The Intestinal Thread of Fate: How the Microbiota Shapes the Story of Liver Disease

Abstract

Metabolic dysfunction–associated steatotic liver disease (MASLD) is a multifactorial condition linked to liver injury, insulin resistance, and disrupted gut–liver interactions. A key aspect of MASLD pathogenesis is the dysfunction of intestinal barriers, including mechanical, immunological, and microbial alterations that amplify liver damage. The disruption of tight junctions and increased intestinal permeability allow microbial products, such as lipopolysaccharides, to enter the bloodstream, triggering liver inflammation via Kupffer cell activation. In MASLD, the gut vascular barrier is also compromised, marked by increased expression of PV-1. Additionally, dysbiosis, driven by high-fat, high-sugar diets, shifts the gut microbiota toward pro-inflammatory species, exacerbating systemic inflammation and intestinal permeability. This imbalance activates Toll-like receptor signaling, which promotes endotoxin-induced liver injury. Gut dysbiosis further impairs lipid metabolism, contributing to hepatic steatosis and MASLD progression. The gut–liver axis plays a critical role, with factors like altered bile acid metabolism and toxic metabolites such as hydrogen sulfide worsening intestinal barrier function and fueling chronic inflammation. This review aims to explore the complex role of the gut–liver axis in MASLD progression, highlighting the mechanisms of intestinal barrier dysfunction, dysbiosis, and microbial contributions to liver injury. It also discusses therapeutic strategies targeting intestinal barriers, including dietary and microbiota-based interventions, while acknowledging the challenges of personalized treatment approaches. Future research should focus on multi-omics technologies and the safety and efficacy of microbiota-targeted therapies in MASLD management.

1. Introduction

Metabolic dysfunction–associated steatotic liver disease (MASLD) represents a recent nosological redefinition in the landscape of liver disorders, introduced to replace the term non-alcoholic fatty liver disease (NAFLD). This semantic update emphasizes the primary role of metabolic disorders in the pathogenesis of the condition [1]. MASLD is defined by the excessive accumulation of triglycerides in the liver, leading to hepatic steatosis without significant alcohol consumption, in the presence of at least one cardiometabolic risk factor, including obesity, insulin resistance (IR), and type 2 diabetes mellitus (T2DM) [1]. The global increase in the prevalence of these conditions has made MASLD one of the leading causes of chronic liver disease, closely linked to the rising incidence of obesity and metabolic syndrome [2]. This condition affects approximately 30% of the global population, with its prevalence exceeding 50% in high-risk groups, such as individuals with T2DM. In Latin America, the prevalence of this condition exceeds 40%, while in Europe and the United States, it ranges between 25% and 31% [3,4,5].

Several risk factors contribute to the onset and progression of MASLD, including genetic predisposition (e.g., PNPLA3 polymorphisms), dietary habits, and sedentary lifestyle [3]. The clinical spectrum of MASLD varies from simple steatosis to metabolic dysfunction–associated steatohepatitis (MASH), which is characterized by hepatocyte injury and inflammation, potentially progressing to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) [3]. Beyond liver-related outcomes, MASLD is a strong predictor of cardiovascular disease (CVD), which remains the leading cause of mortality in affected patients. Additionally, MASLD has been associated with an increased risk of chronic kidney disease (CKD), extrahepatic cancers, and all-cause mortality [6,7,8,9].

The diagnosis of MASLD relies on the presence of hepatic steatosis, which can be detected using imaging modalities such as ultrasound, magnetic resonance imaging (MRI), or controlled attenuation parameter (CAP) in transient elastography [3]. Liver biopsy remains the gold standard for assessing fibrosis and inflammation in advanced cases, although non-invasive biomarkers and fibrosis scores, such as the FIB-4 index and NAFLD fibrosis score, are increasingly utilized in clinical practice [3].

From a pathogenetic perspective, MASLD is a multifactorial condition in which IR, lipotoxicity, oxidative stress, and the activation of innate and adaptive immune responses play crucial roles. These mechanisms contribute to progressive liver damage [10,11,12]. In recent years, the gut–liver axis has taken center stage in studying the pathogenesis and progression of MASLD, attracting growing scientific interest [13]. This axis is characterized by a bidirectional interaction between the gastrointestinal tract and the liver, mediated by complex biological signals, including gut-microbiota-derived metabolites, immune molecules, and neurotransmitters [14,15]. Portal circulation serves as the primary connection point, transporting nutrients, bacterial endotoxins, bile acids, and other bioactive molecules, influencing liver function and contributing to liver disease pathogenesis [16].

Under physiological conditions, the gut microbiota maintains hepatic homeostasis by facilitating digestion, producing beneficial metabolites such as short-chain fatty acids (SCFAs), and modulating intestinal and systemic immune responses [17]. Pathological alterations in gut microbiota composition, known as dysbiosis, can impair intestinal permeability, promoting the translocation of bacteria, lipopolysaccharides (LPS), and other endotoxins into the portal circulation. This phenomenon, referred to as “leaky gut”, triggers hepatic inflammatory responses [18,19,20]. This process contributes to the progression of the disease toward metabolic dysfunction–associated steatohepatitis (MASH) and fibrosis [20,21]. Emerging data underscore the pivotal role of the gut–liver axis in MASLD pathogenesis. Alterations in gut microbiota composition and impaired intestinal permeability stimulate the activation of Kupffer cells and hepatic stellate cells, promoting fibrogenesis and the secretion of pro-inflammatory cytokines [20,21,22]. Additionally, gut-microbiota-derived metabolites, such as trimethylamine N-oxide (TMAO), modulate hepatic lipid metabolism and contribute to the progression of cardiovascular and metabolic diseases, including MASLD [23,24]. Accordingly, this review aims to provide a comprehensive overview of the molecular and cellular mechanisms that underpin the gut–liver interaction in MASLD. We focus on the contributions of intestinal dysbiosis, altered permeability, and bacterial metabolites, and we discuss emerging therapeutic strategies, including probiotics, prebiotics, and novel pharmacologic agents, that target this axis to improve clinical outcomes in MASLD patients.

2. Literature Research Methodology

A comprehensive literature search was conducted to capture a wide spectrum of studies relevant to MASLD and its intricate relationship with the gut microbiota. The search strategy involved querying major databases PubMed/MEDLINE, EMBASE, Scopus, and Web of Science using a combination of keywords and Medical Subject Headings (MeSH) terms. These search terms included, but were not limited to, “MASLD”, “Metabolic Dysfunction-Associated Steatotic Liver Disease”, “gut microbiota”, “intestinal barriers”, “liver disease”, “pathogenesis”, “risk factors”, “dysbiosis”, and “Fecal Microbiota Transplantation”. No restrictions were placed on the publication date to ensure the inclusion of both seminal and contemporary literature, while only studies published in English were considered. In addition to database searches, the reference lists of relevant articles were manually screened to identify further studies that might have been missed initially, driven by the specific themes of the review. Studies were selected based on their relevance to these topics and their methodological rigor.

3. Risk Factors and Predisposition for MASLD

3.1. Genetic and Epigenetic Factors

Genetic and epigenetic predisposition plays a critical role in the development and progression of MASLD and HCC. Advanced genomic studies, such as genome-wide association studies (GWAS) and exome-wide association studies (EWAS), have identified key single nucleotide polymorphisms (SNPs) in genes including PNPLA3, TM6SF2, MBOAT7, GCKR, and HSD17B13. These genetic variants modulate crucial processes such as lipid metabolism, oxidative stress, and hepatic inflammation, influencing disease susceptibility and progression (Table 1) [25,26].

3.1.1. PNPLA3

The PNPLA3 (patatin-like phospholipase domain-containing protein 3) gene encodes a multifunctional enzyme that regulates hepatic lipid metabolism, exhibiting both triglyceride hydrolase and acyltransferase activity on lipid droplets [27]. The I148M variant (rs738409 C>G), resulting from an isoleucine-to-methionine substitution at position 148, was the first genetic variant identified as a major risk factor for NAFLD and has since been strongly associated with MASLD and HCC risk [28]. This mutation impairs PNPLA3 lipase function, leading to triglyceride accumulation in hepatocytes and contributing to steatosis, steatohepatitis, fibrosis, and hepatocellular carcinoma [29]. Experimental studies in murine models indicate that PNPLA3 interacts with CGI-58, a protein essential for lipid droplet degradation. The absence of hepatic CGI-58 exacerbates the effects of the I148M variant, further increasing lipid deposition [30]. Notably, another PNPLA3 polymorphism, rs6006460 (G>T), appears to have a protective role, potentially altering enzymatic activity in a way that mitigates hepatic lipid accumulation and HCC risk [31,32].

