ABSTRACT
Metabolic dysfunction–associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease, has become the most common form of chronic liver disease in children. The spectrum of pediatric MASLD ranges from simple steatosis to steatohepatitis, fibrosis, cirrhosis, and in rare cases, hepatocellular carcinoma. Its pathogenesis involves a complex interplay among genetic, epigenetic, and environmental factors, along with alterations in the gut microbiota and its associated metabolites. Given the staggering prevalence and the distinct etiopathogenesis of pediatric MASLD, characterization of the gut microbiota and microbial products could facilitate the development of diagnostic tools and inform targeted therapeutic strategies. Current research on the gut microbiome in the context of pediatric MASLD is limited by small sample size, inadequate use of liver biopsy, methodological inconsistencies in sequencing, and confounding effects from metabolic comorbidities. In this review, we summarize clinical studies on alterations in the gut microbiota and microbial products (short-chain fatty acids, bile acids, and ethanol) that impact the pathogenesis of pediatric MASLD. We discuss the therapeutic potential of dietary modification, pharmacological treatments, and probiotics in improving disease progression by summarizing current clinical studies. Enhancing our understanding of the gut-liver axis may aid in the development of effective therapeutic strategies for pediatric MASLD.
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Keywords: Metabolic dysfunction–associated steatotic liver disease; Gut microbiome; Liver diseases; Children; Probiotics
INTRODUCTION
Metabolic dysfunction–associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease, affects nearly 10% of children globally, with the prevalence rising to over 30% among children with obesity [
1,
2]. The spectrum of pediatric MASLD ranges from isolated steatosis to metabolic dysfunction-associated steatohepatitis (MASH), which may progress to fibrosis and even cirrhosis, and in rare cases, hepatocellular carcinoma. Pediatric MASLD is strongly associated with extrahepatic comorbidities, such as type 2 diabetes (T2DM), hypertension, dyslipidemia, and obstructive sleep apnea, and contributes to early mortality [
3]. Despite this, there are currently no U.S. Food and Drug Administration (FDA)-approved pharmacotherapies for pediatric MASLD. Histologically, type 1 MASH is predominantly observed in adults and is characterized by steatosis, ballooning degeneration, and perisinusoidal fibrosis. In pediatric populations, MASLD presents as two distinct subtypes: type 1, which shares features with the adult form, and type 2, characterized by steatosis, portal inflammation, and portal fibrosis, with type 2 being the predominant form in children [
4].
Although pediatric MASLD is becoming increasingly prevalent and is associated with substantial adverse health outcomes, the pathophysiological mechanisms driving its initiation and progression remain largely elusive. Emerging evidence suggests that MASLD develops through a complex interaction of genetic susceptibility, dysregulated lipid metabolism, impaired insulin resistance, augmented lipotoxicity and inflammatory responses, compromised gut barrier function, and gut dysbiosis, combined with environmental exposures (
Fig. 1) [
5]. Compared with adult studies, research on gut microbiota and microbial metabolites in pediatric MASLD remains limited and exhibits marked heterogeneity, primarily due to disparities in sample sizes and study methodologies.
In this review, we focus on the pathogenic role of the gut microbiome in pediatric MASLD by summarizing clinical studies that investigate alterations in gut microbiota and microbial products, and by exploring how these changes may contribute to disease progression. Furthermore, we evaluate clinical intervention approaches, including both pharmacological and microbiome-based therapies, for treatment of pediatric MASLD. Consistent with prior literature, we define pediatric MASLD as occurring in individuals under 19 years of age. Only studies involving children and adolescents diagnosed with MASLD based on imaging or histology, and characterized by 16S rRNA or metagenomic sequencing, are included in this review.
THE GUT-LIVER AXIS AND PEDIATRIC MASLD
The human gut harbors trillions of microorganisms, comprising bacteria, fungi, viruses, and archaea, which collectively form a complex ecosystem and play critical roles in host metabolism, immunity, and digestion [
6,
7]. Healthy individuals typically share a core gut microbiota profile dominated by the Bacteroidetes and Firmicutes phyla, representing ~90% of the total microbial population. Despite this high degree of compositional similarity, each individual harbors a unique microbial signature [
8]. Along the gastrointestinal tract, there is considerable compositional variation as the pH and oxygen availability differ from the mouth to the colon [
9]. The gut microbiota of infants displays compositional and functional differences compared with adults, achieving an adult-like microbial architecture at the age of 3 years. A longitudinal study including 903 infants revealed that the gut microbiota development progresses through three stages: a Bifidobacterium-dominated phase (3–14 months), a transitional phase with Bacteroidetes/Proteobacteria shifts (15–30 months), and a stable Firmicutes-rich phase over 31 months [
10]. Importantly, children aged 3–4 years old still had a lower microbial diversity compared with adults, suggesting that the gut microbiome may take longer to establish than previously thought [
11]. Evidence from human and animal studies suggests that disruption of the gut microbiome can have a profound impact on host physiology and immunology.
