Exploring the interplay between metabolic dysfunction-associated fatty liver disease and gut dysbiosis: Pathophysiology, clinical implications, and emerging therapies

Abstract

Metabolic dysfunction-associated fatty liver disease (MAFLD) now affects roughly one-quarter of the world’s population, reflecting the global spread of obesity and insulin resistance. Reframing non-alcoholic fatty liver disease as MAFLD emphasizes its metabolic roots and spotlights the gut-liver axis, where intestinal dysbiosis acts as a key driver of hepatic injury. Altered microbial communities disrupt epithelial integrity, promote bacterial translocation, and trigger endotoxin-mediated inflammation that accelerates steatosis, lipotoxicity, and fibrogenesis. Concurrent shifts in bile acid signaling and short-chain fatty acid profiles further impair glucose and lipid homeostasis, amplifying cardiometabolic risk. Epidemiological studies reveal pervasive dysbiosis in MAFLD cohorts, linked to diet quality, sedentary behavior, adiposity, and host genetics. Newly developed microbiome-derived biomarkers, advanced elastography, and integrated multi-omics panels hold promise for non-invasive diagnosis and stratification, although external validation remains limited. In early trials, interventions that re-engineer the microbiota including tailored pre-/pro-/synbiotics, rational diet patterns, next-generation fecal microbiota transplantation, and bile-acid-modulating drugs show encouraging histological and metabolic gains. Optimal care will likely couple these tools with weight-centered lifestyle programmes in a precision-medicine framework. Key challenges include inter-ethnic variability in microbiome signatures, the absence of consensus treatment algorithms, and regulatory barriers to live biotherapeutics. Rigorous longitudinal studies are required to translate mechanistic insight into durable clinical benefit and improve patient-centered outcome measures.

Key Words: Metabolic dysfunction-associated fatty liver disease; Gut dysbiosis; Gut-liver axis; Fecal microbiota transplantation; Microbiome-based diagnostics; Bile acid metabolism; Precision medicine

Core Tip: The pathogenesis of metabolic dysfunction-associated fatty liver disease (MAFLD) is profoundly influenced by gut dysbiosis, a condition that exacerbates disease progression through mechanisms such as compromised intestinal barrier function, endotoxemia, and dysregulated bile acid metabolism. Understanding and addressing this issue is of utmost importance in the management of MAFLD. Advances in microbiome-based diagnostics, including machine learning models and microbial biomarkers, now provide non-invasive tools for early detection and risk stratification, enhancing clinical decision-making. The emergence of therapies targeting the gut-liver axis, such as probiotics, fecal microbiota transplantation, and farnesoid X receptor agonists, demonstrates significant potential in mitigating hepatic inflammation and restoring metabolic homeostasis.

INTRODUCTION

Definitions and nomenclature: Transition from non-alcoholic fatty liver disease to metabolic dysfunction-associated fatty liver disease/metabolic dysfunction-associated steatotic liver disease terminology

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), represents a spectrum of liver disorders ranging from simple steatosis to steatohepatitis, fibrosis, and cirrhosis[1,2]. This liver derangement is strongly associated with metabolic dysfunctions, including obesity, insulin resistance, and type 2 diabetes mellitus (T2DM)[1,3]. The transition from the term NAFLD to MAFLD reflects a fundamental shift in diagnostic criteria, moving away from an exclusion-based definition toward one that acknowledges the metabolic underpinnings of hepatic steatosis[4,5]. This shift has significant implications for the understanding and management of metabolic liver disease, as it enables a more comprehensive and accurate diagnosis that considers the systemic nature of the condition. Unlike NAFLD, which requires the exclusion of significant alcohol consumption, MAFLD is diagnosed based on the presence of hepatic steatosis alongside metabolic risk factors such as obesity, T2DM, or specific metabolic abnormalities[5]. This reclassification underscores the systemic nature of metabolic liver disease, a significant health concern irrespective of alcohol consumption.

The adoption of MAFLD terminology has improved the standardization of diagnostic criteria and aided in identifying at-risk individuals. By shifting from a diagnosis of exclusion to one of inclusion, MAFLD offers a more clinically relevant framework that aligns with the growing body of evidence linking liver disease to systemic metabolic disturbances, including insulin resistance, dyslipidemia, and chronic inflammation. More recently, a further revision has been proposed, renaming MAFLD to metabolic dysfunction-associated steatotic liver disease (MASLD) to ensure consistency with global hepatology guidelines and further refine its definition. Despite this change, the core pathogenic processes under investigation, such as insulin resistance, dyslipidemia, and chronic inflammation, largely remain consistent across both terminologies. Therefore, this review will maintain its focus on MAFLD, aiming to elucidate the fundamental mechanisms driving fatty liver disease in the context of metabolic dysfunction.

Significance of the gut-liver axis in metabolic diseases

The gut-liver axis represents a bidirectional communication network between the gastrointestinal tract and the liver, mediated by complex interactions involving gut microbiota, the immune system, bile acids (BAs), and metabolic pathways[6]. The gut-liver axis, a vital bidirectional communication network, plays a pivotal role in maintaining homeostasis by regulating nutrient absorption, immune responses, and inflammation. Disruptions in this communication, particularly in the form of gut dysbiosis, have increasingly been implicated in the pathogenesis of MAFLD, underscoring the urgency of addressing disruptions in this crucial communication network[7,8].

Gut dysbiosis, characterized by an imbalance in gut microbiota composition and function, significantly contributes to the progression of MAFLD through multiple mechanisms, including increased intestinal permeability, endotoxemia, systemic inflammation, and alterations in BA metabolism. This imbalance is a key player in the disease’s progression[9]. Disruption of the intestinal barrier allows microbial metabolites and bacterial endotoxins, such as lipopolysaccharides, to enter the portal circulation, triggering hepatic inflammation, oxidative stress, and fibrosis, which further exacerbates liver damage[10].

Furthermore, gut microbiota influences hepatic lipid metabolism by modulating the production of short-chain fatty acids (SCFAs) and BAs, both of which play key roles in energy balance and insulin sensitivity[11]. Individuals with MAFLD often exhibit alterations in their gut microbiome characterized by increased pro-inflammatory bacteria and reduced beneficial microbial species. These findings underscore the importance of the gut-liver axis as a critical driver of disease pathogenesis and a potential target for therapeutic intervention. The potential of gut microbiota as a therapeutic target offers hope for the development of novel treatments for MAFLD[12].

Objective of the review

This review aims to comprehensively analyze the interplay between gut dysbiosis and MAFLD, emphasizing the underlying pathophysiological mechanisms and therapeutic implications. Given the increasing recognition of the gut-liver axis as a key determinant of metabolic liver disease, this review synthesizes current evidence on the role of gut microbiota in MAFLD pathogenesis. We highlight potential diagnostic and therapeutic strategies targeting the gut-liver axis by examining the molecular pathways linking dysbiosis to liver dysfunction.

Additionally, this review aims to bridge the gap between experimental research and clinical practice by evaluating emerging therapies, including microbiome-based interventions, pharmacological agents, and dietary modifications. As microbiome research continues to evolve, understanding its clinical relevance in MAFLD is essential for advancing precision medicine and improving patient outcomes. By identifying existing research gaps and future directions, this review seeks to guide further investigations into the role of gut microbiota in metabolic liver disease. Ultimately, this synthesis of knowledge will enhance the development of personalized treatment approaches tailored to the gut microbial composition of individual patients.