3.1.2. TM6SF2

The TM6SF2 (transmembrane 6 superfamily member 2) gene regulates the hepatic lipid metabolism by influencing triglyceride secretion via very-low-density lipoprotein (VLDL) formation [33]. The E167K variant (rs58542926), involving a glutamate-to-lysine amino acid substitution, reduces VLDL secretion efficiency, leading to hepatic lipid accumulation and promoting the progression of steatosis and fibrosis [34]. Although this polymorphism is associated with lower total cholesterol and LDL levels, it paradoxically increases the risk of advanced fibrosis and MASLD-HCC, highlighting the dual impact of genetic alterations on lipid metabolism and liver disease progression [35,36]. Another variant, rs5842926 (C>T), has also been implicated in modifying lipid metabolism and liver disease susceptibility [35,36].

3.1.3. GCKR

The GCKR (glucokinase regulatory protein) gene encodes a key regulator of glucokinase, an enzyme involved in glucose metabolism. The rs1260326 (C>T) polymorphism enhances glycolysis and de novo lipogenesis, thereby increasing the risk of hepatic lipid accumulation and the progression to MASH and fibrosis [37,38,39].

3.1.4. HSD17B13

The HSD17B13 (hydroxysteroid 17-beta dehydrogenase 13) gene encodes an enzyme associated with hepatic lipid droplets. The rs72613567 (T>TA) variant has been identified as a protective factor against MASLD, particularly in individuals carrying the PNPLA3 I148M risk allele. This variant reduces hepatic inflammation and fibrosis and is associated with a lower risk of HCC development [40,41].

3.1.5. Epigenetic and Epitranscriptomic Modifications

Beyond genetics, epigenetic mechanisms, including DNA methylation and histone modifications, influence gene expression and the hepatic microenvironment, further modulating disease susceptibility and progression [42]. Emerging evidence highlights the role of epitranscriptomic modifications, such as N6-methyladenine (m6A), in regulating MASLD-HCC progression [42,43]. Enzymes such as METTL3 (a “writer” of m6A modifications) and YTHDF1 (a “reader” of m6A marks) modulate the expression of key genes and activate oncogenic pathways. Notably, the EZH2/interleukin-6 (IL-6) signaling axis has been implicated in immune dysfunction and tumor progression [44].

The complex interplay of genetic, epigenetic, and epitranscriptomic factors significantly contributes to the pathogenesis of MASLD and its progression to HCC. These mechanisms represent crucial targets for innovative diagnostic and therapeutic strategies aimed at improving clinical outcomes in high-risk patients.

3.2. Metabolic Consequences and Other Contributing Factors

MASLD is closely linked with metabolic syndrome, a condition defined by the presence of at least three of the following five criteria—central obesity (increased waist circumference), hypertriglyceridemia, low levels of high-density lipoprotein cholesterol (HDL-C), impaired fasting glucose or T2DM, and arterial hypertension—which collectively serve as primary risk determinants for liver pathologies [45,46]. In this context, each component contributes through distinct yet interrelated mechanisms that not only initiate hepatic steatosis but also drive its progression to more advanced states, such as MASH and fibrosis (Table 1).

Central obesity plays a pivotal role in MASLD pathogenesis. The hypertrophy of visceral adipocytes leads to reduced insulin sensitivity and altered adipocytokine secretion, resulting in an increased release of free fatty acids (FFAs) into the circulation [47]. Once in the bloodstream, these FFAs are transported via the portal vein to the liver, where they accumulate in hepatocytes through processes that include de novo lipogenesis, diminished hepatic lipid metabolism, and augmented exogenous lipid supply. This lipid overload triggers mitochondrial dysfunction and enhances reactive oxygen species (ROS) production, while toxic lipid metabolites—such as ceramides and diacylglycerols—induce endoplasmic reticulum (ER) stress, activate inflammatory cascades, and lead to hepatocyte cell death [48,49].

Insulin resistance (IR), a hallmark of metabolic syndrome, further exacerbates hepatic lipid deposition by pro de novo lipogenesis and impairing fatty acid oxidation [50,51]. This metabolic imbalance sets off a vicious cycle characterized by chronic hyperglycemia and hyperinsulinemia, which in turn amplify pro-inflammatory and pro-fibrotic pathways, thereby escalating liver damage. Moreover, hyperinsulinemia impedes lipolysis in adipocytes, resulting in a further increase in circulating FFAs that continue to fuel hepatic steatosis [52].

The metabolic dysregulation is compounded by hypertriglyceridemia, which contributes to the production of toxic lipid species that intensify oxidative stress and inflammation. Elevated triglyceride levels are also associated with the formation of small, dense VLDL and LDL particles with high atherogenic potential, linking these lipid abnormalities to both liver and cardiovascular disease [53,54]. Concurrently, reduced levels of HDL-C compromise reverse cholesterol transport and, in the presence of systemic inflammation and IR, lead to a transformation of HDL into pro-inflammatory particles. Alterations in HDL composition, including diminished levels of apolipoproteins ApoAI and ApoAII, further exacerbate systemic inflammation and promote hepatic lipid accumulation [55,56,57]. Additional metabolic disturbances such as impaired fasting glucose and T2DM are closely tied to IR and contribute to a pro-inflammatory state that worsens liver injury and perpetuates the cycle of hyperinsulinemia and intrahepatic lipid accumulation [58,59,60]. Likewise, arterial hypertension—mediated by the chronic activation of the renin–angiotensin–aldosterone system and increased expression of angiotensin type 1 receptors—leads to vasoconstriction, vascular fibrosis, and reduced nitric oxide synthesis. These vascular changes not only disrupt hepatic microcirculation but also exacerbate the intricate interplay between metabolic, cardiovascular, and hepatic dysfunctions [61,62].

3.3. Prevalence of Lean/Non-Obese MASLD and Medication-Induced MASH

Recent studies suggest that approximately 10–15% of patients with MASLD are lean or non-obese, demonstrating that liver disease can develop even in the absence of overt obesity [12] (Table 1). Although these individuals typically exhibit a normal body mass index, they may harbor distinct metabolic abnormalities, such as increased visceral adiposity, subtle insulin resistance, or dyslipidemia, that predispose them to hepatic fat accumulation and progression to steatohepatitis [12]. This lean/non-obese phenotype of MASLD may involve unique pathophysiological mechanisms, including specific genetic and epigenetic factors, that differentiate it from the classic presentation of MASLD associated with metabolic syndrome [8,12]. As such, these cases require tailored diagnostic approaches, such as advanced imaging modalities and metabolic profiling, to accurately assess liver fat content and associated risks. In turn, therapeutic strategies may also need to be customized, focusing on both lifestyle interventions and targeted pharmacological treatments that address these specific metabolic derangements [8,12].

In parallel, medication-induced MASH represents another important, though less common, etiology of liver injury [46]. Certain drugs, including tamoxifen, corticosteroids, and various antipsychotic agents, have been implicated in the development of hepatic steatosis and the progression to MASH [46]. The underlying mechanisms for medication-induced MASH may involve direct hepatotoxicity, alterations in lipid metabolism, or disruptions in insulin signaling pathways, which can mimic the metabolic disturbances observed in MASLD associated with metabolic syndrome [46]. Therefore, a comprehensive clinical evaluation should include a detailed medication history to identify potential drug-related contributions to liver disease. Recognizing medication-induced MASH is crucial, as it may necessitate a modification of the therapeutic regimen, such as dose adjustment or switching to an alternative medication, along with supportive measures to protect liver function [46].

In summary, the close relationship between metabolic syndrome and MASLD underscores the need for integrated approaches to mitigate metabolic risk factors. Targeted interventions, including lifestyle modifications, weight reduction, and pharmacological therapies to improve insulin sensitivity and reduce systemic inflammation, are essential to prevent disease progression and improve clinical outcomes.

Table 1. Risk factors and predisposition for MASLD.

