Gut microbiota in non-alcoholic fatty liver disease: Pathophysiology, diagnosis, and therapeutics

Abstract

Non-alcoholic fatty liver disease (NAFLD), also referred to as metabolic-associated fatty liver disease, is among the most prevalent chronic liver conditions. In some cases, NAFLD may lead to liver inflammation and non-alcoholic steatohepatitis, which can eventually progress to liver cirrhosis and hepatocellular carcinoma. The pathophysiology of NAFLD is complex, involving both genetic and environmental factors. NAFLD is a multisystem disease linked to a higher likelihood of developing metabolic disorders such as type 2 diabetes, obesity, and cardiovascular and chronic kidney diseases. The gut-liver axis represents a key connection between the gut microbiota and the liver, and its disruption has been linked to NAFLD. Growing evidence underscores the significant role of gut microbiota in the onset and progression of NAFLD, with alterations in the gut microbiome and impaired gut barrier function. Studies have identified key microbiota signatures and metabolites linked to NAFLD, implicating oxidative stress, endotoxemia, and inflammatory pathways that further strengthen the connection between gut microbiota and NAFLD. Modulation of gut microbiota through diet and microbiota-centered therapies, such as next-generation probiotics and fecal microbiota transplantation, holds promise for treating NAFLD. In this review, we explore the key link between gut microbiota and the development and progression of NAFLD, as well as its potential applications in the diagnosis and treatment of the disease.

Key Words: Non-alcoholic fatty liver disease; Gut microbiota; Gut-liver axis; Short-chain fatty acids; Bile acids; Mediterranean diet; Ketogenic diet; Probiotics; Prebiotics; Fecal microbiota transplantation

Core Tip: Non-alcoholic fatty liver disease (NAFLD) is among the most prevalent chronic liver conditions that has been recognized as an emerging global health problem. Evidence suggests that the gut microbiota plays a critical role in the pathophysiology of NAFLD by influencing liver inflammation through its metabolites. Identifying gut microbial and metabolic signatures opens new avenues for early diagnosis and therapeutic interventions. Microbiota-targeted therapies, including diet, probiotics, prebiotics, synbiotics, and fecal microbiota transplantation, hold promise for alleviating NAFLD and improving patient outcomes.

INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD), also referred to as metabolic-associated fatty liver disease (MAFLD), is defined as an excessive accumulation of intracellular hepatic fat without consuming significant amounts of alcohol[1]. The novel and non-stigmatizing nomenclature for NAFLD is metabolic dysfunction-associated steatotic liver disease (MASLD)[2], although its use in current literature is limited. Therefore, the term NAFLD is being adhered to in the present manuscript. NAFLD has been recognized as an emerging global health problem and a leading cause of chronic liver diseases[3]. NAFLD affects 25% to 30% of the global population[4], with a prevalence of 25% and 34% in Western countries and Asia, respectively[5,6]. NAFLD results in liver inflammation, which progresses to non-alcoholic steatohepatitis (NASH) and ultimately to liver cirrhosis and hepatocellular carcinoma (HCC)[7]. NAFLD is the third leading cause of HCC[8]. Although only 10% of individuals with NAFLD develop liver cirrhosis and HCC, the disease imposes a significant economic burden[9]. NAFLD is considered a multisystem disease due to its association with a higher risk of metabolic disorders such as type 2 diabetes, obesity, and cardiovascular and chronic kidney diseases[7,10]. Although NAFLD is strongly linked to metabolic syndrome[11,12], it may develop even in the absence of metabolic syndrome[13]. NAFLD is also associated with an increased risk for sarcopenia, which is the loss of skeletal muscle mass, strength, and function[14]. NAFLD often progresses asymptomatically, with clinical symptoms appearing in patients only in the last-stage of liver disease[15]. The primary pathological feature of NASH is inflammation, which significantly contributes to the onset of NASH and its advancement to cirrhosis and HCC.

The pathophysiology of NAFLD is complex, involving both genetic and environmental factors[16]. Factors contributing to NAFLD include obesity, lack of physical activity, high-fat diet (HFD), de novo lipogenesis, lipotoxicity, insulin resistance, epigenetic impairment, gut barrier dysfunction, and gut microbiota dysbiosis[4,17-21]. Insulin resistance and obesity are crucial pathophysiological factors in the onset and progression of NAFLD, which is often considered the hepatic manifestation of metabolic syndrome.

The current methods for NAFLD diagnosis include biopsy, imaging techniques such as fibroscan, ultrasound, and magnetic resonance imaging, and serum biomarkers. Biopsy is invasive and carries potential risks, while imaging techniques and serum biomarkers have limited diagnostic accuracy and poor predictive value[22-25]. Therefore, there is an urgent need for robust and accurate noninvasive tools for the early NAFLD diagnosis. This review explores the relationship between the gut microbiota and the onset and progression of NAFLD. We also discuss the potential of microbiota in the diagnosis, treatment, and prevention of NAFLD.

GUT-LIVER AXIS

The gut-liver axis is the bidirectional relationship between the gut and the liver, which are connected through the hepatic portal vein, bile tract, and systemic circulation and involve various signals and interactions that influence both gastrointestinal (GI) and liver health[26]. This complex communication network plays a crucial role in metabolic processes, immune responses, and the overall maintenance of metabolic homeostasis. The gut-liver axis also forms a key link between the gut microbiota and the liver. The liver receives most of its blood supply from the hepatic portal vein and the hepatic artery. Nearly 70%-75% of the blood supplied to the liver is delivered through the hepatic portal vein, which transports nutrient-rich blood from the GI tract and spleen to the liver. In addition to nutrients, the hepatic portal vein also transports bacteria and toxins to the liver. The metabolites and products of the gut microbiota reach the liver via the hepatic portal vein, while bioactive molecules such as bile acids (BAs), produced by the liver, reach the intestine to regulate gut microbiota[27]. The gut-liver axis is critical in NAFLD pathogenesis[28] and its disruption is increasingly recognized as a significant contributor to the disease. Therefore, understanding the gut-liver axis and its role in NAFLD pathogenesis has important clinical implications. The multiple-hit hypothesis also supports the role of the gut-liver axis in NAFLD development, proposing that the onset and progression of the disease are due to a combination of various contributing factors. Moreover, the interaction between the “mucus layer-soil” and “gut bacteria-seed” is an important factor that determines the pathogenesis of NAFLD[29].

GUT MICROBIOTA AND NAFLD PROGRESSION

The human gut microbiota consists of approximately 40 trillion microorganisms that inhabit the human GI tract[30,31], weighing approximately 1-2 kg[32]. Gut microbiota includes bacteria, archaea, fungi, protozoa, and viruses[33]. While 40% of the core microbiota is shared among humans, the remaining 60% usually varies[30]. Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Fusobacteria are the dominant bacterial phyla in humans[34]. The dominant phyla in the colon are Bacteroidetes and Actinobacteria, while the dominant phylum in the small intestine is Firmicutes[35,36].

The gut microbiota is essential for normal gut physiology and homeostasis and is closely linked to health and disease[37-41]. The gut microbiota is also associated with energy metabolism and storage in the liver. Published data agree on the role of gut microbiota in the development of metabolic disorders such as obesity[42,43], diabetes[44], cardiovascular[45], and chronic liver diseases[46]. Obesity is the most prevalent metabolic disorder. The gut microbiota plays a crucial role in the onset of obesity and associated metabolic disorders[47]. Gut microbiota is known to affect liver lipid metabolism, particularly de novo lipogenesis[48]. Gut microbes ferment complex carbohydrates to produce short-chain fatty acids (SCFAs), which stimulate de novo lipogenesis[49] and regulate host energy metabolism[50,51]. Gut microbes also stimulate the accumulation of triglycerides in adipocytes[37]. Moreover, gut microbes enhance lipid and monosaccharide absorption. For example, colonizing the microbiota from conventionally raised mice into adult germ-free mice results in a 60% increase in body fat and insulin resistance, even if there is a reduction in food intake[37].

A growing body of evidence highlights the crucial role of the gut microbiota and their metabolites in the onset and progression of NAFLD[28,52]. Under normal conditions, the intestinal barrier prevents the movement of microbes and their products to the liver. An alteration in gut microbiota, known as dysbiosis, is caused due to an imbalance between symbiotic and opportunistic microbes. Many factors contribute to dysbiosis, including high-fat and high-sugar diets, lack of fiber, indiscriminate use of antibiotics, infections, and inflammation. Dysbiosis results in impairing intestinal tight junctions. This results in increased intestinal permeability, allowing harmful bacteria and their metabolites to enter the liver, thereby stimulating hepatic immune cells and triggering inflammatory pathways and NAFLD[53-55]. Bacterial components, particularly lipopolysaccharide (LPS), a cell component of Gram-negative bacteria, cause metabolic endotoxemia[56] by triggering hepatic expression of toll-like receptor 4 (TLR4) and secretion of interleukin-8 (IL-8), causing inflammatory reactions[57]. These inflammatory reactions further alter the intestinal microecosystem, leading to a vicious cycle of NAFLD progression[58]. Identifying gut microbiota signature and metabolic profiles in NAFLD patients may provide novel biomarkers for accurate diagnosis and staging of the disease.

MICROBIOTA SIGNATURES IN NAFLD

Various methods are available to study microbial diversity[59]. Next-generation sequencing methods are the methods of choice to explore microbial diversity from the human gut. Gut microbial diversity is a crucial determinant of human health. Several studies have indicated that NAFLD is associated with reduced gut microbial diversity[60,61]. Astbury et al[61] reported reduced α- and β-diversity in NASH patients, with or without cirrhosis, compared to healthy controls. They also observed an abundance of the genus Collinsella and a decrease in Ruminococcaceae in NASH subjects. A cross-sectional study with 87 children with biopsy-proven NAFLD found a reduced α-diversity of fecal microbiomes in children with NAFLD and NASH with a higher abundance of Prevotella copri in those with severe fibrosis[62]. Another cross-sectional study involving 73 participants found decreased bacterial α-diversity in subjects with NAFLD, with a higher Firmicutes-to-Bacteroidetes ratio and reduced abundance of Bacteroidetes, Prevotella, Gemmiger, and Oscillospira[63]. Wang et al[64] found that NAFLD was associated with reduced diversity and a phylum-level changes in the fecal microbiome. There was a 24% reduction in Firmicutes, particularly SCFA-producing genera, and a 20% increase in Bacteroidetes. A recent meta-analysis encompassing 54 studies with 8894 participants reported a moderate decrease in α-diversity indices such as Shannon and Chao1 among NAFLD patients[65].

