Functional constipation (FC) represents a prevalent bowel disorder worldwide, affecting individuals across all age groups. It is defined by recurrent symptoms of infrequent stools, difficulty in stool passage, or a sensation of incomplete evacuation. Beyond its gastrointestinal manifestations, FC has been associated with various systemic conditions, including colorectal cancer and cardiovascular diseases (Wu X. et al., 2024). In pediatric populations, organic causes of constipation are exceedingly rare, with epidemiological studies indicating that over 90% of cases are attributed to functional origins. Globally, the prevalence of FC in children, when diagnosed according to the Rome IV criteria, is estimated at 14.4% (Tran and Sintusek, 2023). This condition stands as the most common gastrointestinal disorder in children, with the majority of cases emerging during infancy and toddlerhood (Chogle et al., 2016; Malowitz et al., 2016; Benninga et al., 2016). The long-term implications of untreated or inadequately managed childhood constipation are significant. It has been reported that approximately 25% of affected individuals may experience persistent symptoms extending into adulthood, leading to substantial impairments in quality of life (Pijpers et al., 2010). Early recognition and appropriate intervention are therefore critical in mitigating both the immediate and long-term burdens of this condition.

The infant gut microbiota plays a pivotal role in shaping health across various developmental stages and exerts long-term influences throughout life (Pantazi et al., 2025; Sarkar et al., 2021). In this study, we conducted a cross-sectional analysis involving children aged 0–2 years, recruiting participants with FC as well as those without FC (referred to as BC for balanced/healthy controls). Fecal and urinary samples were collected from these participants and analyzed. This dual approach was utilized to characterize the gut microbiota profiles in FC children and to elucidate the underlying metabolic mechanisms associated with this condition. The findings may provide critical insights for developing targeted interventions and improving clinical outcomes in pediatric populations affected by FC.

Within the first 2 years of life, a critical period for human development, trillions of microbes and their associated genes undergo assembly and stabilization within the human body, forming the foundation of the gut microbiome (Robertson et al., 2019). Accumulating evidence underscores dysbiosis of the intestinal flora as a pivotal etiological factor in pediatric FC. Recent metagenomic studies have further illuminated the central role of bacterial composition and metabolic potential in the pathophysiology and progression of FC (de Meij et al., 2016; Mancabelli et al., 2017). Building upon these insights, we undertook an investigation into the intestinal microbiota and urinary metabolomics profiles of Chinese children aged 0–2 years diagnosed with FC. This study was designed to systematically characterize the microbial community structure and its associated metabolic signatures in this specific population, thereby providing a targeted understanding of the microbiome-metabolome interactions underlying pediatric FC during early childhood development.

Although the FC and BC groups did not show significant differences in terms of alpha diversity, LEfSe analysis revealed significant differences in the composition of the gut microbiota between the two groups. Specifically, five genera had significantly higher relative abundance in the FC group than in the BC group; these were Alistipes, Eggerthella, Prevotella, Collinsella, and Subdoligranulum. Genera with higher relative abundance in the BC group included Veillonella and Selenomonas. Alistipes, a prominent genus within the gut microbiota, exerts dual regulatory effects on intestinal motility through tryptophan metabolism. This genus competitively utilizes tryptophan via the kynurenine pathway, thereby limiting substrate availability for serotonin synthesis through the 5-hydroxytryptophan (5-HTP) pathway (Tyrrell et al., 2011). The resultant serotonin deficiency impairs enteric nervous system signaling, directly contributing to delayed colonic transit-a hallmark of FC. Clinical observations reveal elevated mucosal colonization of Alistipes in FC patients, correlating with diminished 5-HTP levels and prolonged intestinal transit times (Mearin et al., 2016; Sugitani et al., 2021). Notably, therapeutic interventions involving Bifidobacterium species demonstrate constipation-alleviating effects through a tripartite mechanism: enhancing acetate production, promoting Lactobacillus proliferation, and specifically suppressing Alistipes overgrowth (Wang L. et al., 2017). This microbial modulation rebalances the tryptophan-serotonin axis while improving luminal SCFA profiles, suggesting Alistipes abundance serves as both a biomarker and potential therapeutic target in FC pathophysiology (Natividad et al., 2018; Muralidharan et al., 2021).

