, we found that a microbial metabolite hyodeoxycholic acid (HDCA, 3α,6α-dihydroxy-5β-cholanic acid), negatively correlates with the severity of diarrhea symptoms and peripheral 5-HT levels, suggesting HDCA is potentially beneficial for managing IBS-D and regulating 5-HT abnormalities. Through fecal microbiota transplantation, we further confirmed that HDCA-enriched fecal microbiota plays a crucial role in regulating 5-HT production and GI motility. In addition, we showed HDCA inhibits 5-HT production in \u003cem\u003eex vivo\u003c/em\u003e mice colonic tissues.\u003c/p\u003e\n\u003cp\u003eTherefore, we investigated the action and underlying molecular mechanisms of HDCA and HDCA-enriched traditional medicine PULVIS FELLIS SUIS (PFS) on GI symptoms in a mouse model of IBS-D. Notably, we showed HDCA and PFS alleviated diarrhea symptoms and inhibited 5-HT production in mice with experimental IBS by directly inhibiting the TPH1 activity, a rate-limiting enzyme for 5-HT biosynthesis. Our findings suggest that HDCA is a promising microbe-derived therapeutic agent for IBS-D through inhibiting peripheral 5-HT biosynthesis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHDCA was negatively associated with serum 5-HT in patients with IBS-D\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify metabolites that correlated with 5-HT production in IBS-D patients, we first conducted sub-group analysis in IBS-D patients using a 75% cut-off value of serum 5-HT we determined in healthy controls (HC). Accordingly, IBS-D patients were grouped as IBS-D 5-HT\u003csup\u003e+\u003c/sup\u003e (\u0026gt;75% cut-off value of 5-HT in HC) group and IBS-D 5-HT\u003csup\u003econtrol\u003c/sup\u003e group (\u003cu\u003e\u0026lt;\u003c/u\u003e75% cut-off value of 5-HT in HC) and we showed serum levels of 5-HT in IBS-D 5-HT\u003csup\u003e+\u003c/sup\u003e patients were significantly higher compared with subjects from IBS-D 5-HT\u003csup\u003econtrol\u003c/sup\u003e group and HC group (p\u0026lt;0.001 in all cases, Figure.1A).\u003c/p\u003e\n\u003cp\u003eWe then determined changes in the fecal metabolome of IBS-D 5-HT\u003csup\u003e+\u003c/sup\u003e patients in comparison with HC group and IBS-D 5-HT\u003csup\u003econtrol\u003c/sup\u003e group to further investigate the correlation between gut-microbial metabolites in feces, serum 5-HT and GI symptoms. As shown in Figure.1B, significant differences in metabolomic profiles were found in IBS-D 5-HT\u003csup\u003e+\u003c/sup\u003e group compared with IBS-D 5-HT\u003csup\u003econtrol\u003c/sup\u003e group and HC group. Notably, significant changes in fecal bile acid profiles were found in IBS-D 5-HT\u003csup\u003e+\u003c/sup\u003e patients as shown by reduction of hyocholic acid (HCA) species including hyodeoxycholic acid (HDCA), taurohyocholic acid (THCA), taurohyodeoxycholic acid (THDCA), as well as increment of chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and lithocholic acid (LCA) (Figure.1C). Accordingly, we determined the correlation between fecal bile acid profiles, serum 5-HT and diarrhea symptoms indexes. Among all bile acid species, we showed that only fecal HCA species were negatively correlated with peripheral 5-HT and diarrhea indexes (r=-0.3, -0.27 and -0.36, p\u0026lt;0.05 in all cases, Figure.1D).\u003c/p\u003e\n\u003cp\u003eAmong all HCA species, we showed that only HDCA was significantly reduced in both serum and fecal samples from IBS-D 5-HT\u003csup\u003e+\u003c/sup\u003e patients compared with HC group among all HCA species (p\u0026lt;0.001 in both cases, Figure.1E-F). Correlation analysis revealed fecal and serum HDCA was negatively associated with serum 5-HT levels and diarrhea indexes in IBS-D patients with the lowest r-value and p-value (r=-0.41, -0.46, -0.23, -0.31, -0.37, and -0.27, p\u0026lt;0.05 in all cases, Figure.1G). Accordingly, we also validated the negative correlation between HDCA, diarrhea severity and 5-HT levels in another IBS-D cohort (r\u0026lt;-0.3, p\u0026lt;0.01 in all cases in 5-HT\u003csup\u003e+\u003c/sup\u003e IBS-D subgroup, Figure.2). These results established the clinical relevance of HDCA on 5-HT abnormalities in IBS-D and demonstrated that targeting HDCA is a potential therapeutic strategy for diarrhea management in IBS-D through regulating 5-HT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHDCA-associated fecal microbiota from IBS-D patients regulated peripheral 5-HT and GI motility in antibiotics-treated mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand whether endogenous HDCA is modulated by gut microbiota, we determined changes in HCA species in both germ-free mice and antibiotics-treated mice (Figure.3A). Notably, we showed both HCA and HDCA and their glycine and taurine-conjugated forms GHCA, THCA, GHDCA and THDCA are significantly reduced in both serum and fecal samples from both germ-free mice and antibiotics-treated mice compared with mice raised in an SPF environment (p\u0026lt;0.01 in both cases, Figure.3B-M), revealing endogenous HDCA is manipulated by gut microbiota.