Structure-Based Design of Xanthine-Imidazopyridines and -Imidazothiazoles as Highly Potent and In Vivo Efficacious Tryptophan Hydroxylase Inhibitors.

Tryptophan hydroxylases catalyze the first and rate-limiting step in the biosynthesis of serotonin, a well-known neurotransmitter that plays an important role in multiple physiological functions. A reduction of serotonin levels, especially in the brain, can cause dysregulation leading to depression or insomnia. In contrast, overproduction of peripheral serotonin is associated with symptoms like carcinoid syndrome and pulmonary arterial hypertension. Recently, we developed a class of TPH inhibitors based on xanthine-benzimidazoles, characterized by a tripartite-binding mode spanning the binding sites of the cosubstrate pterin and the substrate tryptophan and by chelation of the catalytic iron ion. Herein, we describe the structure-based development of a second generation of xanthine-imidiazopyridines and -imidazothiazoles designed to inhibit TPH1 in the periphery while preventing the interaction with TPH2 in the brain. Lead compound 32 (TPT-004) shows superior pharmacokinetic and pharmacodynamic properties as well as efficacy in preclinical models of peripheral serotonin attenuation and colorectal tumor growth.

Influence of variants in serotonin modulating genes on the risk, aggressiveness, and prognosis of oropharynx cancer.

BACKGROUND: Serotonin (5-HT) is involved in regulating tumor growth, as well as psychiatric disorders. It is synthesized by tryptophan hydroxylase (TPH) and acts through 5-HT receptors (HTRs). Single-nucleotide variations (SNVs) in TPH1 rs623580 (T>A), TPH2 rs4570625 (G>T), and HTR1D rs674386 (G>A) may affect 5-HT levels. However, the effect of these SNVs on oropharynx carcinoma (OPC) is unknown. METHODS: DNA from 251 patients with OPC and 254 controls was analyzed by RT-PCR. Transcriptional activity of TPH1 rs623580 and HTR1D rs674386 was studied by luciferase assays. Multivariate statistical tests were utilized to evaluate group differences and survival outcomes. RESULTS: TPH1 TT was more frequent in patients than in controls (OR: 1.56, p = 0.03). Patients with HTR1D GG/GA showed invasive tumors (p = 0.01) and shorter survival (HR: 1.66, p = 0.04). TPH1 TT (0.79-fold, p = 0.03) and HTR1D GG (0.64-fold, p = 0.008) presented lower transcriptional activity. CONCLUSION: Our data suggest that SNVs in 5-HT modulating genes can influence OPC.

The Role of C1473G Polymorphism in Mouse Triptophan Hydroxylase 2 Gene in the Acute Effects of Ethanol on the c-fos Gene Expression and Metabolism of Biogenic Amines in the Brain.

Tryptophan hydroxylase 2 is a key enzyme in the synthesis of the neurotransmitter serotonin, which plays an important role in the regulation of behavior and various physiological functions. We studied the effect of acute ethanol administration on the expression of the early response c-fos gene and metabolism of serotonin and catecholamines in the brain structures of B6-1473C and B6-1473G congenic mouse strains differing in the single-nucleotide substitution C1473G in the Tph2 gene and activity of the encoded enzyme. Acute alcoholization led to a significant upregulation of the c-fos gene expression in the frontal cortex and striatum of B6-1473G mice and in the hippocampus of B6-1473C mice and caused a decrease in the index of serotonin metabolism in the nucleus accumbens in B6-1473C mice and in the hippocampus and striatum of B6-1473G mice, as well as to the decrease in the norepinephrine level in the hypothalamus of B6-1473C mice. Therefore, the C1473G polymorphism in the Tph2 gene has a significant effect of acute ethanol administration on the c-fos expression pattern and metabolism of biogenic amines in the mouse brain.

Glutamine increases stability of TPH1 mRNA via p38 mitogen-activated kinase in mouse mastocytoma cells.