The liver possesses a unique dual blood supply system that receives 70–80% of its blood from the portal vein and the remaining 20–30% from the hepatic artery [
12]. On the other hand, the bile acids produced by the liver are secreted into the intestine through the biliary system. The commensal gut microbiota helps maintain liver health under physiological conditions [
13], yet may produce deleterious metabolites and promote liver injury in pathological states [
14-
16]. When the gut barrier is compromised, the liver is the first organ exposed to microorganisms and microbial products originating from the intestine (
Fig. 2). A meta-analysis of 14 studies (6 in adults and 8 in children) showed increased intestinal permeability in both children and adults with MASLD compared with healthy controls, as assessed by urinary recovery of orally administered non-metabolizable sugars or intestinal zonula occludens protein levels [
17]. Furthermore, intestinal permeability was higher in children with steatohepatitis compared with those with steatosis only, and the lactulose/mannitol ratio was found to correlate with the disease severity [
18]. The mechanisms by which an impaired intestinal barrier contributes to adult MASLD pathogenesis have been reviewed in detail elsewhere [
5,
19]. However, potential mechanistic differences between pediatric and adult MASLD remain to be elucidated.
GUT MICROBIOME IN HUMAN MASLD
Gut microbiome in adult MASLD
Most studies investigating the gut microbiome in patients with MASLD have focused on characterizing the composition and function of intestinal bacteria. However, findings across different studies have been inconsistent, likely due to limited sample size, absence of validation cohorts, failure to adjust for metabolic cofactors, and inaccurate characterization of liver severity [
5]. Nevertheless, studies consistently demonstrate reduced microbial diversity and altered microbial composition in patients with MASLD, characterized by a decrease in anti-inflammatory microbes and an increase in pro-inflammatory microbes [
20]. A meta-analysis of 15 studies comprising 1,265 individuals (577 MASLD and 688 controls) demonstrated a distinct gut microbial profile in patients with MASLD, featuring higher abundance of
Escherichia, Prevotella, and
Streptococcus coupled with lower levels of
Coprococcus, Faecalibacterium, and
Ruminococcus [
21]. In non-obese MASLD, Lee et al. [
22] showed that specific taxa were associated with fibrosis severity and were more prominent in the disease group, such as
Ruminococcaceae and
Veillonellaceae. To investigate the relationship between gut microbiota and fibrosis progression, Loomba et al. [
23] compared patients with mild to moderate fibrosis (stages 0–2) to those with advanced fibrosis (stages 3–4) in the context of MASLD. They found that patients with advanced fibrosis had increased levels of Proteobacteria and
Escherichia coli, along with decreased levels of Firmicutes.
Beyond bacteria, the gut fungal component (mycobiome) has also been characterized in adults with MASLD. Interestingly, the gut fungal diversity showed inconsistent results in different studies: internal transcribed spacer 2 sequencing revealed increased fungal diversity in Chinese MASLD patients versus controls [
24], contrasting with metagenomic findings [
25]. Meanwhile, studies on German cohorts found no significant difference [
26]. At the genus level, the abundances of
Talaromyces, Cladophialophora, and
Sordaria were increased in patients with MASLD and showed positive correlations with both liver injury markers (alanine aminotransferase [ALT] and gamma glutamyl-transferase) and lipid metabolism parameters (total cholesterol and triglycerides). At the species level, Demir et al. [
26] showed an increased abundance of
Candida albicans, Mucor sp., Penicillium sp., and
Babjeviella inositovora associated with MASLD severity. Finally, characterization of intestinal virome revealed reduced viral diversity and decreased
Lactococcus phage proportions in patients with MASLD activity score (MAS) 5–8 vs. MAS 0–4 [
27]. Future studies are needed to elucidate the causal relationship between alterations of viral taxa and MASLD pathogenesis.
Research on the gut microbiome in MASLD has primarily focused on adult populations, with substantially fewer studies conducted in children and adolescents, as summarized in
Table 1 and
Figure 3 [
28-
42]. Previous research suggests that maternal programming factors (e.g., obesity, gestational diabetes mellitus, medicine, and diet) significantly shape the gut microbiota of infants, with early microbial alterations linked to pediatric MASLD pathogenesis [
43]. However, current conclusions are primarily derived from germ-free mouse models transplanted with stool samples from infants born to obese mothers [
44]. The causal mechanisms connecting maternal risk factors to specific microbial taxa alterations in pediatric MASLD development require further investigation. Existing studies in pediatric populations are mainly limited by small sample sizes, the challenges of performing liver biopsy, and the unclear genetic background. Nonetheless, previous studies consistently reported reduced α-diversity and increased levels of
Prevotella in children with MASLD compared with healthy controls and obese children without MASLD.