Category	Specific Factor	Main Pathogenetic Mechanisms	Clinical Effects/Risks	Ref.
Genetic	PNPLA3 (I148M, rs738409, G allele)	Impaired lipase activity; accumulation of triglycerides in hepatocytes; lipid droplet remodeling dysfunction	Steatosis, NASH, Fibrosis, Increased risk of HCC	[25,26,27,28,29,30,31,32]
	PNPLA3 (rs6006460, G>T variant)	Modulates risk in protective direction	Potential protective role against hepatic lipid accumulation	[25,26,27,28,29,30,31,32]
	TM6SF2 (E167K, rs5842926, C>T variant)	Reduced VLDL secretion; hepatic lipid accumulation; lower total cholesterol and LDL levels	Progression to advanced hepatic fibrosis, MASLD-HCC	[33,34,35,36]
	GCKR (rs1260326, C>T variant)	Enhanced glycolysis and de novo lipogenesis; increased hepatic lipid accumulation	Progression to MASH and fibrosis	[37,38,39]
	MBOAT7 polymorphisms	Alters phospholipid remodeling; increases hepatic fat accumulation and inflammation	Associated with fibrosis and progression of MASLD	[25,26]
	HSD17B13 (rs72613567, T>TA variant)	Loss-of-function variant; reduced hepatic inflammation and fibrosis, especially in PNPLA3 I148M carriers	Protective against MASLD progression and HCC	[40,41,42]
Epigenetic	DNA methylation	Alters gene expression; promotes pro-inflammatory and pro-fibrotic pathways	Progression from MASLD to MASH, fibrosis, HCC	[43]
	Histone modifications	Modifies chromatin structure; impacts transcriptional activity of genes related to inflammation and fibrosis	Increased susceptibility to MASLD and progression to advanced liver disease	[43]
Epitranscriptomic	m6A RNA modification	Regulates RNA stability and translation; affects lipid metabolism and oncogenic pathways	Promotes MASLD progression and HCC development	[44]
	METTL3 (m6A writer) and YTHDF1 (m6A reader)	Modulate gene expression; activate oncogenic signals (e.g., EZH2, IL-6)	Immune dysfunction, enhanced tumorigenesis, risk of HCC	[43,44,45]
Metabolic Syndrome	Central obesity (increased waist circumference)	Visceral adipocyte hypertrophy; increased FFAs; portal delivery of lipids to the liver	Steatosis, mitochondrial dysfunction, ROS production, hepatocyte apoptosis	[46,47,48,49,50]
	Insulin resistance (IR)	Promotes de novo lipogenesis; impairs fatty acid oxidation; perpetuates hyperglycemia and hyperinsulinemia	Inflammatory and fibrotic liver damage; progression to MASH	[51,52,53,54]
	Hypertriglyceridemia	Increases toxic lipid species; generates small dense VLDL and LDL particles with high atherogenic potential	Oxidative stress, inflammation, liver and cardiovascular disease	[55,56,57,58]
	Low HDL-C	Impaired reverse cholesterol transport; pro-inflammatory HDL phenotype with reduced ApoAI and ApoAII levels	Systemic inflammation; hepatic lipid accumulation	[56,57,58]
	Impaired fasting glucose/Type 2 Diabetes Mellitus (T2DM)	Chronic hyperinsulinemia; promotes pro-inflammatory state; exacerbates hepatic lipid deposition	Accelerated liver injury, steatosis, fibrosis	[63,64,65]
	Arterial hypertension	Activation of RAAS; vascular fibrosis; decreased NO synthesis; impaired hepatic microcirculation	Worsens metabolic, cardiovascular, and hepatic dysfunction	[66,67,68,69,70]
Lean/Non-obese MASLD	Increased visceral adiposity	Excess intra-abdominal fat despite normal BMI; metabolic abnormalities (IR, dyslipidemia)	Hepatic fat accumulation, progression to steatohepatitis and fibrosis	[8,12]
	Subtle insulin resistance	Mild impairment in insulin action, undetected by conventional markers	Contributes to hepatic lipid accumulation	[50,51]
	Dyslipidemia	Elevated triglycerides, low HDL-C	Promotes hepatic steatosis	[53,54,55,56,57]
	Specific genetic and epigenetic factors unique to lean MASLD	Genetic variants distinct from classic MASLD	Different pathogenetic pathways; tailored diagnostic and therapeutic approaches	[8,12]
Drug-induced MASH	Tamoxifen	Induces hepatic steatosis via altered lipid metabolism and mitochondrial dysfunction	Drug-induced MASLD/MASH	[46]
	Corticosteroids	Increases gluconeogenesis and lipogenesis; promotes IR	Hepatic steatosis, progression to MASH	[46]
	Antipsychotics (e.g., olanzapine)	Promotes weight gain, IR, and dyslipidemia; direct hepatotoxic effects	MASLD/MASH secondary to pharmacologic agents	[46]
Other Contributing Factors	High-calorie diet	Excess caloric intake; stimulates de novo lipogenesis	Hepatic steatosis, progression to MASLD	[45,46,47,48,49,50,51,52,53,54]
	Physical inactivity	Reduced energy expenditure; promotes weight gain and IR	Increased risk of MASLD	[45,46,47,48,49,50,51,52,53,54]
	Intestinal dysbiosis	Alters gut–liver axis; increases intestinal permeability and endotoxemia	Inflammation, hepatic injury, progression to MASLD	[15]

Table 1. Risk factors and predisposition for MASLD.

Category	Specific Factor	Main Pathogenetic Mechanisms	Clinical Effects/Risks	Ref.
Genetic	PNPLA3 (I148M, rs738409, G allele)	Impaired lipase activity; accumulation of triglycerides in hepatocytes; lipid droplet remodeling dysfunction	Steatosis, NASH, Fibrosis, Increased risk of HCC	[25,26,27,28,29,30,31,32]
	PNPLA3 (rs6006460, G>T variant)	Modulates risk in protective direction	Potential protective role against hepatic lipid accumulation	[25,26,27,28,29,30,31,32]
	TM6SF2 (E167K, rs5842926, C>T variant)	Reduced VLDL secretion; hepatic lipid accumulation; lower total cholesterol and LDL levels	Progression to advanced hepatic fibrosis, MASLD-HCC	[33,34,35,36]
	GCKR (rs1260326, C>T variant)	Enhanced glycolysis and de novo lipogenesis; increased hepatic lipid accumulation	Progression to MASH and fibrosis	[37,38,39]
	MBOAT7 polymorphisms	Alters phospholipid remodeling; increases hepatic fat accumulation and inflammation	Associated with fibrosis and progression of MASLD	[25,26]
	HSD17B13 (rs72613567, T>TA variant)	Loss-of-function variant; reduced hepatic inflammation and fibrosis, especially in PNPLA3 I148M carriers	Protective against MASLD progression and HCC	[40,41,42]
Epigenetic	DNA methylation	Alters gene expression; promotes pro-inflammatory and pro-fibrotic pathways	Progression from MASLD to MASH, fibrosis, HCC	[43]
	Histone modifications	Modifies chromatin structure; impacts transcriptional activity of genes related to inflammation and fibrosis	Increased susceptibility to MASLD and progression to advanced liver disease	[43]
Epitranscriptomic	m6A RNA modification	Regulates RNA stability and translation; affects lipid metabolism and oncogenic pathways	Promotes MASLD progression and HCC development	[44]
	METTL3 (m6A writer) and YTHDF1 (m6A reader)	Modulate gene expression; activate oncogenic signals (e.g., EZH2, IL-6)	Immune dysfunction, enhanced tumorigenesis, risk of HCC	[43,44,45]
Metabolic Syndrome	Central obesity (increased waist circumference)	Visceral adipocyte hypertrophy; increased FFAs; portal delivery of lipids to the liver	Steatosis, mitochondrial dysfunction, ROS production, hepatocyte apoptosis	[46,47,48,49,50]
	Insulin resistance (IR)	Promotes de novo lipogenesis; impairs fatty acid oxidation; perpetuates hyperglycemia and hyperinsulinemia	Inflammatory and fibrotic liver damage; progression to MASH	[51,52,53,54]
	Hypertriglyceridemia	Increases toxic lipid species; generates small dense VLDL and LDL particles with high atherogenic potential	Oxidative stress, inflammation, liver and cardiovascular disease	[55,56,57,58]
	Low HDL-C	Impaired reverse cholesterol transport; pro-inflammatory HDL phenotype with reduced ApoAI and ApoAII levels	Systemic inflammation; hepatic lipid accumulation	[56,57,58]
	Impaired fasting glucose/Type 2 Diabetes Mellitus (T2DM)	Chronic hyperinsulinemia; promotes pro-inflammatory state; exacerbates hepatic lipid deposition	Accelerated liver injury, steatosis, fibrosis	[63,64,65]
	Arterial hypertension	Activation of RAAS; vascular fibrosis; decreased NO synthesis; impaired hepatic microcirculation	Worsens metabolic, cardiovascular, and hepatic dysfunction	[66,67,68,69,70]
Lean/Non-obese MASLD	Increased visceral adiposity	Excess intra-abdominal fat despite normal BMI; metabolic abnormalities (IR, dyslipidemia)	Hepatic fat accumulation, progression to steatohepatitis and fibrosis	[8,12]
	Subtle insulin resistance	Mild impairment in insulin action, undetected by conventional markers	Contributes to hepatic lipid accumulation	[50,51]
	Dyslipidemia	Elevated triglycerides, low HDL-C	Promotes hepatic steatosis	[53,54,55,56,57]
	Specific genetic and epigenetic factors unique to lean MASLD	Genetic variants distinct from classic MASLD	Different pathogenetic pathways; tailored diagnostic and therapeutic approaches	[8,12]
Drug-induced MASH	Tamoxifen	Induces hepatic steatosis via altered lipid metabolism and mitochondrial dysfunction	Drug-induced MASLD/MASH	[46]
	Corticosteroids	Increases gluconeogenesis and lipogenesis; promotes IR	Hepatic steatosis, progression to MASH	[46]
	Antipsychotics (e.g., olanzapine)	Promotes weight gain, IR, and dyslipidemia; direct hepatotoxic effects	MASLD/MASH secondary to pharmacologic agents	[46]
Other Contributing Factors	High-calorie diet	Excess caloric intake; stimulates de novo lipogenesis	Hepatic steatosis, progression to MASLD	[45,46,47,48,49,50,51,52,53,54]
	Physical inactivity	Reduced energy expenditure; promotes weight gain and IR	Increased risk of MASLD	[45,46,47,48,49,50,51,52,53,54]
	Intestinal dysbiosis	Alters gut–liver axis; increases intestinal permeability and endotoxemia	Inflammation, hepatic injury, progression to MASLD	[15]