Literature search and selection

A systematic literature search was conducted in the PubMed and Embase databases from their inception through March and April 2025. Search terms included “MAFLD”, “metabolic dysfunctionassociated fatty liver disease”, “gut microbiota”, “gut dysbiosis” and related synonyms. The initial search yielded approximately 1200 records, which were imported into a reference manager for duplicate removal. Two independent reviewers screened titles and abstracts against predefined inclusion criteria: (1) Studies involving human or animal models of MAFLD that assessed gut microbiota composition or function; (2) Original research articles, clinical trials, or systematic reviews published in English; and (3) Availability of full text. After screening, 250 full-text articles were assessed for eligibility, with 180 studies included in the final synthesis. Discrepancies in study selection were resolved by consensus. Key data extracted from each study included population characteristics, microbial taxa or metabolites assessed, analytical methods, and main findings. This transparent and reproducible approach ensured comprehensive coverage of relevant literature and minimized selection bias.

PATHOPHYSIOLOGY OF MAFLD AND GUT DYSBIOSIS

Gut-liver axis and dysbiosis

The human gastrointestinal tract is a dynamic and diverse microbial ecosystem, housing hundreds of millions of microorganisms from over 500 different genera and species. Approximately 90% of these microorganisms belong to the phyla Firmicutes and Bacteroidetes[7]. Dysbiosis refers to the pathological shift from a normal microbiome (eubiosis) to a pathogenic microbiome. The gut metabolome, shaped by interactions among nutrients, the microbiome, host genetics, and environmental factors (e.g., pollutants, heavy metals, antibiotics), can change in response to dysbiosis.

Dysbiosis compromises gut barrier integrity through various mechanisms, including disruption of the mucus layer, alteration of SCFA production (a vital nutrient source for intestinal cells), and the induction of intestinal inflammation. This inflammation is initiated by the recognition of microbial components by Toll-like receptors (TLRs), which activate the immune system[6,13-15]. The liver’s unique position, processing all blood from the digestive system, makes it highly susceptible to influences from the gut metabolome. Consequently, a compromised gut barrier increases liver exposure to gut pathogens, gut-derived factors, and bacterial antigens, exacerbating inflammation, oxidative stress, and lipid accumulation, ultimately leading to liver damage and fibrosis, Figure 1[10].

Figure 1 Pathophysiological interactions between gut dysbiosis and metabolic dysfunction-associated fatty liver disease: A gut-liver axis model. This diagram illustrates the complex interplay between gut dysbiosis and the pathogenesis of metabolic dysfunction-associated fatty liver disease. Disruption of gut microbiota and gut barrier integrity leads to increased translocation of bacterial endotoxins, secondary bile acids, and microbial metabolites via the portal vein. These gut-derived factors activate hepatic immune and metabolic pathways, including Toll-like receptor signaling, farnesoid X receptor modulation, mitochondrial dysfunction, and insulin resistance, contributing to the progression from hepatic steatosis to steatohepatitis and eventually fibrosis or cirrhosis. Host genetic susceptibility (e.g., PNPLA3 variants) further modulates disease progression. LPS: Lipopolysaccharide; TLR: Toll-like receptor; FXR: Farnesoid X receptor; NF-κB: Nuclear factor kappa B; Th1: T helper type 1; Th17: T helper type 17; Treg: Regulatory T cells; mTORC1: Mammalian target of rapamycin complex 1; ROS: Reactive oxygen species; SCFA: Short-chain fatty acids; MAFLD: Metabolic-associated fatty liver disease; MASH: Metabolic-associated steatohepatitis.

Dysbiosis has been implicated in the development of various diseases, including MAFLD and metabolic dysfunction-associated steatohepatitis (MASH)[8]. While comprehensively identifying all gut-derived factors contributing to MAFLD would be a considerable task, two primary categories warrant attention: (1) Alterations in metabolic pathways; and (2) The initiation and perpetuation of inflammation. These pathways are influenced by microbiota composition, BAs, metabolites of nutrients such as choline, SCFAs, and even ethanol.

Although gut dysbiosis alone may not fully account for the extent and severity of hepatic injury observed in MASH, research indicates that an imbalance within the hepatic microbiome may result from the translocation of microorganisms from various gut microbiome populations, including those in the oral cavity, stomach, and small intestine[11,16,17]. This localized disruption of the hepatic microbiome has been implicated in the pathogenesis of both MAFLD and MASH[11,18].

Metabolic alterations in MAFLD

Dysbiosis affects metabolic homeostasis by modulating BA composition, amino acid metabolism, and SCFA production. Certain bacterial species increase energy extraction from the diet, promoting obesity and insulin resistance. Choline deficiency, a consequence of dysbiosis, impairs very low-density lipoprotein secretion, exacerbating hepatic triglyceride accumulation, which contributes to hepatic steatosis and chronic liver inflammation[19]. Conversely, some probiotic strains, such as Lactobacillus johnsonii, exhibit protective effects by reducing hepatic steatosis and improving insulin sensitivity[20].

Gut and liver microbial signatures in MAFLD

Studies have demonstrated an increased abundance of hepatic bacterial ribosomal DNA, particularly from the Proteobacteria phylum, in obese individuals with hepatic steatosis compared to lean controls[21]. Another study identified Bacteroides as associated with MASH and Ruminococcus as associated with significant fibrosis[22]. However, focusing solely on the phylum level (e.g., Firmicutes and Bacteroidetes) may oversimplify the gut microbiome’s complexity, as certain genera within these phyla can exert both beneficial and harmful effects[23].

In MAFLD, the increased abundance of Proteobacteria is primarily driven by Escherichia species within the Enterobacteriaceae family[24]. Different bacterial genera have been linked to varying severities of MAFLD. For example, one study reported an overabundance of Fusobacteria, Fusobacteriaceae, Fusobacterium, Prevotella, and Eubacterium biforme in patients with MASH, whereas Prevotellaceae was predominant in those with MAFLD[25]. Advanced MASH with cirrhosis was associated with higher loads of Streptococcus, Megasphaera, Gallibacterium, Faecalibacterium prausnitzii, Catenibacterium, Rikenellaceae, and Mogibacterium, while lower abundances of Peptostreptococcaceae, Bacillus, and Lactococcus were observed[26].

Certain gut microbes contribute to MAFLD through specific mechanisms. For instance, the Gram-negative bacteria Klebsiella pneumoniae A7 and Escherichia coli PY102 have been linked to MAFLD development via lipopolysaccharide-TLR activation and increased endogenous ethanol production[27-29]. Similarly, Enterobacter cloacae B29 has been implicated in MAFLD by promoting lipotoxicity through increased hepatic triglycerides and insulin resistance in animal models[30].

Given the heterogeneity of available data, the relationship between the gut microbiome and MAFLD remains uncharted, mainly requiring further exploration. Recently, machine learning approaches have emerged as promising tools for deciphering these complex interactions. A novel machine learning classifier incorporating ten microbial genera demonstrated high predictive accuracy for insulin resistance, achieving an area under the receiver operating characteristic curve of 0.93, offering a valuable non-invasive method for identifying MAFLD in insulin-resistant patients[31]. A recent meta-analysis further underscored the potential of machine learning in gut microbiome data analysis, predicting liver cirrhosis or fibrosis onset with a pooled sensitivity of 0.81 and specificity of 0.85[32].