In a pioneering study by Zhu et al. [
28], 16S rRNA sequencing was applied to characterize gut microbiota profiles in healthy controls (n=16), obese children without fatty liver (n=25), and obese children with biopsy-confirmed MASH patients (n=22). In children with MASH, the distinct microbial signature was characterized by increased levels of Proteobacteria,
Enterobacteriaceae, and
Escherichia, as well as elevated blood ethanol levels, likely resulting from the expansion of ethanol-producing bacteria. In 2017, Del Chierico et al. [
30] analyzed gut microbiota using fecal samples from healthy controls (n=54), obese children without fatty liver (n=8), and MASLD patients (n=53, 26 with MASH). Compared with controls, MASLD patients showed an increased abundance of Actinobacteria and decreased abundance of Bacteroidetes. Additionally, MASH patients were characterized by reduced levels of
Oscillospira and increased levels of
Dorea and
Ruminococcus. In 2018, Stanislawski et al. [
31] analyzed the associations among gut microbiota, diet, and hepatic fat fraction (HFF) in adolescents with (n=8) or without (n=99) MASLD. Despite the small sample size in the MASLD group, they found that lower α-diversity was associated with a higher HFF. A combined model including
Bilophila and
Paraprevotella abundance, monounsaturated fat intake, and body mass index (BMI) z-scores explained 32% of the variance in HFF. In 2022, Zhou et al. [
38] investigated the gut microbiota in 58 children and adolescents, including healthy controls (n=15), obese children without MASLD (n=16), and with MASLD (n=27). HFF was determined by magnetic resonance imaging proton density fat fraction (MRI-PDFF). Metagenomic sequencing of fecal samples showed higher levels of
Megamonas, especially
Megamonas hypermegale and
Megamonas rupellensis, in MASLD patients compared with non-MASLD subjects, suggesting a potential link between MASLD progression and selective enrichment of
Megamonas species.
Beyond observational studies, recent studies have investigated the mechanisms through which specific microbial taxa contribute to the pathogenesis of pediatric MASLD. In a prospective cohort study involving 87 children with biopsy-proven MASLD and 37 children with obesity as controls, Schwimmer et al. [
32] found that the α-diversity was lowest in patients with MASH and that a higher abundance of
Prevotella copri was associated with more severe liver fibrosis. The association between
P. copri and fibrosis severity was further validated in a meta-analysis of shotgun metagenomic sequencing data from 9 studies and an additional recruited cohort in pediatric population. This analysis showed that the abundance of
P. copri was higher in MASH patients with grade 2 fibrosis compared with those with grade 1 or grade 0. Machine-learning models could discriminate pediatric from adult MASH with an AUROC of 97%, with key discriminatory taxa including
P. copri (clade A),
Romboutsia timonensis, and
Phocaeicola dorei [
45]. The pathogenic role of
P. copri in pediatric MASLD was investigated in juvenile mice fed with high-fat diet (HFD) from 3 to 8 weeks of age. Animal experiments showed that
P. copri supplementation exacerbated HFD-induced liver steatosis and gut dysbiosis in juvenile mice. Mechanistically,
P. copri-produced 5-aminopentanoic acid exacerbated steatosis by promoting lipogenesis and fatty acid uptake, while reducing hepatic very-low-density lipoprotein export [
46]. Beyond
P. copri, Wei et al. [
39] demonstrated that
Enterococcus spp. were enriched in children with obesity and MASLD. Notably, patient-derived
Enterococcus faecium B6 exacerbated HFD-induced MASLD progression in juvenile mice (3-4 weeks old). Mechanistic studies revealed that
E. faecium B6 activated peroxisome proliferator-activated receptor γ via its metabolite tyramine, leading to increased lipid accumulation, inflammation, and fibrosis.
In addition to characterizing changes in the gut microbiota, metagenomic sequencing also revealed alterations in microbial function associated with the pathogenesis of pediatric MASLD. Pro-inflammatory pathways such as lipopolysaccharide biosynthesis and flagellar assembly were enriched in more severe disease states (MASH or MASH with fibrosis), suggesting a microbial contribution to hepatic inflammation and fibrosis [
32]. Meanwhile, short-chain fatty acids (SCFAs) production pathways—including fermentation of pyruvate, acetyl-CoA, and hexitols—showed altered patterns, reflecting changes in microbial energy metabolism and fermentation capacity [
32,
37,
41]. Additionally, shifts in fatty acid β-oxidation, amino acid metabolism (particularly branched-chain and aromatic amino acids), and folate transformation pathways were observed in children with MASLD, indicating broader disruptions in microbial metabolic functions [
33,
37,
41]. Although a growing body of evidence has highlighted the association between gut dysbiosis and the progression of pediatric MASLD, future studies are needed to elucidate the causal roles of specific microbial taxa, as well as the contributions of fungi and viruses.
