Gut microbiota differences, metabolite changes, and disease intervention during metabolic - dysfunction - related fatty liver progression

Abstract

In the current era, metabolic dysfunction-associated steatotic liver disease (MASLD) has gradually developed into a major type of chronic liver disease that is widespread globally. Numerous studies have shown that the gut microbiota plays a crucial and indispensable role in the progression of MASLD. Currently, the gut microbiota has become one of the important entry points for the research of this disease. Therefore, the aim of this review is to elaborate on the further associations between the gut microbiota and MASLD, including the changes and differences in the microbiota between the healthy liver and the diseased liver. Meanwhile, considering that metabolic dysfunction-associated fatty liver and metabolic dysfunction-associated steatohepatitis are abnormal pathological states in the development of the disease and that the liver exhibits different degrees of fibrosis (such as mild fibrosis and severe fibrosis) during the disease progression, we also conduct a comparison of the microbiota in these states and use them as markers of disease progression. It reveals the changes in the production and action mechanisms of short-chain fatty acids and bile acids brought about by changes in the gut microbiota, and the impact of lipopolysaccharide from Gram-negative bacteria on the disease. In addition, the regulation of the gut microbiota in disease and the production and inhibition of related disease factors by the use of probiotics (including new-generation probiotics) will be explored, which will help to monitor the disease progression of patients with different gut microbiota compositions in the future and carry out personalized targeted therapies for the gut microbiota. This will achieve important progress in preventing and combating this disease.

Key Words: Metabolic dysfunction-associated steatotic liver disease; Gut microbiota; Short-chain fatty acids; Bile acids; Lipopolysaccharides; Probiotics

Core Tip: This review elaborates in detail on the comparison of the gut microbiota between metabolic dysfunction-associated steatotic liver disease patients and healthy individuals, between metabolic dysfunction-associated fatty liver and metabolic dysfunction-associated steatohepatitis, as well as between the stages of mild fibrosis and significant fibrosis. It reveals the changes in the production and action mechanisms of short-chain fatty acids, bile acids, and lipopolysaccharides brought about by the changes in the gut microbiota. In addition, it also explores the regulation of the gut microbiota in the disease by using probiotics and the production and inhibition of related disease factors.

INTRODUCTION

As living standards get better and people's diets change, unhealthy eating habits high in fat and calories have increasingly normalized. Gradually, metabolic dysfunction-associated steatotic liver disease (MASLD) has become one of the principal chronic liver diseases across the globe, and the quantity of MASLD patients continues to ascend year after year[1]. MASLD is a chronic condition that occurs without alcohol consumption or with minimal alcohol intake and manifests as the accretion of fat in hepatocytes. This condition encompasses a range of liver diseases, such as metabolic dysfunction-associated fatty liver (MAFL), metabolic dysfunction-associated steatohepatitis (MASH), liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Based on numerous studies of MASLD, the development of it involves a widely accepted "two-hit theory". This theory includes the "first hit" of liver lipid accumulation and the "second hit" of inflammation and fibrosis caused by oxidative stress and the release of pro - inflammatory factors. As researchers gain a deeper understanding of MASLD, the "multiple - hit" hypothesis, which is based on the "first hit" involving fat tissue accumulation and encompasses the roles of insulin resistance (IR), inflammatory factors, and the gut microbiota, is gradually being accepted[2]. The causes leading to these consequences are closely related to gut microbiota dysbiosis, which is considered a key factor in the pathology of MASLD. It increases the synthesis of triglycerides and the liver's uptake of free fatty acids. This leads to excessive accumulation of triglycerides, causing liver lipid peroxidation and the deposition of reactive oxygen species (ROS), which in turn causes hepatocellular damage[3]. Whether it is the widely accepted "two-hit" or the emerging "multiple hits", the composition and changes in the gut microbiota play considerably important roles in both[4].