Expression changes for tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in serotonin synthesis, by environmental glutamine (GLN) were examined in mouse mastocytoma-derived P815-HTR cells. GLN-treated cells exhibited a robust increase in TPH1 mRNA after a 6 h exposure to GLN. 6-Diazo-5-oxo-L-norleucine (DON), a glutamine-utilizing glutaminase inhibitor, significantly inhibited the GLN-induction of TPH1 mRNA. Nuclear run-on assays and mRNA decay experiments demonstrated that the primary mechanism leading to increased TPH1 mRNA levels was not due to transcriptional changes, but rather due to increased TPH1 RNA stability induced by GLN. Treatment with GLN also led to activation of p38 MAP kinase, but not p42/44 MAPK. In addition, SB203580, a p38 MAP kinase specific inhibitor, completely abolished the GLN-mediated increase of TPH1 mRNA levels, suggesting the pathway stabilizing TPH1 mRNA might be mediated by the activated p38 MAP kinase pathway. Additionally, SB203580 significantly reduced the stability of TPH1 mRNA, and this reduction of the stability was not affected by GLN in the culture medium, implying a sequential signaling from GLN being mediated by p38 MAP kinase, resulting in alteration of TPH1 mRNA stability. TPH1 mRNA stability loss was also dependent on de novo protein synthesis as shown by treatment of cells with a transcriptional/translational blocker. We provide evidence that TPH1 mRNA levels are increased in response to increased exogenous GLN in mouse mastocytoma cells via a stabilization of TPH1 mRNA due to the activity of the p38 MAP kinase.

Prevention of Loperamide-Induced Constipation in Mice and Alteration of 5-Hydroxytryotamine Signaling by Ligilactobacillus salivarius Li01.

Although Ligilactobacillus salivarius Li01 (Li01) has shown much promise in preventing multiple gastrointestinal diseases, the potential of the probiotic in alleviating constipation and the related mechanisms remain unclear. In this study, the effects of Li01 were evaluated in a loperamide-induced constipation mouse model. The results demonstrated that Li01 intervention can relieve constipation symptoms by improving water content, quantity, and morphology of feces and act as an intestinal barrier structure protector. Furthermore, Li01 can modulate gut motility (gastrointestinal transit rate), the fluid transit-associated expression of aquaporins, and the serum parameters vasoactive intestinal peptide, substance P, and somatostatin. Constipation significantly increased the levels of 5-hydroxytryotamine (5-HT) in serum (p < 0.01) and decreased the levels in the intestine (p < 0.001). Due to its function of elevating the expression of tryptophan hydroxylase 1, this was reversed after Li01 treatment. Li01 also promoted the expression of 5-HT receptor 3 and 4, indicating that the 5-HT signaling pathway may play a critical role in the mechanism by which Li01 alleviate constipation symptoms. Additionally, Li01 significantly altered the gut microbiota composition by enhancing the ratio of Firmicutes/Bacteroidetes and increasing the abundance of Rikenellaceae_RC9 genera. Based on the above results, Li01 may have the potential to effectively alleviate constipation by regulating the 5-HT pathway and alteration of the gut microbiota.

Serotonin-deficient neonatal mice are not protected against the development of experimental bronchopulmonary dysplasia or pulmonary hypertension.

Serotonin (5-hydroxytryptamine, 5-HT) is a potent pulmonary vasoconstrictor and contributes to high pulmonary vascular resistance in the developing ovine lung. In experimental pulmonary hypertension (PH), pulmonary expression of tryptophan hydroxylase-1 (TPH1), the rate limiting enzyme in 5-HT synthesis, and plasma 5-HT are increased. 5-HT blockade increases pulmonary blood flow and prevents pulmonary vascular remodeling and PH in neonatal models of PH with bronchopulmonary dysplasia (BPD). We hypothesized that neonatal tph1 knock-out (KO) mice would be protected from hypoxia-induced alveolar simplification, decreased vessel density, and PH. Newborn wild-type (WT) and tph1 KO mice were exposed to normoxia or hypoxia for 2 weeks. Normoxic WT and KO mice exhibited similar alveolar development, pulmonary vascular density, right ventricular systolic pressures (RVSPs), and right heart size. Circulating (plasma and platelet) 5-HT decreased in both hypoxia-exposed WT and KO mice. Tph1 KO mice were not protected from hypoxia-induced alveolar simplification, decreased pulmonary vascular density, or right ventricular hypertrophy, but displayed attenuation to hypoxia-induced RVSP elevation compared with WT mice. Tph1 KO neonatal mice are not protected against hypoxia-induced alveolar simplification, reduction in pulmonary vessel density, or RVH. While genetic and pharmacologic inhibition of tph1 has protective effects in adult models of PH, our results suggest that tph1 inhibition would not be beneficial in neonates with PH associated with BPD.