The gut microbiota composition in Rett syndrome patients with constipation demonstrates a significant enrichment of Eggerthella, a potentially pathogenic genus associated with intestinal barrier dysfunction. This dysbiotic state generates a unique SCFA profile independent of constipation status, suggesting intrinsic microbial metabolic reprogramming linked to Eggerthella proliferation (Strati et al., 2016). Mechanistically, Eggerthella exhibits proinflammatory properties through induction of intestinal epithelial inflammation and compromised barrier integrity, potentially exacerbating gastrointestinal manifestations including bleeding and bloating (Gardiner et al., 2015). Concurrently, Prevotella demonstrates microenvironmental adaptability to inflammatory conditions, with its increased abundance in constipated mice models and human cohorts reflecting a symbiotic relationship with proinflammatory states. This genus may perpetuate intestinal dysmotility through sustained inflammatory signaling (Liu et al., 2019; Li et al., 2024). The microbial ecology analysis reveals functional dichotomy between Veillonella and Selenomonas in gut homeostasis. While Veillonella’s dominance in healthy small intestinal ecosystems aligns with its elevated abundance in control groups, its metabolic reliance on organic acid fermentation may paradoxically exacerbate constipation through gas-mediated intestinal distension. In contrast, Selenomonas demonstrates therapeutic potential through glucose fermentation pathways, as evidenced by its post-transplant enrichment in fecal microbiota transplantation studies correlating with improved colonic motility and mitigation of constipation-related neuropsychiatric comorbidities (van den Bogert et al., 2013; Yang et al., 2023). These findings collectively highlight the complex interplay between specific bacterial taxa (Eggerthella, Prevotella) driving inflammatory-metabolic dysfunction and counterregulatory genera (Selenomonas) mediating intestinal homeostasis restoration, providing mechanistic insights into microbial contributions to constipation pathophysiology.

The liquid-quantity coupledomics approach utilized in this study revealed a distinct metabolite profile in children with FC. A total of 75 differential metabolites were identified, along with several associated metabolic pathways. Among these, oleamide, a fatty acid amide derived from oleic acid, emerged as a critical regulator of enteric dynamics. Oleamide has been demonstrated to inhibit intestinal motility by activating the cannabinoid receptor CB1 in murine models (Capasso et al., 2005). Traditional Chinese medicine, specifically MaZiRenWan, has been shown to mitigate oleamide-induced reductions in intestinal motility. This effect is likely mediated through enhanced degradation of oleamide via colonic fatty acid amide hydrolase, thereby improving intestinal motility in FC patients (Huang et al., 2020). Other metabolites identified in this study, such as palmitamide and n-octanoylsphingosine, currently lack established correlations with FC, warranting further investigation into their potential roles in the pathophysiology of this condition. These findings underscore the importance of oleamide and its regulatory mechanisms in FC, while highlighting the therapeutic potential of targeted interventions aimed at modulating fatty acid amide metabolism.

Glutathione serves as a critical antioxidant in the body, capable of scavenging free radicals and reducing oxidative reactions, thereby preserving cellular integrity. Additionally, glutathione plays a key role in promoting metabolism, aiding in the elimination of waste and toxins, and enhancing the body’s immune function. Notably, glutathione metabolism also influences intestinal barrier function by modulating oxidative stress and regulating the expression of pro-inflammatory cytokines. In constipated rats, the content of glutathione was decreased. After treatment with traditional Chinese medicine, its content increased, suggesting a correlation with constipation. A novel strain BC99 exerted an anti-constipation effect in adults, which may be related to glutathione metabolism (Wu Y. et al., 2024).