Several studies have reported phylum-level changes in NAFLD[66,67] and have identified gut microbiome signatures associated with the disease[60,68-70] (Table 1). An important feature associated with NAFLD and other metabolic disorders is a decline in the abundance of commensal bacteria indicative of a healthy gut, such as Akkermansia muciniphila and Faecalibacterium prausnitzii[71-75] and an increased abundance of endotoxin-producing bacteria, such as Enterobacter, Escherichia coli, and Klebsiella pneumoniae[76-82]. Escherichia coli O157:H7 has been shown to have a damaging effect on tight junctions, thereby increasing intestinal permeability[82]. Jiang et al[83] found an increased abundance of Escherichia, Lactobacillus, Streptococcus, and Anaerobacter in NAFLD patients compared to healthy subjects. The abundance of Bacteroides also increases significantly in patients with NASH and fibrosis[84]. A significant association exists between Ruminococcus and hepatic steatosis[85], and the abundance of Ruminococcus is notably increased in patients with fibrosis[84]. Contrary to this, Da Silva et al[86] found a lower abundance of Ruminococcus in simple steatosis and NASH patients. Ruminococcus gnavus produces an inflammatory polysaccharide that stimulates the production of inflammatory cytokines by dendritic cells[87]. Ruminococcus gnavus is therefore implicated in inflammatory diseases such as Crohn’s disease and may also play a key role in NAFLD development.

Table 1 Gut microbiota alterations associated with non-alcoholic fatty liver disease.

Number of participants	Microbiota alterations	Ref.
NASH (n = 65), HC (n = 76)	Increased Collinsella, decreased Ruminococcaceae in NASH	[61]
NAFLD (n = 87), obesity without NAFLD (n = 37)	High abundance of Prevotella copri in severe fibrosis	[62]
Obese youth with NAFLD (n = 44), without NAFLD (n = 29)	Higher Firmicutes-to-Bacteroidetes ratio and lower abundance of Bacteroidetes, Prevotella, Gemmiger, and Oscillospira in NAFLD	[63]
NAFLD (n = 43), HC (n = 83)	Reduction in Firmicutes and Clostridia, increase in Bacteroidetes and Bacteroidia, decrease in Lachnospiraceae, Ruminococcaceae, Lactobacillaceae, and Peptostreptococcaceae in NAFLD	[64]
HS (n = 56), HC (n = 49)	Fewer Lachnospiraceae and Ruminococcaceae, enrichment of Acidaminococcus, Escherichia spp., Bacteroides spp.	[66]
Stage 0-2 NAFLD-related fibrosis (n = 72), stage 3 or 4 NAFLD-related fibrosis (n = 14)	Eubacterium rectale and Bacteroides vulgatus abundant in mild/moderate NAFLD, Bacteroides vulgatus and Escherichia coli most abundant in advanced fibrosis. Ruminococcus obeum CAG:39, Ruminococcus obeum, and Eubacterium rectale significantly lower in advanced fibrosis	[67]
NAFLD (n =25), HC (n = 22)	Escherichia_Shigella, Lachnospiraceae_Incertae_Sedis, and Blautia more abundant, Prevotella decreased in NAFLD. Higher abundance of genus Blautia and the corresponding Lachnospiraceae family in NASH. Higher abundance of Escherichia_Shigella and the corresponding Enterobacteriaceae family in fibrosis	[60]
NASH (n = 46), HC (n = 38)	Significant reduction in Akkermansia muciniphila and increase in Enterobacteriaceae in NASH	[74]
Total (n = 1148), NAFLD (n = 205)	Significant reduction in family Ruminococcaceae and the genus Faecalibacterium in NAFLD	[75]
NAFLD-cirrhosis (n = 27), non-NAFLD control (n = 54)	Enrichment in Negativicutes, reduction in Clostridia	[68]
SS (n = 11), NASH (n = 22), HC (n = 17)	Lower percentage of Bacteroidetes (Bacteroidetes to total bacteria counts) in NASH than SS and HC	[69]
Severe steatosis (n = 36), fibrosis (n = 13)	Significantly reduction in relative abundance of fecal Clostridium sensu stricto in liver fibrosis	[70]
NAFLD (n = 60), chronic viral hepatitis (n = 32), HC (n = 50)	Escherichia coli predominant bacterium in NAFLD	[79]
NAFLD (n = 29), HC (n = 25)	Expansion of Escherichia_Shigella in NAFLD	[81]
NAFLD (n = 57)	Bacteroides abundance significantly increased, Prevotella abundance decreased in NASH and F ≥ 2. Ruminococcus abundance significantly higher in F ≥ 2 patients	[84]
Total (n = 1355), steatosis (n = 472)	Coprococcus, Ruminococcus gnavus increased in steatosis	[85]
SS (n = 15), NASH (n = 24), HC (n = 28)	Less abundance of Bacteroidetes and Firmicutes, more abundance of Lactobacillus and Lactobacillaceae in NAFLD compared to HC. Lower abundance of Ruminococcus, Faecalibacterium prausnitzii and Coprococcus in both NASH and SS patients compared to HC	[86]
NAFLD (n = 13), obese no NAFLD (n = 11), HC (n = 26)	More abundance of Gammaproteobacteria and Prevotella in NAFLD	[88]
NASH (n = 16), HC (n = 8)	More abundance of Bacteroidetes (Bacteroides and Prevotella genus) in NASH	[89]
MAFLD (n = 32), HC (n = 30)	Increased abundance of Prevotella, Bacteroides, and Escherichia-Shigella in MAFLD	[90]
Obese with NAFLD (n = 36), obese without NAFLD (n = 17), HC (n = 20)	Decreased abundance of Blautia, Alkaliphilus, Flavobacterium, and Akkermansia in obese subjects, with or without NAFLD, and increased abundance of Streptococcus in NAFLD	[92]
NAFLD (n = 90), HC (n = 90)	Increase Slackia, Dorea formicigenerans Methanobrevibacter and Phascolarctobacterium in NAFLD	[93]
MAFLD (n = 81), HC (n = 25)	Enrichment of Dorea, Lactobacillus and Megasphaera in MAFLD	[94]
Mild NAFLD (n = 33), moderate NAFLD (n = 20), severe NAFLD (n = 30), HC (n = 21)	Enrichment of Faecalibacterium, Subdoligranulum, Haemophilus, and Roseburia in NAFLD	[97]
NAFL (n = 14), NASH (n = 18), HC (n = 27)	Higher abundance of Fusobacteria and Fusobacteriaceae in NASH compared to NAFL and HCs	[98]
NAFLD-related cirrhosis and HCC (n = 21), NAFLD-related cirrhosis without HCC (n = 20), HC (n = 20)	Higher abundance of Enterobacteriaceae and Streptococcus and a reduction in Akkermansia in cirrhosis. Higher abundance of Bacteroides and Ruminococcaceae, reduction in Bifidobacterium in HCC	[8]
NAFLD (n = 98), first-degree relatives (n = 105)	Enrichment of Streptococcus and Megasphaera in NAFLD-cirrhosis	[99]
NAFLD-HCC (n = 32), NAFLD-cirrhosis (n = 28), non-NAFLD control (n = 30)	Significant enrichment of Bacteroides xylanisolvens, Ruminococcus gnavus, and Clostridium bolteae in NAFLD-HCC and NAFLD-cirrhosis	[100]

NASH: Non-alcoholic steatohepatitis; HC: Healthy controls; NAFLD: Non-alcoholic fatty liver disease; HS: Hepatic steatosis; SS: Simple steatosis; HCC: Hepatocellular carcinoma; MAFLD: Metabolic dysfunction-associated fatty liver disease; NAFL: Non-alcoholic fatty liver.

An increased abundance of Prevotella has been observed in patients with NAFLD and NASH[88,89]. Contrary to this, several studies have found a lower abundance of Prevotella in NAFLD, NASH, and fibrosis patients[60,84] and a higher abundance in healthy individuals[83]. Further research is warranted to establish Prevotella abundance in fatty liver diseases. A study involving 32 MAFLD patients found that there was an increase in the relative abundance of Fusobacteriota, Bacteroidota, Pseudomonadota, and a decrease in Bacillota. At the genus level, there was an increase in the relative abundances of Escherichia-Shigella, Prevotella, and Bacteroides[90]. In a meta-analysis, Li et al[91] concluded that an increase in Streptococcus, Escherichia, and Prevotella, and a decrease in Ruminococcus, Coprococcus, and Faecalibacterium could represent the microbial signature of NAFLD.

Nistal et al[92] compared the gut microbiota of obese patients, with or without NAFLD, to healthy controls. They found a decreased abundance of Blautia, Alkaliphilus, Flavobacterium, and Akkermansia in obese subjects, with or without NAFLD, and an increased abundance of Streptococcus in patients with NAFLD. A community-based cohort study identified microbial signatures associated with NAFLD risk such as Slackia, Dorea formicigenerans, Methanobrevibacter, and Phascolarctobacterium[93]. A study involving 81 MAFLD patients found that Dorea, Lactobacillus, and Megasphaera were enriched in the MAFLD group, while Ruminococcus obeum and Alistipes were more abundant in healthy individuals[94]. A two-sample Mendelian randomization analysis revealed that Christensenellaceae, Lactobacillaceae, and Intestinibacter were negatively correlated, while Actinomycetales, Coriobacteriia, Ruminococcaceae_UCG005, and Oxalobacteraceae were positively correlated with NAFLD[95]. A recent study established a causal relationship between the abundance of eight gut microbiota species and progression of NAFLD[96]. These include the order Actinomycetales, NB1n, the family Ruminococcaceae, Actinomycetaceae, and Oxalobacteraceae, which were positively correlated; the Christensenellaceae R7 group, Lactobacillaceae, and Intestinibacter were negatively correlated with the onset of NAFLD. Recently, Yang et al[97] reported that four bacterial genera, Haemophilus, Faecalibacterium, Roseburia, and Subdoligranulum, along with acetic acid and butyric acid, effectively distinguished NAFLD patients from healthy individuals.

In addition to microbiota signatures associated with NAFLD, several studies have also explored microbiota signatures associated with NASH, fibrosis, and cirrhosis. Rau et al[98] found that patients with NASH exhibited a greater abundance of Fusobacteria and Fusobacteriaceae compared to NAFLD and healthy controls. Ponziani et al[8] found that the fecal microbiota of patients with NAFLD-related cirrhosis showed an increased abundance of Enterobacteriaceae and Streptococcus and a decreased abundance of Akkermansia. Caussy et al[99] reported an enrichment of Streptococcus and Megasphaera in NAFLD-cirrhosis. Studies have found that the patients with moderate to severe fibrosis had an increased abundance of Bacteroides, Ruminococcus, Acidaminococcus, Veillonella spp., and Enterobacteriaceae but a decreased abundance of Faecalibacterium prausnitzii, Eubacterium spp., and Prevotella[60,67,68]. Behary et al[100] found that Bacteroides xylanisolvens, Clostridium bolteae, and Ruminococcus gnavus had higher abundance in both NAFLD-HCC and NAFLD-cirrhosis. Moreover, Veillonella parvula and Bacteroides caecimuris could discriminate NAFLD-HCC from NAFLD cirrhosis.