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on this finding, we performed HDCA-associated fecal microbiota transplantation (FMT) from IBS-D patients in recipient mice after treatment with antibiotics. To determine the causal effects of HDCA-associated fecal microbiota on serum 5-HT and GI motility, fecal microbiota from IBS-D patients with high and low HDCA levels based on a top/bottom 25% cut-off value (labeled as HDCA\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eand HDCA\u003csup\u003e-\u003c/sup\u003e) was orally administered to recipient antibiotic-treated mice (Figure.4A). Fecal HDCA levels from donor samples in HDCA\u003csup\u003e+\u003c/sup\u003e FMT group was significantly higher compared with HDCA\u003csup\u003e-\u0026nbsp;\u003c/sup\u003eFMT group (p\u0026lt;0.001, Figure.4B). Notably, we showed GI motility was also regulated by HDCA-enriched FMT as evidenced by prolonged GI transit time and decreased defecation frequency, although no significant changes in fecal moisture content (p\u0026lt;0.05 in both cases, Figure.4C-D,\u0026nbsp;n.s., Figure.4E). Moreover, peripheral 5-HT was also found significantly reduced in HDCA\u003csup\u003e+\u003c/sup\u003e FMT group compared with HDCA\u003csup\u003e-\u0026nbsp;\u003c/sup\u003eFMT group as shown by 5-HT levels in serum, ileum and proximal colonic tissues (p\u0026lt;0.001 in both cases, Figure.4F-H). In alignment with fecal HCA species profiles from donor samples in HDCA\u003csup\u003e+\u003c/sup\u003e FMT group and HDCA\u003csup\u003e-\u0026nbsp;\u003c/sup\u003eFMT group, we showed HDCA significantly increased in serum and ileum samples from recipient mice in HDCA\u003csup\u003e+\u003c/sup\u003e FMT group compared with HDCA\u003csup\u003e-\u0026nbsp;\u003c/sup\u003eFMT group (p\u0026lt;0.001 in both cases, Figure.4I-J). These data suggested that HDCA-associated fecal microbiota regulate GI motility and peripheral 5-HT levels, highlighting the therapeutic potential of HDCA for the treatment of diarrhea in IBS-D.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHDCA and Refined PULVIS FELLIS SUIS (RPFS) alleviated diarrhea and inhibited 5-HT production in a mouse model of IBS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then employed \u003cem\u003ein vitro\u003c/em\u003e models, including QGP-1 cells along with mice intestinal segment cultures, to study the effects of HDCA on 5-HT production. Notably, we showed HDCA significantly inhibited 5-HT production in these models in a dose-dependent manner and time-dependent manner in QGP-1 cells (p\u0026lt;0.05, Figure.5A) and intestinal segment cultures from mice (p\u0026lt;0.05 in both cases, Figure.5B), suggesting HDCA is a potential candidate for the management of diarrhea through inhibiting 5-HT production. Subsequently, we evaluated the effects of Refined PULVIS FELLIS SUIS (RPFS), a traditional Chinese medicine enriched with HCA species, on 5-HT production. Our data showed RPFS extract containing 38.67% of total bile acids and 12.46% of total bile acids are HCA species (Figure.5C). We then showed RPFS significantly inhibited 5-HT production in mice intestinal segment cultures in a dose-dependent manner (p\u0026lt;0.05 in both cases, Figure.5D), revealing the therapeutic potential of RPFS, a commonly used non-toxic traditional medicine, on IBS-D via inhibiting 5-HT. Although RPFS also contains a high amount of UDCA and CDCA species, we did not find UDCA or CDCA affect 5-HT levels in QGP-1 cells at different dosages in the physiological range (n.s., Figure.5E-F), suggesting only HCA species in RPFS exhibited inhibitory effects on 5-HT production.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further evaluate the therapeutic effects of HDCA and RPFS on IBS-D, we employed a mouse model of IBS induced by TNBS, which is characterized by diarrhea and elevated 5-HT levels (Figure.6A). Notably, both HDCA and RPFS effectively alleviated diarrhea in TNBS-treated mice, as shown by prolonged GI transit time, reduced defecation frequency, and fecal water content (p\u0026lt;0.01 in both cases, Figure.6B-D). HDCA and RPFS significantly reduced 5-HT levels in both ileum and proximal colon in TNBS-treated mice, as demonstrated by LC-MS analysis and immunofluorescence (IF) staining of 5-HT (p\u0026lt;0.01 in both cases, Figure.6E-H). Moreover, 5-HT level in brain tissues is found significantly reduced in TNBS-treated mice (p\u0026lt;0.01, Figure.6J), but no significant changes in HDCA or 5-HT levels were found in brain tissues in the treatment of HDCA or RPFS in TNBS-treated mice, indicating HDCA is not distributed to brain tissues and affected brain 5-HT levels after oral administration (n.s., Figure.6I-J). Considering 5-HT inhibition is associated with alterations of mental disorder\u003csup\u003e8\u003c/sup\u003e, we evaluated general locomotor activity in\u0026nbsp;TNBS-treated mice in the treatment of HDCA and we showed that both HDCA in high dose and RPFS treatment did not result in depression or anxiety symptoms in TNBS-treated mice (n.s., Figure.7A-D), revealing safety evidence of HDCA and RPFS treatment for IBS-D. Collectively, our findings demonstrated that HDCA and RPFS improved diarrhea phenotypes and inhibited peripheral 5-HT production in TNBS-treated mice.