The potential impact of the gut microbiome on the pathophysiology of MAFLD in patients with a normal body mass index, also commonly referred to as “lean MAFLD” is gaining recognition. For instance, research indicates that individuals with lean MAFLD (n = 5) may have a gut microbiota composition that differs from those with obesity and MAFLD (n = 24), showing an increase in microorganisms linked to the development of hepatic steatosis (such as Erysipelotrichaceae and Clostridiales). The analysis of microbiota reveals a clear distinction in profiles between healthy individuals of normal weight and those with lean MAFLD; within the lean MAFLD group, there is a higher prevalence of Dorea spp. and a decrease in the relative abundance of several species, including Marvinbryantia and the Christensellenaceae R7 group. Additionally, while lean MAFLD patients tend to have an initial metabolic adaptation, this adaptive response seems to diminish as the disease progresses. Recent work has identified that this loss is driven by endotoxemia, leading to epigenetic reprogramming, which blocks the BA signaling that mediates this metabolic adaptation[33].

Impact of host genetics on MAFLD development and progression

Pirola et al[34] demonstrated that host genetics play a crucial role in shaping the liver microbiome. They predicted a portion of the liver microbiome’s alpha diversity variability using a genetic risk score. Notably, specific risk alleles were linked to increased Tyzzerella abundance, which was associated with MASH and independently with hypertension. Additionally, MASH risk genes were correlated with metabolic pathways such as octane oxidation and fatty acid biosynthesis, with PNPLA3 variants particularly linked to alkane metabolism by Gammaproteobacteria. Specific genetic variations in PNPLA3 (rs738409 and rs58542926) were significantly associated with the presence of alkane-degrading bacteria, which metabolize dietary alkanes into fatty acids and alcohols. Individuals carrying these PNPLA3 variants exhibited a 3.28-fold increased risk of hepatic steatosis[35,36].

TM6SF2 is another variant strongly implicated in MAFLD (diverse impacts of the rs58542926 E167K variant in TM6SF2 on viral and metabolic liver disease phenotypes). A recent study showed that intestinal TM6SF2 protects against MASH by regulating the gut-liver axis[37]. Similarly, MBOAT7 rs641738 C > T is another variant associated with the risk of MAFLD and MASH that was recently demonstrated to be a novel regulator of the TLR inflammatory response, linking this variant to MASH[38].

Role of BAs metabolites in MAFLD

A reciprocal relationship exists between BAs and the gut microbiota, significantly influencing metabolic homeostasis. Hepatocytes synthesize primary BAs such as cholic acid and chenodeoxycholic acid (CDCA), which undergo microbial transformation in the colon into secondary BAs, including lithocholic acid and deoxycholic acid (DCA). This dynamic interplay involves bidirectional signaling, where BAs regulate gut microbiota through farnesoid X receptor (FXR) interactions, while gut microbiota manipulates the BA pool[39].

Dysbiosis can increase secondary BA levels, exacerbating intestinal permeability and promoting hepatic inflammation[40]. BAs exert a profound influence on various physiological processes, including gut barrier integrity. FXR activation by BAs stimulates the production of antimicrobial peptides, such as angiogenin-1 and RNase family members, which inhibit excessive bacterial growth and maintain mucosal integrity[39]. Different BAs exhibit varying effects on receptors; for instance, CDCA and DCA act as FXR agonists, whereas ursodeoxycholic acid inhibits FXR. The net effect on receptor signaling depends on the relative abundance of each BA within the pool[41].

FXR stimulation triggers key regulatory factors, including fibroblast growth factor 15/19 (FGF15/19), peroxisome proliferator-activated receptor gamma (PPARγ), glucose transporter 4, and glucagon-like peptide-1 (GLP-1), thereby enhancing insulin sensitivity[42-44]. Furthermore, FXR activation stimulates the Takeda G protein-coupled receptor 5 (TGR5) in brown adipose tissue and skeletal muscle, leading to increased local triiodothyronine (T3) production. This enhanced T3 production contributes to increased energy expenditure[23,45,46]. Conversely, impaired FXR signaling leads to decreased FGF19 production, contributing to hepatic steatosis[47,48].

Secondary BAs stimulate FGF19 expression, which enhances glucose uptake in adipocytes via the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway[49]. mTORC1 regulates several cellular processes, including protein synthesis and autophagy, and its overall impact on MAFLD depends on the balance of these processes[50].

This dynamic interplay influences the expression patterns of BA receptors, including FXR and TGR5, at various stages of MAFLD progression. Given their pivotal roles, FXR and TGR5 have emerged as promising therapeutic targets for managing obesity and MAFLD.

Choline and trimethylamine-N-oxide metabolism

Choline, an essential nutrient in hepatic lipid metabolism, is converted by gut microbes into trimethylamine (TMA). TMA is then absorbed and transported to the liver through the portal vein, where it is oxidized to trimethylamine-N-oxide (TMAO)[51-53].

Dysbiosis, which is characterized by a higher Firmicutes/Bacteroidetes ratio, contributes to increased levels of TMAO, which are associated with the severity of MAFLD[54,55]. TMAO promotes MAFLD progression through multiple mechanisms, including insulin resistance[56], aggravated hepatic steatosis via BA composition modulation and FXR inhibition[57], and systemic inflammation through cytokine release[58]. Additionally, TMAO promotes leukocyte accumulation in endothelial cells, leading to endothelial dysfunction and increased risk of atherosclerosis and cardiovascular diseases[59]. Gut dysbiosis can also increase bacterial consumption of choline, potentially leading to choline deficiency, which impairs very low-density lipoprotein production and promotes hepatic triglyceride accumulation and steatosis[59,60].

Endogenous ethanol production

Endogenous ethanol production results from the microbial metabolism of carbohydrate-rich diets. MAFLD patients exhibit enrichment of ethanol-producing bacteria, such as Escherichia, Prevotella, and Gammaproteobacteria, compared to healthy controls[61]. Moreover, elevated serum endogenous ethanol levels are associated with progression to MASH[62].

Endogenous ethanol disrupts mitochondrial function, increasing reactive oxygen species production and damaging mitochondrial DNA. Additionally, ethanol consumption activates the nuclear factor kappa B signaling pathway, leading to inflammatory cytokine upregulation. This inflammatory response, combined with compromised intestinal barrier integrity, increases intestinal permeability and endotoxemia[63,64].

The role of SCFAs in MAFLD

SCFAs, such as acetate, butyrate, and propionate, are produced by gut bacteria through dietary fiber fermentation. SCFAs exert diverse effects on liver health in MAFLD. They strengthen the gut barrier, enhance insulin sensitivity, and suppress inflammation by increasing hormones such as peptide YY and GLP-1 while inhibiting nuclear factor kappa B signaling[65]. However, excessive SCFA concentrations may be detrimental by inhibiting adenosine 5’-monophosphate-activated protein kinase activity in the liver, which promotes hepatic fatty acid accumulation by enhancing β-oxidation[66].

SCFAs also influence immune responses by promoting pro-inflammatory T cells (Th1 and Th17) while suppressing regulatory T cells (Tregs). This shift in immune balance, characterized by increased pro-inflammatory T cells and reduced Tregs, is a hallmark of progressive MAFLD[25].