4. Pathogenesis of MASLD

The pathogenesis of MASLD was initially described by the “two-hit hypothesis”, which proposed hepatic lipid accumulation (first hit) followed by oxidative stress and inflammation (second hit) [63]. However, this model has been recently surpassed by the “multiple-hit hypothesis”, recognizing the simultaneous and complex interplay of metabolic, genetic, immunological, and environmental factors [64].

Systemic inflammation is a cornerstone in the progression of MASLD, bridging metabolic dysfunction and chronic liver damage. While hepatic lipid accumulation is the initial event, progression to MASH and fibrosis depends heavily on immune system activation and chronic inflammatory states [65]. In individuals with MASLD, systemic inflammation not only contributes to liver damage but also exacerbates IR and endothelial dysfunction [66]. Elevated levels of cytokines such as tumor necrosis factor-α (TNF-α), IL-6, and IL-1β sustain low-grade chronic inflammation, amplified by the secretion of pro-inflammatory chemokines like monocyte chemoattractant protein-1 (MCP-1) from hypertrophic adipocytes [66,67].

At cellular level, IR promotes increased adipocyte lipolysis, resulting in a heightened influx of FFAs to the liver. The subsequent accumulation of FFAs in hepatic mitochondria generates ROS, fostering lipotoxicity and triggering both inflammatory and fibrotic processes [69,70]. Mitochondrial overload and ROS production contribute to hepatocyte necrosis and activation of stellate cells, which deposit collagen and drive fibrotic progression [71]. Oxidative stress, primarily from mitochondrial fatty acid oxidation, peroxisomal activity, and endoplasmic reticulum (ER) stress, inflicts damage on lipids, proteins, and DNA, thereby disrupting cellular functions and perpetuating innate immune responses [70,71,72,73]. Toxic lipid peroxidation produces toxic molecules such as MDA and 4-HNE, further damaging cellular membranes and amplifying inflammatory responses [74]. Oxidative stress activates hepatic stellate cells (HSCs), leading to the production of collagen and other extracellular matrix components, driving fibrotic progression [71]. In addition, mitochondrial dysfunction—marked by impaired oxidative phosphorylation and the induction of apoptotic processes—exacerbates both lipid accumulation and fibrogenesis [70,75]. Strategies aimed at enhancing mitochondrial biogenesis and employing selective antioxidants have thus emerged as promising therapeutic avenues [76].

Oxidative stress and redox dysregulation in the ER impair proper protein folding, activating the unfolded protein response (UPR). When UPR becomes maladaptive, it results in hepatocyte apoptosis, further exacerbating fibrosis [77,78]. Compounding these events, impaired lysosomal function, often a consequence of lipid accumulation that destabilizes lysosomes and results in the release of proteolytic enzymes such as cathepsin B, promotes inflammation and cell death [79]. Defective autophagy and reduced lysosomal acid lipase activity exacerbate intrahepatic lipid accumulation, self-perpetuating the cycle of liver damage [80,81].

Apoptosis, regulated by Bcl-2 family proteins, is a crucial process in MASLD progression. Pro-apoptotic proteins like Bax and Bak induce mitochondrial membrane permeabilization, causing cytochrome c release and caspase activation [82,83]. Lipid accumulation activates “BH3-only” proteins that interact with Bcl-2, intensifying hepatocyte apoptosis and accelerating progression to MASH and fibrosis [84,85]. Additionally, ferroptosis, a form of cell death mediated by iron-dependent lipid peroxidation, has emerged as a key mechanism in MASLD. Reduced activity of glutathione peroxidase 4 (GPX4) in the liver impairs ROS neutralization, exacerbating lipid accumulation and fibrosis [86,87]. The use of ferroptosis inhibitors, such as liproxstatin-1, has shown promising effects in preventing liver damage and improving lipid homeostasis, suggesting a potential therapeutic target [88,89].

The immune system plays a central role in MASLD progression. Kupffer cell activation by danger signals, such as LPS and lipid peroxidation products, induces the secretion of pro-inflammatory cytokines (TNF-α, IL-6) and amplifies hepatic oxidative stress [90,91]. Neutrophils also contribute to liver damage by releasing proteolytic enzymes and ROS. The formation of neutrophil extracellular traps (NETs) is associated with fibrosis and hepatocarcinogenesis [92]. T cells and NK cells play immunomodulatory roles: CD8+ T cells promote chronic liver damage, while NK cells limit fibrosis through direct cytotoxicity. However, their functionality can be compromised in chronic inflammation settings, facilitating disease progression [93].

Collectively, these complex cellular and molecular events drive the progression of MASLD from simple steatosis to MASH and advanced fibrosis. Notably, emerging evidence highlights the gut–liver axis as a critical link between hepatic injury and intestinal barrier dysfunction. Disruptions in the intestinal barrier, increasing permeability and allow microbial products such as LPS to enter the portal circulation, further exacerbate hepatic inflammation and fibrogenesis. By facilitating the translocation of pro-inflammatory molecules from the compromised gut barrier to the liver, the gut–liver axis plays a central role in triggering and sustaining inflammatory and fibrotic responses [15]. A deeper understanding of the pathogenic mechanisms underlying MASLD offers new opportunities for targeted therapeutic interventions. Strategies that modulate oxidative stress, improve mitochondrial function, inhibit ferroptosis, and regulate immune responses could represent innovative approaches to prevent disease progression and improve clinical outcomes.

5. Intestinal Barriers

The intestine is one of the largest and most dynamic mucosal surfaces in the human body, serving as a crucial barrier against pathogenic microorganisms and toxic substances. In addition to its role in digestion and nutrient absorption, the intestine acts as the first line of defense against external threats, playing a key role in the interaction between the external environment and the host’s internal microenvironment [94]. The gut–liver axis, mediated by the portal circulation, represents a critical anatomical and functional connection for regulating metabolic and immune flows. This bidirectional relationship allows for dynamic interaction between the intestine and liver, modulating systemic metabolism and immunity [95].