\u003c/p\u003e\n\u003cp\u003eWe then determined levels of HCA species in both ileum and proximal colon from TNBS-treated mice after the treatment of HDCA in various dosages and RPFS. Notably, we showed HDCA, THDCA and GHDCA but not HCA, THCA and GHCA were significantly increased in ileum and proximal colon tissues in the treatment of HDCA in a dose-dependent manner (p\u0026lt;0.05 in all cases, Figure.8A-L). Similarly, HDCA, GHDCA and THDCA were found upregulated in ileum and proximal colon tissues in the treatment of RPFS (p\u0026lt;0.05 in all cases, Figure.8A-L). These results revealed that treatment of HDCA and RPFS at indicated dosages resulted in around 50-300\u0026micro;M HDCA levels in the colonic tissues of TNBS-treated mice, while HDCA can be transformed into both taurine and glycine-conjugated HDCA (20-200\u0026micro;M) \u003cem\u003ein vivo\u003c/em\u003e, suggesting peripheral 5-HT inhibition in the treatment of HDCA and RPFS can be mediated by HDCA, THDCA and GHDCA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHDCA suppressed 5-HT biosynthesis by inhibiting TPH1 activity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTPH1 is a rate-limiting enzyme responsible for the 5-HT production by catalyzing the conversion of tryptophan into 5-hydroxytryptophan (5-HTP), the precursor of 5-HT\u003csup\u003e9\u003c/sup\u003e. Through a TPH1 inhibitor screening assay, we preliminarily showed HDCA inhibited TPH1 activity in a dose-dependent manner (Figure.9A).\u0026nbsp;To understand the underlying mechanisms of HDCA action on 5-HT production, we determined the molecular interactions between human TPH1 recombinant protein and HDCA species including HDCA, THDCA and GHDCA using surface plasmon resonance (SPR). It is noteworthy that HDCA exhibited rapid binding to the TPH1 with an equilibrium dissociation constant (K\u003csub\u003eD\u003c/sub\u003e) value of 97\u0026micro;M, indicating a direct binding between human TPH1 activity with HDCA (Figure.9B-C). In contrast, both THDCA and GHDCA exhibited no binding to TPH1 with K\u003csub\u003eD\u003c/sub\u003e values of 6510\u0026micro;M and 8670\u0026micro;M\u0026nbsp;(Figure.9D-E). We further showed inhibitory effects of HDCA on TPH1 in HEK 293 cells transfected with wildtype rhTPH1 with an IC\u003csub\u003e50\u003c/sub\u003e value of 265.7\u0026micro;M for HDCA (p\u0026lt;0.001, Figure.9F), while THDCA and GHDCA failed to inhibit 5-HTP synthesis within physiological dosage (n.s., Figure.9F). Herein, we revealed HDCA suppressed 5-HT biosynthesis by inhibiting TPH1 activity, thus alleviating diarrhea symptoms in patients with IBS-D.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo address the binding sites of HDCA toward the TPH1 protein, we performed molecular docking and showed HDCA binds to the TPH1 protein at the following amino acid residues: Cys364, Pro268, Pro238, Leu236, Tyr312, Phe313 and Phe241 (Figure.10A) with a binding free energy of -45.40 kcal/mol (Table.S6). Accordingly, we constructed a HEK 293 cell line transfected with TPH1 plasmids containing mutations at those key residues involved in HDCA to determine the molecular mechanisms of HDCA binding to TPH1 (Figure.10B). Notably, HDCA displays a markedly suppressed binding affinity to the mutant forms of rhTPH1 Cys364 in comparison to the wild-type protein TPH1 (n.s., Figure.10C-J), suggesting Cys364 is a key binding site of HDCA for its inhibitory effects on TPH1. We also evaluated whether other receptors including FXR\u003csup\u003e10\u003c/sup\u003e, LXR\u003csup\u003e11\u003c/sup\u003e, PXR, VDR\u003csup\u003e12\u003c/sup\u003e, GPR119, TGR5\u003csup\u003e13\u003c/sup\u003e and PPARa\u003csup\u003e14\u003c/sup\u003e, are possibly involved in the HDCA action on 5-HT production, but we did not find agonism or antagonism of these receptors by small molecules affected inhibitory effects of HDCA on 5-HT production in QGP-1 cells (n.s., Figure.11A-L). Therefore, we demonstrated that HDCA and RPFS, as potent gut microbe-derived TPH1 inhibitors, effectively alleviated diarrhea in mice with experimental IBS.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePeripheral 5-HT has been found significantly increased in IBS-D, which could be attributed to increased 5-HT biosynthesis, reduced 5-HT reuptake and EC cell hyperplasia. Currently, treatment strategies for IBS-D approved by the U.S. FDA that focus on modulating 5-HT levels or its activity at various receptors are limited\u003csup\u003e15\u003c/sup\u003e. To date, the U.S. FDA has approved only telotristat ethyl (LX-1032), an inhibitor of both TPH1 and TPH2, for the management of carcinoid syndrome-related diarrhea\u003csup\u003e16\u003c/sup\u003e. Alosetron (5-HT3 receptor antagonist) has been used to treat IBS-D in women, but severe adverse effects including increased abdominal pain, constipation and ischemic colitis have been reported\u003csup\u003e17\u003c/sup\u003e. Drugs that inhibit 5-HT synthesis such as LX-1031 are being explored for their potential to reduce 5-HT production in the gut, potentially alleviating symptoms of IBS-D. However, reducing peripheral 5-HT can lead to side effects such as constipation, depression, or mood changes. Achieving selective inhibition of peripheral 5-HT without affecting central nervous system 5-HT levels can be challenging.\u0026nbsp;Therefore, there is a significant medical need for the development of inhibitors of 5-HT biosynthesis without substantial side effects in the treatment of IBS-D. Notably, we demonstrated that HDCA exclusively inhibits peripheral 5-HT level without affecting mental behaviors, which offers an advantage in treating IBS-D, particularly when co-morbid with anxiety or depression.