MICROBIAL PRODUCTS AND PEDIATRIC MASLD
Short-chain fatty acids
SCFAs, the primary end products of microbial fermentation of nondigestible proteins and fibers, serve critical roles in maintaining gut barrier integrity, providing energy sources for enterocytes and colonocytes, and regulating immune systems. Acetate, butyrate, and propionate are the predominant SCFAs in the gut, comprising over 95% of the total [
47]. Previous studies suggest that the most abundant SCFA-producing bacteria belong to the members of the
Lachnospiraceae and
Ruminococcaceae families. Notably,
Eubacterium rectale, Faecalibacterium prausnitzii, certain
Roseburia species, and
Anaerostipes have been well-characterized as butyrate producers [
48]. By activating the G-protein-coupled receptors (GPCRs) GPR41 and GPR43, which are expressed in various tissues (colon, adipose tissue, skeletal muscle, and liver), SCFAs provide key precursors for lipogenesis and gluconeogenesis, potentially contributing to the pathogenesis of MASLD [
49].
In fecal samples from pediatric populations, children with MASLD exhibited significantly lower levels of formate, acetate, and valerate relative to controls, while levels of butyrate and propionate remain unchanged [
29]. In contrast, Du et al. [
42] analyzed SCFA levels in fecal samples from healthy controls and obese children with and without MASLD, finding no significant differences in the overall proportions of SCFAs or in the levels of individual SCFAs across the groups. Given that both studies only compared MASLD patients with controls, whether SCFA levels differ between metabolic dysfunction-associated steatotic liver (MASL) and MASH needs further investigation. Future studies with larger sample size and assessments of circulating SCFAs are needed to elucidate their role in the pathogenesis of pediatric MASLD and to confirm previously reported inconsistencies.
Bile acids play critical roles in emulsifying dietary lipids and regulating host lipid and glucose metabolism by binding to host nuclear receptors and GPCRs. Primary bile acids are synthesized by hepatocytes from cholesterol through a series of reactions catalyzed by cytochrome P450 enzymes in the liver [
50]. Microbial deconjugation is carried out by bacteria with bile salt hydrolase (BSH) activity that prevents reuptake of bile acids from the terminal ileum via the apical sodium dependent bile acid transporter. In the colon, deconjugated primary bile acids that escape reuptake are metabolized into secondary bile acids through hydroxylation, epimerization, esterification, and desulfation [
51]. Bile acid synthesis and transport can be regulated by a negative feedback mechanism mediated by the farnesoid X receptor (FXR), which is mainly expressed in the liver and intestine. In the liver, FXR activation occurs in response to increased levels of intracellular bile acids, resulting in suppression of bile acid synthesis, enhancement of bile acid transport, and reduction in bile acid uptake. In contrast, intestinal FXR activation promotes bile acid efflux into the blood while inhibiting bile acid synthesis via secretion of fibroblast growth factor (FGF)19 (or FGF15 in mice) in the terminal ileum [
52]. As the gut microbiota participates in secondary bile acid synthesis, bile acids, in turn, can modulate the composition of the gut microbiota.
In children, patients with non-fibrotic MASLD had lower serum levels of total bile acids compared with healthy controls, primarily due to a reduction in glycine-conjugated bile acids. In contrast, although serum bile acid levels were elevated in fibrotic MASLD patients relative to those with non-fibrotic MASLD, they remained lower than the levels observed in the control group. Notably, serum levels of FGF19 were reduced in patients with MASLD and were positively correlated with ursodeoxycholic acid levels [
53]. Furthermore, children with MASH exhibited lower hepatic FXR expression and serum FGF19 levels than those with MASL and healthy controls, emphasizing the role of the FXR–FGF19 axis in disease progression [
54]. Building on this, Yu et al. [
55] integrated metagenomics and metabolomics analyses using fecal samples and demonstrated a reduction in secondary bile acids, along with alterations in gut microbiota with BSH activity. Additionally, fecal levels of lithocholic acid were positively correlated with the abundance of
Eubacterium and
Ruminococcaceae bacteria, while negatively correlated with the abundance of
E. coli. Furthermore, feeding 15-day-old piglets a high-fructose, HFD for 10 weeks led to reduced FGF19 expression in the distal ileum and decreased FGF19 levels in the serum compared with controls. Taken together, these studies emphasize the role of bile acids and FXR-FGF19 signaling in the pathogenesis of pediatric MASLD, suggesting that targeting bile acid metabolism may offer potential therapeutic benefits.