Numerous studies across different NAFLD patient populations have reported discrepant microbiota signatures, making it difficult to establish a consensus on the microbiota associated with NAFLD. These discrepancies found in microbiota signatures across studies are mainly attributed to sequencing and diagnostic tools, geographical locations, ethnicity, population characteristics, and disease spectrum[11]. Nevertheless, comparing these studies may provide useful insights into the microbiota signatures that can effectively help diagnose NAFLD onset and progression.

MICROBIOTA, TOLL-LIKE RECEPTORS, AND NAFLD

Gut microbiota-derived products, called microorganism-associated molecular patterns (MAMPs), play a crucial role in NAFLD progression[101]. MAMPs include LPS, peptidoglycan, flagellin, lipoproteins, lipoteichoic acid, and DNA. These MAMPs trigger pattern-recognition receptors, such as toll-like receptors (TLRs). TLR signaling is triggered in NAFLD, and TLRs are involved in the development of steatosis and fibrosis[102]. TLRs associated with NASH include TLR2, TLR4, TLR5, and TLR9[103-105]. TLR4- or TLR9-deficient mice fed on HFD or choline-deficient diet were effectively protected from hepatic steatosis[103,106], suggesting a strong association between TLR activation and NAFLD.

TLR2 is the receptor for peptidoglycan and lipoteichoic acid, which are components of the cell walls of Gram-positive bacteria. TLR2-deficient mice on a choline-deficient amino acid-defined diet showed lower expression of proinflammatory cytokine and were resistant to diet-induced steatohepatitis[107]. TLR4, the receptor for LPS, activates the MyD88/nuclear factor kappa B (NF-κB) cascade, triggering the secretion of pro-inflammatory cytokines like IL-6 and tumor necrosis factor-α (TNF-α), leading to inflammation and liver fibrosis[58]. TLR4 can also activate NF-κB through the toll/IL-1 receptor domain-containing adapter protein[108]. Several studies have reported an increase in LPS and TLR4 in the liver of NASH subjects[109-111]. Wild-type mice on a standard diet receiving continuous low doses of LPS developed hepatic steatosis and insulin resistance[56]. Similarly, NAFLD mice receiving LPS injections showed an increase in proinflammatory cytokines and liver injury[112,113]. Humans with NAFLD also show elevated LPS levels[114]. Decreasing plasma LPS levels with antibiotics can decrease disease severity in NAFLD patients[115], suggesting the role of LPS and TLR4 signaling in NAFLD progression. TLR5 recognizes bacterial flagellin and is crucial in maintaining the integrity of intestinal epithelium. TLR5 deletion is associated with spontaneous colitis in mice[116]. TLR5 knockout mice show alterations in gut microbiota and develop obesity and steatosis[105]. TLR5 plays a critical role in NAFLD progression by regulating gut microbiota, inflammation, and endothelial-to-mesenchymal transition via the MYD88/TWIST1 pathway[117]. TLR9 is the receptor for bacterial DNA. TLR9 activation is associated with liver injury and NASH[118]. TLR9-deficient mice on a choline-deficient amino acid-defined diet showed suppression of diet-induced insulin resistance and weight gain and less inflammation and steatosis than wild-type mice[103].

TLR signaling induces the secretion of pro-inflammatory cytokines, TNF-α and IL-1β, key mediators in the onset and progression of NAFLD. This is evident from the fact that mice deficient in TLR2, 4, and 9 expressed low proinflammatory cytokines[106,107]. Hepatic macrophages (Kupffer cells) are the primary source of these proinflammatory cytokines[119]. TNF-α not only regulates lipid metabolism but also impairs insulin signaling, resulting in insulin resistance and elevated insulin levels[120]. It also promotes cholesterol accumulation in hepatocytes[121]. Elevated insulin promotes hepatic lipogenesis and the influx of free fatty acid into hepatocytes, while insulin resistance promotes the release of fatty acids from adipocytes and prevents their uptake by adipocytes[122]. Fat-laden hepatocytes have elevated expression of apoptosis signal-regulating kinase-1, c-Jun N-terminal kinase, and pro-apoptotic genes such as Bax, mediating TNF-α-induced apoptosis[123]. In line with these findings, TNF-α signaling-deficient mice are found to be resistant to diet-induced NAFLD[124]. IL-1β regulates lipid metabolism and promotes the accumulation of fats in hepatocytes[125], resulting in NAFLD. Consistent with these findings, IL-1β signaling-deficient mice were found to be effectively protected against steatosis and liver fibrosis[126].

MICROBIOTA METABOLITES IN NAFLD

The microbiota-derived metabolites can either activate or suppress signaling pathways, thereby influencing the progression of NAFLD. These metabolites, implicated in NAFLD, include SCFAs, BAs, trimethylamine (TMA) metabolites, and tryptophan metabolites (Figure 1). Aragonès et al[127] used the term “liquid biopsy” to describe circulating microbiota metabolites associated with NAFLD, which may serve as a noninvasive diagnostic tool for NASH. Detecting these microbiota-derived metabolites offers a novel, noninvasive diagnostic strategy for NAFLD. Several studies have identified metabolite signatures useful for diagnosing and monitoring NAFLD progression. A metabolic analysis of 112 patients revealed 15 serum metabolites strongly associated with early-stage NAFLD[128]. Another study involving 161 NAFLD patients and 149 healthy controls, identified 24 differentially present metabolites between the two groups[129]. Metabolites such as prasterone, indoxylsulfuric acid, sebacic acid, arachidonic acid, and pregnenolone sulfate could help distinguish NAFLD patients. An animal study identified a depletion of indole derivatives such as indole-3-propionic acid (IPA), indole-3-acetic acid (IAA), methyl IAA, 5-hydroxyindole-3-acetic acid, and indole-3-acrylic acid, and an increase in taurine-conjugated BAs such as taurocholic acid and taurochenodeoxycholic acid in NAFLD[130]. Yang et al[90] identified 22 common differential metabolites in the feces and serum of MAFLD patients. Their analysis revealed that taurocholic acid and lithocholic acid were decreased, while glycocholic acid was increased in the serum of MAFLD patients. In the feces, allolithocholic acid, taurodeoxycholic acid, dehydrocholic acid, and 7-ketolithocholic acid were found to be decreased in MAFLD patients.

Figure 1 Gut microbiota metabolites in non-alcoholic fatty liver disease progression. Figure created with BioRender.com. LPS: Lipopolysaccharide; TLR: Toll-like receptor; IL-6: Interleukin-6; TNF: Tumor necrosis factor; Th17: T helper type 17 cell; SCFA: Short-chain fatty acids; NF-κB: Nuclear factor kappa B; IAA: Indole-3-acetic acid; IPA: Indole-3-propionic acid; K. pneumoniae: Klebsiella pneumoniae; E. coli: Escherichia coli; TMA: Trimethylamine; TMAO: Trimethylamine N-oxide; MCP-1: Monocyte chemoattractant protein-1.

SCFAs

SCFAs, including propionate, acetate, and butyrate, are produced through microbial fermentation of complex carbohydrates[131]. SCFAs are responsible for the beneficial effects of gut microbiota. SCFAs are crucial for maintaining the integrity of the intestinal barrier and decreasing intestinal inflammation[132]. For example, butyrate has been shown to restore mucosal barrier function in heat- and detergent-induced colonic injury in rats[133]. SCFAs also modulate colonic regulatory T cell and exhibit anti-inflammatory properties[134]. Specifically, butyrate and propionate can reduce the levels of pro-inflammatory cytokines[135]. Additionally, SCFAs activate the G-protein-coupled receptors expressed on colonic epithelial cells, hepatic cells, and adipocytes[50,136,137]. The interaction of SCFAs with GPR43 has been shown to reduce inflammation[136]. Furthermore, SCFAs can protect from the accumulation of body fat and liver lipids in mice[138] as well as provide protection against diet-induced obesity and insulin resistance[139]. Given these effects, SCFAs may play a protective role in the development NAFLD[140].

Butyrate exerts an anti-inflammatory effect by inhibiting T helper type 17 cells through the activation of regulatory T cells in the intestine, thereby reducing pro-inflammatory signaling pathways[141]. Gut dysbiosis lead to a reduction in butyrate levels, which in turn contributes to low-grade inflammation. Butyrate also plays crucial role in preserving intestinal integrity. In NAFLD, decreased butyrate levels increase gut permeability, facilitating bacterial and LPS translocation[28]. Studies done in animal models have also showed that oral administration of butyrate enhances the intestinal barrier integrity and reduces pathological liver alterations[142-146]. In mice with HFD-induced NAFLD, butyrate mitigates inflammation and fat accumulation by promoting the growth of beneficial bacteria and enhancing the GI barrier[146]. Furthermore, butyrate intervention in HFD-fed mice increase SCFA-producing bacteria, reduces pathogenic bacteria and serum endotoxin levels, and inhibits the expression of proinflammatory cytokines[147]. Administration of sodium butyrate can prevent the progression from simple steatosis to steatohepatitis[145].

Administration of sodium acetate has been shown to have a hepatoprotective effect against nicotine-induced hepatic steatosis[148]. Acetate also reduces hepatic and intramuscular triglyceride content and inhibits lipid accumulation by suppressing fatty acid synthesis and enhancing fatty acid oxidation[149]. Administration of sodium propionate, sodium acetate, or sodium butyrate to methionine- and choline-deficient (MCD) diet-fed NASH mice alleviates NASH by lowering serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), liver cholesterol, and triglycerides[143].

BAs

Primary BAs are produced by the liver and are secreted into the duodenum with bile. BAs and the gut microbiota engage in a bidirectional interaction[150]. The gut microbiota converts primary BAs into secondary BAs. The intestinal bacteria, particularly Bifidobacterium, Lactobacillus, Clostridium, Bacteroides, and Listeria, produce bile salt hydrolases that deconjugate BAs[151]. The deconjugated BAs are then converted into secondary BAs by 7α-dehydroxylase, primarily produced by Clostridium and Eubacterium.