\u003c/p\u003e\n\u003cp\u003eIn the present study, we delineated the therapeutic potential of HDCA in the treatment of IBS-D through inhibiting 5-HT biosynthesis. Our results showed that serum and fecal HDCA levels are negatively correlated with serum 5-HT and the severity of GI symptoms in IBS-D. We employed FMT studies to demonstrate that HDCA-associated microbiota is a key regulator of 5-HT production in recipient mice. The reduction of HDCA in IBS-D patients compared with HC and the regulatory role of HDCA-associated fecal microbiota on defecation frequency and fecal moisture emphasize the role of HDCA as not only a biomarker for IBS-D but also a potential therapeutic agent. Based on these observations, our preclinical studies in the TNBS-induced IBS mouse model further revealed the therapeutic roles of HDCA and RPFS in alleviating GI symptoms by inhibiting TPH1, the key enzyme in peripheral 5-HT biosynthesis. Taken together, our findings showed that HDCA is a promising drug candidate for IBS-D and other 5-HT-targeted diseases via inhibition of 5-HT biosynthesis. Clinical trials for TPH1 inhibitors have focused on conditions including carcinoid syndrome, IBS-D, pulmonary arterial hypertension (PAH), fibromyalgia, obesity, and metabolic disorders\u003csup\u003e18\u003c/sup\u003e. Herein, both HDCA and RPFS as potent TPH1 inhibitors can be further evaluated for their efficacy and safety profiles in these conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePFS, as a natural source of HDCA, has a long-standing history in traditional medicine for the treatment of diarrhea\u003csup\u003e18\u003c/sup\u003e. Considering that the levels of HCA and HDCA species in humans are much lower than those in pigs, the supplementation of exogenous PFS enriched with HDCA presents a promising strategy for managing IBS-D. In addition to their effects on IBS-D, HDCA and PFS have been discovered to bring numerous advantages in treating metabolic disorders such as obesity, non-alcoholic fatty liver disease (NAFLD), metabolic disorders and diabetes\u003csup\u003e12,14\u003c/sup\u003e. Given that inhibiting the 5-HT synthesis in the periphery has been demonstrated to improve obesity and metabolic dysfunction\u003csup\u003e19\u003c/sup\u003e, our research also offers a mechanism of action that explains the advantageous effects of HDCA on metabolic disorders.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsidering the heterogeneous pathogenesis of IBS-D\u003csup\u003e20\u003c/sup\u003e and following results from this research, we are actively examining whether other pharmacological mechanisms are involved in the ameliorating effects of HDCA on GI symptoms in mouse models of IBS-D. For example, we will examine whether HDCA modulates gut-microbial metabolites (bile acids, short-chain fatty acids, amino acid-derived metabolites), neurotransmitters (histamine, glutamine, GABA, norepinephrine, dopamine, acetylcholine), neuroendocrine factors (GLP-1, PYY, NGF) and proteases to improve diarrhea and visceral hypersensitivity in different mouse models of IBS-D in the future.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, our investigation elucidates a pivotal molecular mechanism for IBS-D in which HDCA reduction is positively associated with diarrhea severity and 5-HT overproduction in IBS-D. We have revealed the therapeutic role of HDCA and RPFS, as TPH1 inhibitors, in modulating 5-HT biosynthesis, thus providing effective relief of GI symptoms in IBS-D. Furthermore, we highlight that a gut microbiota-HDCA-5HT axis is an important regulator for the management of IBS-D. These findings present a promising candidate for 5-HT-focused therapies in IBS-D and related GI disorders.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResource availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLead contact\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFurther information and requests for resources and reagents can be directed to and will be fulfilled by the lead contact Lixiang Zhai (\u003cu\[email protected]\u003c/u\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMaterials and data availability\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eReagents and resource details are provided in Table.S1. The clinical data used in this study were from the accession number PRJNA1139229 in NCBI, and are available for public access.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eHuman study\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe determined the changes in bile acid (BA) profiles and metabolic metabolites in 5-HT signaling in IBS-D patients from a clinical study of healthy controls and IBS-D patients which was approved by the Research Ethics Committee of Hong Kong Baptist University (HASC/15-16/0300 and HASC/16-17/0027) (ClinicalTrials.gov Identifier: NCT02822677 and NCT03457324). Briefly, IBS-D patients (n=345) meeting Rome IV diagnostic criteria and age and gender-matched healthy volunteers (n=91) as controls were recruited. Written consent was obtained from each subject prior to sample collection and analysis. Biological samples, including serum and feces of all participants, were collected and transported to the laboratory in dry ice and stored at -80\u0026nbsp;until further LC-MS analysis as previously described\u003csup\u003e3,12\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMouse study\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe mouse study was approved by the Research Ethics Committee of Hong Kong Baptist University at Hong Kong Baptist University (REC/23-24/0346). 6-8 weeks old C57BL/6J mice were purchased from the Jicui Yaokang Co., Ltd. (Jiangsu Province, China) and raised in Animal Unit of Centre for Chinese Medicine Drug Development Limited. Mice were housed with a 12-h light/dark cycle at a controlled temperature of around 25℃ and a controlled humidity of 60% with free access to standard rodent diet and water.