Loss of Pleckstrin homology like domain, family A, member 1 promotes type â ¡ alveolar epithelial cell apoptosis in chronic obstructive pulmonary disease emphysematous phenotype via interaction with tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon.

Emphysematous phenotype is the most important phenotypic component of chronic obstructive pulmonary disease and is associated with substantial morbidity and mortality. The current pharmaceutical treatments and therapeutic procedures do not reduce pulmonary damage in patients with emphysematous phenotype. Therefore, it is important to identify effector molecules that can be used as interfering targets in such patients. Apoptosis of type II alveolar epithelial cells plays a key role in the phenotypic formation. This study aimed to further explore the molecular mechanisms involved in this process. The number of type II alveolar epithelial cells was significantly reduced due to increased apoptosis in patients with emphysematous phenotype compared to those with non-emphysematous phenotype. Pleckstrin homology like domain, family A, member 1 (PHLDA1) was mainly distributed in type II alveolar epithelial cells in both groups but was markedly reduced in patients with emphysematous phenotype. Overexpression of PHLDA1 prevented cigarette smoke extract-stimulated apoptosis of type II alveolar epithelial cells, whereas its knockdown worsened the apoptosis. PHLDA1 binding ability to tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (YWHAE) was weakened after exposure to cigarette smoke extract, with decreased PHLDA1 level lowering the abundance of YWHAE and attenuating the binding ability of YWHAE to p-Bad. These results demonstrate that considerable apoptosis of type II alveolar epithelial cells occurs in patients with emphysematous phenotype, and PHLDA1 may act as an effective antiapoptotic factor via YWHAE. Moreover, PHLDA1 may serve as a potential interfering target, providing insights into therapeutic strategies for emphysematous phenotype.

Generation of a TPH2-EGFP reporter cell line for purification and monitoring of human serotonin neurons in vitro and in vivo.

Generation of serotonin neurons (SNs) from human pluripotent stem cells (hPSCs) provides a promising platform to explore the mechanisms of serotonin-associated neuropsychiatric disorders. However, neural differentiation always yields heterogeneous cell populations, making it difficult to identify and purify SNs in vitro or track them in vivo following transplantation. Herein, we generated a TPH2-EGFP reporter hPSC line with insertion of EGFP into the endogenous tryptophan hydroxylase 2 (TPH2) locus using CRISPR-Cas9-mediated gene editing technology. This TPH2-reporter, which faithfully indicated TPH2 expression during differentiation, enabled us to obtain purified SNs for subsequent transcriptional analysis and study of pharmacological responses to antidepressants. In addition, the reporter system showed strong EGFP expression to indicate SNs, which enabled us to explore in vitro and ex vivo electrophysiological properties of SNs. In conclusion, this TPH2-EGFP reporter cell line might be of great significance for studies on human SN-related development and differentiation, drug screening, disease modeling, and cell replacement therapies.

Structure-Based Design of Xanthine-Benzimidazole Derivatives as Novel and Potent Tryptophan Hydroxylase Inhibitors.

Tryptophan hydroxylases catalyze the first and rate-limiting step in the synthesis of serotonin. Serotonin is a key neurotransmitter in the central nervous system and, in the periphery, functions as a local hormone with multiple physiological functions. Studies in genetically altered mouse models have shown that dysregulation of peripheral serotonin levels leads to metabolic, inflammatory, and fibrotic diseases. Overproduction of serotonin by tumor cells causes severe symptoms typical for the carcinoid syndrome, and tryptophan hydroxylase inhibitors are already in clinical use for patients suffering from this disease. Here, we describe a novel class of potent tryptophan hydroxylase inhibitors, characterized by spanning all active binding sites important for catalysis, specifically those of the cosubstrate pterin, the substrate tryptophan as well as directly chelating the catalytic iron ion. The inhibitors were designed to efficiently reduce serotonin in the periphery while not passing the blood-brain barrier, thus preserving serotonin levels in the brain.