BAs help preserve intestinal barrier function, thereby preventing bacterial translocation[152]. Acting as signaling molecules, BAs bind to various nuclear receptors[153,154], which play an important role in energy regulation. Dysbiosis in the gut microbiota decreases the synthesis of secondary BAs, which dysregulates nuclear receptors, contributing to the development of NAFLD[152]. BAs regulate hepatic steatosis by activating farnesoid X receptor (FXR) and Takeda G protein-coupled receptor 5 (TGR5)[155]. Intestinal FXR stimulates the release of fibroblast growth factor 15/19 (FGF15/19), which acts on the liver, muscle, and adipose tissue[156]. Overexpression of FGF15 reduces fat mass in mice[157], whereas its knockout promotes hepatic steatosis in HFD-fed mice[158]. Disruption of intestine-specific FXR has been shown to reduce hepatic triglyceride accumulation in a murine model of HFD-induced NAFLD[159]. FXR activation decreases fatty acid uptake and synthesis while increasing fatty acid oxidation[160]. It also triggers peroxisome proliferator-activated receptor α (PPARα) expression, promoting β-oxidation and decreasing lipid accumulation[161]. Mice lacking FXR exhibit elevated serum BAs, cholesterol, triglycerides, and hepatic cholesterol and triglycerides[162]. TGR5 activation promotes the release of glucagon-like peptide-1 from intestinal enteroendocrine cells, enhancing insulin secretion and improving glucose tolerance[163]. TGR5 also suppresses the NF-κB pathway, and TGR5 knockout mice exhibit more severe hepatic necrosis and inflammation[164]. Furthermore, TGR5 activation by BAs inhibits nod-like receptor protein 3 inflammasome, thereby elevating inflammation[165].

Serum concentrations of both primary and secondary BAs are elevated in patients with NAFLD[166]. NASH is characterized by decreased secondary BAs, increased total primary BAs, and elevated conjugated to unconjugated chenodeoxycholate, cholate, and total primary BAs[167]. A metabolomic study revealed alterations in the plasma BAs in NAFLD patients, with plasma 7-keto deoxycholic acid levels associated with advanced-stage hepatic fibrosis and plasma 7-keto lithocholic acid linked to NASH[168]. Increased taurocholate has also been associated with higher grades of steatosis. Elevated levels of BAs and propionate have been reported in non-obese NAFLD patients with fibrosis[169]. A cohort study found that the ratio of circulating conjugated chenodeoxycholic acids to muricholic acids was elevated in NASH patients[170]. Recently, Wang et al[171] showed that tauroursodeoxycholic acid, a synthetic bile salt, reduced obesity and hepatic lipid accumulation, while also enhancing intestinal barrier function and gut microbial diversity.

Tryptophan and its metabolites

Tryptophan is an essential amino acid that must be supplemented through diet. Gut microbes directly influence the three major Trp metabolism pathways, which produce serotonin, kynurenine, and indole derivatives[172]. Tryptophan metabolites can play either a positive or negative role in NAFLD. Alterations in tryptophan and its metabolites have been observed in NAFLD-cirrhosis[68]. Gut microbiota metabolizes unabsorbed tryptophan into indole and its derivatives, such as IPA and IAA, which improve gut barrier function and modulate the immune response via aryl hydrocarbon receptor[173]. Indole and its derivatives are also known for their anti-inflammatory effects[174]. Numerous studies have highlighted the anti-inflammatory potential of indole. Knudsen et al[175] showed that indole reduced markers of hepatic damage and hepatic expression of genes involved in inflammation and macrophage activation in ob/ob mice. Beaumont et al[176] demonstrated that the oral administration of indole reduced LPS-induced increase in proinflammatory mediators and prevented LPS-induced changes in cholesterol metabolism. Zhao et al[177] reported that IPA inhibited NF-κB signaling and reduced the levels of proinflammatory cytokines. Additionally, studies have also shown that indole enhances intestinal integrity, and there is a negative correlation between indole and hepatic fat content[178,179]. NAFLD has been linked to reduced production of indole and its derivatives[179,180]. IPA levels were found to be lower in liver fibrosis patients compared to controls[181]. Administration of IPA can increase the expression of tight junction proteins, maintain intestinal epithelium homeostasis, reduce hepatic inflammation and liver injury, and modulate gut microbiota in HFD-fed rats[177]. It has also been shown to lower fasting blood glucose levels, plasma insulin levels, and insulin resistance in mice[182]. A recent study demonstrated lower levels of IPA and IAA in the feces of patients with hepatic steatosis compared to healthy controls[183]. Moreover, the administration of IPA and IAA improved hepatic steatosis and inflammation in an animal model by inhibiting the NF-κB signaling pathway.

Ritze et al[184] found that oral tryptophan supplementation attenuated experimental NAFLD in mice, probably by stabilizing the intestinal barrier and improving the dysregulated intestinal serotonergic system. IAA administration can reduce HFD-induced oxidative stress, plasma total cholesterol, low-density lipoprotein cholesterol (LDL-C), and inflammatory response of the liver in a mouse model[185]. A study involving 58 obese patients who underwent sleeve gastrostomy found that the levels of IAA increased post-surgery, and plasma IAA levels were negatively correlated with liver fat attenuation[186]. Another indole derivative, NecroX-7, was shown to reduce serum AST and ALT, hepatic steatosis and fibrosis, and lipid peroxidation in ob/ob mice[187].

Serotonin [5-hydroxytryptamine (5-HT)] is a tryptophan metabolite produced by the action of tryptophan hydroxylase. Studies have identified 5-HT as a risk factor in the pathological progression of NASH. Wang et al[188] showed that 5-HT increased lipid accumulation and expression of lipogenesis-related genes, while the absence of 5-HT prevented hepatic lipid accumulation and the expression of inflammatory factors. Choi et al[189] demonstrated that inhibiting serotonin synthesis alleviates hepatic steatosis by reducing liver 5-HT (serotonin) receptor 2A signaling. Moreover, tryptophan hydroxylase and 5-HT (serotonin) receptor 2A knockdown mice exhibit resistance to HFD-induced hepatic steatosis. In contrast to 5-HT’s pro-inflammatory effects, its metabolite melatonin has been shown to have anti-inflammatory properties. Melatonin reduces serum and liver triglyceride levels and inhibits LPS-induced hepatic fat buildup in mice[190]. It also improves NAFLD phenotypes, and reduces the expression of pro-inflammatory cytokines, lowers oxidative stress levels, ALT, fasting plasma glucose, body and liver weight, and LDL-C[191,192]. In a randomized controlled trial involving seventy-four patients with NAFLD, melatonin significantly decreased levels of pro-inflammatory cytokines, hepatic inflammation, and improved fat metabolism parameters[193]. Another randomized, double-blind, placebo-controlled clinical trial involving forty-five patients found that melatonin significantly improved fatty liver grade, ALT, AST, weight, waist, abdominal circumference, and both systolic and diastolic blood pressure[194].

TMA and its derivatives

The gut microbiota ferments dietary nutrients like choline and carnitine to produce TMA, which is subsequently converted into TMA N-oxide (TMAO) in the liver[195]. TMAO has been associated with several chronic diseases, such as diabetes, insulin resistance, liver steatosis, hypertension, atherosclerosis, heart failure, and cancer. The association between TMAO and NAFLD was first established by Chen et al[196], who found that TMAO levels in NAFLD patients were 4.17 times higher than in healthy controls. Elevated TMAO levels are linked to obesity and the severity of MAFLD[197]. TMAO promotes inflammation in adipose tissue[198,199], increases fasting insulin resistance and pro-inflammatory cytokine monocyte chemoattractant protein-1, and decreases anti-inflammatory cytokine IL-10 in adipose tissue[199]. It has also been shown to increase triglyceride levels and the expression of liver fibrosis-related genes, particularly KRT17, in fatty liver cells in vitro[200]. A cohort study of 5292 participants found that the plasma TMAO levels were linked to all-cause mortality in patients with NAFLD[201]. TMAO exacerbates hepatic steatosis by modulating BA metabolism. A case-control study involving biopsy-proven NAFLD patients identified a positive correlation between TMAO levels and serum levels of total BAs[202]. This study also found that TMAO administration deteriorated liver function and elevated hepatic triglyceride and lipogenesis in HFD-fed mice. TMAO binds to the endoplasmic reticulum stress kinase protein kinase RNA-like endoplasmic reticulum kinase and triggers the unfolded protein response (UPR)[203]. UPR is also triggered by the buildup of misfolded proteins in the endoplasmic reticulum and is known to be activated in various liver diseases, including hepatic steatosis[204]. These findings suggest that TMAO plays a key role in the progression and development of NAFLD through UPR activation.

TMAO is an early biomarker of NAFLD, with a specific cut-off level identifying individuals at high risk of the disease[205]. A cohort study of 357 Mexican obese patients with varying stages of NAFLD found a significant association between TMAO and choline levels and the risk of NASH[206]. In contrast, Zhao et al[207] reported that oral TMAO reduced ALT, AST, and hepatic and serum cholesterol levels, restored gut flora diversity, and attenuated steatohepatitis in HFD and high-cholesterol diet-fed rats. A newly identified metabolite, N,N,N-trimethyl-5-aminovaleric acid, has been found to be elevated in patients with hepatic steatosis[208].

Endogenous ethanol

Ethanol is produced in the small intestine by gut microbiota acting on dietary carbohydrates and is subsequently processed by the liver through the action of alcohol dehydrogenases. A potential link exists between blood ethanol levels and alterations in the gut microbiota[209]. Zhu et al[78] reported significantly elevated blood ethanol levels and an increased abundance of alcohol-producing bacteria, such as Escherichia, in NASH patients compared to healthy individuals and obese non-NASH patients. Michail et al[88] found notably higher ethanol levels in children with NAFLD compared to controls. Engstler et al[210] also observed significantly elevated fasting ethanol levels in children with NAFLD compared to controls. Further studies in mice suggested that elevated ethanol levels may be due to insulin-dependent impairments in alcohol dehydrogenase activity rather than increased endogenous ethanol synthesis. In a prospective clinical study, Meijnikman et al[211] measured ethanol levels in 146 individuals and used selective ADH inhibition to show that elevated portal vein ethanol concentrations, driven by gut microbiota (e.g., Lactobacillaceae), increase with NAFLD progression and play a significant role in its pathogenesis. Additionally, the high-alcohol-producing bacterium Klebsiella pneumoniae has been associated with 60% of individuals with NAFLD[212] and oral gavage or fecal microbiota transplantation (FMT) with Klebsiella pneumoniae can induce NAFLD in recipient mice.

DIET, MICROBIOTA, AND NAFLD

Diet not only affects the progression of various metabolic disorders but also the gut microbiota composition and intestinal barrier function, suggesting that an unhealthy diet may contribute to NAFLD progression[213]. Several studies have shown that a Western diet increases, while a Mediterranean diet (MD) decreases, intrahepatic fat accumulation[214,215]. Western diet induces NASH in male mice, exhibiting features matching human steatohepatitis, such as hepatic inflammation, steatosis, and fibrosis[216]. Additionally, Western diet increases the risk of NAFLD by 56%, while the Prudent and MDs reduce the risk by 22% and 23%, respectively[217].