The human intestine is divided into two main sections: the small intestine (comprising the duodenum, jejunum, and ileum) and the large intestine (including the colon, sigmoid colon, and rectum). The morphological and functional structure of these two sections is highly specialized. The small intestine, with its villi and crypts, offers a vast absorptive surface supported by specialized epithelial cells such as enterocytes and enteroendocrine cells, essential for selective molecular transport [96,97]. In contrast, the large intestine, characterized by a flatter surface, maintains barrier integrity through the continuous renewal of epithelial cells generated in the crypts of Lieberkühn, including goblet cells, Paneth cells, and stem cells [98,99]. The intestinal barrier, composed of epithelial cells tightly connected by tight junctions, a mucus layer, and immune components, regulates the interaction between the gut microbiota and the mucosal immune system, playing an integrated role in defending against pathogens and toxins [100]. The selective permeability of this barrier enables the absorption of essential nutrients while limiting the passage of toxins and pathogenic microorganisms. However, environmental factors, gut microbiota dysbiosis, and inflammatory conditions can impair this function, leading to increased intestinal permeability, known as “leaky gut” [94]. This phenomenon facilitates the translocation of pro-inflammatory molecules, such as LPS, into the portal circulation. LPS, a component of Gram-negative bacterial membranes, activates hepatic immune cells, such as Kupffer cells, inducing local and systemic inflammation that contributes to the progression of MASLD into MASH and liver fibrosis [101]. Recent evidence indicates a correlation between plasma LPS levels and the severity of liver disease in patients with MASLD, underscoring the pathogenic role of altered intestinal permeability [102,103]. The liver, receiving 70% of its blood supply from the portal vein, is exposed to the metabolic products of the gut microbiota, such as SCFAs, secondary bile acids, and other microbial metabolites. These compounds modulate hepatic metabolism and immune responses, directly influencing the pathophysiology of liver diseases [95]. The integrated and interdependent system characterizing the gut–liver axis provides a fundamental framework for understanding liver disease pathogenesis, opening new perspectives for developing targeted therapeutic strategies.

The intestine employs a complex system of barriers divided into three main levels: mechanical, immune, and microbial. These barriers work synergistically to preserve intestinal homeostasis, prevent pathogenic invasion, and ensure the selective absorption of nutrients [104]. The mechanical barrier includes the intestinal epithelium, mucus-producing goblet cells, Paneth cells that secrete antimicrobial peptides, and enteric glial cells [105,106,107]. Intestinal epithelial cells (IECs), arranged in a monolayer connected by junctional complexes such as tight junctions, adherens junctions, and desmosomes, ensure the barrier’s selectivity [108]. The mucus layer, particularly developed in the colon, acts as a chemical filter preventing direct contact between microorganisms and the intestinal epithelium. Gel-forming mucins, such as MUC2, are the main structural components of mucus, creating a protective network that harbors the commensal microbiota [109]. The immune barrier consists of innate and adaptive immune cells as well as IECs with immune functions. IECs release cytokines such as IL-10 and antimicrobial peptides to modulate immune homeostasis and prevent excessive bacterial proliferation [110]. Dendritic cells, regulatory T cells (Tregs), and plasma cells in the lamina propria contribute to immune tolerance, partly through the production of secretory immunoglobulin A (SIgA) [111,112].

The gut microbiota, comprising trillions of microorganisms, serves as a biological barrier by competing with pathogens for resources and producing beneficial metabolites, such as SCFAs and secondary bile acids [113,114]. Alterations in microbiota balance, referred to as dysbiosis, are associated with inflammatory, metabolic, and autoimmune diseases, emphasizing the importance of maintaining a healthy microbiota to preserve intestinal barrier integrity [115].

6. Gut Microbiota and Microbiome

Life on Earth has evolved from a common unicellular ancestor, giving rise to the remarkable biological complexity observed today, with sophisticated cellular and systemic organization. Within this context, humans emerge as a superorganism, integrated with trillions of symbiotic bacteria that coexist synergistically with the host’s eukaryotic cells. This dynamic entity, termed the “holobiont”, encompasses both the human genome and the microbial genome, collectively referred to as the “hologenome”. Variation within this system can occur through changes in either the host or microbial genome, maintaining a balance between stability and adaptability. In this context, humans function as a “superorganism”, coexisting with trillions of symbiotic microorganisms that form a dynamic entity known as the “holobiont”. This holobiont integrates both the human genome and the microbial genome, collectively referred to as the “hologenome”. Variations within this system can occur due to changes in either the host or the microbial genome, balancing stability with adaptability [116,117].

Following the completion of the Human Genome Project in 2001, it became evident clear that a comprehensive deeper understanding of human biology requires exploring interactions between the human host organism and its microbiota [118,119,120]. Subsequent initiatives, such as the Human Microbiome Project (HMP) and the Metagenomics of the Human Intestinal Tract (MetaHIT), revolutionized research by focusing on four main microbial colonization sites: the mouth, gut, vagina, and skin. Among these, the gut microbiota has garnered particular interest due to its clinical relevance [121]. The human gut, with a mucosal surface area of approximately 200–300 m², harbors a diverse community ten trillion symbionts belonging to about 50 bacterial phyla and 100–1000 species of symbiotic microorganisms, encompassing around 50 bacterial phyla and hundreds of species. The term “microbiota” refers to the collection of microorganisms colonizing a specific habitat, such as the gut, and includes bacteria, viruses, fungi, and archaea. The “microbiome”, on the other hand, represents the entire refers to the complete genetic repertoire of these microbial communities and their interactions with the host environment [122]. While interconnected, the microbiota reflects the physical entities of the microorganisms, whereas the microbiome expresses their functional potential. While the microbiota reflects the physical organisms, the microbiome captures their functional potential [123,124].

The analysis study of gut microbiota employs culture-dependent and culture-independent methods. Traditional culture-dependent techniques, first developed by Robert Koch in 1881, involve isolating and characterizing microorganisms but are limited in scope, capturing only 30–50% of the intestinal bacteria due to growth challenges. Advances in enriched culture media and pre-incubation methods have expanded the ability to isolate previously uncultivable species [125]. Furthermore, culturomics, combining extensive cultivation with mass spectrometry and 16S rRNA sequencing, has led to the identification of over 1000 prokaryotic species, including novel ones [126]. Culture-independent methods, initially based on Sanger sequencing and 16S rRNA analysis, have evolved with metagenomic sequencing and next-generation sequencing (NGS) technologies, improving efficiency and reducing costs [127]. More recently, third-generation technologies, such as PacBio and MinION, have enabled rapid detection of epigenetic modifications and pathogens [128].

The human gastrointestinal tract presents distinct microenvironments that exert a profound influence on the composition and distribution of the resident microbiota [129,130,131,132]. These ecological niches vary significantly along the gastrointestinal axis, primarily due to differences in pH, oxygen tension, nutrient availability, and immune surveillance mechanisms. The stomach represents the first critical barrier to microbial colonization. Its highly acidic environment, with a pH typically ranging between 1 and 3, creates an inhospitable setting for most microorganisms. Nevertheless, a limited number of acid-resistant species are capable of colonizing this region. Among them, Streptococcus, Neisseria, and Lactobacillus are the predominant genera identified, belonging mainly to the phyla Firmicutes, Bacteroidetes, and Proteobacteria (

Table 2. Gut microbiota composition across intestinal sections.

Intestinal Section	Predominant Bacteria	Main Functions	Ref.
Stomach	Streptococcus, Neisseria, Lactobacillus	Acid resistance, limited diversity	[129]
Small Intestine	Aerobic Gram-positive (duodenum), anaerobes (ileum)	Nutrient absorption, gradual transition	[130,131]
Large Intestine	Firmicutes, Bacteroidetes.	Fermentative role; Fermentation of complex carbohydrates, SCFA synthesis	[132,133]

SCFA: short-chain fatty acid.

) [129]. Despite the low bacterial density and diversity in the stomach, these microorganisms play pivotal roles in maintaining mucosal homeostasis and contributing to the early phases of digestion. Progressing distally, the small intestine offers a more favorable environment for microbial growth, though the density and diversity of microorganisms vary considerably along its length. In the duodenum, bacterial concentrations are relatively low, ranging from 10³ to 10⁴ colony-forming units per milliliter (CFU/mL), primarily composed of Gram-positive aerobic species [130]. As the luminal content moves towards the ileum, bacterial density markedly increases, reaching up to 10⁹ CFU/mL. This transition is accompanied by a compositional shift favoring anaerobic and Gram-negative bacteria, which thrive in the progressively less oxygenated environment [130,131]. The small intestine microbiota plays essential roles in nutrient absorption, the metabolism of bile acids, and the modulation of the mucosal immune system. The large intestine, or colon, constitutes the most densely populated microbial habitat within the human body, harboring an estimated 10¹² CFU/mL [132]. It serves as the principal site for the fermentation of indigestible carbohydrates and the synthesis of SCFAs, such as acetate, propionate, and butyrate, which are critical for energy homeostasis and epithelial integrity [132,133]. The colonic microbiota is dominated by species within the Firmicutes and Bacteroidetes phyla, with these taxa playing vital roles in regulating local immune responses and maintaining intestinal barrier function.