Short-chain fatty acids (SCFAs), which are mainly constituted by acetate, propionate, and butyrate, are among the metabolic by-products of the gut microbiota. and help maintain the homeostasis of the gut microbiota, induce fat oxidation, and reduce the impact on the liver caused by fat accumulation and the lipid oxidative stress response. Moreover, they strengthen the integrity of intestinal epithelial cells, inhibit the occurrence of the "intestinal leakage" phenomenon, and can also achieve insulin sensitivity by activating G protein-coupled receptors (GPRs)[5]. In addition to producing SCFAs, the gut microbiota also protects the liver by secreting related hydrolases that affect the regulation of bile acids (BAs) in the gut-liver axis. These hydrolases prevent the accumulation of BAs in the liver that can cause hepatocyte damage, promote the metabolism of triglycerides, and activate anti-inflammatory factors in the enterohepatic circulation of BAs, which inhibit the activity of hepatic stellate cells (HSCs) and inflammatory responses. If harmful bacteria within the gut microbiota, such as some gram-negative bacteria, proliferate excessively, an increase in the concentration of lipopolysaccharides (LPS) can occur. An imbalanced gut microbiota disrupts intestinal barrier function, allowing pathogens and LPS to move along the gut-liver axis to the liver, activating the production of many proinflammatory factors, which exacerbates the inflammatory response in MASLD[6].

Therefore, understanding the regulatory bacteria related to the metabolic products of the gut microbiota that affect the development and progression of MASLD and understanding the changes in their abundance in patients constitute an important basis for the microbial approach to prevent or treat this disease. In this review, we conduct a detailed comparison between the gut microbiota profiles of MASLD patients and those of healthy individuals. Through this comparison, we aim to elucidate how alterations in the gut microbiota and its associated metabolites exert an impact on the disease. Additionally, we explore the regulatory effects of probiotics on the disease, thereby providing insights into potential therapeutic strategies.

CHANGES IN THE GUT MICROBIOTA ASSOCIATED WITH MASLD

The human intestinal microbiota (IM) is composed of a variety of microorganisms, including bacteria, archaea, and eukaryotes, with bacteria making up the vast majority of the IM. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria accounted for 98% of the bacterial species. In the IM of adults, the proportion of Firmicutes is 60%-80%, and that of Bacteroidetes is approximately 20%-40%[3,7]. At the genus level, the relative abundance of Prevotella, Bacteroides, Escherichia-Shigella (E. Shigella), Megamonas, Fusobacterium, and Lachnospiraceae increased, whereas the relative abundance of Faecalibacterium, Romboutsia, Blautia, Agathobacter, Clostridium_sensu_stricto_1, Ruminococcus, and Coprococcus decreased[8]. Based on the aforementioned basic composition of the gut microbiota, we compared the changes in the microbiota at different stages of the disease (Figure 1).

Figure 1 The changes in healthy livers, livers at different stages of diseases, and local liver cells, as well as the changes in some gut microbiota in the pathological states of the liver at various stages. MASLD: Metabolic dysfunction-associated steatotic liver disease; MAFL: Metabolic dysfunction-associated fatty liver; MASH: Metabolic dysfunction-associated steatohepatitis. Created from BIOGDP.com[93].

Comparison of the intestinal flora between MASLD patients and healthy people

An increasing number of studies have demonstrated that there are significant changes in β- diversity between MASLD patients and healthy subjects, indicating a phenomenon of gut microbiota dysbiosis. At the phylum level, compared with healthy individuals, MASLD patients presented a relative decrease in the abundance of Firmicutes and an increase in Bacteroidetes and Proteobacteria[9], with a reduced ratio of Firmicutes/Bacteroidetes.

At the family level, the abundance of Dubosiella, Fusobacteriaceae, and Prevotellaceae increased, whereas the abundance of Ruminococcaceae and Odoribacteraceae significantly decreased[10,11]. The abundance of Bacteroidaceae increased with increasing severity of MASLD, while Prevotellaceae and Erysipelotrichaceae decreased[11,12]. At the genus level, the relative abundance of Prevotella, Bacteroides, E. Shigella, Megamonas, Fusobacterium, and Lachnospiraceae increased, whereas the relative abundance of Clostridium_sensu_stricto_1, Agathobacter, Romboutsia, Faecalibacterium, Blautia, Ruminococcus, and Coprococcus decreased[13]. Faecalibacterium is primarily composed of Faecalibacterium prausnitzii (F. prausnitzii); Prevotella is mainly Prevotella copri (P. copri) and Prevotella stercorea, with P. copri being associated with chronic inflammation, such as colitis, IR, and metabolism, and may also hinder the reproduction of beneficial bacteria[14]. A decrease in Ruminococcus, Coprococcus, and F. prausnitzii is associated with MASLD and is independent of body mass index and IR.