Tryptophan degradation enzymes and Angiotensin (1-7) expression in human placenta.

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are key enzymes for tryptophan degradation, regulating immune tolerance during pregnancy. The intrauterine renin-angiotensin system is also involved in the progression of a healthy pregnancy. Angiotensin(1-7) maintains the integrity of fetal membranes via counteracting the pro-inflammatory actions of Angiotensin II. No data are available on placental Angiotensin(1-7) co-expression with TDO. We aimed to characterize TDO mRNA expression and its localization in different areas of the placenta of physiological pregnancies delivered at term; its co-expression with Angiotensin(1-7) and its correlation with the plasma kynurenine/tryptophan (Kyn/Trp) ratio was investigated. This prospective observational study included a nonconsecutive series of 20 singleton uncomplicated pregnancies delivered vaginally. TDO mRNA was expressed in both maternal and fetal sides of the placentas and TDO protein also in the villi and it was co-expressed with IDO1 in almost half of the placental cells at these sites. The percentage of TDO+ and IDO1+ cells appeared to be influenced by maternal pre-gestational smoking and newborn weight. A strong correlation was found between the percentage of TDO+ and IDO1+ cells in the villi. TDO+ cells also expressed Angiotensin(1-7), with a higher percentage on the fetal side and in the villi compared to the maternal one. Kyn/Trp plasma ratio was not correlated with IDO and TDO expression nor with the patient's characteristics. Collectively, our data indicate that TDO is detectable in placental tissue and is co-expressed with IDO and with Angiotensin(1-7)+ on the fetal side and in the villi.

Peptide OM-LV20 protects astrocytes against oxidative stress via the 'PAC1R/JNK/TPH1' axis.

Stroke can lead to severe nerve injury and debilitation, resulting in considerable social and economic burdens. Due to the high complexity of post-injury repair mechanisms, drugs approved for use in stroke are extremely scarce, and thus, the discovery of new antistroke drugs and targets is critical. Tryptophan hydroxylase 1 (TPH1) is involved in a variety of mental and neurobehavioral processes, but its effects on stroke have not yet been reported. Here, we used primary astrocyte culture, quantitative real-time PCR, double immunofluorescence assay, lentiviral infection, cell viability analysis, Western blotting, and other biochemical experiments to explore the protective mechanism of peptide OM-LV20, which previously exhibited neuroprotective effects in rats after ischemic stroke via a mechanism that may involve TPH1. First, we showed that TPH1 was expressed in rat astrocytes. Next, we determined that OM-LV20 impacted expression changes of TPH1 in CTX-TNA2 cells and exhibited a protective effect on the decrease in cell viability and catalase (CAT) levels induced by hydrogen peroxide. Importantly, we also found that TPH1 expression induced by OM-LV20 may be related to the level of change in the pituitary adenylate cyclase-activating peptide type 1 receptor (PAC1R) and to the JNK signaling pathways, thereby exerting a protective effect on astrocytes against oxidative stress. The protective effects of OM-LV20 likely occur via the 'PAC1R/JNK/TPH1' axis, thus highlighting TPH1 as a novel antistroke drug target.

Efficacy, safety and unmet needs of evolving medical treatments for carcinoid syndrome.