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnimal experiment 1: Measurement of HCA species\u0026nbsp;in germ-free mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the changes of HCA species in germ-free mice and normal mice, germ-free mice were purchased from Nanjing GemPharmatech Co., Ltd. (Jiangsu Province, China) and were housed in sterile plastic isolators with fully blocked exposure to microorganisms, with the intent of keeping them free of detectable bacteria, viruses, and eukaryotic microbes at the Laboratory Animal Facility of Nanjing GemPharmatech Co., Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnimal experiment 2: Fecal microbiota transplantation from IBS-D patients with either high or low fecal HDCA levels in an antibiotic-treated mouse model\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e6-8 weeks male wild-type mice were gavaged with 10 mL/kg antibiotics mixture containing 50 mg/kg vancomycin, 100 mg/kg neomycin, 100 mg/kg metronidazole, 100 mg/kg ampicillin, 50 mg/kg streptomycin for 10 consecutive days to establish an antibiotics-treated mouse model following a previous study\u003csup\u003e2\u003c/sup\u003e. The gut microbiota deletion was induced by the administration of the antibiotic mixture and confirmed by DNA gel electrophoresis of fecal samples.\u003c/p\u003e\n\u003cp\u003eThe antibiotics-treated mice were randomly divided into 4 groups: HDCA-enriched\u0026nbsp;(HDCA\u003csup\u003e+\u003c/sup\u003e) and HDCA-reduced (HDCA\u003csup\u003e-\u003c/sup\u003e) fecal microbiota from IBS-D patients (n=5/group). For fecal samples of humans, about 2 g of fresh fecal samples were added with 5 times sterilized phosphate-buffered saline (PBS, m/v) and homogenized as fecal microbiota suspension. The oral gavage of fecal microbiota suspension (equal to 6mg for each mouse in 200μL PBS) was conducted for 10 days daily after finishing the antibiotics treatment. The serum and fecal levels of HDCA were measured on day 5 and 10 to determine whether the fecal microbiota transplantation affects HDCA levels in recipient mice. Defecation frequency, GI transit, fecal moisture content and visceral hypersensitivity were measured on day 10 and day 11 to determine whether fecal microbiota transplantation affects diarrhea-related phenotypes in recipient antibiotics-treated mice.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnimal experiment 3: Evaluation of HDCA/Refined\u0026nbsp;\u003c/em\u003e\u003cem\u003ePULVIS FELLIS SUIS treatment on a mouse model of IBS induced by TNBS\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e6-8 weeks old C57BL/6J mice were fasted overnight and provided with 5% glucose in drinking water. After weighing, mice were anesthetized using 2% isoflurane. A 100 μL TNBS solution in 30% ethanol was then slowly administered into the colon using a 1 mL syringe and 3.5F catheter. The catheter was then gently removed, and the mice were positioned head-down for 5 min. Subsequently, the mice were maintained in a head-down vertical position for an additional 5 min. The control group received 100 μL saline. Mice were weighed on day 1 to 7, 14, and 21 following TNBS administration. Diarrhea-related phenotypes including defecation frequency, gastrointestinal (GI) transit and fecal moisture content were measured on day 14 to examine the establishment of the IBS model.\u003c/p\u003e\n\u003cp\u003eOn day 28, mice model of IBS induced by TNBS were divided into 4 groups: (1) TNBS group (n=5): mice were gavaged with vehicle control (0.5% CMC-Na); (2) 25 mg/kg HDCA (n=5): mice were administered with HDCA by oral gavage at a dose of 25 mg/kg/day; \u0026nbsp;(3) 50 mg/kg HDCA (n=5): mice were administered with HDCA by oral gavage at a dose of 50 mg/kg/day; \u0026nbsp;(4) 75 mg/kg HDCA (n=5): mice were administered with HDCA by oral gavage at a dose of 75 mg/kg/day; (5) Refined PULVIS FELLIS SUIS (n=5): mice were administered with Refined PULVIS FELLIS SUIS by oral gavage at a dose of 100 mg/kg/day; (6) Alosetron: mice were administered with alosetron by oral gavage at a dose of 3 mg/kg/day. Both diarrhea-related phenotypes were measured as previously described\u003csup\u003e2\u003c/sup\u003e after 14 days of HDCA and Refined PULVIS FELLIS SUIS treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro study\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eQGP-1 cells\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eQGP-1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). QGP-1 cells were incubated in a humidified atmosphere with 95% air and 5% CO\u003csub\u003e2\u003c/sub\u003e at 37 ℃. When reached 80% confluence, cells were seeded to a 96-well plate at a density of 5x10\u003csup\u003e4\u003c/sup\u003e cells/well. After 24 hours, cells were incubated with different concentrations of HDCA, GHDCA, and THDCA for 24 hours for the determination of 5-HT production in the culture medium.