The overall composition of the gut microbiota is shaped by a complex interplay of genetic, environmental, and dietary factors. Among the dominant phyla consistently observed in healthy individuals are Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrucomicrobia [134]. These microbial communities contribute to key physiological processes, including energy harvest, immune modulation, and protection against pathogenic invasion. Notably, specific genera such as Clostridium, Lactobacillus, Bacteroides, and Akkermansia muciniphila have been identified as crucial players in maintaining metabolic balance and immune homeostasis [134,135,136].

The gut microbiota represents a highly complex and dynamic ecosystem that performs several critical functions essential for maintaining host health (

Table 3. Critical functions of the gut microbiota and their impact on host health.

Function	Description	Ref.
Nutrient Metabolism	Fermentation of dietary fibers, producing SCFAs (acetate, butyrate, propionate) essential for energy homeostasis and inflammatory modulation. Synthesis of essential vitamins, such as vitamin K and B-group vitamins.	[114,137]
Intestinal Barrier Integrity	Modulation of tight junction proteins and increased mucin production to prevent pathogen translocation.	[138]
Immune Modulation	Regulation of immune development and tolerance through interactions with dendritic cells, regulatory T cells, and SIgA production.	[136,139]
Production of Bioactive Metabolites	Beyond SCFAs, the microbiota produces metabolites such as TMAO and phenolic compounds, influencing systemic metabolism, including liver functions.	[140]

SCFAs: short-chain fatty acids, TMAO: trimethylamine N-oxide, SIgA: secretory immunoglobulin A.

). Among its primary roles is the metabolism of nutrients. The microbial community actively ferments dietary fibers, resulting in the production of SCFAs, such as acetate, butyrate, and propionate. These metabolites are fundamental for sustaining energy homeostasis and modulating inflammatory responses at both the intestinal and systemic levels [137]. In addition, the gut microbiota contributes to the biosynthesis of essential vitamins, notably vitamin K and members of the B-group vitamin family, which play pivotal roles in coagulation, cellular metabolism, and neurological function [114]. Another vital function of the gut microbiota is the maintenance of intestinal barrier integrity. By modulating the expression and organization of tight junction proteins and promoting mucin production, the microbiota fortifies the mucosal barrier, thereby preventing the translocation of pathogenic microorganisms and harmful antigens into the systemic circulation [138]. This barrier function is crucial for preserving intestinal homeostasis and preventing inflammatory disorders. Furthermore, the gut microbiota plays an indispensable role in the regulation of immune responses. It orchestrates the development and maturation of the host’s immune system, fostering tolerance to commensal organisms while maintaining vigilance against potential pathogens. Interactions between the microbiota and immune components such as dendritic cells, regulatory T cells, and the production of secretory immunoglobulin A (SIgA) are fundamental to this modulatory activity [136,139]. Beyond its metabolic and immunological functions, the gut microbiota is also responsible for producing a wide range of bioactive metabolites. In addition to SCFAs, microbial metabolism gives rise to compounds such as TMAO and various phenolic derivatives. These metabolites exert significant effects on systemic metabolism, influencing processes that include lipid metabolism, glucose homeostasis, and liver function [140].

The diversity and functional complexity of the gut microbiota underscore its integral role in sustaining both intestinal and systemic health. The intricate interplay between host physiology and microbial activity not only supports gut homeostasis but also has far-reaching implications for overall metabolic regulation. This highlights the gut microbiota as a promising target for therapeutic strategies aimed at preventing and managing a broad spectrum of diseases.

7. Alterations in Intestinal Barriers in MASLD Pathogenesis

MASLD is closely associated with intestinal barrier dysfunction, including tight junction (TJ) alterations, increased intestinal permeability, and dysbiosis. These factors interact in a complex pathological cycle, amplifying inflammation and liver damage.

The intestinal mechanical barrier serves as the first line of defense against pathogens, and its impairment is a critical element in MASLD pathogenesis. Disruption of the mucus layer and protective chemical compounds compromise intestinal homeostasis, favoring bacterial overgrowth and increasing intestinal permeability [141]. Recent studies have demonstrated that epithelial barrier dysfunction in patients with MASH and animal models is linked to chronic mucosal inflammation. Under these conditions, tight junction proteins undergo contraction and cytoplasmic dislocation, which heightens mucosal permeability, allowing for the translocation of bacteria and microbial products, such as LPS [141,142]. Once in the portal circulation, LPS reaches the liver and stimulates Kupffer cells, triggering the release of pro-inflammatory cytokines that drive liver injury [143].

The gut vascular barrier (GVB) plays a crucial role in preventing bacterial dissemination. In individuals with MASLD, increased expression of PV-1, a GVB permeability marker, reflects compromised integrity. Studies have shown that during MASH progression, enteric pathogens interfere with the WNT/β-catenin pathway in endothelial cells, exacerbating liver damage [144,145]. The intestinal immunological barrier, primarily composed of SIgA, lymphocytes, and plasma cells, plays a key role in modulating mucosal immunity. SIgA helps neutralize pathogens by specifically binding to Gram-negative bacteria. However, in patients with MASLD, immunological barrier functionality is compromised, promoting bacterial translocation and inflammation [146,147].

The gut–liver axis is central to MASLD pathogenesis. Damage-associated molecular patterns (DAMPs) and microbe-associated molecular patterns (MAMPs) activate sterile inflammatory responses in the liver, contributing to hepatic damage [148]. Elevated serum IgA levels have been observed in patients with MASLD, correlating with advanced fibrosis and increased activation of plasma cells in secondary lymphoid organs [149,150]. In murine models, hepatic B lymphocytes are stimulated by intestinal microbial factors which exacerbate inflammation and liver fibrosis [151]. Similarly, patients with MASLD exhibit reduced regulatory T cells (Tregs) expressing FOXP3 and increased Th1 and CD8+ T cells in the intestinal lamina propria [15]. Mast cells (MCs), which are present in the intestinal barrier, also contribute to dysfunction. These cells release cytokines, histamine, and proteases, including trypsin and chymase, that degrade ZO-1 protein, reduce JAM-A expression, and compromise epithelial integrity [152,153]. Histamine, in particular, has been shown to increase intestinal permeability and bacterial translocation, aggravating inflammation [153,154].

The intestinal microbial barrier is essential for maintaining microbiota homeostasis and preventing pathogenic colonization. However, a Western diet (WD), rich in saturated fats, cholesterol, and refined carbohydrates, is associated with dysbiosis and reduced populations of commensal bacteria, favoring the proliferation of pro-inflammatory species such as Gram-negative bacteria [155,156]. These changes increase intestinal permeability and activate Toll-like receptor (TLR) family signaling, promoting LPS-mediated inflammation and endotoxin activity [157,158]. Obesity and hypercaloric diets also induce dysbiosis and metabolic alterations, characterized by reduced microbial diversity and a lower Firmicutes/Bacteroidetes ratio in individuals with obesity compared to lean controls [159,160]. In patients with MASLD, the intestinal microbiome exhibits an altered “signature”, characterized by increased Proteobacteria, Enterobacteria, Escherichia, and Bacteroides species, alongside reduced Firmicutes species [160,161]. These microbial shifts not only compromise the intestinal barrier but also exacerbate the gut–liver inflammatory cycle.

The alterations in intestinal barriers (

Table 4. Mechanisms of intestinal barrier dysfunction in MASLD.

Type of Damage	Description	Consequences on MASLD	Ref.
Mechanical	Alterations in tight junctions, increased permeability	Bacterial translocation, activation of Kupffer cells	[141,142,143,144,145]
Immunological	Reduced production of SIgA, increased pro-inflammatory cytokines	Systemic inflammation, liver damage	[146,147,148,149,150,151,152,153,154]
Microbial	Decreased microbial diversity, increased pathogenic bacteria	Dysbiosis, activation of the gut–liver inflammatory cycle	[155,156,157,158,159,160,161]

SIgA: secretory immunoglobulin A.

)—mechanical, immunological, and microbial—play a pivotal role in MASLD pathogenesis, contributing to chronic inflammation and liver damage. While the underlying mechanisms are complex and interdependent, understanding these dynamics is essential for developing targeted therapeutic interventions aimed at restoring intestinal homeostasis and modulating inflammatory responses associated with MASLD.

8. Gut Dysbiosis in the Pathogenesis of MASLD

Gut dysbiosis is a pivotal factor in the pathogenesis of MASLD, influencing key processes such as hepatic metabolism, intestinal barrier integrity, and the activation of systemic inflammatory signaling. The gut microbiota plays a crucial role in maintaining metabolic homeostasis, but alterations in its composition and function can increase intestinal permeability. This allows bacteria and microbial products, such as LPS, to enter the portal circulation, activating Kupffer cells via TLRs. This cascade triggers an inflammatory response that exacerbates liver damage [162,163].