Ethanol is naturally produced as a metabolic byproduct of fermentation in certain bacteria and fungi. It can be metabolized by enzymes in the intestinal epithelial cells or transported into the liver via the portal vein [
56]. Previous studies showed that the serum levels of ethanol were higher in adults with MASH, especially in the portal vein. Transplantation of fecal microbiota from MASH patients with ethanol-producing
Klebsiella pneumoniae into mice recapitulated MASH phenotypes, which were ameliorated following bacteriophage therapy [
57]. In children with MASH, the blood ethanol levels were significantly elevated accompanied by an increased abundance of
Escherichia, highlighting the association between endogenous ethanol production and the pathogenesis of steatohepatitis. [
28] Consistently, Michail et al. [
29] reported elevated fecal ethanol levels in children with MASLD compared with obese children and healthy controls, and there was no difference between the obese and healthy groups. In contrast, Zhu et al. [
28] found no significant difference in fecal ethanol levels between children with MASLD and healthy controls. Future studies are needed to elucidate the role of ethanol-producing microbes in the pathogenesis of pediatric MASLD, particularly given the observed elevation of serum ethanol levels in affected children. Targeting these microbes with bacteriophages may offer novel therapeutic strategies; for example, phage therapy specifically against high alcohol-producing
K. pneumoniae has been shown to alleviate steatohepatitis in HiAlc Kpn-caused MASH mouse model [
57]. These findings suggest a potential translational approach that could be explored in pediatric settings.
MANAGEMENT OF PEDIATRIC MASLD
There is no FDA-approved pharmacotherapy for pediatric MASLD, so lifestyle modifications, including diet, physical activity, sleep quality, and mental health, remain the mainstay of disease management [
58]. Additionally, the effectiveness of bariatric surgery and pharmacological treatments has been evaluated by various studies, yet their outcomes remain inconsistent. In this section, we review clinical studies on diet modifications, pharmacological treatment options, and microbiome-based therapies conducted in children with MASLD.
Although obesity is an important risk factor for the development of MASLD, increased energy storage has been considered an adaptive response for the body in situations of food scarcity [
59]. Several studies have evaluated the effects of dietary interventions and nutrient modifications in children with MASLD. Akbulut et al. [
60] compared the effects of a Mediterranean diet and a low-fat diet on improving hepatic steatosis and insulin resistance in children with MASLD. After a 12-week trial, they found that both diets reduced liver fat content with no significant difference between the two approaches, while the Mediterranean diet was more effective in improving insulin resistance. Beyond overall dietary patterns, specific macronutrients also play crucial roles in pediatric MASLD pathogenesis. Recent data suggest that consumption of high dietary sugar is associated with the development of obesity, T2DM, MASLD, and cardiovascular disease [
61]. Since fructose is exclusively metabolized in the liver, its overconsumption can increase de novo lipogenesis (DNL), which further promotes hepatic steatosis. Clinical evidence supports the metabolic benefits of reducing sugar intake: a clinical study showed that short-term fructose restriction for 9 days decreased liver fat, visceral adipose tissue, and DNL, while improving insulin kinetics in children with obesity [
62]. Moreover, an 8-week randomized clinical trial demonstrated that a low sugar diet significantly improved hepatic steatosis and reduced levels of ALT in adolescent boys [
63]. Beyond inducing hepatic steatosis by promoting DNL independent of body weight changes, fructose may also impair intestinal barrier function, resulting in endotoxemia and subsequent hepatic dysfunction [
64,
65]. In healthy adults, high-fructose syrup decreased the abundance of Firmicutes while increasing Bacteroidetes. The increase in Bacteroidetes was positively correlated with plasma low-density lipoprotein cholesterol levels, whereas Firmicutes showed a negative correlation. This highlights the link between high-fructose intake and dysregulation of host lipid metabolism [
66]. Emerging evidence suggests that dietary components other than fructose, such as dairy fat, may also modulate MASLD progression. A clinical study in children at risk for MASLD demonstrated an inverse correlation between dairy fat consumption and liver fat content measured by MRI-PDFF. Furthermore, plasma levels of odd chain fatty acids (C15:0) and monomethyl branched chain fatty acids (iso-C17:0) were inversely correlated with hepatic steatosis [
67]. Therefore, current evidence demonstrates that dietary interventions may modulate pediatric MASLD progression through multifaceted mechanisms involving metabolism, gut barrier function, and microbiota. Future research should focus on defining optimal nutrient combinations and developing personalized nutritional strategies for clinical management.
Clinical investigations of pharmacological interventions for pediatric MASLD have primarily focused on four therapeutic approaches: insulin sensitizers, antioxidants, polyunsaturated fatty acids, and microbiome-based therapies [
68]. Among insulin-sensitizing agents, metformin remains the only one that has been systematically evaluated in the pediatric populations. The TONIC study, conducted by the MASH Clinical Research Network, investigated the therapeutic effects of vitamin E (400 IU twice daily) and metformin (500 mg twice daily) in pediatric patients with biopsy-proven MASLD and ALT levels exceeding 60 U/L. The study showed that neither vitamin E nor metformin exhibited superiority over placebo in achieving sustained ALT reduction over 96 weeks. Although metformin significantly improved hepatocyte ballooning compared with placebo, it had no significant effects on steatosis, inflammation, or fibrosis by histology. In contrast, vitamin E treatment significantly reduced MAS and showed significantly higher MASH resolution rates versus placebo [
69]. Cysteamine bitartrate, originally developed for the treatment of cystinosis, has been investigated as a potential therapeutic agent for pediatric MASLD. In a pilot study of 13 children with biopsy-proven MASLD and elevated ALT (≥60 U/L), 24-week cysteamine bitartrate treatment significantly reduced both ALT and aspartate aminotransferase (AST) levels, along with increased adiponectin concentrations [
70]. In the multicenter, double-blind CyNCh trial, 169 children with MAS ≥4 were randomly assigned to receive cysteamine bitartrate or placebo for 52 weeks. Although the primary outcome-defined as a ≥2-point improvement in MAS without fibrosis progression- did not differ between the two treatment groups, the cysteamine bitartrate group showed reductions in ALT and AST levels, as well as in lobular inflammation. Interestingly, a four-fold increase in histological improvement was observed among patients with lower body weight (≤65 kg), suggesting a potential weight-dependent therapeutic efficacy. Moreover, children with borderline zone 1 MASH pattern had 4 times the odds of having histologic improvement with cysteamine bitartrate compared with placebo, while this therapeutic effect was not observed in children with borderline zone 3 MASH, emphasizing the importance of considering the histological subtypes in clinical studies [
71].