MD is rich in nutrients, particularly monounsaturated fatty acids, polyunsaturated fatty acids, and fiber, all of which have beneficial effects on lipid metabolism and fatty liver disease[218]. Randomized controlled trials have confirmed the efficacy of MD in reducing total cholesterol, serum triglycerides, hepatic steatosis, and liver cirrhosis in NAFLD patients[219-221] (Table 2). Additionally, MD has also been shown to improve insulin resistance[222,223]. MD is considered a safe and effective means of treating NAFLD patients. A meta-analysis further confirmed that MD significantly reduced the fatty liver index and insulin resistance in patients with NAFLD[224]. In a randomized clinical trial involving children, MD and a low-fat diet reduced hepatic steatosis, hepatic enzymes, and insulin resistance[225]. Another randomized clinical trial with adolescents similarly demonstrated a significant reduction in hepatic steatosis, serum transaminase levels, and insulin resistance with MD and a low-fat diet[226].

Table 2 Clinical trials on dietary interventions in non-alcoholic fatty liver disease.

Number of participants	Dietary intervention	Outcome	Ref.
n = 74	Melatonin	Significant decrease in levels of pro-inflammatory cytokines, inflammation in liver and improvement in parameters of fat metabolism	[193]
n = 45	Melatonin	Significant improvement in the grade of fatty liver, ALT, AST, weight, waist and abdominal circumference and systolic and diastolic blood pressure	[194]
n = 18	MD	Significant reduction in mean body weight, waist circumference, ALT and AST	[220]
n = 49	MD or LFD	Reduction in hepatic steatosis and liver enzymes with MD and LFD. Improvements in total cholesterol, serum triglyceride, and glycated hemoglobin with MD	[221]
n = 56	MD or ML	Weight reduction with MD and ML. Significant improvement in ALT levels and liver stiffness with ML and liver stiffness with MD	[222]
n = 44	MD or LFD	Significant decrease in hepatic steatosis, serum transaminase levels, and insulin resistance with MD and LFD	[227]
n = 27	KD or LFD	Significantly reduction in liver fat with both KD and LFD	[233]
n = 37	High animal or plant protein diet	Reduction in liver fat within 6 weeks, no change in body weight	[251]
n = 40	Low free sugar diet	Reduction in hepatic steatosis and ALT level	[249]
n = 29	Low dietary sugar diet	Reduction in hepatic de novo lipogenesis, hepatic fat and fasting insulin	[250]

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; MD: Mediterranean diet; LFD: Low-fat diet; ML: Mediterranean lifestyle; KD: Keto diet.

The ketogenic diet (KD) involves a significant reduction in carbohydrate intake. Ketosis is achieved when carbohydrate intake is below 50 g per day, regardless of fat and calorie intake[227]. KD has been shown to reduce intrahepatic triglycerides by 31% and hepatic insulin resistance by 58%[228]. However, various risk factors are associated with KD. For example, Muyyarikkandy et al[229] showed that long-term KD intervention can aggravate hepatic mitochondrial dysfunction and oxidative stress in mice. Anekwe et al[230] found that KD resulted in worsened hyperlipidemia and elevated liver enzymes in a 57-year-old woman. Despite these risk factors, studies have found that carbohydrate restriction can help modulate lipid metabolism, improving NAFLD with efficacy similar to that of low-fat diets[231]. A randomized controlled trial further demonstrated that KD and low-fat diet can significantly reduce liver fat in NAFLD patients in the short term[232].

A high dietary fiber intake improves metabolic parameters associated with NAFLD[233] and promotes the growth of beneficial bacteria, such as Bifidobacterium, Lactobacillus, and Bacteroidetes, and SCFA-producing bacteria[234,235]. In contrast, the MCD diet has been shown to significantly reduce Bifidobacterium and Lactobacillus in feces[236]. Choline affects the composition of the gut microbiota[237]. Choline plays a critical role in lipid metabolism and prevents fat buildup in the liver. Additionally, choline is also metabolized by gut microbes to dimethylamine and TMA, which are subsequently converted into TMAO in the liver, leading to hepatic inflammation and damage[238]. Furthermore, choline-deficient diet has been associated with the development of NASH and dysbiosis of the gut microbiota[239-241].

HFD is associated with an increased abundance of Proteobacteria, such as Desulfovibrionaceae and Enterobacteriaceae[56,242]. Proteobacteria can increase LPS levels or disrupt the intestinal barrier[243,244], contributing to the development of NAFLD. A high intake of dietary cholesterol accelerates the development of spontaneous NAFLD and triggers liver inflammation in mice[245]. A higher fat fraction has been linked to lower α-diversity of the gut microbiota[246]. In contrast, a low intake of dietary saturated fatty acids in humans is associated with increased microbial diversity, irrespective of fiber intake, while a high intake of polyunsaturated fatty acid has a protective effect against steatosis without affecting the microbiota[247]. Diets such as HFD, MCD, or fructose-rich diet modify gut microbiota and impair intestinal permeability, leading to increased circulating LPS levels[106], thus inducing NAFLD in mice. Additionally, a diet low in free sugars has been shown to decrease hepatic steatosis and ALT levels without causing any adverse effects[248]. Furthermore, reducing dietary sugar has been shown to lower hepatic de novo lipogenesis, hepatic fat, and fasting insulin[249].

A diet high in animal or plant protein has been shown to reduce liver fat by 36% to 48% without affecting body weight[250]. Such a diet can also lower hepatic enzymes, reduce markers of inflammation, decrease serum keratin 18 levels and improve insulin sensitivity. Recently, Zhou et al[251] demonstrated that isoleucine-restricted diets could reverse HFD-induced weight gain, hepatic inflammation, increased fasting glucose levels, and improved hepatic insulin resistance, lipid metabolism, and gluconeogenesis disorder.

Berberine, a quaternary ammonium compound found in some plants, exhibits potent antibacterial activity. Studies suggest that berberine can modulate gut microbiota and offers therapeutic potential for treating metabolic diseases[252,253]. Similarly, resveratrol, a natural polyphenol found in many plants such as grapes may help prevent metabolic diseases by lowering blood glucose and lipid levels[254]. This effect is partly attributed to its ability to modify gut microbiota.

MICROBIOTA-TARGETED THERAPEUTICS FOR NAFLD

Currently, there are no Food and Drug Administration-approved drug therapies for NAFLD. Lifestyle modifications, including dietary interventions and exercise, and pharmacotherapy, including the use of metformin, thiazolidinediones, statins, and ursodeoxycholic acid, are the only known treatments of NAFLD[255]. Restoring gut homeostasis in NAFLD patients through microbiota-targeted therapies has emerged as a promising alternative approach for managing and treating the disease (Figure 2). Prebiotics, probiotics, and synbiotics have been shown to improve hepatic alterations associated with hypercholesterolemia by modulating the expression of genes related to β-oxidation and lipogenesis[256]. Strategies aimed at modulating the gut microbiota through prebiotics, probiotics, synbiotics, and FMT may offer potential therapeutic options for managing NAFLD (Table 3).

Figure 2 Microbiota-targeted therapies for non-alcoholic fatty liver disease treatment. Figure created with BioRender.com. IL-6: Interleukin-6; TLR: Toll-like receptor; TNF: Tumor necrosis factor; PPAR: Peroxisome proliferator-activated receptor; NAFLD: Non-alcoholic fatty liver disease; LPS: Lipopolysaccharide; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; SCFA: Short-chain fatty acids.

Table 3 Clinical trials on microbiota-targeted interventions in non-alcoholic fatty liver disease.

Number of participants	Intervention	Outcome	Ref.
n = 20	L. rhamnosus GG	Significant decrease in ALT and antipeptidoglycan-polysaccharide antibodies, no effect on BMI Z score and visceral fat	[280]
n = 44	VSL#3	Improvement in NAFLD and reduction in triglycerides and insulin resistance	[311]
n = 28	L. bulgaricus and S. thermophilus	Reduction in the levels of ALT, AST, and GGT	[315]
n = 20	Lepicol probiotic (L. plantarum, L. delbrueckii, L. acidophilus, L. rhamnosus and B. bifidum)	Reduction in intrahepatic triglyceride and serum AST, no change in waist circumference, BMI, and glucose and lipid levels	[316]
n = 72	Probiotic yogurt containing L. acidophilus La5 and B. lactis Bb12	Reduction in the levels of ALT, AST, total cholesterol, and LDL cholesterol	[317]
n = 42	Lactocare (L. casei, L. acidophilus, L. rhamnosus, L. bulgaricus, B. breve, B. longum, S. thermophilus)	Reduction in insulin, insulin resistance and inflammation markers such as TNF-α and IL-6	[318]
n = 64	Probiotic mixture (L. acidophilus ATCC B3208, B. lactis DSMZ 32269, B. bifidum ATCC SD6576, and L. rhamnosus DSMZ 21690)	Reduction in waist circumference, cholesterol, triglycerides, LDL-C, ALT and AST. No change in weight, BMI, and BMI Z score	[319]
n = 65	Probiotic mixture (L. acidophilus, L. rhamnosus, L. paracasei, Pediococcus pentosaceus, B. lactis, and B. breve)	Reduction in body weight, total body fat, and intrahepatic fat	[320]
n = 58	“Symbiter” containing 14 probiotic belonging to Bifidobacterium, Lactobacillus, Lactococcus, and Propionibacterium	Reduction in fatty liver index, liver stiffness, serum AST, GGT, TNF-α and IL-6	[321]
n = 39	Multi-strain probiotics containing six different Lactobacillus and Bifidobacterium spp.	Stabilization of the mucosal immune function. No effect on hepatic steatosis and fibrosis levels	[322]
n = 40	Akkermansia muciniphila	Improvement in insulin sensitivity, reduction in insulinemia, plasma total cholesterol, levels of the markers for liver dysfunction and inflammation	[334]
n = 22	Inulin-type fructans	Increased fecal Bifidobacterium. No effect on liver fat contents, liver function tests, and metabolic and inflammatory mediators	[375]
n = 197	Resistant starch	Reduction in intrahepatic triglyceride content and liver enzymes	[382]
n = 66	Synbiotic (B. longum with FOS)	Significant reduction in inflammatory markers, LDL cholesterol, steatosis, and NASH activity index	[395]
n = 52	Protexin (L. casei, L. rhamnosus, L. acidophilus, L. bulgaricus, B. breve, B. longum, S. thermophilus with prebiotic FOS)	Significant attenuation in inflammatory markers, such as ALT, AST, GGT, high-sensitivity CRP, TNF-α, and NF-κB p65	[397]
n = 50	Protexin (L. casei, L. rhamnosus, L. acidophilus, L. bulgaricus, B. breve, B. longum, S. thermophilus with prebiotic FOS)	Reduction in inflammatory mediators, hepatic steatosis and fibrosis	[398]
n = 104	Synbiotic (FOS and B. animalis subspecies lactis BB-12)	Alteration in fecal microbiota. No effect on liver fat content or markers of liver fibrosis	[406]
n = 21	Fecal microbiota transplantation	Reduction in small intestinal permeability. No improvement in insulin resistance and hepatic proton density fat fraction	[418]

L. rhamnosus: Lactobacillus rhamnosus; L. bulgaricus: Lactobacillus bulgaricus; S. thermophilus: Streptococcus thermophilus; L. acidophilus: Lactobacillus acidophilus; L. plantarum: Lactiplantibacillus plantarum; L. paracasei: Lactobacillus paracasei; L. casei: Lactobacillus casei; B. breve: Bifidobacterium breve; B. longum: Bifidobacterium longum; B. bifidum: Bifidobacterium bifidum; FOS: Fructo-oligosaccharides; B. animalis: Bifidobacterium animalis; B. lactis: Bifidobacterium lactis; ALT: Alanine aminotransferase; BMI: Body mass index; NAFLD: Non-alcoholic fatty liver disease; AST: Aspartate aminotransferase; GGT: Gamma-glutamyl transferase; LDL: Low-density lipoprotein; TNF: Tumor necrosis factor; IL: Interleukin; LDL-C: Low density lipoprotein-cholesterol; NASH: Non-alcoholic steatohepatitis; CRP: C-reactive protein; NF-κB: Nuclear factor kappa B.