Diet is one of the main drivers of gut microbial dysbiosis. A Western diet (WD), rich in refined sugars and saturated fats, reduces microbial diversity and promotes the overgrowth of Gram-negative bacteria such as Bacteroidetes, while decreasing beneficial bacteria such as Prevotella [151,164]. This microbial imbalance disrupts the production of SCFAs, which play vital roles in regulating gut barrier integrity and inflammation. While butyrate supports intestinal health and reduces inflammation, acetate, the most abundant SCFAs, has obesogenic effects and contributes to hepatic lipid accumulation [165]. In addition, diets high in saturated fats and taurine-conjugated bile salts promote the production of hydrogen sulfide (H₂S), a toxic molecule that further damages the intestinal barrier and exacerbates inflammation, accelerating MASLD progression [166].

The gut microbiota also influences lipid metabolism through the activation of lipoprotein lipase (LPL) and modulation of AMP-activated protein kinase (AMPK), which regulates lipid oxidation and metabolic balance. Furthermore, microbial communities chemically modify bile acids, affecting hepatic glucose and lipid metabolism [167].

As gut dysbiosis compromises the intestinal barrier, bacterial translocation is facilitated, allowing bacterial components and endotoxins to enter the portal circulation. These elements reach the liver, where they activate Kupffer cells via TLRs, amplifying the inflammatory response and promoting liver damage [168]. Upon arrival in the liver, gut-derived endotoxins, primarily LPS, bind to TLR4 on Kupffer cells, initiating a MyD88-dependent signaling cascade [167,168]. This cascade activates nuclear factor-κB, leading to the transcription of numerous pro-inflammatory cytokines such as TNF-α and pro-IL-1β [167,168]. Importantly, TLR activation also primes the assembly of the NLRP3 inflammasome, a multiprotein complex that serves as a critical mediator of inflammation [167,168]. A secondary signal, often in the form of ROS or other metabolic stressors, is then required to fully activate the inflammasome [167,168]. Once activated, caspase-1 cleaves pro-IL-1β and pro-IL-18 into their active forms, IL-1β and IL-18, which further amplify hepatic inflammation and contribute to the progression of liver injury in MASLD [167,168]. This TLR–inflammasome axis thus provides a crucial mechanistic link between gut barrier dysfunction, microbial dysbiosis, and the chronic inflammatory state observed in MASLD. Moreover, microbial fermentation of carbohydrates generates endogenous ethanol, which is transported to the liver via the portal vein. This ethanol exacerbates oxidative stress and mitochondrial dysfunction, worsening hepatic steatosis. Elevated levels of alcohol-producing bacteria have been found in individuals with MASLD, highlighting the significance of this pathogenic pathway [169,170].

Patients with MASLD exhibit reduced microbial diversity, with a marked increase in Gram-negative bacteria and a decrease in beneficial species such as Prevotella. Recent studies have suggested that targeted dietary interventions, such as supplementation with oat beta-glucan and alanyl-glutamine dipeptides, can restore microbial balance and improve the pathological features of MASLD [171]. Gut dysbiosis also impacts immune responses, compromising the intestinal barrier and facilitating bacterial translocation. This contributes to chronic systemic inflammation, driven by cytokines like TNF-α, which further exacerbates IR and liver damage [105,171] Additionally, reduced secretion of IgA worsens intestinal permeability and mucosal dysfunction [172].

The gut–liver axis—characterized by interactions between the intestinal epithelial, vascular, and immune barriers and the hepatic system—plays a central role in MASLD. Dysregulation of this axis, driven by microbial dysbiosis and increased intestinal permeability, promotes TLR activation and chronic hepatic inflammation [173,174]. Changes in the Firmicutes/Bacteroidetes ratio, reductions in beneficial species like Akkermansia muciniphila and Faecalibacterium prausnitzii, and the accumulation of pro-inflammatory metabolites such as TMAO and H₂S are strongly associated with MASLD progression [39,175,176]. Gut dysbiosis is thus a critical contributor to MASLD pathogenesis, influencing inflammatory, metabolic, and immune responses (Figure 1). Therapeutic strategies aimed at restoring microbial balance—such as probiotics, prebiotics, and dietary interventions—hold promise for mitigating disease progression. However, further research is needed to develop personalized interventions that fully leverage the potential of the gut microbiota in managing MASLD.

9. Dietary Approach and Gut Microbiota Modulation in the Management of MASLD: Evidence and Perspectives

9.1. Dietary Interventions in MASLD

A weight loss of 7–10% from the initial body weight is recommended to achieve a negative energy balance and improve hepatic outcomes. This objective can be attained through a hypocaloric diet, with an energy deficit of 500–1000 kcal per day, combined with a structured exercise program [177,178]. Although the role of ultra-processed foods and alcohol in MASLD remains debated, emerging evidence favors the Mediterranean diet (MD). Its nutritional profile—rich in dietary fiber, monounsaturated and polyunsaturated fatty acids, and bioactive compounds with anti-inflammatory and antioxidant properties—has demonstrated efficacy in reducing hepatic steatosis and improving metabolic parameters, even in patients with type 2 diabetes mellitus (T2DM) [3,179,180]. Dietary modifications have a profound impact on the gut microbiota. Chronic consumption of a high-fat diet in murine models has been shown to increase the Firmicutes/Bacteroidetes ratio, a feature associated with enhanced metabolic efficiency and hepatic lipid accumulation [181]. Conversely, a fiber-rich diet exerts protective effects by attenuating hepatic inflammation and promoting the proliferation of beneficial bacterial species such as Akkermansia muciniphila [182]. Moderate coffee intake has also been associated with a protective role against MASLD and its progression to MASH and fibrosis. The proposed mechanisms include enhanced glutathione synthesis, reduced oxidative stress via the scavenging of reactive oxygen species (ROS), and modulation of gut microbiota composition, specifically, the restoration of the Firmicutes/Bacteroidetes ratio and promotion of probiotic species such as Bifidobacterium [183,184,185].

9.2. Role of Exercise in Modulating the Gut Microbiota

Exercise is a well-established non-pharmacological intervention that exerts beneficial effects on metabolic outcomes and modulates the gut microbiota. Studies conducted in athletes and healthy individuals have demonstrated that regular physical activity increases microbial diversity, with a higher relative abundance of Akkermansia muciniphila observed in lean individuals compared to those with elevated body mass index (BMI) [186]. In animal models, exercise has been shown to reduce certain microbial taxa (e.g., Lactobacillaceae, Proteobacteria, Bacteroidetes, Flavobacterium, Alkaliphilus), decrease the Firmicutes/Bacteroidetes ratio, and increase populations within the phylum Verrucomicrobia and the family Turicibacteraceae [187]. When combined with a high-fat diet, exercise provides benefits beyond caloric restriction alone, improving insulin sensitivity, lowering LDL cholesterol levels, and reducing hepatic fat accumulation and serum triglycerides [188].

9.3. Antibiotics and Gut Microbiota Modulation in MASLD

Antibiotics have been explored as a therapeutic option to modulate the gut microbiota in MASLD, although the evidence remains inconclusive. Preclinical studies have demonstrated that norfloxacin and neomycin can improve hepatic function by altering microbiota composition and reducing bacterial translocation; however, a randomized controlled trial (RCT) in obese patients with MASLD did not demonstrate significant metabolic benefits [189]. In contrast, the combination of metronidazole and inulin has been associated with significant reductions in serum alanine aminotransferase (ALT) levels compared to placebo [190]. Rifaximin, one of the most extensively studied non-absorbable antibiotics, has been shown to have modest effects on fecal bile acid profiles in patients with cirrhosis [191]. More recently, an RCT demonstrated that six months of rifaximin therapy (1100 mg/day) resulted in significant reductions in serum ALT (from 64.6 ± 34.2 to 38.2 ± 29.2; p = 0.01) and aspartate aminotransferase (AST) levels (from 66.5 ± 42.5 to 41.8 ± 30.4; p = 0.02), as well as improvements in insulin resistance, pro-inflammatory cytokines, endotoxemia, cytokeratin-18 (CK-18), and the NAFLD steatosis score [192]. Nonetheless, the effects of rifaximin in MASLD remain variable: while some studies have reported improvements in ALT levels, reductions in endotoxins, and lower pro-inflammatory cytokine concentrations [193], others have not observed significant benefits [194].