More recently, the effects of
N-acetyl cysteine (NAC) were evaluated in 13 children with MAS>2. After 16 weeks of treatment, NAC improved inflammation markers, oxidative stress, and insulin resistance. Additionally, NAC treatment led to significant decreases in liver enzymes levels, liver fat fraction, and ultrasound-assessed liver stiffness [
72]. Given that findings from adult MASLD patients may not be directly extrapolated to pediatric populations, future clinical trials on pharmacological interventions should focus on larger, well-characterized pediatric cohorts to establish evidence-based therapeutic strategies.
Current clinical trials evaluating probiotic (live bacteria) interventions in children with MASLD have predominantly employed combinations of
Lactobacillus and
Bifidobacterium strains. One commonly studied probiotic is VSL#3, which comprises eight specific bacterial strains:
Lactobacillus casei subsp. paracasei, Lactobacillus plantarum, Lactobacillus acidophilus, and
Lactobacillus delbrueckii subsp. bulgaricus; Bifidobacterium longum, Bifidobacterium infantis, and
Bifidobacterium breve; and
Streptococcus thermophilus [
73]. As of the publication date, 4 clinical trials have explored the therapeutic potential of probiotics in children with MASLD. In 2011, a pilot study including children with MASLD (n=20) demonstrated that 8-week supplementation with
Lactobacillus rhamnosus GG (12 billion CFU/day) significantly reduced ALT levels independent of BMI changes [
74]. In a randomized triple-blind trial, 64 obese children with ultrasound-diagnosed MASLD received probiotic capsules (containing
L. acidophilus ATCC B3208,
B. lactis DSMZ 32269,
B. bifidum ATCC SD6576, and
L. rhamnosus DSMZ 21690) or placebo for 12 weeks. At the end of the trial, both ALT and AST levels decreased, and normal liver sonography was reported in 53.1% patients in the intervention group versus 16.5% in the placebo group [
75]. In obese children with biopsy-confirmed MASLD, a 48-week VSL#3 intervention led to significant reductions in both MASLD severity and BMI, although no statistically significant differences in ALT levels were observed compared with the placebo group [
76]. Importantly, combining VSL#3 with lifestyle modification led to greater improvements in fatty liver grade, anthropometric measures, and metabolic parameters in obese children than either intervention alone, suggesting this dual approach may offer an effective treatment strategy [
77].
Various studies in obese children showed that specific probiotic strains, including
L. rhamnosus bv-77,
L. salivarius, and
B. animalis, significantly reduced BMI and improved the blood lipid content. In contrast, other strains such as
B. bifidum,
B. longum, and
L. acidophilus showed no effects on either lipid metabolism or glucose homeostasis [
78]. Given that weight loss remains the cornerstone therapeutic strategy for pediatric MASLD, clinical evidence from probiotic studies in obese children may provide valuable mechanistic insights for disease management.
CONCLUSIONS, CHALLENGES, AND FUTURE PERSPECTIVES
Pediatric MASLD, as the leading cause of chronic liver disease in children, presents a growing public health burden and is associated with significant premature mortality along with increased risks of both hepatic and extrahepatic comorbidities. Although gut dysbiosis is considered to participate in the onset and progression of MASLD in both adults and children, the unique physiological and histological features in children should be considered when applying microbiome-based interventions.
Despite growing research on pediatric MASLD, critical knowledge gaps persist regarding disease mechanisms, along with ongoing challenges in diagnostic precision and treatment strategies. One of the major challenges in studying pediatric MASLD is the lack of well-validated animal models. Although several studies have attempted to establish mouse models of pediatric MASLD through HFD feeding or chemical induction in juvenile mice, these models often fail to fully recapitulate the metabolic and microbial characteristics observed in humans [
79-
81]. In addition to diet-induced models, humanized mice, generated through fecal microbiota transplantation into germ-free mice, have also been utilized to study gut microbiota-disease interactions. However, the colonization potential of human microbial taxa in mice requires careful evaluation, given the substantial differences in microbial communities between the two species [
82].
