Research on the role of the amino acid TPH is receiving increasing attention. TPH is found in protein-rich foods such as egg, milk, oats, cheese, nuts and seeds. The majority of TPH is absorbed within the small intestine, but excessive intake of protein and amino acids (6-18 g/day) can make some protein reach the colon (31). SCFAs are the major metabolites produced by microbial fermentation of dietary fiber. They are vital in maintaining metabolism, neurological function and immune systems (32). Dietary fiber is consumed from the diet, and it cannot be digested and absorbed directly by the gastrointestinal system. Instead, it escapes digestion in the stomach and small intestine, and undergoes fermentation by anaerobic microbes in the cecum and colon. This fermentation process produces SCFAs, mainly including acetate, propionate and butyrate salts (33,34). SCFAs are readily absorbed from the gut into the circulation and directly reach the liver. Previous research has shown that injecting butyrate via portal vein results in a 70% increase in non-rapid eye movement sleep in mice (35,36). This finding supports the connection between gut microbiota and related metabolites with sleep disorders (37). These metabolites can be absorbed and utilized by the host's gut and are measurable in the host's circulation. However, some microbial metabolites can promote health, while others can be toxic and detrimental to health. A previous study linked insufficient sleep to reduced SCFAs production by the gut microbiome. Another study found a positive association between sleep duration and the concentrations of total SCFAs, acetate and propionate in stool (38). These results suggest that shorter sleep duration is associated with lower SCFA production, providing some evidence that SCFAs may influence sleep. Furthermore, studies have indicated that diet may influence sleep through melatonin and its biosynthesis from TPH (39).

TPH is the only substance needed to make 5-HT, and in germ-free mice, 5-HT levels were 2.8-fold lower. This reduction can partly be due to microbial processes (40). 5-HT, a neurochemical molecule that exhibits diurnal variations, is associated with the hypothalamic pathways that promote sleep and regulate glucose homeostasis (41). Both gut microbiota and enterochromaffin cells can influence 5-HT synthesis by controlling the rate-limiting enzyme TPH hydroxylase (42). Subsequently, 5-HT is converted by different enzymes into either melatonin or 5-hydroxyindoleacetic acid, with melatonin being the primary substance responsible for regulating sleep initiation and circadian rhythms (43). Certain specific bacterial strains, including Lactobacillus, Lactococcus, Prevotella, Streptococcus thermophilus, Escherichia coli, K-12, Morganella morganii, Klebsiella pneumoniae and Staphylococcus aureus, can produce 5-HT and other biogenic amines from TPH (44). This impacts gut motility and secretion, and can also affect 5-HT levels in the brain, potentially influencing mood and cognitive functions (45).

In addition, SCFAs can influence the serotonergic system. 5-HT activity is primarily influenced by the extracellular availability of 5-HT, which is regulated by the 5-HT transporter (SERT) (46). A previous study found that SCFAs affected the activity of intestinal SERT (47). In this study, the activity and expression levels of SERT were decreased by propionate and acetate, concurrently elevating the levels of specific 5-HT receptors that amplified 5-HT signaling. By contrast, butyrate enhanced both the activity and expression levels of SERT, and upregulated anti-inflammatory molecules such as interleukin (IL)-10 (47). Additionally, it was observed that low levels of acetate, propionate and butyrate has an impact on SERT activity, whereas increased concentrations did not influence SERT functionality. Although butyrate appeared to have no effect on receptor expression, propionate and acetate both notably increased the mRNA levels of 5-HTR1A, 2B and 7 (48). The 5-HTR1A receptor, which is expressed in intestinal epithelial cells and enteric neurons, can activate the release of 5-HT from enterochromaffin cells (49).

In conclusion, dietary intake of TPH is crucial for 5-HT synthesis, and influences emotions and cognitive functions through the actions of gut microbiota, highlighting the importance of the gut-brain axis in maintaining overall health.

The VN serves as the principal conduit for transmitting internal organ information to the CNS. Earlier research indicated that the VN may be involved in conveying information from the gut microbiota to the brain (50). Stimuli from chemicals or microbes in the gastrointestinal tract can activate Trpa1⁺ EECs, which subsequently transmit signals to the vagal ganglia. Previous research shows that intestinal epithelial cells have developed specialized EECs, specifically enterochromaffin cells (51). EECs are distributed throughout the whole gastrointestinal tract and respond to various luminal stimuli by secreting hormones or neurotransmitters in a calcium-dependent mechanism (52). Therefore, the link between EECs and neurons establishes a direct pathway through which the intestinal epithelium can convey sensory information to the brain. Recent research has shown that bacterial TPH degradation metabolites, such as indole or Indole-3-acetaldehyde (IAld), activate the vagal ganglia through the EEC Trpa1 signaling pathway. Additionally, by measuring fresh tissue slices from the intestines of humans and mice exposed to indole, it was observed that indole significantly induced the secretion of 5-HT in both human and mouse intestines, while a Trpa1 inhibitor blocked this effect (53) (Fig. 1).