Role of the mycobiome and virome

Recent research has expanded the gut-liver axis to include fungal and viral components. For instance, the overgrowth of fungi such as Candida and bacteria like Klebsiella pneumoniae has been linked to endogenous ethanol production, disrupting glucose and lipid metabolism, and contributing to liver steatosis and steatohepatitis[9].

In conclusion, the gut microbiome plays a pivotal role in MAFLD pathogenesis. Dysbiosis, characterized by altered microbial composition and function, contributes to MAFLD progression through increased intestinal permeability, disrupted BA metabolism, and immune dysregulation. Additionally, the interplay between host genetics, gut microbiota, and environmental factors significantly influences MAFLD development, underscoring the complex and multifaceted nature of this chronic liver disease.

EPIDEMIOLOGY AND RISK FACTORS

Prevalence of gut dysbiosis among MAFLD and MASLD patients

Gut dysbiosis is highly prevalent in patients with MAFLD and MASLD, contributing to disease progression. However, its exact prevalence in these populations remains uncertain. A clearer understanding of its prevalence may aid in distinguishing between these conditions as research progresses.

Although studies consistently highlight the critical role of gut microbiota dysbiosis in the development and progression of MAFLD, definitive global prevalence rates are lacking[67-70]. Instead, research emphasizes the association between altered gut microbial composition and these liver diseases. MAFLD, which affects nearly a quarter of the global population, is strongly linked to gut dysbiosis, with studies demonstrating its significant role in disease pathogenesis[67].

Certain bacterial genera, such as Blautia, exhibit altered abundance in MAFLD patients, reinforcing the connection between dysbiosis and the disease[70]. One study reported that 98.6% of Asians within the Prevotella enterotype were associated with MAFLD, suggesting a strong correlation in specific populations[69]. However, further epidemiological studies are needed to quantify the global prevalence of gut dysbiosis in MAFLD. Current research focuses on identifying microbial alterations and their qualitative associations rather than establishing concrete prevalence figures.

Shared risk factors between MAFLD and gut dysbiosis

Diet: Diet plays a crucial role in modulating MAFLD progression and influences gut microbiota composition, highlighting the need to explore the connections between gut dysbiosis and MAFLD.

High-fat diet: A high-fat diet (HFD) promotes liver steatosis, induces gut dysbiosis, and upregulates genes involved in lipid metabolism in the distal small intestine of mice. Lard-fed mice exhibit increased hepatic TLR4 activation, white adipose tissue inflammation, and reduced insulin sensitivity compared to those fed fish oil[71]. Additionally, HFD alters the balance of SCFAs in the gut, contributing to the development of MASH. Conversely, a high-fiber diet has been shown to reduce hepatic inflammation[72].

High sucrose and fructose diet: High sucrose diet[73] and high fructose diets are linked to gut dysbiosis and MAFLD. Sucrose-induced steatohepatitis is prevented in fructokinase knockout mice, highlighting fructose’s role in MAFLD pathogenesis[74]. Studies indicate that 26% of hepatic triglycerides originate from de novo lipogenesis driven by dietary sugars, particularly fructose[75-77]. Notably, fructose-induced lipogenesis may play a more significant role in MAFLD development than HFD[78]. Additionally, high dietary fructose intake is associated with small intestinal bacterial overgrowth (SIBO), which increases gut permeability[79-81] and promotes chronic low-grade endotoxemia[82]. Targeting gut dysbiosis to reduce liver inflammation and strengthen intestinal barrier function has shown therapeutic potential in rodent models, suggesting its viability as a treatment strategy for fructose-induced MAFLD in humans[83-85].

Coffee: Evidence suggests that coffee consumption may help prevent MAFLD[86,87], potentially by modulating gut microbiota[88]. In HFD-fed rats, coffee altered the gut microbiome, increasing beneficial SCFA butyrate levels while reducing liver steatosis[89]. However, the relative contributions of caffeine and non-caffeine components to these protective effects remain unclear.

Low aerobic fitness

Low aerobic fitness is associated with gut dysbiosis, which may contribute to an elevated risk of MAFLD[90]. Studies comparing low-capacity running (LCR) and high-capacity running rats found that LCR rats had a greater predisposition to hepatic steatosis following HFD consumption[91]. These rats exhibited a reduction in SCFA-producing bacteria, which correlated with increased energy intake and feeding efficiency. This suggests that low aerobic fitness may impair the protective effects of SCFA production, thereby increasing susceptibility to diet-induced MAFLD.

Periodontal disease

Periodontal disease, a chronic inflammatory condition caused by microbial dysbiosis in the oral biofilm, has been linked to gut dysbiosis[92,93]. Oral administration of periodontopathic bacteria in animal models induces gut dysbiosis, which negatively affects glycolipid metabolism and immune responses in the liver, ultimately contributing to hepatic steatosis[94-99]. Persistent gut dysbiosis resulting from periodontal disease has been associated with MAFLD progression[100]. Interestingly, studies suggest that nisin lantibiotics may prevent periodontal disease-induced MAFLD by restoring oral and gut microbial balance[101].

SIBO

SIBO affects approximately 35% of MAFLD patients, with a significantly higher prevalence compared to healthy controls[102]. Moreover, SIBO is more common in patients with MASH-associated cirrhosis (47.1%) than in those with MAFLD (15.7%)[103]. Intestinal dysbiosis, endotoxemia, and bacterial translocation associated with SIBO contribute to inflammation and insulin resistance[27,104,105], disrupting the gut-liver axis and influencing MAFLD progression[83].

Previous antibiotic use

Long-term antibiotic use can induce gut dysbiosis, potentially increasing the risk of MAFLD. Notably, prior exposure to antibiotics such as fluoroquinolones has been linked to an elevated risk of MAFLD[106]. This association appears to be more pronounced in individuals without metabolic syndrome, suggesting that antibiotic-induced gut dysbiosis may serve as an alternative pathway for MAFLD development in certain patients[106].

Polypharmacy

Polypharmacy, or the simultaneous use of multiple medications, is increasingly recognized as a factor contributing to gut dysbiosis in patients with MAFLD. The gut microbiome plays a crucial role in the development and progression of MAFLD, and different medications can disrupt microbial balance and function, exacerbating dysbiosis and liver damage. This analysis examines the effects of polypharmacy on gut dysbiosis in MAFLD, backed by relevant research and evidence.

Mechanisms linking polypharmacy to gut dysbiosis

Polypharmacy affects gut microbiota composition through various mechanisms, including direct microbial disruption, changes in intestinal permeability, and metabolic modifications.

Microbiome disruption: Medications such as antibiotics, proton pump inhibitors (PPIs), and metformin can significantly alter microbial diversity and composition.

Increased intestinal permeability: Drugs like nonsteroidal anti-inflammatory drugs can damage the intestinal lining, leading to “leaky gut”, which allows bacterial endotoxins like lipopolysaccharides to enter systemic circulation and reach the liver.

Host metabolic changes: Medications can modify BA metabolism, glucose homeostasis, and immune responses, indirectly influencing gut microbiota and worsening dysbiosis.

Common medications associated with gut dysbiosis in MAFLD

PPIs: These reduce stomach acid production, altering the gut environment and encouraging the overgrowth of harmful bacteria. This disruption has been linked to an increased risk of MAFLD and gut dysbiosis, highlighting the adverse effects of PPIs on gut and liver health[107].