This review reports on the currently available medical treatment options for the control of symptoms due to carcinoid syndrome in patients with neuroendocrine tumors. The efficacy and adverse events (AEs) of approved drugs such as somatostatin analogues (SSA), telotristat ethyl (TE) and interferon-alpha, are reviewed. Somatostatin analogues remain the standard treatment of carcinoid syndrome based on the high expression of somatostatin receptors and the resulting inhibition of secretion of bioactive compounds; their use is associated with relatively mild AEs, involving mainly the gastrointestinal system, and being usually transient. Although dose escalation of SSA remains an unapproved option, it is clinically implemented to alleviate symptoms in refractory carcinoid syndrome and supported by the most recent guidelines. The side effects associated with the increased dose are in general mild and consistent with standard dose of SSA. Telotristat ethyl, an oral inhibitor of tryptophan hydroxylase, the rate-limiting enzyme in serotonin biosynthesis, represents a rather novel innovative treatment option in patients with carcinoid syndrome suffering from diarrhea and complements the standard therapy of SSA. Given the low toxicity profile, TE may be considered an early add-on treatment to SSA in patients with uncontrolled carcinoid syndrome. However, further prolonged follow-up of patients treated with TE may be needed to exclude potential AEs, such as liver toxicity or depressed mood, in patients with long-term treatment. Interferon alpha is a cytokine with direct inhibitory effect on hormone secretion and tumor cell proliferation and an approved therapy in carcinoid syndrome but is associated with significant AEs in the majority of the patients requiring frequently dose reduction. The finding of a more favorable tolerability of pegylated interferon needs to be confirmed in a prospective study.

Ahi1 regulates serotonin production by the GR/ERß/TPH2 pathway involving sexual differences in depressive behaviors.

BACKGROUND: Depression is one of the most common psychiatric diseases. The monoamine transmitter theory suggests that neurotransmitters are involved in the mechanism of depression; however, the regulation on serotonin production is still unclear. We previously showed that Ahi1 knockout (KO) mice exhibited depression-like behavior accompanied by a significant decrease in brain serotonin. METHODS: In the present study, western blot, gene knockdown, immunofluorescence, dual-luciferase reporter assay, and rescue assay were used to detect changes in the Ahi1/GR/ERß/TPH2 pathway in the brains of male stressed mice and male Ahi1 KO mice to explain the pathogenesis of depression-like behaviors. In addition, E2 levels in the blood and brain of male and female mice were measured to investigate the effect on the ERß/TPH2 pathway and to reveal the mechanisms for the phenomenon of gender differences in depression-like behaviors. RESULTS: We found that the serotonin-producing pathway-the ERß/TPH2 pathway was inhibited in male stressed mice and male Ahi1 KO mice. We further demonstrated that glucocorticoid receptor (GR) as a transcription factor bound to the promoter of ERß that contains glucocorticoid response elements and inhibited the transcription of ERß. Our recent study had indicated that Ahi1 regulates the nuclear translocation of GR upon stress, thus proposing the Ahi1/GR/ERß/TPH2 pathway for serotonin production. Interestingly, female Ahi1 KO mice did not exhibit depressive behaviors, indicating sexual differences in depressive behaviors compared with male mice. Furthermore, we found that serum 17ß-estradiol (E2) level was not changed in male and female mice; however, brain E2 level significantly decreased in male but not female Ahi1 KO mice. Further, ERß agonist LY-500307 increased TPH2 expression and 5-HT production. Therefore, both Ahi1 and E2 regulate the ERß/TPH2 pathway and involve sexual differences in brain serotonin production and depressive behaviors. CONCLUSIONS: In conclusion, although it is unclear how Ahi1 controls E2 secretion in the brain, our findings demonstrate that Ahi1 regulates serotonin production by the GR/ERß/TPH2 pathway in the brain and possibly involves the regulation on sex differences in depressive behaviors. Video Abstract.

Tryptophan hydroxylase 1 drives glioma progression by modulating the serotonin/L1CAM/NF-κB signaling pathway.