In the experiment conducted by Yang et al[15], the severity of MASLD was divided into three subtypes: Mild, moderate, and severe. In all three subtypes, the amounts of Faecalibacterium, Subdoligranulum, Haemophilus, and Roseburia were reduced. In the mild group, Weissella and Butyricicoccus were reduced, whereas in the moderate and severe groups, Eubacterium_hallii_group, Lachnospiraceae_UCG_004, Intestinibacter, Rothia, Erysipelotrichaceae_UCG_003, and Eubacterium_ventriosum_group were significantly reduced.

Comparison of the intestinal flora between MASH patients and MASH patients

Two pathological states comprise MASLD, namely, MAFL and MASH, which can be distinguished by liver biopsy to determine the presence of ballooning hepatocytes. They are typically associated with increased obesity and metabolic abnormalities. The former is considered an early manifestation of MASLD, whereas the latter is considered a severe stage that may progress to liver fibrosis, cirrhosis, and even HCC[16,17]. Among patients with MAFL, approximately 10%-20% progress to MASH, and one-third of MASH patients develop cirrhosis[18]. Thus, it is of particular significance to investigate the alterations in the gut microbiota throughout the different stages of the development of both MAFL and MASH.

In studies, it has been observed that with increasing severity of MASLD, there are no significant differences in the gut microbiome when classified by phylum; therefore, analysis will focus on the family level, where differences begin to emerge[8,12].

In one study, Boursier et al[12] compared 57 subjects using the 16S ribosomal RNA gene sequencing method. Compared with those in healthy individuals, the relative abundances of Bacteroides and Fusobacterium in MAFL patients were greater, whereas the abundance of Prevotella was lower. At the family level, Clostridiaceae, Ruminococcaceae, Enterobacteriaceae, and Erysipelotrichaceae were more concentrated in the early MASH group. As MASH progresses, the abundances of Fusobacteriaceae and Ruminococcaceae continue to decrease, while the abundances of Spirochaetes, Alistipes, Dorea, Bosea, Campylobacter, and Megamonas increase, and liver fibrosis may occur at this time. Enterobacteriaceae, Romboutsia, and Erysipelatoclostridium were enriched in the early stage but began to decrease in the middle stage of MASH development[11,19]. When the critical state of MASH progresses to fibrotic MASH, Mucispirillum and Dubosiella are enriched, whereas Akkermansia and Romboutsia are almost depleted[11]. Some studies have shown that, compared with those in the MAFL group, the abundances of Fusobacterium, Prevotella, Eubacterium biforme under the genus Eubacterium, and Megamonas in the MASH group were significantly greater, whereas the abundances of Parabacteroides and Dialister were lower[10]. However, another study revealed that the abundance of Prevotella in MASH patients was decreased and inversely proportional to the abundance of Bacteroides[12].

No/mild liver fibrosis stage (F0/F1) and significant liver fibrosis stage (≥ F2)

With respect to the dynamic changes in the livers of patients with MASLD, there are still certain limitations in merely differentiating the changes in the gut microbiota between fatty liver disease and hepatitis. Therefore, it is necessary to analyze whether fluctuations in the gut microbiota occur during the non-/mild liver fibrosis stage (F0/F1) and the significant liver fibrosis stage (≥ F2).