Previous research has shown that sleep deprivation (SD) may impair the function of the intestinal barrier (54), triggering oxidative stress and causing damage to the intestinal mucosa (55). The expression of tight junction proteins (occludin and ZO-1) in the intestinal tissue decreases, leading to an increase in intestinal permeability. After the disruption of the intestinal barrier, TLR4 recognizes lipopolysaccharide (LPS), it binds to myeloid differentiation factor 88, ultimately activating the IκB kinase complex. This leads to the degradation of IκB, the release of NF-κB, and the initiation of transcription of inflammatory genes, resulting in the transcription of pro-inflammatory factors IL-6, IL-1β and TNF-α (56), leading to an increase in their levels in both the brain and the intestine (57). IL-1β, TNF-α and IL-6 are the most extensively studied pro-sleep and pro-inflammatory cytokines that regulate sleep (24,58), and poor sleep quality is also positively associated with an increase in IL-8 (59).

In a previous study, the antioxidant fullerene nano-antioxidants (FNAO) improved the sleep of sleep-deprived zebrafish by regulating the redox balance in the intestine. In addition, in mice that received oral administration of FNAO, the levels of the tight junction protein occludin in the intestine increased, while the levels of the pro-inflammatory cytokines IL-6, IL-1β and TNF-α in the intestine decreased, significantly improving sleep in mice (57). All these findings indicate that pro-inflammatory immune response and oxidative stress in the intestine are closely related to sleep. Continuous disruption of the intestinal barrier leading to an increase in intestinal permeability; microbes, LPS, peptidoglycans and pathogen-associated molecular patterns can be recognized by peripheral macrophages; and this recognition promotes the release of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) into the bloodstream, thus triggering systemic inflammation (60,61). Moreover, These molecules then cross the intestinal barrier (IB) and BBB to reach the brain parenchyma, where they are exposed to microglia. This is an example of microbiome-derived microbial-associated molecular patterns. Microglia and astrocytes are activated through the TLR4-myeloid differentiation factor 88-NF-κB signaling pathway, thereby generating neuroinflammation that affects sleep (62-64). The suprachiasmatic nucleus (SCN) in the hypothalamus is the central regulator of human's circadian rhythm and is crucial for controlling the metabolic rhythm in mice (65). Extensive evidence shows that pro-inflammatory mediators inhibit the expression of clock genes and their targets in both the SCN and peripheral tissues, affecting the biological clock and thus disrupting the circadian rhythm. In addition, microglia and astrocytes are the brain's primary immune and inflammatory cells, respectively, playing key roles in antigen presentation and the production of both pro-inflammatory and anti-inflammatory factors (66,67). Chronic SD activates these cells, indicating that they could serve as potential targets for reducing neuroinflammation and oxidative stress in the brain following SD (68). In a previous study reporting both the melatonin supplementation and butyrate supplementation groups reversed the changes in neuroinflammation and cell apoptosis induced by SD (69). The levels of Iba1-positive cells, as well as IL-6 and TNF-α in the CA1, CA3 and dentate gyrus regions of the hippocampus were significantly lower compared to those in the sleep deprivation group. This is likely related to the inhibition of the activation of NF-κB in intestinal cells, which partially suppresses the production of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6, as well as the inhibition of microglial activation (70-72).

In addition, various TPH metabolites produced by the gut microbiota, such as indole and indole-3-acetic acid can significantly weaken the activation of NF-κB in macrophages induced by LPS. It can also significantly reduce the increase of pro-inflammatory cytokines in intestinal epithelial cells induced by TNF-α and LPS, as well as decrease the activation of IL-6 and IL-1β in cells stimulated by LPS (Fig. 2) (73).