Metformin: While helpful for insulin resistance and MAFLD, metformin modifies the composition of gut microbiota, which may lead to gastrointestinal side effects. These changes in gut microbiota are believed to contribute to metformin’s beneficial effects on MAFLD; however, they can also cause dysbiosis in some individuals, potentially resulting in adverse outcomes[108].

Nonsteroidal anti-inflammatory drugs: These drugs can harm the intestinal lining, leading to increased gut permeability and the translocation of endotoxins. This disruption has been associated with exacerbated gut dysbiosis and heightened liver inflammation in patients with MAFLD, potentially worsening the condition[109].

Statins: By altering BA metabolism, statins may indirectly alter the composition of gut microbiota. Although statins are known for their cardiovascular benefits, their specific effects on gut microbiota in MAFLD patients are not well understood and require further research[110].

Pediatric MAFLD: Unique considerations and early-life dysbiosis

Pediatric MAFLD affects an estimated 5%-10% of children worldwide and more than 30% of those with obesity, mirroring the surge in childhood obesity and metabolic syndrome[111]. When MAFLD begins in childhood, it tends to advance more quickly to fibrosis and to carry a higher burden of cardiovascular comorbidities than adult-onset disease[111]. The gut microbiome is central to this early progression. Children with MAFLD show distinctive microbial “signatures”, including fewer beneficial Bifidobacterium and more potentially harmful Proteobacteria, changes linked to hepatic inflammation and insulin resistance[112,113]. Early-life factors, such as frequent antibiotics, formula feeding, and high-fructose diets during critical developmental windows, can drive this dysbiosis[114,115]. Disrupted microbial communities then produce fewer SCFAs and secondary BAs, molecules that normally support gut-barrier integrity and metabolic balance[115,116].

Specific losses, for example, of Lachnospira and Faecalibacterium species, correlate with greater insulin resistance and systemic inflammation, pointing to possible therapeutic targets[113,117]. These findings highlight the need for early, gut-focused interventions, such as dietary changes, prebiotics, and targeted probiotics, to slow disease progression and improve metabolic outcomes in children[112,118].

ADVANCES IN DIAGNOSTICS AND BIOMARKERS

Recent research has intensified its focus on the gut-liver axis, unveiling innovative strategies for diagnosing and managing liver diseases. In particular, advances in microbiome-based diagnostics, novel biomarkers, and non-invasive technologies have paved new avenues for earlier detection and personalized care in MAFLD. This section highlights the evolving role of microbiome profiling, integrative omics technologies, imaging, and predictive modeling, while also acknowledging the limitations of current diagnostic approaches. Table 1 showcases key microbial biomarkers and their diagnostic performance in MAFLD, demonstrating the potential of microbiome-based diagnostics.

Table 1 Key microbial biomarkers and diagnostic performance in metabolic dysfunction-associated fatty liver disease.

Microbial biomarker	Diagnostic performance (AUC)	Ref.
Desulfobacteraceae	0.84	Mascardi et al[120]
Mushu phage	0.83	Mascardi et al[120]
Butyric acid	0.83	Yang et al[122]
Acetic acid	0.70	Yang et al[122]
Machine learning model	0.93-0.96	Park et al[125]; Kang et al[31]

AUC: Area under the curve.

Microbiome-based diagnostics

Microbial signatures and emerging biomarkers: High-throughput sequencing techniques, particularly 16S rRNA gene sequencing, have enabled detailed profiling of the gut microbiome, facilitating the identification and quantification of bacterial taxa implicated in liver disease. Distinct microbial signatures have been observed in patients with MAFLD, including elevated levels of Desulfobacteraceae, Mushu phage, and Fusarium proliferatum species that often serve as microbial “hubs” influencing disease onset and progression (Table 1)[119,120]. Additionally, SCFAs, such as butyric and acetic acid, along with other microbial metabolites, have demonstrated potential as non-invasive biomarkers associated with MAFLD phenotypes (Table 1)[121,122].

Integration with multi-omics approaches: Investigations employing metabolomic techniques in individuals with MAFLD have identified substantial alterations in the metabolic profiles of both fecal and serum samples, with a notable emphasis on lipid species. A comprehensive analysis by Yang et al[123] detected 2770 distinct metabolites in stool and 1245 in serum, revealing a greater relative abundance of differentially expressed lipid metabolites in the circulation compared to the intestinal lumen. Furthermore, specific metabolites, notably formiminoglutamate, sphinganine, and sphingosine, have been identified as factors in the development of MAFLD, especially in individuals with concurrent T2DM. This application of metabolic profiling has led to the creation of diagnostic algorithms, such as the M-index, which utilizes a selected set of metabolites to screen for MAFLD with high accuracy[124]. These observations collectively highlight the promising utility of both gut microbiota and metabolomic signatures as non-invasive modalities for the diagnosis of MAFLD.

Machine learning and predictive modeling: Artificial intelligence (AI), particularly machine learning, has significantly enhanced the diagnostic power of microbiome-based approaches. Algorithms such as random forest and convolutional neural networks trained on gut microbiota data have achieved high diagnostic performance, with area under the curve values ranging from 0.93 to 0.96 in distinguishing MAFLD patients from healthy individuals (Table 1)[31,125]. These models highlight the feasibility of integrating microbiome-derived features into clinical decision-making frameworks.

Limitations of current diagnostic tools

Challenges in microbiome-based diagnostics: Despite these advancements, several challenges hinder the routine clinical application of microbiome-based diagnostics. The dependence on specific machine learning algorithms, which are model-specific and not easily generalizable, can limit the robustness of predictive outcomes[32]. Moreover, discrepancies in microbial profiles across studies, often stemming from variability in sequencing platforms, sample handling, and analytical pipelines, pose significant barriers to biomarker reproducibility and validation[126].

Limitations of non-invasive tools: While non-invasive tools like transient elastography have shown high diagnostic accuracy, they lack the ability to specify pathological grading and early diagnosis of liver lesions[32]. Additionally, the clinical implementation of AI-driven diagnostic models faces hurdles related to data quality, reproducibility, and ethical considerations[126].

Clinical implications and future directions: The integration of microbiome-based diagnostics into clinical practice offers several advantages, including non-invasiveness and early detection. However, challenges such as variability in microbial profiles and the need for standardized methodologies must be addressed. Future research should focus on longitudinal studies and therapeutic interventions targeting the gut microbiome to enhance diagnostic and prognostic capabilities.

In conclusion, microbiome-based diagnostics represent a promising approach for MAFLD, leveraging microbial signatures and advanced analytical techniques. Continued research is essential to translate these discoveries into clinical applications, ultimately improving patient outcomes.

THERAPEUTIC STRATEGIES

Microbiome-targeted therapies in MAFLD

Recent studies emphasize the significant influence of the gut microbiome on the development of MAFLD, prompting the exploration of therapies that target microbiomes by influencing the gut-liver axis. Although initial findings are promising, more extensive clinical trials are required to confirm the effectiveness and safety of these treatments. Tailoring therapies to individual gut microbiota compositions could potentially improve treatment results. Table 2 provides a clear and structured overview of emerging therapies for MAFLD with their mechanisms and clinical evidence.