BACKGROUND: Glioma is one of the main causes of cancer-related mortality worldwide and is associated with high heterogeneity. However, the key players determining the fate of glioma remain obscure. In the present study, we shed light on tumor metabolism and aimed to investigate the role of tryptophan hydroxylase 1 (TPH-1) in the advancement of glioma. METHOD: Herein, the levels of TPH-1 expression in glioma tissues were evaluated using The Cancer Genome Atlas (TCGA) database. Further, the proliferative characteristics and migration ability of TPH-1 overexpressing LN229/T98G cells were evaluated. Additionally, we performed a cytotoxicity analysis using temozolomide (TMZ) in these cells. We also examined the tumor growth and survival time in a mouse model of glioma treated with chemotherapeutic agents and a TPH-1 inhibitor. RESULTS: The results of both clinical and experimental data showed that excess TPH-1 expression resulted in sustained glioma progression and a dismal overall survival in these patients. Mechanistically, TPH-1 increased the production of serotonin in glioma cells. The elevated serotonin levels then augmented the NF-κB signaling pathway through the upregulation of the L1-cell adhesion molecule (L1CAM), thereby contributing to cellular proliferation, invasive migration, and drug resistance. In vivo experiments demonstrated potent antitumor effects, which benefited further from the synergistic combination of TMZ and LX-1031. CONCLUSION: Taken together, these data suggested that TPH-1 facilitated cellular proliferation, migration, and chemoresistance in glioma through the serotonin/L1CAM/NF-κB pathway. By demonstrating the link of amino acid metabolic enzymes with tumor development, our findings may provide a potentially viable target for therapeutic manipulation aimed at eradicating glioma.

MiR-126-5p Promotes Tumor Cell Proliferation, Metastasis and Invasion by Targeting TDO2 in Hepatocellular Carcinoma.

TDO2 is a key enzyme in the kynurenine metabolic pathway, which is the most important pathway of tryptophan metabolism. It has been shown that miRNAs are involved in cell metastasis through interaction with target mRNAs. In this study, we found 645 miRNAs that could be immunoprecipitated with TDO2 through the RNA-immunoprecipitation experiment. miR-126-5p was selected as the research target, which was also confirmed by dual-luciferase reporter assay. Through qRT-PCR analysis, it was verified that the overexpression of miR-126-5p promoted the expression of TDO2, PI3K/AKT and WNT1. Meanwhile, it was verified that overexpression of miR-126-5p can promote intracellular tryptophan metabolism by HPLC. We also verified the effects of miR-126-5p on cell proliferation, migration, and invasion by cck-8, cell colony formation and trans-well assay in both HCCLM3 cells and HepG2 cells. In vivo experiments were also conducted to verify that miR-126-5p promoted tumor formation and growth via immunohistochemical detection of cell infiltration and proliferation to generate markers Ki-67, BAX, and VEGF. In conclusion, our results suggest that miR-126-5p is a biomarker and a potential new treatment target in the progression of HCC via promoting the expression of TDO2.

The Combined Effects of Amyloidosis and Serotonin Deficiency by Tryptophan Hydroxylase-2 Knockout Impacts Viability of the APP/PS1 Mouse Model of Alzheimer's Disease.

BACKGROUND: A decline of brain serotonin (5-HT) is held responsible for the changes in mood that can be observed in Alzheimer's disease (AD). However, 5-HT'ergic signaling is also suggested to reduce the production of pathogenic amyloid-ß (Aß). OBJECTIVE: To investigate the effect of targeted inactivation of tryptophan hydroxylase-2 (Tph2), which is essential for neuronal 5-HT synthesis, on amyloidosis in amyloid precursor protein (APP)swe/presenilin 1 (PS1) ΔE9 transgenic mice. METHODS: Triple-transgenic (3xTg) APP/PS1 mice with partial (+/-) or complete Tph2 knockout (-/-) were allowed to survive until 6 months old with APP/PS1, Tph2-/-, and wildtype mice. Survival and weight were recorded. Levels of Aß42/40/38, soluble APPα (sAßPPα) and sAßPPß, and cytokines were analyzed by mesoscale, neurotransmitters by mass spectrometry, and gene expression by quantitative PCR. Tph2, microglia, and Aß were visualized histologically. RESULTS: Tph2 inactivation in APP/PS1 mice significantly reduced viability, without impacting soluble and insoluble Aß42 and Aß40 in neocortex and hippocampus, and with only mild changes of soluble Aß42/Aß40. However, sAßPPα and sAßPPß in hippocampus and Aß38 and Aß40 in cerebrospinal fluid were reduced. 3xTg-/-mice were devoid of Tph2 immunopositive fibers and 5-HT. Cytokines were unaffected by genotype, as were neocortical TNF, HTR2a and HTR2b mRNA levels in Tph2-/- mice. Microglia clustered around Aß plaques regardless of genotype. CONCLUSION: The results suggest that Tph2 inactivation influences AßPP processing, at least in the hippocampus, although levels of Aß are unchanged. The reduced viability of 3xTg-/-mice could indicate that 5-HT protects against the seizures that can impact the viability of APP/PS1 mice.