Both Firmicutes and Bacteroidetes were the primary phyla in the F0/F1 and ≥ F2 groups. The ratio of Firmicutes to Bacteroidetes in the ≥ F2 group was slightly lower than that in the F0/F1 group. This genus mainly includes Bacteroides, Blautia, Faecalibacterium, Agathobacter, Mucispirillum, Ruminococcus, and Bifidobacterium, which is similar to the gut microbiota composition of the aforementioned MASLD patients and healthy subjects[20]. A number of studies have revealed that the presence of liver fibrosis is related to the enrichment of Enterobacteriaceae, E. Shigella, Acadaminococcus, Sutterella, and Corynebacterium. The enrichment of Enterobacteriaceae is considered to be related to liver fibrosis, steatosis, and inflammation[21]. It was demonstrated in Rau's experiment that, compared with F0/F1 patients, patients with ≥ F2 had a greater frequency of Acadaminococcus and Prevotella, whereas the frequency of Alistipes onderdonkii in the genus Alistipes was significantly lower[10]. A study indicated that patients with ≥ F2 had higher levels of Bacteroides, Ruminococcus, Alistipes, Dorea, and Spirochaeta. Unlike Rau et al's experiment[10], this study revealed that the abundance of Prevotella in patients with ≥ F2 was lower than that in patients with F0/F1, which was consistent with the results of most experiments. Moreover, the abundances of Bacteroides and Ruminococcus increased with increasing degree of fibrosis, and a greater abundance of Ruminococcus was independently associated with the occurrence of fibrosis[12], and a higher abundance of Ruminococcus was independently associated with the occurrence of fibrosis[21]. Another study revealed that patients with ≥ F2 had relatively high abundances of Parabacteroides, E. Shigella, and Fusobacterium. Among them, Fusobacterium was dominant in patients with MASH. E. Shigella is an ethanol-producing bacterium, and a high abundance of E. Shigella can increase the ethanol concentration in patients' bodies, which, like LPS, can act as a toxin to accelerate the development of MASLD[20]. Moreover, it can increase intestinal permeability and portal vein LPS levels and activate inflammation to cause liver damage[21].

IMPACT OF METABOLITES AFFECTED BY THE GUT MICROBIOTA ON THE DEVELOPMENT AND PROGRESSION OF MASLD

The impact of the gut microbiota on the production of SCFAs in MASLD

SCFA production: SCFAs are gut microbiota metabolites produced by the fermentation of dietary fiber by a variety of gut microorganisms. Moreover, it is also a microbial metabolite that has a significant effect on the development of MASLD and MASH. Compared with those in healthy controls, the concentrations of various SCFAs in the blood of MAFL patients were significantly greater, but the SCFA concentrations in the blood of MASH patients were significantly lower than those in the blood of MAFL patients[5]. The concentration of SCFAs in the blood tends to first increase but then decreases with increasing severity of inflammation and fibrosis during the disease process. The reason for this phenomenon may be that at the early stage of the disease, the intestinal barrier begins to be damaged, its permeability increases, and SCFAs permeate the intestinal wall and then enter the bloodstream. Moreover, to counteract the decrease of SCFAs, the body initiates a "self-protection mechanism" to achieve a "compensatory increase" of SCFAs. As the disease progresses, there is a severe imbalance of the gut microbiota, a significant decrease in the concentration of beneficial bacteria that produce SCFAs and the growth of harmful bacteria. Coupled with the metabolic abnormalities caused by liver inflammation and fibrosis, the concentration of SCFAs changes. The change in SCFA concentration is related to the change in the abundance of bacteria capable of producing SCFAs in the gut. In the study conducted by Rau et al[10], it was found that the levels of acetate and propionate were higher in the fecal samples of patients with MASLD. This was associated with the increased abundances of species such as P. copri, Megashpaera, Fusobacterium, Ruminococcus torques and Eubacterium biforme in the feces of MASLD patients, and these species happen to be regarded as SCFA-producing bacteria. In addition, Bifidobacterium bifidum, Bacteroides, Phascolarctobacterium, Blautia, Dorea and Faecalibacterium are also bacteria characterized by SCFA production[5,22,23]. Heinritz et al[24] found in the fecal analysis of the pig model that the abundance of Enterobacteriaceae was negatively correlated with the SCFA concentration, and perhaps an increase in the abundance of Enterobacteriaceae had an inhibitory effect on the growth of SCFA - producing bacteria. It is undeniable that there are still certain limitations in the research on SCFAs and related microbiota in fecal samples. Such samples contain the microorganisms and metabolites from different parts of the entire intestine. After the digestion process and excretion, the composition of the microbiota and substances in them cannot reflect the dynamic changes within the intestine in real time.