Probiotics

Probiotics are living microorganisms that provide multiple health benefits to the host if administered adequately[257]. These health benefits include inhibition of pathogenic bacteria, regulation of the immune system, reduction in blood cholesterol, and enhancement of the intestinal mucosal barrier function[258,259]. Probiotics work by modifying the gut microbiota and producing the metabolites that inhibit pathogenic microbes, thus help prevent intestinal infections[260-262]. The effects of probiotics depend on the factors such as the duration of treatment, cocktail combination, and probiotic dosage. Lactobacillus and Bifidobacterium are among the widely used probiotics and have demonstrated potential in treating NAFLD. Both are known to improve glucose-insulin homeostasis and reduce hepatic steatosis by modulating gut microbiota[263].

Lactobacillus: Studies have shown that Lactobacillus exhibits a hypocholesterolemic effect[264-266] suggesting its potential for use in NAFLD therapeutics. Lactobacillus casei strain Shirota (LcS) has been found to protect against diet-induced NASH and steatosis[236,267] and to improve insulin resistance in diet-induced obesity[268]. Oral administration of LcS increases the levels of Lactobacillus and Bifidobacterium, suppresses MCD-diet-induced NASH, reduces serum LPS, and mitigates inflammation and fibrosis in the liver[236]. Additionally, LcS administration attenuates TLR4 signaling in the liver, thus protecting against the development of fructose-induced NAFLD[268]. Lactobacillus paracasei (L. paracasei) has been shown to alleviate hepatic steatosis in a mouse model of NASH[269]. This effect is attributed to its ability to decrease the expression of TLR4, TNF-α, IL-4, monocyte chemotactic protein-1, PPAR-γ, PPAR-δ, and NADPH oxidase-4.

L. plantarum MB452 has been shown to enhance intestinal barrier function by upregulating genes involved in tight junction signaling[270]. L. plantarum NCU116 administration has demonstrated the ability to restore liver function and reduce fat accumulation, and decrease proinflammatory cytokines in the liver[271]. Oral gavage of L. plantarum ZJUIDS14 to HFD-induced NAFLD mice alleviates liver damage, insulin resistance, and hepatic steatosis. It also strengthens intestinal barrier integrity, regulates gut microbiota, and improves mitochondrial function[272]. Additionally, L. plantarum has been found to increase the hepatic expression of PPAR-α and stimulate AMP-activated protein kinase activation. Administration of L. plantarum NA136 to the insulin-resistant mouse NAFLD model relieves insulin resistance, inhibits pathogenic bacteria, promotes beneficial bacteria, improves intestinal barrier integrity, and reduces inflammation caused by HFD and high fructose diets[273]. Park et al[274] reported that administration of L. plantarum strains to a rat model of HFD/high fructose diet-induced NAFLD reduced hepatic lipid accumulation, expression of de novo lipogenesis-related genes, and serum levels of AST and ALT.

L. rhamnosus GG inhibits NF-κB signaling, reducing the effects of pro-inflammatory cytokines on epithelial barrier integrity[275]. Administration of L. rhamnosus GG has been shown to decrease hepatic fat content, serum triglyceride and cholesterol levels, and expression of pro-inflammatory genes and suppress FXR and FGF15 signaling[276]. It also reduces portal LPS and duodenal IkappaB protein levels while restoring the abundance of duodenal tight junction protein in mice with high-fructose diet-induced NAFLD[277]. L. rhamnosus GG treatment improves hepatic steatosis, liver injury, adipose inflammation, glucose intolerance, and insulin resistance[278]. In a randomized controlled trial involving twenty obese children, administration of L. rhamnosus GG significantly decreased ALT levels and antipeptidoglycan-polysaccharide antibodies without affecting body mass index (BMI) Z score and visceral fat[279].

L. gasseri BNR17 is known to reduce body weight, white adipose tissue weight, serum leptin, and insulin, while increasing the expression of fatty acid oxidation-related genes[280]. Naudin et al[281] found that oral gavage of L. lactis subsp. cremoris to mice on a Western diet resulted in reduced hepatic steatosis, inflammation, and weight gain; lower serum cholesterol and BMI; and increased glucose tolerance. L. coryniformis subsp. torquens T3 has been shown to inhibit obesity and liver inflammation, while alleviating hepatic fat buildup in mice with NAFLD[282]. Additionally, L. coryniformis can reduce the abundance of harmful bacteria, enhance the intestinal barrier, and increase SCFA content. L. johnsonii BS15 protects HFD-induced NAFLD mice from hepatic steatosis and hepatocyte apoptosis[283]. BS15 also lowers serum LPS levels, inhibits insulin resistance, and decreases intestinal permeability. Oral administration of L. reuteri GMNL-263 decreases levels of serum glucose, insulin, lipids, and hepatic injury markers, along with improving liver lipid profile in high fructose-induced hepatic steatosis in mice[284]. GMNL-263 also reduces TNF-α and IL-6 levels in adipose tissue and increases the number of Bifidobacterium and Lactobacillus. The therapeutic effects of Lactobacillus are not limited to live bacteria; heat-killed L. brevis SBL88 has been shown to significantly reduce hepatic fat droplets without altering the gut microbiota but by modulating lipid and insulin metabolism[285]. Despite the potential of Lactobacillus in NAFLD treatment, some studies have shown contrasting results. In a double-blind placebo-controlled study, it was found that L. acidophilus did not have any effect on serum lipid levels[286]. Furthermore, many studies have observed an increased abundance of Lactobacillus in patients with NAFLD[94].

Bifidobacterium: Bifidobacterium is another widely used probiotic. Studies have found a decrease in the abundance of Bifidobacterium in NAFLD patients[78,242]. Therefore, restoring the abundance of Bifidobacterium through probiotic supplementation could be a potential alternative treatment for NAFLD. Bifidobacterium has been shown to improve visceral fat, cholesterol levels, and insulin resistance[287,288]. It can alleviate NAFLD through the activation of the sirtuin inhibitor (sirtuin 1)-mediating signaling pathway[289].

B. animalis ssp. lactis GCL2505 has demonstrated an anti-metabolic syndrome effects in HFD-fed mice. Oral administration of GCL2505 has been shown to reduce visceral fat accumulation, improve glucose tolerance, and increase acetate levels[290]. Administration of B. adolescentis and B. pseudocatenulatum CECT 7765 has been shown to reduce visceral fat accumulation, liver steatosis, serum cholesterol, triglyceride, glucose, and IL-6 and decrease insulin sensitivity in HFD-fed rats[287,291]. B. longum has proven to be more efficient than L. acidophilus in attenuating liver fat accumulation[292]. An et al[293] found antiobesity and lipid-lowering effects of three Bifidobacterium spp. (B. longum SPM 1207, B. longum SPM 1205, and B. pseudocatenulatum SPM 1204) in HFD-induced obese rats. A recent study found that B. lactis Probio-M8 could improve liver damage and hepatic steatosis in HFD-fed rats[294]. Furthermore, M8 modulated the gut microbiota by increasing the abundance of Bifidobacterium and decreasing the abundance of Bilophila, Lachnoclostridium, GCA-900066225, and Phascolarctobacterium.

Saccharomyces boulardii: Saccharomyces boulardii is a fungal probiotic that provides a conducive environment for the growth of Lactobacillus and Bifidobacterium[295]. S. boulardii modulates gut microbiota and the immune system, exhibits antitumor activity, inhibits pathogen growth and inflammation, and maintains the SCFA levels and intestinal barrier integrity[296-298]. Several studies have highlighted the potential of S. boulardii in NAFLD treatment. Everard et al[299] found that oral administration of S. boulardii in obese mice for 4 weeks led to reduced body weight, fat mass, and hepatic steatosis and modulated gut microbial composition. Yang et al[300] reported that S. boulardii gavage to MCD diet-fed mice resulted in improved liver function, less hepatic steatosis, decreased inflammation, increased microbiota diversity, and maintained gut integrity.

Probiotic cocktails: Many studies have explored the potential of a combination of two or more probiotic strains in NAFLD treatment. A cocktail of B. infantis, L. acidophilus, and Bacillus cereus has been shown to delay the progression of NAFLD by suppressing the LPS/TLR4 signaling[301]. Oral administration of L. lactis and Pediococcus pentosaceus can mitigate NAFLD progression by modulating the gut environment[130]. Consumption of kefir, a probiotic drink with more than 50 species of lactic acid bacteria and yeast, prevents obesity and NAFLD by modulating gut microbiota and promoting fatty acid oxidation[302]. Oral administration of Symbiter, a concentrated biomass of 14 probiotic bacteria combined with omega-3, has been shown to decrease hepatic triglyceride and hepatic steatosis[303].