Administration of a Probiotic Mixture Ameliorates Cisplatin-Induced Mucositis and Pica by Regulating 5-HT in Rats.

Probiotic-based therapies have been shown to be beneficial for chemotherapy-induced mucositis. Previous research has demonstrated that a probiotic mixture (Bifidobacterium brevis, Lactobacillus acidophilus, Lactobacillus casei, and Streptococcus thermophilus) can ameliorate chemotherapy-induced mucositis and dysbiosis in rats, but the underlying mechanism has not been completely elucidated. We aimed to determine the inhibitory effects of the probiotic mixture on cisplatin-induced mucositis and pica and the underlying mechanism, focusing on the levels of 5-hydroxytryptamine (5-HT, serotonin) regulated by the gut microbiota. A rat model of mucositis and pica was established by daily intraperitoneal injection of cisplatin (6 mg/kg) for 3 days. In the probiotic+cisplatin group, predaily intragastric injection of the probiotic mixture (1 × 109 CFU/kg BW) was administrated for 1 week before cisplatin injection. This was then followed by further daily probiotic injections for 6 days. Histopathology, pro-/anti-inflammatory cytokines, oxidative status, and 5-HT levels were assessed on days 3 and 6. The structure of the gut microbiota was analyzed by 16S rRNA gene sequencing and quantitative PCR. Additionally, 5-HT levels in enterochromaffin (EC) cells (RIN-14B cell line) treated with cisplatin and/or various probiotic bacteria were also determined. The probiotic mixture significantly attenuated kaolin consumption, inflammation, oxidative stress, and the increase in 5-HT concentrations in rats with cisplatin-induced intestinal mucositis and pica. Cisplatin markedly increased the relative abundances of Enterobacteriaceae_other, Blautia, Clostridiaceae_other, and members of Clostridium clusters IV and XIVa. These levels were significantly restored by the probiotic mixture. Importantly, most of the genera increased by cisplatin were significantly positively correlated with colonic 5-HT. Furthermore, in vitro, the probiotic mixture had direct inhibitory effects on the 5-HT secretion by EC cells. The probiotic mixture protects against cisplatin-induced intestine injury, exhibiting both anti-inflammatory and antiemetic properties. These results were closely related to the reestablishment of intestinal microbiota ecology and normalization of the dysbiosis-driven 5-HT overproduction.

Administration of cyclophosphamide to rats induces pica and potentiates 5-hydroxytryptamine synthesis in the intestine without causing severe intestinal injury.

The effects of cyclophosphamide on 5-hydroxytryptamine (5-HT) synthesis in the intestinal tissue of rats were investigated. Rats received 120 mg/kg cyclophosphamide intraperitoneally as a single administration, and kaolin and food intake was measured by an automatic monitoring apparatus. Ileal tissues were collected at either 24 or 72 h after administration. Cyclophosphamide caused a significant increase in kaolin intake at the acute and the delayed phases and was associated with a decrease in food intake, and body weight. Cyclophosphamide had no significant effect on intestinal mucosal morphology, or inducible nitric oxide synthase and cyclooxygenase-2 expression in the intestine. Cyclophosphamide significantly increased tryptophan hydroxylase 1 (TPH1) mRNA expression, number of anti-TPH antibody-positive cells, and 5-HT content in the intestine. Cyclophosphamide also significantly increased the expression of Tac1 mRNA, encoding preprotachykinin-1, which is a preprotein of substance P, and the number of anti-substance P antibody-positive cells in the intestine. Cyclophosphamide significantly increased Lgr5, Bmi1, and Atoh1 mRNA levels, which are markers for the proliferation and differentiation of stem cells. This study demonstrated that cyclophosphamide induced pica in rats, and potentiated 5-HT synthesis associated with hyperplasia of substance P-containing enterochromaffin cells without causing severe intestinal injury.