Aromatic hydrocarbon receptors (AHR) act as a key mediator in brain signaling pathways for TPH metabolites. In recent years, bacterial TPH metabolites have been extensively studied as ligands for AHR, a transcription factor that is commonly found in immune system cells. A study has indicated that activated AHR can modulate innate and adaptive immune responses in a ligand-specific manner (74). It is associated with various chronic diseases, particularly inflammatory conditions. TPH metabolites derived from the gut microbiota, such as indole-3-sulfonic acid, indole-3-acetic acid (IAA), indoxyl sulfate (IS), indole-3-propionic acid (IPA), indole-3-aldehyde (I3A) and IAld, can activate AHR (75). TPH metabolites, including IAA, IS, IPA, I3A and IAld, transmit signals through astrocytic AHR, activating TGF-α or inhibiting vascular endothelial growth factor (VEGF)-β (76,77). TGF-α derived from microglia exerts neuroprotective effects through the epidermal growth factor (ErbB1) receptor in astrocytes. On the other hand, VEGF-B produced by microglia and other sources, when activating Flt-1 in astrocytes, can promote CNS inflammation (78). Type-I interferon (IFN-I) signaling in astrocytes collaborates with TPH microbial metabolites to activate AHR (79). Activated AHR subsequently suppresses the activation of NF-κB by inducing the expression of suppressor of cytokine signaling 2 (Socs2) through cytokine signaling (80). In addition, AHR plays a key role in blocking the inflammatory and neurotoxic effects of IFN-α receptor 1 (IFNAR-1) (79). Therefore, the IFN-I-AHR-Socs2-NF-κB pathway indicates that targets interferon α and β receptor subunit 1 signaling could potentially be used to treat CNS inflammation (Fig. 3).

Microbial-derived indole metabolites can bidirectionally regulate the release of the appetite hormone GLP-1 (81). GLP-1, when present in the CNS, shows potential neuroprotective effects and helps improve neuronal survival. Specifically, when exposed to physiological levels of indole, GLP-1 secretion from colonic enteroendocrine L cells increases (82). GLP-1 can enhance brain-derived neurotrophic factor (BDNF) in the brain (83). A previous study investigated the role of BDNF in cognition, inflammation and neurodegenerative diseases (84). The role of GLP-1 in inhibiting CNS cell apoptosis, promoting neuronal growth, reducing cell proliferation and decreasing oxidative damage has been revealed (85). Indole interacts with enteroendocrine L cells, leading to the release of GLP-1. Before being released by the intestine, GLP-1 is rapidly deactivated by dipeptidyl peptidase-4. It has been speculated that GLP-1 acts locally on the terminals of the vagal afferent nerves (86). Based on the input from the VN, GLP-1 then signals to brain circuits and the nucleus tractus solitarius (NTS). The NTS can project to multiple sleep-regulating brain regions impact sleep (Fig. 3) (87).

The biological clock is a key timer in the interaction between the host and microbiota. It is a 24 h biological oscillator that coordinates changes in behavior and physiology to anticipate environmental fluctuations within the 24 h day-night cycle. The biological clock is primarily controlled by the SCN in the hypothalamus, which is influenced by light-dark cycles, sleep-wake patterns and feeding rhythms. The SCN, in turn, coordinates peripheral clocks, regulating tissue-specific clock-controlled genes. The gut microbiome's daily rhythms synchronize with those of distant tissues and organs, including the gastrointestinal tract and liver, as well as key physiological processes. By integrating both nutritional and hormonal elements, this process modulates gene expression and regulates the biological rhythm of the host (88,89).

In birds, the circadian rhythm regulation system includes positive clock genes such as circadian locomotor output cycles kaput (CLOCK) and cBMAL1 (cBMAL1 and cBMAL2), as well as negative clock genes such as CPER (CPER2 and CPER3) and cCRY (cCRY1 and cCRY2) (90). Disturbances in the gut microbiome's circadian rhythms and its metabolites may influence the central biological clock, as well as the rhythmic expression of the aforementioned genes in the liver and gastrointestinal tract (91). The regulation of host biological clock gene expression is primarily mediated by the gut microbiome through its derived metabolites, including SCFAs and their receptor genes, such as free fatty acid receptor 2 (FFAR2) and FFAR3 (92).

Previous research showed that an intermittent light cycle enhanced the circadian rhythms of cBMAL1, cBMAL2, cCRY1 and cCRY2 in the hypothalamus, increased the expression of cCLOCK, cBMAL1 and cCRY2 in the liver, and upregulated the expression of seven clock genes (including cBMAL1 and FFAR2) in the cecal wall. Such intermittent light cycle also significantly altered the composition and metabolic function of the cecal microbiome via the melatonin pathway. Under intermittent light cycle treatment, the concentrations of SCFAs and the abundance of SCFA-producing genera, such as Odoribacter splanchnicus, were significantly increased. Correlation analysis indicated a positive correlation between the presence of Enterococcus and the expression of cBMAL1 and FFAR2 in the cecal wall, as well as cBMAL2 expression in the hypothalamus. cBMAL2 expression in the hypothalamus showed a clear circadian rhythm under an intermittent light cycle (93). It was therefore hypothesized that SCFAs may further feedback and enhance peripheral and central rhythms by activating SCFA receptor genes (such as FFAR2) in the cecal wall.