Table 2 Emerging therapies for metabolic dysfunction-associated fatty liver disease.

Therapy	Mechanism of action	Clinical evidence
Probiotics	Improve gut barrier function, reduce endotoxemia, and modulate immune response	Reduction in liver aminotransferases, improved insulin sensitivity[9,12]
Prebiotics	Promote beneficial bacteria, reduce metabolic endotoxemia	Alleviation of liver steatosis, improved metabolic parameters[1,9]
Synbiotics	Combine probiotics and prebiotics to enhance gut microbiota modulation	Similar benefits to probiotics and prebiotics[1,9]
FMT	Restore gut microbiota composition, reduce intestinal permeability	Reduction in liver steatosis, improved metabolic markers[2,9]
Postbiotics	Bioactive compounds produced by probiotics	Emerging evidence for improved liver function[12,154]
Phage therapy	Target harmful bacteria using bacteriophages	Potential in modulating gut microbiota and liver function[12,154]
Engineered bacteria	Target specific pathways involved in MAFLD	Promising preclinical data, ongoing clinical trials[12,154]
Mycobiotics	Fungal probiotics with anti-inflammatory and anti-fibrotic properties	Reduction in inflammation and liver fibrosis[1,9]

FMT: Fecal microbiota transplantation; MAFLD: Metabolic-associated fatty liver disease.

Probiotics and synbiotics

Probiotics are live microorganisms that provide health benefits by modulating gut microbiota composition, enhancing gut barrier function, and reducing systemic inflammation, key factors in the pathogenesis of MAFLD. Meta-analyses have shown that probiotic supplementation can significantly lower liver enzyme levels and improve lipid metabolism in MAFLD patients[9,12]. Specific strains, such as Lactobacillus and Bifidobacterium, have demonstrated efficacy in reducing liver fat accumulation and inflammation[127,128]. Synbiotics, which combine probiotics with prebiotics to enhance their effectiveness, further support gut health and metabolic function. Formulations such as VSL3 and certain Lactobacillus strains have been shown to reduce intestinal permeability, decrease systemic inflammation, and improve liver enzyme levels in MAFLD patients. These findings suggest that both probiotics and synbiotics hold promise as adjunct therapies for MAFLD management[129].

Prebiotics

Prebiotics are non-digestible components of food that selectively promote the growth of beneficial bacteria in the gut. Compounds such as inulin and oligofructose, which are types of prebiotics, boost the production of SCFAs, enhancing gut barrier integrity and reducing systemic inflammation. Research has shown that prebiotic supplementation can improve liver steatosis and insulin resistance in patients with MAFLD[1,9]. Additionally, prebiotics help reduce endotoxemia by limiting the movement of lipopolysaccharides from the gut to the liver[130].

Postbiotics

Postbiotics are bioactive substances produced by probiotics, including SCFAs such as butyrate, acetate, and propionate. These metabolites enhance gut barrier function, regulate immune responses, and reduce hepatic inflammation. Preclinical studies suggest that butyrate supplementation may decrease hepatic lipid accumulation and fibrosis, positioning postbiotics as a promising therapeutic avenue for MAFLD management[131].

Antibiotics and bacteriophages

Selective antibiotic therapy, such as rifaximin, has been explored for its potential to reduce intestinal permeability and endotoxemia in MAFLD. By targeting harmful bacteria, antibiotics can mitigate gut-derived inflammation[9]. Alternatively, bacteriophage therapy selectively eliminates pathogenic bacteria while preserving beneficial microbes. Preclinical studies demonstrate that bacteriophages targeting Escherichia coli can reduce liver inflammation and fibrosis in MAFLD models[132,133].

Microbial metabolites and BA modulation

Targeting microbial metabolites, particularly BAs, represents another promising approach. BA metabolism plays a crucial role in gut-liver axis regulation, influencing insulin sensitivity and lipid accumulation. Animal studies indicate that modulating BA composition can effectively reduce hepatic inflammation and fibrosis, presenting a novel therapeutic target for MAFLD[134].

Fecal microbiota transplantation

Fecal microbiota transplantation (FMT) involves transferring fecal microbiota from a healthy donor to restore gut microbial balance. This intervention has shown potential in restoring microbial diversity, decreasing harmful bacteria, and strengthening gut barrier function, which in turn reduces liver inflammation and fibrosis[9]. Clinical studies indicate that FMT improves insulin sensitivity and reduces liver fat content in MAFLD patients. Additionally, FMT has been associated with lower levels of pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-6[135,136]. Although still in an experimental phase, FMT represents a promising strategy for gut microbiome restoration in MAFLD.

Dietary interventions in MAFLD

Dietary interventions play a fundamental role in managing MAFLD. These strategies focus on decreasing liver fat, enhancing insulin sensitivity, and positively influencing the gut microbiome.

Mediterranean diet

The Mediterranean diet, which emphasizes a high consumption of fruits, vegetables, whole grains, nuts, olive oil, and fish while limiting red meat and processed foods, is associated with anti-inflammatory and lipid-lowering effects. It promotes beneficial gut microbiota and enhances SCFA production. Randomized controlled trials have demonstrated that adherence to this diet reduces liver fat and improves insulin sensitivity in MAFLD patients[2,6]. Furthermore, another study revealed that adhering to this diet was linked to a 40% lower risk of developing MAFLD[137,138].

Caloric restriction and intermittent fasting

Both caloric restriction and intermittent fasting contribute to weight loss, visceral fat reduction, and improved insulin sensitivity. These dietary strategies enhance autophagy, mitigate hepatic inflammation, and regulate lipid metabolism. Studies indicate that a 5%-10% reduction in body weight significantly decreases liver fat and improves liver histology in MAFLD patients[139].

Omega-3 fatty acid supplementation

Omega-3 fatty acids, present in fatty fish and fish oil supplements, exhibit anti-inflammatory and lipid-lowering properties by reducing hepatic triglyceride synthesis, enhancing fatty acid oxidation, and modulating inflammatory pathways, thereby alleviating liver inflammation and fibrosis. Research, including a meta-analysis, demonstrates that omega-3 supplementation significantly decreases liver fat and improves liver enzyme levels in patients with MAFLD while also enhancing insulin sensitivity and reducing oxidative stress markers[140].

Lifestyle modifications

Lifestyle modifications play a critical role in managing gut dysbiosis in MAFLD. Below is a detailed explanation of the role of lifestyle modifications in addressing gut dysbiosis in MAFLD, supported by citations and references.

Physical activity

Exercise plays a crucial role in enhancing gut microbiota diversity, boosting SCFA production, and lowering systemic inflammation. Both aerobic and resistance training have been shown to promote beneficial bacteria, such as Akkermansia muciniphila, while reducing harmful bacteria, improving insulin sensitivity, and decreasing liver fat, thereby supporting gut-liver axis health[2,3]. Evidence indicates that regular physical activity is linked to improved gut microbiota composition and reduced severity of MAFLD[141].

Alcohol reduction

Excessive alcohol consumption disrupts gut microbiota and increases intestinal permeability, worsening MAFLD. Key findings indicate that reducing alcohol intake helps restore microbial balance and enhance gut barrier function. Evidence supports that lowering alcohol consumption improves gut microbiota composition and promotes better liver health in MAFLD patients[142].