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIntestinal segments\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe intestinal tissues from 6-8 week-old mice were collected and cut into small pieces (0.5 cm) and placed in a 12-well cell culture plate. Intestinal segments were incubated at 37 ℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% air, and cultivated with RPMI-1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin, followed by treatment with HDCA and Refined PULVIS FELLIS SUIS with various concentrations as indicated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMetabolomics analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor fecal and colonic samples, about 50 mg of feces or colonic tissues were extracted with 10-fold 95% methanol (m/v) containing 125 ng/mL L-Tryptophan-d\u003csub\u003e5\u003c/sub\u003e, 50 ng/mL cholic acid-d\u003csub\u003e4\u003c/sub\u003e and 50 ng/mL deoxycholic acid-d\u003csub\u003e4\u003c/sub\u003e as internal standards. The samples were then homogenized in TissueLyser II for 2 min and stored in 4 ℃ fridges overnight. After centrifugation at 15, 000 rpm for 15 min at 4 ℃ on the second day, the supernatant was collected for LC-MS/MS analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor serum samples, about 80 µL serum samples were extracted with methanol containing internal standards (125 ng/mL L-Tryptophan-d\u003csub\u003e5\u003c/sub\u003e, 50 ng/mL cholic acid-d\u003csub\u003e4\u003c/sub\u003e and 50 ng/mL deoxycholic acid-d\u003csub\u003e4\u003c/sub\u003e) in a ratio of 1:4 (v/v) and stored overnight at -20 ℃. After centrifugation at 15, 000 rpm for 15 min at 4 ℃, the supernatant was collected for LC-MS/MS analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor brain samples, about 100 mg of brain tissues were extracted with 2-fold 95% methanol (m/v) containing internal standards, and then homogenized in TissueLyser II for 2 min and stored at -20 ℃ overnight. After centrifugation at 15, 000 rpm for 15 min at 4 ℃, 100 µL supernatant was transferred was used for LC-MS/MS analysis.\u003c/p\u003e\n\u003cp\u003eAn Agilent 1290 Infinity II UPLC system coupled with a triple quadrupole (QQQ) 6470 mass spectrometer was used for targeted metabolomics analysis. An Agilent ECLIPSE PLUS C18 column (2.1 × 5 mm, 1.8 μm) with a pre-column was used. For the LC-MS-QQQ analysis of 5-HT, the mobile phases were solution A (water containing 0.1% formic acid) and solution B (acetonitrile containing 0.1% formic acid). The gradient was set as follows: 2% B (0-0.5 min), 2%-30% B (0.5-4 min), 30%–100% B (4-6 min), 100% B (6-8 min), 100%-2% B (8-8.5 min), and maintained at 2% B (8.5-10 min). For the LC-MS-QQQ analysis of HDCA and HCA species, the mobile phases were solution A (water containing 0.1% formic acid) and solution B (acetonitrile containing 0.1% formic acid). The gradient was as follows: 15% B (0-2 min), 15%-25% B (2-4 min), 25%-35% B (4-20 min), 35%-50% B (20-30 min), and 50%-100% B (30-33 min). The column temperature was maintained at 40°C, the flow rate was set as 0.4 mL/min, and the sample injection volume was set as 2 μL. The TQ MS was operated in positive/negative ion mode with an electrospray ionization (ESI) source separately. The ESI capillary voltage was set to 4000 V and 3500 V respectively, and the nozzle voltage to 1000 V. The nitrogen drying gas flow rate was 10 L/min, heated to 350°C. The sheath gas temperature was 350°C, with a flow rate of 8 L/min, and the nebulizer pressure was 45 psi. The mass spectrometry was performed in multiple reaction monitoring (MRM) mode. MS data were collected and processed using Agilent software, with data acquisition information provided in\u0026nbsp;Table.S2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSurface plasmon resonance analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed on a Biacore 8k (GE Healthcare, Sweden). The target recombinant human protein TPH1 was diluted to 40 μg/mL using 10 mM acetate at pH 4.0 and covalently immobilized on parallel channels of CM5 sensor chip surface for 10 min. The CM5 chip surface was activated by 0.4 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.1 M N-hydroxysuccinimide for 7 min at a flow rate of 10 μL/min. The immobilized chip was blocked with 1 M ethanolamine for 7 min at a flow rate of 10 μL/min. The HDCA was diluted in running buffer containing phosphate buffered saline (PBS), 1mM dithiothreitol, 0.5% P20 and 5% dimethylsulfoxide (DMSO) at a series of concentrations 2.5, 5, 10, 25, 50, 100 μM, respectively. The solution of HDCA was injected for 3 min at 30 μL/min, and the dissociation was followed for 1.5 min. All data were analyzed using kinetic models with Biacore 8k Evaluation Software. A TPH1 inhibitor LX-1031 and tryptophan were used as positive controls for the binding experiment of HDCA.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMolecular docking\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDocking analysis was performed by Schrödinger v.2023 software with default parameters. The protein structure of TPH1 (PDB ID: 5JD6) and HDCA structure (CAS: 83-49-8) were uploaded to the software. MOE was applied for the interaction analysis between TPH1 and HDCA.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCloning strategies\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe full-length human TPH1 gene (NM_004179.3) was from Pcr-bluntII-TOPO-TPH1 plasmid (Miaoling). TPH1 was recombined into PLV3-CMV-MCS-3xFLAG-Puro vector and one FLAG tag at the amino terminus through BmaHI and NheI. The plasmids were confirmed by Sanger sequencing by BGI. 50 μl PCR reactions were performed using Phusion\u003csup\u003e®\u003c/sup\u003e high-fidelity DNA polymerase (New England Biolabs). The cycling protocol was 98°C, 30 sec, then 35 cycles of 98°C 15s, 55°C 30s, 72°C 1min. The final extension is 72°C, 10min.\u0026nbsp;The resulting PCR products are digested using BmaHI and NheI (New England Biolabs). DNA ligation reactions were performed in a final volume of 10 μl using T4 DNA ligase following the manual from New England Biolabs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSite-directed mutagenesis by PCR\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAmplify the parental plasmid (PLV3-CMV-MCS-3xFLAG-Puro vector) containing TPH1. 