Current research has primarily focused on bacterial communities, while the composition and functional roles of nonbacterial microorganisms remain largely unexplored. Future studies should elucidate the potential contributions of non-bacterial microorganisms, including their independent effects and cross-kingdom interactions, in pediatric MASLD pathogenesis. Moreover, no studies have systematically compared the gut microbial profiles between histologic subtypes (type 1 vs. type 2) in pediatric MASLD, future investigations should address this gap, as identifying subtype-specific microbial signatures could enable non-invasive diagnostic stratification and personalized therapeutic approaches. In addition, nearly all microbiome-associated studies in pediatric MASLD have been conducted in obese children. The gut microbial characteristics of non-obese pediatric MASLD remain poorly understood and warrant dedicated investigation. Given the strong heritable component of pediatric MASLD, identifying genetic variants through genetic predisposition screening may aid in early diagnosis and personalized management. Considering the significant influence of maternal programming on the gut microbiome of infants, investigating how maternal risk factors contribute to pediatric MASLD pathogenesis via gut microbiome modulation may offer novel insights into disease mechanisms. Finally, the potential influence of age and sex on gut microbiota composition and MASLD progression in children remains largely uncharacterized. Future research should systematically evaluate age- and sexrelated microbial variations to uncover potential mechanisms and guide individualized prevention and treatment strategies for pediatric MASLD.
FOOTNOTES
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Authors’ contributions
Lu Jiang drafted the manuscript. Lan-Duoduo Du prepared the figures and table. Jing Zeng, Hui-Kuan Chu, and Zhong Peng revised the manuscript. Jian-Gao Fan wrote and revised the manuscript.
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Acknowledgements
This study was supported by National Key R&D Program of China (2022YFA1305600), Noncommunicable Chronic Diseases-National Science and Technology Major Project (2023ZD0508700), National Natural Science Foundation of China (82100950, 82470602, 82470600, 82170593), and Natural Science Foundation of Shanghai (23ZR1452600). The updated MASLD terminology has been applied retrospectively to earlier NAFLD studies given the disease concordance. Figures were created with Biorender.com.
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Conflicts of Interest
The authors have no conflicts to disclose.
Figure 1.Schematic representation of risk factors in the pathogenesis of pediatric MASLD. Prenatal factors (maternal obesity, gestational diabetes mellitus, medicine, and diet) and postnatal factors (formula feeding and abnormal birth anthropometrics) may indirectly affect adipogenesis and metabolic programming in infants, thereby increasing susceptibility to the development and progression of MASLD. DNL, de novo lipogenesis; ER, endoplasmic reticulum; FFA, free fatty acids; MASH, metabolic dysfunction-associated steatohepatitis; MASLD, metabolic dysfunction–associated steatotic liver disease; ROS, reactive oxygen species.
Figure 2.Dysregulated interactions between gut microbiome and liver in pediatric MASLD. Genetic predisposition works in concert with epigenetic and environmental factors (diet and sedentary lifestyle) to promote gut dysbiosis and gut barrier dysfunction. When the gut barrier is compromised, microorganisms, PAMPs, and microbial metabolites can reach the liver via the portal vein. In the liver, Kupffer cells are activated, triggering a cascade of proinflammatory responses that lead to steatosis, cell death, inflammation, and fibrosis. Alternatively, direct damage to hepatocytes may also occur. HSCs, hepatic stellate cells; KCs, Kupffer cells; MASLD, metabolic dysfunction– associated steatotic liver disease; PAMPs, pathogen-associated molecular patterns.
Figure 3.Comparison of gut microbiota changes between children and adults associated with MASLD progression. Alterations in gut bacteria at the genus and species levels are compared across different conditions during MASLD progression. All pediatric MASLD cases included in this figure are obese children. In children, comparisons are made between MASL and HCs, MASH and HCs, and between MASH and MASL. In adults, comparisons of MASL vs. HCs, MASH vs. MASL, and advanced (stage 3–4) vs. moderate fibrosis (stage 0–2) are conducted within obese MASLD populations. Gut microbial changes in non-obese MASLD individuals with different fibrosis severities (stage 0–3) are also included for broader comparison. HCs, healthy controls; MASH, metabolic dysfunction-associated steatohepatitis; MASL, metabolic dysfunction-associated steatotic liver; MASLD, metabolic dysfunction–associated steatotic liver disease.