In addition, in the positive feedback loop of the circadian clock, CLOCK and BMAL1 proteins promote the activation of downstream genes by acetylating histones. Acetylation loosens chromatin, making the genes more accessible to transcription factors and RNA polymerase, which helps initiate transcription (94,95). Deacetylation is often carried out by histone deacetylases (HDACs), and cryptochrome (CRY) proteins maintain histone acetylation by inhibiting HDAC activity, thereby enhancing transcriptional activity. On the other hand, increased HDAC activity suppresses the expression of clock genes. Similarly, SCFAs such as butyrate, propionate and isovalerate strongly inhibit HDAC activity, increasing histone acetylation levels. After oral administration of SCFAs, the circadian rhythm phase in the peripheral tissues of mice changes (96). Using oscillation experiments with LUC-type intestinal cells over 72 h, a study revealed that acetate, butyrate, isovalerate and propionate induces phase delays. The expression of intestinal BMAL1-ELuc showed significant cyclical changes, which closely resembled the phase delay pattern induced by HDAC inhibitors such as trichostatin A and suberoylanilide hydroxamic acid (97). This study supported the idea that SCFAs and microbial metabolites alter the host's clock through HDAC inhibition.

Nuclear receptor subfamily 1 group D member 1 (NR1D1), also known as Rev-erb, is a core component of the molecular circadian clock, regulating the cellular circadian rhythm. A previous study found that, in mice and human submandibular gland cells treated with butyrate, the number of NR1D1-positive cells and the expression level of NR1D1 decreased. As a result, NFIL3, which is negatively regulated by NR1D1, showed increased expression levels under NR1D1 inhibition. Notably, the expression level of nuclear factor, interleukin 3 regulated (NFIL3) was elevated in butyrate-treated cells (98).

Indole can act as an agonist to induce the activation of AHR. Depending on their molecular structure, TPH metabolites can function as either agonists or antagonists to influence AHR (99). The gut microbiota serves as a source of different AHR signals, and evidence suggests that the gut microbiome can influence the circadian rhythm of the host (45). The genes involved in the circadian rhythm are known as 'clock genes', including BMAL1 CLOCK, neuronal PAS domain protein 2 and NR1D1. The interaction between CLOCK/BMAL1 heterodimer and period homolog and CRY proteins is the main feedback loop driving the circadian rhythm. AHR is a PAS domain protein, and BMAL1/CLOCK proteins can form heterodimers with AHR (100). This interaction can disrupt the oligomerization of core clock proteins and impair their transcriptional activation, leading to a disruption of the circadian rhythm (101). Evidence suggests that activation of AHR signaling inhibits the expression of circadian clock genes and, therefore, impairs the circadian rhythm in various experimental models. These studies demonstrate that AHR can function as an inhibitory factor of the circadian rhythm in central and peripheral clocks (Fig. 4) (102).

A previous study found that the BBB is essential for maintaining the homeostasis of nutrients, ions and other molecules in the brain. Its permeability is regulated by SD and can independently influence sleep changes (103). TPH can be broken down via the 5-HT and kynurenine pathways, and is closely associated with the CNS. TPH can enter the CNS through L-type amino acid transporter 1 to cross the BBB (104). In addition, the positive impact of SCFA-producing gut microbiota on BBB integrity has been demonstrated in germ-free mouse models and in mice treated with antibiotics that alter the abundance of specific bacterial families. In mice treated with five non-absorbable antibiotics, the mRNA levels of occludin and ZO-1 in brain microvessels were reduced, while BBB permeability increased. These antibiotics reduced the relative abundance of SCFA-producing bacteria in the gut. However, after fecal microbiota transplantation from pathogen-free gut microbiota to antibiotic-treated mice, the mice restored tight junction protein expression and BBB integrity (105). Another study also showed that, in mice treated with an antibiotic mixture, the levels of acetate, propionate and butyrate in the colon were reduced, along with a decline in object recognition memory. Additionally, the mRNA expression levels of claudin-5 and occludin in the amygdala and hippocampus decreased (106). Similar results were obtained in mice undergoing anesthesia/surgery treated with an antibiotic mixture. Administration of a Lactobacillus bacterial mixture restored the expression levels of claudin-5, occludin and ZO-1 in the hippocampus, and improved BBB permeability (107). These experiments suggest that SCFAs produced by the microbiome play a key role in restoring and maintaining BBB integrity.