9.4. Probiotics, Prebiotics, and Synbiotics in MASLD Management

Probiotics, prebiotics, and synbiotics are emerging as promising adjunctive therapies in MASLD. Probiotics are live microorganisms that confer health benefits by reducing hepatic inflammation, oxidative stress, and fat accumulation. Prebiotics, which are non-digestible food components, selectively stimulate the growth and/or activity of beneficial gut bacteria. Synbiotics combine both approaches, enhancing probiotic survival and efficacy. Although not curative, these interventions may complement standard MASLD management strategies [195]. For instance, a randomized controlled trial demonstrated that six months of probiotic supplementation significantly improved the AST-to-platelet ratio index in patients with non-alcoholic steatohepatitis (NASH) [195]. Another study found that 12 weeks of probiotic treatment reduced hepatic inflammation, although histological evidence remains limited [196]. Conversely, an RCT showed that 24 weeks of probiotic supplementation did not yield additional cardiovascular benefits over placebo in patients with MASH [197]. Studies evaluating prebiotics have shown mixed results. Oligofructose supplementation (16 g/day for eight weeks) significantly reduced AST levels but had no effect on serum triglycerides [23]. A systematic review highlighted methodological limitations across studies, reducing the strength of evidence supporting prebiotic use in MASLD management [198]. A recent systematic review suggests that probiotics and prebiotics may improve metabolic and inflammatory parameters, including BMI, cholesterol, triglycerides, HOMA-IR, transaminases, gamma-glutamyl transferase (GGT), and pro-inflammatory cytokines [199]. Synbiotics, in particular, have demonstrated beneficial effects on BMI, AST, ALT, GGT, and the NAFLD fibrosis score, as confirmed by a network meta-analysis of 26 RCTs [200]. Additional research has reported improvements in inflammatory markers (e.g., TNF-α and C-reactive protein) and liver histological parameters following synbiotic treatment. Specifically, 24 weeks of synbiotic supplementation with Bifidobacterium longum and fructo-oligosaccharides led to significant reductions in hepatic inflammation and histological activity in patients with NASH [201]. Another study observed reduced steatosis on ultrasound after eight weeks of synbiotic therapy, although ALT, AST, and CRP levels remained unchanged [202]. Moreover, a study in lean MASLD demonstrated that 28 weeks of synbiotic supplementation significantly reduced hepatic fibrosis, steatosis, and inflammatory markers [203].

9.5. Fecal Microbiota Transplantation in MASLD

Fecal microbiota transplantation (FMT) is an emerging therapeutic strategy that involves transferring fecal material from a healthy donor to a recipient, with the goal of restoring intestinal eubiosis. FMT specifically targets the gut–liver axis by improving intestinal barrier integrity and reducing systemic inflammation [204,205,206]. In clinical settings, a randomized controlled trial (RCT) demonstrated that a single allogenic FMT infusion improved small intestinal permeability—as assessed by the lactulose/mannitol test—in patients with MASLD, although it did not significantly impact hepatic steatosis or insulin resistance [207]. Conversely, a pilot study administering FMT over three consecutive days reported a modest but statistically significant reduction in hepatic steatosis [208]. Long-term follow-up studies in patients with cirrhosis have shown sustained clinical and cognitive improvements, including reductions in hepatic encephalopathy episodes and hospitalizations [209]. Donor characteristics are critical determinants of FMT efficacy. Evidence suggests that FMT from lean donors improves insulin sensitivity in individuals with metabolic syndrome, whereas FMT from obese donors may transiently impair insulin sensitivity [210,211]. FMT can be administered via oral capsules, nasogastric tubes, enemas, or colonoscopy, with the latter often considered the most effective method for ensuring microbiota engraftment [212,213]. Notably, oral-capsule-based FMT has demonstrated safety and efficacy in modulating gut microbiota composition and reducing circulating levels of taurocholate, a bile acid implicated in hepatic injury and fibrosis [214,215,216].

Targeting gut microbiota through dietary interventions, exercise, antibiotics, probiotics, synbiotics, and fecal microbiota transplantation represents a promising avenue for MASLD management (

Table 5. Summary of therapeutic approaches targeting gut microbiota in MASLD management.

Therapeutic Approach	Intervention and Key Findings	Mechanism and Outcomes	Ref.
Dietary Interventions	Hypocaloric Diet and Exercise: Achieving 7–10% weight loss via a 500–1000 kcal/day deficit improves hepatic outcomes. Mediterranean Diet (MD): Rich in fiber, monounsaturated/polyunsaturated fats, and bioactive compounds; reduces hepatic steatosis and improves metabolic parameters. Moderate Coffee Intake: Associated with reduced oxidative stress and modulation of gut microbiota.	Promotes a negative energy balance and improves metabolic profiles. MD’s anti-inflammatory and antioxidant properties support hepatic and gut health, including a favorable gut microbiota composition (e.g., increased Akkermansia muciniphila).	[177,178,179,180,181,182,183,184,185]
Exercise	Regular physical activity in both human and animal studies. Observed increase in microbial diversity, particularly beneficial species (e.g., Akkermansia muciniphila), and modulation of gut taxa.	Enhances insulin sensitivity, lowers LDL cholesterol, and reduces hepatic fat accumulation and serum triglycerides. Modulates the gut microbiota by decreasing the Firmicutes/Bacteroidetes ratio and influencing specific taxa.	[186,187,188]
Antibiotics	Use of norfloxacin and neomycin in preclinical studies improves hepatic function by altering microbiota and reducing bacterial translocation. Combination of metronidazole and inulin shows significant reductions in serum ALT. Rifaximin therapy (1100 mg/day for 6 months) improves liver enzymes and inflammatory markers, although results are variable.	Alters gut microbiota composition, reducing harmful bacterial translocation. Rifaximin modulates fecal bile acid profiles, improves insulin resistance, and reduces pro-inflammatory cytokines, though its efficacy may vary across studies.	[189,190,191,192,193,194]
Probiotics, Prebiotics, and Synbiotics	Probiotics: Supplementation improves the AST-to-platelet ratio and may reduce hepatic inflammation (varied durations). Prebiotics: Oligofructose (16 g/day for 8 weeks) reduces AST levels, though effects on triglycerides are minimal. Synbiotics: Combined treatments have shown improvements in BMI, liver enzymes (AST, ALT, GGT), NAFLD fibrosis score, and inflammatory markers.	Modulate gut microbiota to reduce hepatic inflammation and oxidative stress. Enhance growth of beneficial bacteria, thereby improving metabolic parameters and reducing liver fat accumulation. Some trials report mixed results, underlining the need for further research.	[195,196,197,198,199,200,201,202,203]
Fecal Microbiota Transplantation (FMT)	FMT administered via oral capsules, nasogastric tubes, enemas, or colonoscopy. Single or multiple infusions have shown improvement in small intestinal permeability, modest reductions in hepatic steatosis, and long-term benefits in cirrhosis (e.g., reduced hepatic encephalopathy episodes).	Restores intestinal eubiosis and improves barrier integrity. Reduces systemic inflammation by modulating the gut–liver axis. Donor characteristics (lean vs. obese) are crucial for efficacy, influencing outcomes such as insulin sensitivity.	[204,205,206,207,208,209,210,211,212,213,214,215,216]

). While emerging evidence supports their metabolic and hepatic benefits, further research is needed to define optimal therapeutic strategies and long-term outcomes.

10. Conclusions

In recent years, the gut–liver axis has emerged as a central player in MASLD pathogenesis, where alterations in intestinal barriers increase permeability, promote bacterial translocation, and trigger hepatic inflammation and fibrosis. Dysbiosis, characterized by a rise in pro-inflammatory bacteria and harmful metabolites (e.g., LPS, TMAO), exacerbates metabolic dysfunction and liver injury through TLR activation and NLRP3 inflammasome assembly.

Lifestyle modifications, particularly a Mediterranean diet, physical activity, and weight loss, remain the cornerstone of MASLD management. Targeted microbiota-based interventions (probiotics, prebiotics, synbiotics, FMT) show promise, but further large-scale RCTs are needed to confirm their efficacy.

Advances in multi-omics technologies (metagenomics, metabolomics, proteomics) offer new insights into microbiota–host interactions, facilitating biomarker discovery and personalized therapies [217,218]. Integrating clinical, genetic, and microbiome data may revolutionize MASLD management, paving the way for precision medicine. The gut microbiota is an intricate determinant of liver health, and its modulation represents both a scientific challenge and a therapeutic opportunity in MASLD.