Kamishoyosan (a Japanese traditional herbal formula), which effectively reduces the aggressive biting behavior of male and female mice, and potential regulation through increase of Tph1, Tph2, and Esr2 mRNA levels.

Kamishoyosan (KSS), a Japanese traditional herbal formula, is used to treat symptoms related to the autonomic nervous system in men and women; it is especially known for improving the symptoms of irritability (e.g., bad temper and persistent anger). Although clinical and ethological studies of KSS have been conducted, its efficacy in reducing irritability remains to be validated. In the present study, male and female ddY-strain mice were isolation-reared for 8 weeks (from the third postnatal week) to induce pathologically aggressive biting behavior (ABB), which was used as an indicator of irritability. The ABB of mice toward metal rods was measured using the Aggressive Response Meter. An intraperitoneal administration of KSS (100 mg/kg) effectively reduced ABB in male and female mice at 2 h after the administration; however, this effect was canceled by prior administration of WAY-100635 [a 5-hydroxytryptoamine (5-HT)-1A receptor antagonist; 0.5 mg/kg] and bicuculline (a type-A gamma-aminobutyric acid receptor antagonist; 1.0 mg/kg). Additionally, tamoxifen, ICI-182780, and G-15 (all estrogen receptor antagonists) inhibited the action of KSS in a dose-dependent manner. Furthermore, gene expression of tryptophan hydroxylase (Tph) 1 and Tph2 were increased and 5-HT immunofluorescence was slightly increased in the dorsal raphe nucleus (DRN) of isolation-reared mice administered with KSS. Collectively, these results indicate that KSS effectively reduces ABB in isolation-reared male and female mice through stimulation of 5-HT production in the DRN. Our findings also suggest that gene expression of estrogen receptor (Esr) 2 increased in the DRN might be associated with the reduction of ABB.

Overproduction of Gastrointestinal 5-HT Promotes Colitis-Associated Colorectal Cancer Progression via Enhancing NLRP3 Inflammasome Activation.

Chronic inflammation is a key driver for colitis-associated colorectal cancer. 5-hydroxytryptamine (5-HT), a neurotransmitter, has been reported to promote inflammation in the gastrointestinal tract. However, the mechanism behind this remains unclear. In this study, we found that 5-HT levels, as well as the expression of tryptophan hydroxylase 1 (TPH1), the 5-HT biosynthesis rate-limiting enzyme, were significantly upregulated in colorectal tumor tissues from patients with colorectal cancer, colorectal cancer mouse models, and colorectal cancer cell lines when compared with normal colorectal tissues or epithelial cell lines. Colorectal cancer cell-originated 5-HT enhanced NLRP3 inflammasome activation in THP-1 cells and immortalized bone marrow-derived macrophages (iBMDM) via its ion channel receptor, HTR3A. Mechanistically, HTR3A activation led to Ca2+ influx, followed by CaMKIIα phosphorylation (Thr286) and activation, which then induced NLRP3 phosphorylation at Ser198 (mouse: Ser194) and inflammasome assembling. The NLRP3 inflammasome mediated IL1ß maturation, and release upregulated 5-HT biosynthesis in colorectal cancer cells by inducing TPH1 transcription, revealing a positive feedback loop between 5-HT and NLRP3 signaling. Silencing TPH1 or HTR3A by short hairpin RNA slowed down tumor growth in an established CT26 and iBMDM coimplanted subcutaneous allograft colorectal cancer mouse model, whereas treatment with TPH1 inhibitor 4-chloro-DL-phenylalanine or HTR3A antagonist tropisetron alleviated tumor progression in an azoxymethane/dextran sodium sulfate-induced colorectal cancer mouse model. Addressing the positive feedback loop between 5-HT and NLRP3 signaling could provide potential therapeutic targets for colorectal cancer.