Table 1.Study cohorts of gut microbiota in pediatric MASLD research
Table 1.
|
Study subject |
Age (yr) |
Diagnosis of fatty liver |
Sequencing method |
Major finding |
|
Healthy controls (n=16); obesity (n=25); MASH (n=22) |
Mean: control=14.4; obesity=12.7; MASH=13.6 |
Liver biopsy |
16S rRNA sequencing |
↑Proteobacteria, Enterobacteriaceae, and Escherichia (MASH vs. obesity) [28] |
|
Healthy controls (n=26); obesity (n=11); MASLD (n=13) |
Mean: control=13.3; obesity=13.2; MASLD=13.6 |
Ultrasonography and elevated transaminases |
16S rRNA sequencing |
↑Prevotella (MASLD vs. healthy controls/obesity) [29] |
|
Healthy controls (n=54); obesity (n=8); MASL (n=27), MASH (n=26) |
7–16 |
Ultrasonography; liver biopsy |
16S rRNA sequencing |
↓Oscillospira (MASLD vs. healthy controls); ↑Ruminococcus, Dorea, and Streptococcus (MASH vs. healthy controls) [30] |
|
Control (n=99); MASLD (n=8) |
12–19 |
MRI-PDFF |
16S rRNA sequencing |
Bilophila, Paraprevotella, dietary intake of MUFAs, and BMI z-scores explained 32.0% of the variation in HFF [31] |
|
Overweight/obesity (n=37); MASLD (n=87) |
8–17 |
Liver biopsy |
16S rRNA sequencing; metagenomic sequencing |
↑ Prevotella copri abundance was associated lower α-diveristy and more severe fibrosis [32] |
|
Healthy controls (n=15); obesity (n=18); MASLD (n=25) |
9–17 |
MRI-PDFF |
Metagenomic sequencing |
↓Bacteroidetes (Alistipes) (MASLD vs. healthy controls), ↓Faecalibacterium prausnitzii (MASLD vs. obesity) [33] |
|
Obesity (n=29); MASLD (n=44) |
Mean: obesity=12.9 MASLD=13.3 |
MRI-PDFF |
16S rRNA sequencing |
↑Firmicutes to Bacteroidetes ratio; ↓Bacteroidetes, Prevotella, Gemmiger, and Oscillospira (MASLD vs. obesity) [34] |
|
Obesity (n=25); MASL (n=25), MASH (n=25) |
7–16 |
Ultrasonography |
16S rRNA sequencing |
↑Harmful bacteria (MASL vs. obesity); |
|
↓Anti-inflammatory/probiotics (MASL vs. obesity), butyrate-producing bacteria (MASH vs. obesity) [35] |
|
Healthy controls (n=63); MASLD (n=63) |
6–7 |
Ultrasonography |
16S rRNA sequencing; metagenomic sequencing |
↑Actinobacteria; ↓Bacteroidetes, Verrucomicrobia, and Akkermansia (MASLD vs. healthy controls) [36] |
|
Obesity (n=18); MASLD (n=18) |
Mean: obesity=12.22 MASLD=12.54 |
MRI-PDFF |
Metagenome sequencing |
↑Fusicatenibacter saccharivorans, Romboutsia ilealis, and Actinomyces sp. ICM47; ↓Bacteroides thetaiotamicro (MASLD vs. obesity) [37] |
|
Healthy controls (n=15); obesity (n=16); MASLD (n=27) |
10–17 |
MRI-PDFF |
Metagenomic sequencing |
↑Megamonas (M. hypermegale and M. rupellensis) (MASLD vs. healthy controls/obesity) [38] |
|
Obesity (n=78); MASLD (n=78) |
6–18 |
Ultrasonography |
16S rRNA sequencing |
↑Enterococcus (MASLD vs. obesity); E. faecium B6 promotes MASLD in mice [39] |
|
Obesity (n=53); MASL (n=53), MASH (n=39) |
7–16 |
Ultrasonography |
16S rRNA sequencing |
↑Faecalitalea, Negativibacillus, ↓Helicobacter, Morganella, Odoribacter (MASL vs. obesity); |
|
↓Butyricicoccus, Haemophilus, Negativibacillus, Odoribacter, Roseburia, Sellimonas (MASH vs. MASL) [40] |
|
Healthy controls (n=35); MASLD (n=79): MASL (n=5), MASH (n=8), Unclassified (n=66) |
7–18 |
Ultrasonography; liver biopsy (n=13) |
16S rRNA sequencing; metagenomic sequencing |
Lachnoclostridium, Escherichia-Shigella, and F. prausnitzii were positively correlated with MASLD indices [41] |
|
Healthy controls (n=14); obesity (n=12); MASLD (n=10) |
6–16 |
Ultrasonography |
16S rRNA sequencing |
Anaerostipes and A. hadrus were identified as biomarkers (obesity vs. MASLD); ↑Ruminococcus torques in MASLD with hepatic E values≥6.2 kPa [42] |
Abbreviations
aspartate aminotransferase
U.S. Food and Drug Administration
G-protein-coupled receptors
metabolic dysfunction-associated steatohepatitis
metabolic dysfunction–associated steatotic liver disease activity score
metabolic dysfunction-associated steatotic liver
metabolic dysfunction–associated steatotic liver disease
magnetic resonance imaging proton density fat fraction
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