VSL#3, a widely used mixture of eight live freeze-dried probiotic strains, has been confirmed as an effective therapeutic option for NAFLD in both animal and human studies. VSL#3 can reduce hepatic fatty acid content, improve liver histology, and limit liver damage associated with NAFLD in mice[304,305]. It has also been shown to improve HFD-induced hepatic steatosis and insulin resistance and attenuate IkappaB kinase signaling and TNF-α in mice[306]. Additionally, VSL#3 has also been found to improve insulin resistance in liver and adipose tissues and protect against the development of steatohepatitis and atherosclerosis[307]. In a Western diet-fed NASH mouse model, VSL#3 supplementation was shown to reduce hepatic fat content, improve insulin sensitivity, and modulate the gut microbiota by enriching butyrate-producing Ruminococcus and Faecalibacterium and reducing Bacteroidaceae, Porphyromonadaceae, and Helicobacteraceae[308]. Loguercio et al[309] reported that the administration of VSL#3 had a positive effect on patients with NAFLD and alcoholic liver cirrhosis, with an improvement in hepatic enzymes. A double-blind randomized controlled trial of VSL#3 on obese children with biopsy-proven NAFLD found that VSL#3 improved NAFLD and reduced triglycerides and insulin resistance[310]. Contrary to the positive effects of VSL#3 in NAFLD treatment, many studies reported alternative findings. Solga et al[311] reported that VSL#3 increased hepatic fat buildup in four NAFLD patients, while Velayudham et al[312] found that VSL#3 did not protect against steatosis and inflammation in NASH, although it could modulate liver fibrosis.

In addition to live probiotic cocktails, cell-free and fermentation extracts of various probiotics have also been explored for treatment of NAFLD. Administration of cell-free supernatants of four probiotic strains, L. salivarius Ls-33, L. acidophilus NCFM, B. lactis HN019, and B. lactis 420 has been shown to increase tight junction integrity[82]. A probiotic mixture consisting of L. acidophilus, L. plantarum, Bifidobacterium bifidum, Bacillus subtilis fermentation extract, Aspergillus oryzae fermentation extract, and maltodextrin has been shown to improve lipid profiles, and reduce TNF-α and IL-6 levels in rats fed a HFD and sucrose diet[313].

Clinical data and randomized controlled trials have supported the efficacy of probiotics in the treatment of NAFLD[314-321] (Table 3). Systematic reviews and meta-analyses have further highlighted the potential of probiotics in treating metabolic diseases, including NAFLD[322-325]. However, a systematic review by Tarantino and Finelli[326] concluded that drawing a definitive conclusions regarding the therapeutic efficacy of probiotics and prebiotics in NAFLD and obesity remains challenging due to the lack of high-quality clinical studies.

Next-generation probiotics: Next-generation probiotics (NGPs) are unconventional probiotics that have demonstrated enhanced therapeutic effects in NAFLD. Akkermansia muciniphila is a mucin degrader and an important component of healthy gut microbiota, which plays a crucial role in preserving the gut barrier[72] and produces SCFAs[327]. A. muciniphila is a promising NGP that is inversely associated with metabolic disorders such as obesity, diabetes, and cardiometabolic diseases[328]. It has been shown to prevent fatty liver disease and decrease serum triglycerides in mice, reinforce the epithelial barrier, and reduce fat deposition and obesity in humans and mice[329,330]. It has also been found to efficiently reverse hepatic steatosis, inflammation, and liver injury in mice[331]. The beneficial effects of A. muciniphila are attributed to increased mitochondrial oxidation, BA metabolism, and L-aspartate levels in the liver, as well as modulation of the gut microbiota. Kim et al[332] found that oral administration of A. muciniphila led to a significant reduction in serum triglyceride, ALT, and IL-6 in obese mice. In a randomized controlled trial, it was found that oral administration of A. muciniphila improved insulin sensitivity and decreased insulinemia, lowered plasma total cholesterol, and reduced the markers of liver dysfunction and inflammation[333]. Daily oral supplementation of 10¹⁰A. muciniphila has been found to be safe and well tolerated. A recent study also showed that A. muciniphila outperformed VSL#3 in reducing hepatic fat content in NAFLD mice[334].

Faecalibacterium prausnitzii is a constituent of the healthy gut microbiota in humans, known for its ability to ferment dietary fibers and produce SCFAs. F. prausnitzii synthesizes a metabolite called microbial anti-inflammatory molecule, which helps maintain intestinal barrier function and elevates the expression of tight junction proteins[335]. When combined with Roseburia intestinalis and Bacteroides faecis, F. prausnitzii can further enhance the epithelial barrier integrity[336]. Oral administration of F. prausnitzii in HFD-fed mice has been shown to decrease hepatic fat content, lower hepatic enzymes, reduce adipose tissue inflammation, and increase fatty acid oxidation and adiponectin signaling[337].

Clostridium butyricum MIYAIRI 588 is a butyrate-producing bacterium traditionally used in humans for the treatment of diarrhea and constipation. MIYAIRI 588 has been shown to prevent NAFLD progression by reducing hepatic fat deposition and improving triglyceride content and insulin resistance[338,339]. Escherichia coli Nissle 1917 promotes the mucosal integrity of intestinal epithelial cells by up-regulating zonula occludens-1 expression in mice[340].

Parabacteroides distasonis has emerged as a promising agent for NAFLD, primarily through its modulation of metabolic pathways and the gut-liver axis. Wei et al[341] demonstrated that P. distasonis alleviates hepatic steatosis and inflammation in mice by inhibiting pro-inflammatory signaling and restoring gut barrier function through its metabolite pentadecanoic acid. Additionally, pentadecanoic acid has been shown to reduce liver fat content and weight in the TANGO randomized controlled trial[342]. It also boosts fatty acid oxidation via γ-linolenic acid production, activating PPARα signaling[343]. Beyond NAFLD, P. distasonis has demonstrated therapeutic potential in obesity and type 2 diabetes by improving glucose homeostasis, insulin sensitivity, and systemic inflammation through the production of SCFAs and succinate[344]. Recent studies have highlighted the therapeutic potential of other bacteria that can be used as NGPs, such as Limosilactobacillus mucosae (L. mucosae)[345], Bacteroides thetaiotaomicron[346], and Lactiplantibacillus plantarum ZDY2013[347]. L. mucosae is effective in reducing body weight, liver weight, adipose tissue weight, total cholesterol, triacylglycerol, and LDL-C in NAFLD mice[345]. Additionally, L. mucosae can decrease the relative abundance of Ruminococcaceae and increase the abundance of Akkermansia. Bacteroides thetaiotaomicron has been shown to reduce fat accumulation, hyperlipidemia, insulin resistance, and body weight, and prevent hepatic steatohepatitis and liver injury in HFD-fed mice[346]. It also modulates the gut microbial composition and decreases the Firmicutes to Bacteroidetes ratio. L. plantarum ZDY2013 administration improves insulin resistance, reduces fat accumulation in the liver, restores liver function, and regulates oxidative stress[347].

Prebiotics

Prebiotics are dietary components, particularly indigestible carbohydrates that maintain a healthy gut microbiota by providing an optimal environment for the growth of beneficial microbes[348,349]. Studies have shown that prebiotic administration increases the plethora of beneficial microbes such as Akkermansia, Faecalibacterium, Bifidobacterium, Lactobacillus, and Roseburia, while decreasing pathogenic microbes[350-354]. Complex carbohydrates, such as fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), inulin, polydextrose, and fructans, are fermented by specific gut microbiota to produce SCFAs.

FOS is known to improve liver pathology, inhibit adipocyte enlargement, and increase fecal concentrations of butyrate and acetate and serum concentrations of propionate[355]. Additionally, FOS administration decreases the expression of genes related to inflammation and lipid metabolism. Studies have shown that dietary FOS can decrease steatohepatitis by restoring intestinal epithelial barrier function and normal GI microflora[356]. Pachikian et al[357] reported that FOS reduced hepatic triglyceride buildup in mice and altered gut microflora by increasing caecal Bifidobacterium spp. and decreasing Roseburia spp. Matsumoto et al[358] reported that FOS could decrease hepatic steatosis, liver inflammation, and expression of TLR4 in an MCD mouse model of NASH. This was accompanied by an increase in fecal SCFAs and immunoglobulin A concentrations. FOS and choline treatment were shown to reduce total mean fat in the liver and heart, thus alleviating hepatic and cardiac steatosis[359]. Oligofructose has been found to decrease plasma LPS, cytokines, and the expression of inflammatory markers in obese mice[360]. Furthermore, oligofructose improves tight-junction integrity and reduces intestinal permeability. Administration of oligofructose in NASH patients has been shown to improve liver steatosis, increase the abundance of Bifidobacterium, and decrease the abundance of Clostridium cluster XI and I[361]. A pilot study also found that oligofructose could reduce insulin level and hepatic enzymes without decreasing plasma lipid levels in NASH patients[362].

GOS increases the abundance of Bifidobacterium[363]. A blend of GOS and FOS (9:1) has been shown to increase both the abundance and proportion of bifidobacteria in healthy infants[364]. A double-blind, randomized study involving 45 overweight adults having ≥ 3 risk factors linked to metabolic syndrome found that GOS administration increased the number of fecal bifidobacteria and fecal secretory immunoglobulin A, while decreasing total cholesterol, insulin, plasma C-reactive protein, and fecal calprotectin[365].

Inulin is a fructan found naturally in a variety of plants. It can decrease plasma triacylglycerol concentrations and hepatic lipogenesis[366] and increase the abundance of Bifidobacterium[367]. Inulin administration results in the production of SCFAs and reduces the expression of genes involved in lipogenesis[368]. It also improves hepatic steatosis and fibrosis in mice[369]. Aoki et al[369] found that inulin consumption resulted in changes in the gut microbiota, including the enrichment of Bacteroides acidifaciens and Blautia producta, which synergistically increased acetate production, an SCFA that protects against NAFLD via the FFAR2 pathway. In a preclinical study, Catry et al[370] found that feeding inulin-type fructans to Apoe^(-/-) mice modulated the gut microbiota by replenishing Akkermansia and decreasing secondary BA-synthesizing bacteria. Similar studies in humans showed that administration of inulin-propionate ester reduced intrahepatocellular lipid content, intra-abdominal adipose tissue distribution, and weight gain[371]. In a randomized double-blind placebo-controlled study with sixty-two participants with NAFLD, administering inulin following a brief metronidazole therapy effectively lowered ALT levels and helped sustain weight loss achieved through a very-low-calorie diet[372]. Another double-blind, placebo-controlled intervention study with 30 obese women found that inulin-type fructans led to an enrichment of F. prausnitzii and Bifidobacterium, which showed a negatively correlation with serum LPS levels[373]. Wei et al[341] demonstrated that soluble inulin is more effective than insoluble cellulose in suppressing NASH progression in mice by reducing hepatic steatosis, necro-inflammation, fibrosis, and ballooning. They also found that inulin enriched P. distasonis, which uses inulin to produce pentadecanoic acid that restored gut barrier function and reduced serum LPS and liver inflammation. Contrary to these findings, a recent randomized controlled trial involving twenty-two participants reported that inulin-type fructans did not affect liver fat content, liver function test, and metabolic and inflammatory mediators[374]. However, fecal samples showed an increase in Bifidobacterium.