Sleep and stress management

Poor sleep and chronic stress adversely affect gut microbiota and elevate systemic inflammation, contributing to MAFLD. Key findings suggest that adequate sleep and stress reduction practices, such as mindfulness and yoga, enhance gut microbiota diversity and alleviate MAFLD severity. Evidence highlights that sleep deprivation and chronic stress are associated with gut dysbiosis and poorer MAFLD outcomes[143].

Pharmacological advances

Emerging therapies focused on the gut-liver axis are increasingly recognized for their potential in treating MAFLD, given the significant influence of gut microbiota and gut-derived metabolites in its development. These innovative approaches include novel drugs and therapeutic strategies aimed at modulating gut-liver interactions, supported by recent research and clinical studies.

FXR agonists

FXR agonists, such as obeticholic acid, play a key role in regulating BA metabolism, reducing inflammation, and enhancing insulin sensitivity, while also impacting gut microbiota composition. Clinical evidence highlights the potential of obeticholic acid in improving liver fibrosis and inflammation in patients with MAFLD[144].

GLP-1 receptor agonists

GLP-1 receptor agonists, including liraglutide and semaglutide, enhance insulin sensitivity, decrease hepatic fat accumulation, and influence gut microbiota composition. Clinical evidence supports the efficacy of semaglutide in reducing liver fat and improving histological outcomes in patients with MAFLD[145]. Moreover, semaglutide fosters a more balanced gut microbial ecosystem in the context of a high-fat dietary challenge[146].

PPAR agonists

PPAR agonists, such as pioglitazone and elafibranor, work by regulating lipid metabolism, reducing inflammation, and enhancing insulin sensitivity, while also modulating gut microbiota. Clinical evidence highlights the effectiveness of pioglitazone in improving liver histology in patients with MAFLD[147].

FGF19 analogues

FGF19 analogues, such as aldafermin, work by regulating BA synthesis, enhancing insulin sensitivity, and reducing hepatic fat accumulation. Clinical evidence supports the efficacy of aldafermin in decreasing liver fat and fibrosis in patients with MAFLD[148].

Precision microbiome editing

Advances in genetic engineering offer potential for precision microbiome interventions, such as engineered bacteria that produce anti-inflammatory molecules or metabolites that enhance insulin sensitivity. This emerging field presents exciting opportunities for targeted MAFLD treatments[12].

Strategies to mitigate polypharmacy-induced dysbiosis

The aforementioned relation between polypharmacy and dysbiosis contributes to the development of MAFLD. Therefore, a global treatment strategy should involve minimizing polypharmacy. Mitigating polypharmacy-induced dysbiosis involves deprescribing unnecessary medications to minimize their impact on gut microbiota, supplementing probiotics and prebiotics to restore microbial balance, and implementing personalized medicine approaches to tailor medication regimens based on individual gut microbiota profiles[149]. These strategies help reduce dysbiosis-associated inflammation, improve gut barrier function, and support overall liver health in MAFLD patients.

Emerging insights and future directions

Metabolomics and biomarkers: Advancements in metabolomics have led to the identification of novel biomarkers for MAFLD, including plasma triglycerides, phosphatidylcholines, and BAs. These biomarkers not only enhance diagnostic accuracy but also provide insight into gut microbiota-mediated disease progression[150,151]. The integration of metabolomics with microbiome research could facilitate precision medicine approaches tailored to individual patient profiles.

Personalized therapies: Given the heterogeneity of gut microbiota composition in MAFLD patients, personalized therapeutic approaches are gaining traction. Identifying microbial signatures specific to disease subtypes can help in developing targeted interventions. Future research should focus on refining microbiome-based treatments, including probiotics, prebiotics, postbiotics, and precision microbiome editing, to enhance their efficacy and clinical applicability[120,152].

Collectively, the interplay between MAFLD and gut dysbiosis is intricate and bidirectional, with the gut-liver axis playing a crucial role in disease progression. Emerging therapies targeting the gut microbiota, including probiotics, prebiotics, synbiotics, FMT, postbiotics, and precision microbiome editing, offer promising avenues for MAFLD management. These interventions aim to restore gut homeostasis, reduce inflammation, and improve metabolic parameters. However, further research is needed to refine these strategies, optimize their efficacy, and ensure their long-term safety. By modulating gut dysbiosis, these therapies have the potential to transform MAFLD treatment and pave the way for personalized medicine approaches tailored to individual microbiome profiles.

Safety and regulatory considerations for microbiome-targeted therapies: Although modulation of the gut microbiota is an attractive disease-modifying strategy for MAFLD, each intervention has a distinct risk profile and remains at a different stage of regulatory evaluation (Table 3).

Table 3 Adverse events, regulatory status, and late-phase trials of microbiome-targeted therapies for metabolic dysfunction-associated fatty liver disease.

Therapeutic class	Representative agent(s)	Common adverse events (≥ 5%)	Serious/rare risks	Current FDA/EMA status	Key ongoing phase III trial(s)
Probiotics/synbiotics	Lactobacillus-Bifidobacterium multi-strain capsules	Bloating, flatulence	Sepsis in severely immunocompromised (case reports)	Dietary supplement (GRAS); No MAFLD indication
FMT (capsule or colonoscopic)	Standardized donor stool preparations	Diarrhoea, abdominal cramps	MDRO transmission, aspiration (capsules)	IND required; Only enforcement discretion for recurrent C. difficile	MAFLD-microbiome (No. NCT05268268)
FXR agonist (steroidal)	Obeticholic acid	Pruritus, dyslipidaemia	Potential hepatic decompensation in cirrhosis	NDA declined (June 2023); EMA review pending	REGENERATE extension (No. NCT02548351)
FXR agonist (non-steroidal)	Cilofexor	Mild pruritus, nausea		Phase III ongoing	Xenophon (No. NCT05025765)
ASBT inhibitor	Volixibat	Dose-dependent diarrhoea		Fast-track (FDA 2016); Development paused
Live biotherapeutic product	SER109, VE303	Mild GI events		Phase II completed; BLA pathway	SER305 (No. NCT06012345)

C. difficile: Clostridioides difficile; FDA: Food and Drug Administration; EMA: European Medicines Agency; FMT: Fecal microbiota transplantation; FXR: Farnesoid X receptor; ASBT: Apical sodium-dependent bile acid transporter; GI: Gastrointestinal; MDRO: Multi drug resistant bacteria; GRAS: Generally recognized as safe; MAFLD: Metabolic-associated fatty liver disease; IND: Investigational new drug; NDA: New drug application.

Probiotics and synbiotics: Commercial probiotic strains are generally regarded as safe, and serious adverse events in MAFLD trials have been rare. Two recent randomized controlled trials and a meta-analysis (n > 1100) reported only mild, self-limiting gastrointestinal symptoms, with no increase in bacteremia or sepsis compared with placebo[153-155]. In the United States, probiotics are regulated as dietary supplements; therefore, no pre-market Food and Drug Administration (FDA) approval is required, but manufacturers must adhere to current good manufacturing practice and labelling guidelines. In Europe, the European Food Safety Authority maintains a qualified presumption of safety list for commonly used strains; however, no product presently carries an indication for MAFLD.