50 μl PCR reactions were performed using Phusion® high-fidelity DNA polymerase (New England Biolabs). The cycling protocol was 98°C, 30 sec, then 35 cycles of 98°C 15s, 55°C 30s, 72°C 5min. The final extension is 72°C, 10min. And then PCR products are digested by DpnI at 37°C for 2h. The digested PCR products are\u0026nbsp;transformed into DH5α.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEstablishment of TPH1 overexpression cell line\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe 293T cells were cultured with DMEM supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco) under the conditions of 37 °C, 5% CO2. \u0026nbsp;Briefly, 6 x 10\u003csup\u003e6\u003c/sup\u003e 293 T cells were seeded in a 10 cm dish in complete DMEM medium 24h prior to transfection with plasmid DNA (TPH1, point mutant TPH1 Leu236, Pro238, Phe241, Pro268, Ala 309, Tyr312 and Phe313) at a ratio of 4:3:1. Polyethylenimine (PEI) was used following the manufacturer’s instructions to produce lentivirus particles. Viral harvest was carried out at 48 post-transfection. The harvested medium was subsequently filtered to remove cell debris using a 0.45‐μm filter. Transfected cells were cultured under the same conditions for another 48h before puromycin selection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTPH1 activity assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTPH1 wildtype and TPH1 mutant cells are seeded in 24 well plates overnight. And then the cells are treated with HDCA and LX-1031 for 12h. A TPH1 inhibitor LX-1031 and tryptophan were used as positive controls for the binding experiment of HDCA. About 100 µL medium was extracted with methanol containing internal standards (125 ng/mL L-Tryptophan-d5) in a ratio of 1:4 (v/v) and stored overnight at -20 ℃. After centrifugation at 15, 000 rpm for 15 min at 4 ℃, the supernatant was collected for LC-MS/MS analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eUnless otherwise specified, data are presented as mean ± standard error of the mean (SEM) in figures and text. Normality tests were conducted, followed by either parametric or non-parametric tests as appropriate. Student’s t-tests (paired or unpaired) or one-way ANOVA were used in relevant scenarios as indicated. Data were analyzed using GraphPad Prism 10.0, and \u003cem\u003ep\u003c/em\u003e-values less than 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5-HIAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5-hydroxyindoleacetic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5-HT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSerotonin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5-HTP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5-hydroxytryptophan\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBile acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eEnterochromaffin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eFMT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eFecal microbiota transplantation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGHCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGlycohyocholic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGHDCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGlycohyodeoxycholic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGastrointestinal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHyocholic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHealthy controls\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHDCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHyodeoxycholic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eIrritable bowel syndrome\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIBS-D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eDiarrhea-predominant irritable bowel syndrome\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eK\u003csub\u003eD\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eDissociation constant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNAFLD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNon-alcoholic fatty liver disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePrinciple component analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRPFS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRefined PULVIS FELLIS SUIS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSPR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSurface plasmon resonance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTHCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTaurohyocholic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTHDCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTaurohyodeoxycholic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTNBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2,4,6-trinitrobenzene sulfonic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTPH1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTryptophan hydroxylase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTPH2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTryptophan hydroxylase 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTrp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eL-tryptophan\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is supported by General Research Fund from the University Grants Committee (12100323 to Zhaoxiang Bian), Health@InnoHK Initiative Fund from the Hong Kong SAR Government (ITC RC/IHK/4/7 to Zhaoxiang Bian), the