Lactulose, a synthetic sugar commonly used for the treatment of constipation, promotes the growth of beneficial bacteria[375]. A study on humans has shown that lactulose increases fecal bifidobacterial counts without significantly affecting Lactobacillus, fecal BAs, and neutral sterols[376]. A study by Fan et al[377] found that while lactulose could not completely prevent steatohepatitis development, it could improve hepatic inflammation in rats with steatohepatitis induced by HFD. Chitin-glucan has been shown to decrease hepatic triglyceride accumulation, weight gain induced by HFD, and hypercholesterolemia[378]. Additionally, a combination of prebiotic isomalto-oligosaccharides and antioxidant lycopene has been found to work synergistically in preventing nonalcoholic fatty liver-like symptoms[379]. Dietary isomalt has been shown to attenuate hepatic steatosis, restore intestinal epithelial integrity, improve the abundance of Akkermansia, suppress Acinetobacter and Corynebacterium_1, and reduce fecal acetate concentrations, while elevating propionate and butyrate in rats[380]. A randomized, placebo-controlled clinical trial involving 197 participants found that resistant starch intervention reduced intrahepatic triglyceride content and liver enzymes[381].

Many recent studies have explored the potential of plant polysaccharides and bioactives in NAFLD treatment. Zhang et al[382] demonstrated that polysaccharides from Smilax china reduced serum and hepatic lipid levels, and reversed HFD-induced increases in body, liver, and adipose tissue weights. The polysaccharide intervention upregulated the genes related to lipolysis, downregulated the genes related to lipogenesis, and restored gut microbiota. Huang et al[383] found that large-leaf yellow tea polysaccharides decreased bile salt hydrolase-producing genera and increased taurine metabolism genera and the levels of conjugated BAs. Hao et al[384] reported that Crataegus pinnatifida polysaccharide effectively reduced liver steatosis, decreased serum levels of triglyceride, total cholesterol, AST, ALT, and LDL-C, inhibited expression of genes for fatty acid synthesis, initiated the expression of genes for fatty acid oxidation, and increased levels of total SCFAs in NAFLD mice. Furthermore, the intervention restored gut microflora by decreasing the Firmicutes to Bacteroidetes ratio and increasing the abundance of Akkermansia. Wu et al[385] demonstrated that polysaccharides from Panax japonicas significantly reduced ALT, hepatic fat buildup, and blood lipids. It also reduced the abundance of Staphylococcus, Turicibacter, and Dubosiella and increased the abundance of Lactobacillus, Bacteroides, and Blautia. Zuo et al[386] found that epigallocatechin gallate, found in green tea, inhibited intestinal barrier dysfunction and inflammation, ameliorated NAFLD phenotypes, and improved metabolic disorders in HFD-fed rats. Moreover, it promoted beneficial microbes, particularly SCFA-producing bacteria, such as Lactobacillus, and suppressed Gram-negative bacteria, such as Desulfovibrio.

Synbiotics

Synbiotics, a combination of pro- and prebiotics[349], have shown potential in modulating gut microbiota composition and improving markers of liver health. L. rhamnosus GG and L. plantarum WCFS1, combined with anthraquinone from Cassia obtusifolia L., have been shown to reduce blood lipid levels, improve insulin resistance, and enhance intestinal mucosal barrier function in NAFLD rats[387]. L. paracasei N1115 and FOS have been shown to alleviate HFD-induced hepatic steatosis, slow the progression of cirrhosis, reduce serum triglyceride and cholesterol, fasting blood glucose and insulin, and improve intestinal barrier function by increasing the expression of tight junction components[388]. FOS and a probiotic cocktail, including L. rhamnosus, L. casei, L. bulgaricus, B. longum, and Streptococcus thermophilus, can reduce liver stiffness, ALT, and serum cholesterol[389]. Administration of Lactobacillus reuteri with guar gum and inulin for three months in NASH patients resulted in reductions in steatosis, weight, waist circumference, and BMI, although it did not improve intestinal permeability or LPS levels[390]. Daily consumption of synbiotic yogurt containing B. animalis and inulin has been shown to reduce NAFLD grade and levels of ALT, AST, alkaline phosphatase, and gamma-glutamyl transferase[391]. A synbiotic consisting of L. paracasei, arabinogalactan, and FOS has been shown to improve insulin resistance, slow NAFLD progression, reduce cytokines and TLR expression, preserve gut barrier integrity, and increase the abundance of Enterobacteriales and Escherichia coli in colonic mucosa[392]. A. muciniphila and quercetin administration in the NAFLD mouse model has been shown to attenuate steatosis by modulating gut microbiota and enhancing hepatic expression of BA synthesis[393]. A randomized controlled trial using B. longum with FOS reported significant reductions in inflammatory markers, LDL-C, steatosis, and NASH activity index[394]. Protexin, a synbiotic containing seven probiotic bacteria belonging to lactobacilli, bifidobacteria, and S. thermophilus with prebiotic FOS, has shown promising results in NAFLD treatment[395-398].

Many recent studies have highlighted the therapeutic potential of different synbiotic blends in treating NAFLD. Zhang et al[399] demonstrated that a synbiotic combination of Bifidobacterium and S. thermophilus with inulin improved total triglyceride, cholesterol (LDL and high-density lipoprotein cholesterol), insulin resistance, and restored the intestinal barrier function and inflammation in an early NAFLD mouse model. Ralli et al[400] found that a combination of silymarin, piperine, and fulvic acid, along with a probiotic blend of Bifidobacterium adolescentis, Bifidobacterium bifidum, Lactobacillus casei, and L. rhamnosus, reduced hepatic fat accumulation and improved gut microbiota composition in an NAFLD mouse model. Gao et al[401] reported that aqueous extract from Chrysanthemum morifolium combined with a probiotic blend containing L. rhamnosus GG, L. rhamnosus CNCMl-4036, L. rhamnosus BPL1-15, and B. lactis BPL-1 effectively reduced body weight and hepatic steatosis, improved glucose intolerance and insulin resistance, and modulated the expression of genes involved in lipid metabolism in HFD-fed mice.

Systematic reviews and meta-analyses have also confirmed the utility of synbiotics in NAFLD treatment[402-404]. A recent systematic review and meta-analysis reported that synbiotic treatment in NAFLD patients led to a reduction in liver enzymes, lowered cholesterol and LDL levels, improved glucose profiles, and decreased pro-inflammatory cytokines such as TNF-α[404]. However, contrary to the positive results reported in several studies, a double-blind phase 2 trial involving 104 patients with NAFLD found that a synbiotic of FOS and B. animalis subspecies lactis BB-12 had no effect on markers of liver fibrosis or liver fat content, although it did alter the fecal microbiota[405]. Clinical trials and animal studies have not reported any adverse effect from probiotic or prebiotic treatments, suggesting that they are safe and effective. However, further research is warranted to better understand the impact of probiotics and prebiotics in human interventions.

FMT

FMT is a biotherapeutic procedure that restores and repairs gut microbial diversity[406]. In FMT, microbial populations are transplanted from a healthy donor to an unhealthy recipient[407]. The potential of FMT can be highlighted by studies showing that germ-free mice develop hepatic steatosis on receiving FMT from NASH-affected mice, suggesting that donor characteristics can be transferred to the recipient through FMT[408]. Additionally, germ-free mice that received FMT from HFD- fed mice exhibited intestinal epithelial barrier disruption[409].

The efficacy of FMT has been demonstrated in human and animal studies. Zhou et al[410] found that FMT from healthy mice protected HFD-fed mice from steatohepatitis. The procedure also led to an increased abundance of Christensenellaceae and Lactobacillus, a rise in caecal butyrate concentration, and enhanced expression of intestinal tight junction protein zonula occludens-1. Furthermore, FMT reduced hepatic triglyceride and cholesterol and lowered pro-inflammatory cytokines. FMT from lean donors to recipients with metabolic syndrome has been shown to improve insulin sensitivity, increase abundance of butyrate-producing intestinal microbiota, and result in changes in plasma metabolites[411,412].

Clinical trials have found that FMT is generally safe, with no significant adverse effects, and leads to sustained shifts in the recipient’s microbiota toward that of the donor[413]. Allogenic FMT using feces from metabolic syndrome donors has been shown to decrease insulin sensitivity in recipients with metabolic syndrome[414]. Even a short one week FMT intervention has proven effective in improving liver disease severity indices[415]. In a double-blind randomized controlled pilot study involving 20 male metabolic syndrome patients, a single FMT produced detectable changes in intestinal microbiota composition[416]. Another randomized control trial with 21 NAFLD patients found that while FMT did not improve hepatic proton density fat fraction and insulin resistance, it did reduce small intestinal permeability[417]. Furthermore, vegan allogenic donor FMT not only induced changes in gut microbiota and plasma metabolites but also caused distinct alterations in hepatic DNA methylation profiles[418].

Although FMT is generally considered a safe therapeutic procedure, it is not entirely free from potential adverse effects and complications[419,420]. Most side effects are usually mild, including abdominal pain, diarrhea, constipation, and low-grade fevers, which typically resolve within a few days[421]. However, more severe and life-threatening events, such as sepsis, bacteremia, ileus, or perforation, have also been reported. One notable adverse event involved two recipients developing extended-spectrum beta-lactamase-producing E. coli bacteremia, following FMT, with one of these patients subsequently dying[422]. FMT has also been associated with an increased risk of transmitting microbiome-associated chronic diseases, including autoimmune, GI, and cardiometabolic disorders[423-426]. It therefore becomes an absolute necessity to carry out donor screening before FMT to avoid the transfer of potentially life-threatening infections from the donor’s microbiota.

CONCLUSION

A growing body of evidence suggests a strong link between alterations in gut microbiota and the pathophysiology of NAFLD. Identifying specific microbiota and metabolic profiles could serve as valuable marker for the development of gut microbiota-based diagnostic tests, providing reliable and accurate diagnosis of NAFLD. Studies have identified microbiota and metabolic signatures associated with different stages of NAFLD, including NASH and cirrhosis. However, the variability in microbial signatures across different studies underscores the need for further research. A particularly important area of focus is understanding the role of certain bacteria in progression of NAFLD and exploring their potential diagnostic implications in distinguishing between the early and advanced stages of the disease.

Future perspective

Further research into the role of the gut microbiota in maintaining gut barrier function and regulating the immune system is essential for fully understanding its involvement in NAFLD. Targeted microbiota manipulation through dietary interventions, probiotics, prebiotics, synbiotics, and FMT represents a promising strategy for NAFLD treatment. However, despite the promising results, further research involving larger cohorts are needed to confirm the efficacy and safety of these microbiota-targeted therapies and to develop personalized treatment approaches for NAFLD. Furthermore, integrating machine learning and artificial intelligence with microbial and metabolic signatures could become a powerful tool for early detection and diagnosis of NAFLD.