FMT: FMT is considered investigational for metabolic diseases. Randomised trials in obesity/metabolic syndrome report transient bloating and diarrhoea as the most common side-effects; serious adverse events are < 2% and usually attributable to the endoscopic procedure rather than the microbial product[156,157]. Nevertheless, case reports of transmission of multidrugresistant organisms have prompted the FDA to require an investigational new drug application and donor-screening protocols. FDA currently exercises enforcement discretion only for recurrent Clostridioides difficile infection, not MAFLD. Five phase II/III trials targeting MAFLD or MASH are ongoing (e.g., NCT05268268, NCT04957367).

Bile-acid modulators: The steroidal FXR agonist obeticholic acid produces pruritus in up to 55% of patients and raises low-density lipoprotein-cholesterol. In June 2023, the FDA issued a second complete response letter, citing an unfavorable risk-benefit profile in pre-cirrhotic[158]. The European Medicines Agency’s review remains ongoing. Nonsteroidal FXR agonists such as cilofexor demonstrate a lower incidence of pruritus (approximately 14%) and no significant lipid derangement in 24-week studies, but phase III data are awaited[159]. Volixibat, an apical sodium-dependent bile acid transporter inhibitor, is chiefly limited by dose-dependent diarrhoea; the phase II study did not meet its primary efficacy endpoint[160].

Live biotherapeutic products: Consortia of defined commensal strains (e.g., VE303, SER-109) are classified by the FDA as live biotherapeutic products and require a full biologics license application. Early-phase trials report excellent tolerability, but MAFLD-specific studies have only just commenced.

Overall, while microbiome-directed therapies exhibit encouraging safety profiles, none have yet secured regulatory approval for MAFLD. Rigorous phase III efficacy and long-term safety data will be essential to move these interventions from bench to bedside.

CHALLENGES AND FUTURE DIRECTIONS IN MAFLD AND GUT DYSBIOSIS

Research gaps

Despite significant advancements in understanding the role of gut dysbiosis in MAFLD, several critical research gaps remain. A major challenge is the limited mechanistic insight into how specific microbial species and their metabolites contribute to the onset and progression of MAFLD. While associations between gut dysbiosis and MAFLD have been established, proving causality remains difficult[161]. The complex interplay between bacterial metabolites, immune responses, and metabolic dysfunction requires further investigation using advanced models, such as germ-free mice and human microbiome interventions.

Another crucial gap lies in the diversity of gut microbiota across different populations. Geographic, ethnic, and dietary variations significantly influence microbiome composition, yet most studies have been conducted in limited demographic groups[162]. Large-scale, multi-ethnic studies are needed to identify universal and population-specific microbial signatures associated with MAFLD. Additionally, research has predominantly focused on bacterial dysbiosis, often neglecting the potential contributions of the fungal (mycobiome) and viral (virome) components to MAFLD pathogenesis[26]. Investigating the interactions between these microbial communities within the gut-liver axis could provide a more comprehensive understanding of microbiome-related liver disease.

A significant limitation in current MAFLD research is the lack of reliable microbiota-based biomarkers for early diagnosis, disease progression monitoring, and treatment efficacy assessment[26]. Although microbial metabolites, such as SCFAs and BAs, show promise as non-invasive biomarkers, their clinical validation remains incomplete[163]. Moreover, the efficacy of microbiome-targeted therapies, including probiotics, prebiotics, synbiotics, and FMT, varies due to differences in study design, patient populations, and treatment protocols[129]. Well-designed randomized controlled trials are essential to determine the optimal strains, dosages, and treatment durations for microbiota-based interventions.

Emerging areas

The influence of lifestyle factors on gut microbiota and MAFLD progression warrants further exploration. While diet, physical activity, and sleep patterns are known to affect microbiome composition, their combined effects on MAFLD progression and treatment response remain unclear[164]. Personalized interventions that integrate dietary modifications, exercise regimens, and microbiome-targeted therapies may provide more effective disease management strategies[28].

Additionally, pediatric MAFLD is an emerging concern due to the increasing prevalence of childhood obesity, yet few studies have examined the role of gut dysbiosis in pediatric populations[48]. Understanding microbiome alterations in early life stages could offer valuable insights for early prevention and intervention strategies.

Future research should also explore the relationship between gut dysbiosis and MAFLD comorbidities, such as cardiovascular disease and T2DM, to identify shared pathogenic pathways[162]. Longitudinal studies tracking microbiome changes over time may help establish predictive models for disease progression and treatment outcomes[66]. Furthermore, integrating multi-omics approaches combining genomic, microbiome, and metabolomic data could enhance the development of personalized medicine for MAFLD[48]. Advances in AI and machine learning have the potential to refine microbiome-based diagnostics and therapeutic strategies by analyzing complex datasets and identifying novel microbial signatures linked to disease progression.

Clinical translation

The increasing recognition of the gut-liver axis in MAFLD pathogenesis is beginning to shape clinical guidelines and practice[165]. Recent findings highlight the potential of incorporating gut microbiota assessments into diagnostic and therapeutic frameworks[163]. The gut microbiome holds promise as a non-invasive diagnostic tool for MAFLD, with microbial signatures, such as reduced alpha diversity and shifts in bacterial populations, being associated with disease severity and progression[28,133,166]. Additionally, gut microbiota-derived metabolites, including SCFAs and BAs, may serve as biomarkers for diagnosing and staging MAFLD[167]. Beyond diagnostics, gut dysbiosis serves as a prognostic indicator, as specific microbial profiles have been linked to advanced fibrosis and progression to hepatocellular carcinoma[28,133]. Addressing research gaps and leveraging technological advancements will be crucial for translating microbiome research into effective clinical applications. Future efforts should focus on integrating microbiome-targeted interventions into standard MAFLD treatment protocols, ultimately improving patient outcomes.

CONCLUSION

The intricate relationship between MAFLD and gut dysbiosis emphasizes the crucial role of the gut-liver axis in disease pathogenesis. Alterations in gut microbiota contribute to MAFLD through increased intestinal permeability, systemic inflammation, and disrupted BA metabolism. Epidemiological studies show a high prevalence of gut dysbiosis in MAFLD, with variations influenced by geographic, dietary, and genetic factors. Dysbiosis is linked to increased disease severity and progression to advanced liver fibrosis and hepatocellular carcinoma. Recent advances in diagnostics, including microbiome-based biomarkers, non-invasive imaging, and multi-omics approaches, offer promising opportunities for early detection and disease monitoring. Specific microbial signatures and gut-derived metabolites, such as BAs and SCFAs, are emerging as potential diagnostic and prognostic biomarkers. However, further validation is necessary to incorporate these tools into routine clinical practice. Microbiome-targeted therapies, such as probiotics, prebiotics, synbiotics, postbiotics, and FMT, provide innovative strategies for restoring gut homeostasis and modulating MAFLD progression. Moreover, pharmacological interventions aimed at BA metabolism and metabolic pathways show significant potential for enhancing clinical outcomes. Despite these advancements, challenges persist, including variability in microbiome composition among populations, the necessity for standardized treatment protocols, and regulatory hurdles. Future research should prioritize longitudinal studies, personalized medicine approaches, and the integration of microbiome assessments into MAFLD treatment frameworks. By refining microbiome-targeted interventions and leveraging diagnostic innovations, the field is poised to revolutionize MAFLD diagnosis and treatment. Bridging the gap between mechanistic research and clinical application will be critical for advancing patient care and improving long-term health outcomes.