The sphingolipid metabolic axis plays a pivotal role in pediatric constipation pathophysiology, generating bioactive mediators such as sphingosine-1-phosphate (S1P), phytosphingosine, and dihydrosphingosine. These molecules regulate gastrointestinal motility via dual mechanisms: direct modulation of smooth muscle contractility and neuromodulatory effects mediated by interstitial cells of Cajal (ICC) (Maceyka et al., 2012). Notably, S1P exerts mechanistic significance by activating ICC pacemaker activity through calcium-dependent pathways, essential for colonic smooth muscle slow-wave propagation (Wang et al., 2023). Clinically, reduced S1P levels correlate with ICC network dysfunction and impaired propulsive motility, linking sphingolipid dysregulation to constipation (Mostafa et al., 2010). Supporting evidence from aging models demonstrates analogous gastrointestinal dysmotility associated with dihydrosphingosine depletion, while therapeutic restoration of phytosphingosine enhances cholinergic signaling, exerting prokinetic effects. These findings underscore the critical relationship between the sphingolipid metabolic axis and constipation (Choi et al., 2015; Wang S. et al., 2017).

Thiamine, also known as vitamin B1, is widely distributed in most foods. While it is particularly abundant in cereals, meat, fish, shrimp, and yeast, a significant portion of vitamin B1 is lost during food processing. As a result, many processed foods are fortified with thiamine. The absorption of thiamine primarily occurs in the duodenum, where it binds with magnesium ions to form its active state, thiamine pyrophosphate (TPP). TPP serves as an essential cofactor in critical metabolic pathways, such as the citric acid cycle and the pentose phosphate pathway. Low levels of thiamine can impair mitochondrial function, leading to reduced oxidative metabolism and decreased energy production. Research on malnutrition among elderly individuals in nursing facilities has shown that thiamine deficiency is associated with gastrointestinal issues, including nausea, vomiting, and constipation (Volkert et al., 2011). Du et al. (2024) conducted a large-scale survey involving 10,371 participants, and the results demonstrated a significant negative correlation between thiamine intake and the prevalence of constipation, with a more pronounced association observed in males, as well as individuals without hypertension or diabetes. One possible explanation for this phenomenon is that dietary thiamine intake is closely related to stool softening and an increased frequency of colonic peristalsis.

Additionally, the combination of characteristic metabolites and gut flora provided an excellent discriminatory marker for FC children, with an AUC of 1.000. This highlights the potential utility of metabolomic and microbiomic profiles in diagnosing and understanding the underlying mechanisms of functional constipation in this population. These findings underscore the importance of integrating metabolomics and microbiomics in elucidating the pathophysiology of functional constipation and identifying potential therapeutic targets.

This investigation acknowledges three principal limitations requiring methodological refinement in subsequent research. First, while demographic parameters (age, gender, delivery mode, feeding practice) showed intergroup parity, the inherent biological variability of infant cohorts across a broad age spectrum (0–2 years) may introduce developmental confounders. Future studies employing stricter age stratification could enhance phenotypic homogeneity. Second, constrained sample size and regional recruitment bias potentially compromise statistical power and generalizability. Nationwide multicenter cohorts incorporating longitudinal designs would improve external validity and enable detection of subtle microbiota-host interactions. Third, the inherent taxonomic limitations of 16S rRNA sequencing preclude precise species-level microbial characterization, particularly critical for functionally divergent subspecies within constipogenic genera. Implementation of shotgun metagenomics coupled with metabolomic profiling could resolve this taxonomic ambiguity while elucidating microbial metabolic networks. Complementary mechanistic validation through fecal microbiota transplantation models and pharmacological modulation of identified microbial targets would strengthen causal inference. Addressing these limitations will facilitate translation of observational associations into therapeutic strategies for pediatric functional constipation.

This study establishes an integrated multi-omics framework for characterizing Chinese FC in pediatric populations. Through systematic correlation of gut microbial dynamics with metabolomic perturbations, we identified FC-specific microbial clusters and bioactive metabolites converging on thiamine, glutathione and sphingolipid metabolic dysregulation. The developed microbial-metabolite diagnostic panel demonstrates superior discriminative capacity for FC detection, outperforming conventional symptom-based assessments. This biomarker platform enables non-invasive stratification of constipation through machine learning-driven pattern recognition of multi-omics signatures.

Our findings establish a functional axis connecting microbial sphingolipid metabolism with intestinal neuromuscular regulation, offering three translational applications: (1) the relationship between crucial gut microbiota and oleamide, (2) strategies for metabolic reprogramming of glutathione, and (3) the correlation between mechanism-based metabolites oleamide and glutathione metabolism. This paradigm advances pediatric gastroenterology practice towards microbiome-informed personalized interventions.