General Program from the Guangdong Basic and Applied Basic Research Foundation (2414050003325 to Lixiang Zhai) and Collaborative Research Fund from the University Grants Committee (C2004-23Y to Lixiang Zhai and Lu Zhang). The authors express their gratitude to all the participants in this study and to the Chinese medicine practitioners who will contribute to the study but are not listed as authors. The authors also declare that they have no competing interests. The authors also thank Vincent and Lily Woo Foundation for the funding support of this work and Wu Jieh Yee Institute of Translational Chinese Medicine Research, HKBU for the technical support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ. B., L.Zhai and H.X. are the principal investigators and design this study. L.Zhai, G.B., J.L. and M.W. drafted the manuscript. G.B., S.X., J.L., B.L., Y. L., and M.Z. conducted the \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e studies including animal experiments and cell experiments. G.B. and S.X. conducted the metabolomics analysis. S.X., Y.Z., Z.N., B.L., Y.L., W.Y., and M.Z. manage the clinical specimens collected from all participants. L.Zhao, L.Zhang, C.L. and X.HL.W. provided important suggestions for the study design and helped revise the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests. The authors have filed patent based in part on the findings in this study.\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWechsler, E. V. \u0026amp; Shah, E. D. Diarrhea-Predominant and Constipation-Predominant Irritable Bowel Syndrome: Current Prescription Drug Treatment Options. \u003cem\u003eDrugs\u003c/em\u003e\u003cstrong\u003e81\u003c/strong\u003e, 1953-1968 (2021). https://doi.org/10.1007/s40265-021-01634-7\u003c/li\u003e\n\u003cli\u003eGao, J., Xiong, T. 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D.\u003cem\u003e et al.\u003c/em\u003e Inhibiting peripheral serotonin synthesis reduces obesity and metabolic dysfunction by promoting brown adipose tissue thermogenesis. \u003cem\u003eNature Medicine\u003c/em\u003e\u003cstrong\u003e21\u003c/strong\u003e, 166-172 (2015). https://doi.org/10.1038/nm.3766\u003c/li\u003e\n\u003cli\u003e Enck, P.\u003cem\u003e et al.\u003c/em\u003e Irritable bowel syndrome. \u003cem\u003eNature Reviews Disease Primers\u003c/em\u003e\u003cstrong\u003e2\u003c/strong\u003e (2016). https://doi.org/10.1038/nrdp.2016.14\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"[email protected]","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":"Hyodeoxycholic acid, PULVIS FELLIS SUIS, Irritable bowel syndrome, TPH1, Serotonin, Diarrhea","lastPublishedDoi":"10.21203/rs.3.rs-6769911/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6769911/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGut dysbiosis significantly contributes to the pathogenesis of diarrhea-predominant irritable bowel syndrome (IBS-D) by enhancing serotonin (5-HT) biosynthesis, which exacerbates diarrhea symptoms. Current treatments aimed at peripheral 5-HT signaling in IBS-D are often limited by efficacy and side effects. This study presents hyodeoxycholic acid (HDCA), a gut-microbial metabolite, as a novel therapeutic strategy that directly inhibits peripheral 5-HT synthesis via tryptophan hydroxylase 1 (TPH1). Our research indicates that HDCA levels are notably reduced in IBS-D patients and show a negative correlation with both diarrhea severity and peripheral 5-HT levels. Furthermore, we demonstrate that HDCA and PULVIS FELLIS SUIS, a traditional Chinese medicine abundant in HDCA, effectively alleviate diarrhea symptoms and inhibit peripheral 5-HT production without impacting central 5-HT levels or mood behaviors in mouse models of IBS. Mechanistically, HDCA directly binds to and inhibits TPH1, thereby suppressing peripheral 5-HT biosynthesis, a critical pathological factor in IBS-D. These findings suggest that HDCA is a promising candidate for microbiota-driven therapeutic interventions and gut-brain axis regulation. This study demonstrates the use of a microbiota-derived metabolite and traditional medicine specifically targeting peripheral 5-HT biosynthesis for treating gastrointestinal disorders. Our results pave the way for new IBS treatment strategies and other conditions requiring TPH1 inhibition, offering novel insights and potential clinical applications.\u003c/p\u003e\n\u003cp\u003eClinicalTrials.gov no: NCT02822677 and NCT03457324\u003c/p\u003e","manuscriptTitle":"Inhibition of peripheral serotonin synthesis by a gut microbe-derived TPH1 inhibitor, hyodeoxycholic acid, alleviates irritable bowel syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 15:05:44","doi":"10.21203/rs.3.rs-6769911/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","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":"e21fbe33-5ce7-4cd4-a596-c083b71a5840","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51305210,"name":"Health sciences/Diseases/Gastrointestinal diseases/Functional gastrointestinal disorders/Irritable bowel syndrome"},{"id":51305211,"name":"Health sciences/Biomarkers/Diagnostic markers"},{"id":51305212,"name":"Health sciences/Health care/Therapeutics/Drug therapy/Molecularly targeted therapy"}],"tags":[],"updatedAt":"2026-04-10T06:05:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-18 15:05:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6769911","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6769911","identity":"rs-6769911","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}