In SCFAs, acetate, propionate, and butyrate are predominant, accounting for 90%-95% of the entire SCFA pool, in which acetate production includes two pathways (Table 1). One way is that most bacteria metabolize pyruvate to produce acetate through acetyl-CoA. The second way is that the microbiota represented by Clostridium synthesizes acetyl-CoA from carbon dioxide and further generates acetate. Subsequently, acetate can serve as a substrate and be further metabolized to butyrate by the acetate-CoA transferase of Anaerostipes and F. prausnitzii. In addition, phosphotransbutyrylase and butyrate kinase can also convert it to butyrate. Both of these pathways involve butyryl-CoA. Coprococcus can convert carbohydrates to pyruvate and generate butyryl-CoA through an oxidative decarboxylation reaction. The production of propionate involves three pathways. Bacteroides and Megasphaera elsdenii, among other genera, can synthesize propionyl-CoA from hexoses and pentoses through the succinic acid pathway and the acrylic acid pathway to produce propionate. Salmonella and Roseburia can produce propionate from deoxyhexoses through the propylene glycol pathway[25,26].

Table 1 The production pathways of different short-chain fatty acids.

	Related microorganisms	Way	Mechanism of action
Acetate[5,25,26,29,84,94]	Holdemanella	Acetyl-CoA pathway	(1) Improve mitochondrial modification and activate AMP-activated protein kinase to enhance fat oxidation[23]; (2) Activate GPR43 receptor[27]; and (3) Provide energy for organisms and synthesize cholesterol and lipids[26]
	F. prausnitzii
	Lachnospiraceae
	Ruminococcus
	Bifidobacterium
	Bacteroides
	Streptococcus
	Clostridum	The Wood-ljungdahl pathway
Propionate[5,22,25,26,84,94,95]	F. prausnitzii	Succinate pathway and acrylate pathway	(1) Enhance fatty-acid oxidation and reduce hepatic lipid accumulation[15]; (2) Activate GPR43 and GPR41 receptors[28]; and (3) Participate in the regulatory metabolism of gluconeogenesis[26]
	Prevotella
	Bacteroides
	Megasphaera elsdenii
	Dalister succinatiphilus
	Ruminococcus
	Veillonella
	Phascolarctobacterium
	Coprococcus
	Roseburia	Propylene glycol pathway
	Salmonella
	Akkermansia municiphilla
Butyrate[15,22,25,26,84,95,96]	Eubacteriota	Acetyl-coa pathway	(1) The energy source for the intestinal mucosa, promote the expression of tight junction proteins, and prevent "intestinal leakage"[97]; (2) Reduce the secretion of pro-inflammatory factors[25]; (3) Induce fat oxidation and attenuate the activation of HSC[16,36,37]; and (4) Activate GPR43 and GPR41 receptors[28]
	Clostridial cluster IV and XIVa
	Dalister succinatiphilus
	Oscillibacter
	Dorea
	F. prausnitzii
	Roseburia spp
	Ruminococcus
	Eubacterium rectale
	Coprococcus	Phosphotransbutyrylase/butyrate kinase pathway
	Anaerobutyricum
	Subdoligranulum
	Roseburia

F. prausnitzii: Faecalibacterium prausnitzii; HSC: Hepatic stellate cell.

SCFAs can bind to the GPR41, GPR43, and GPR109A receptors and act as signaling molecules to alleviate MASLD: GPRs are a superfamily of cell surface receptors with a seven-transmembrane helical structure. Among them, GPR41 (FFA3), GPR43 (FFA2), and GPR109A (HM74A) are all receptors that can be bound by SCFAs. They are mainly distributed in adipose tissue, the intestine, and immune cells. Through the signal transduction of these receptors, it helps to reduce lipid accumulation, improve the inflammatory response, and thus prevent the occurrence and further development of MASLD[27].

Acetate, propionate and butyrate can bind to GPR43 to initiate cell-specific signal cascades, increasing the secretion of GLP-1 by intestinal endocrine cells. As an incretin, GLP-1 can regulate insulin sensitivity, lower blood glucose, induce a feeling of satiety, and control appetite and body weight[28]. In addition, it can promote fat and glucose metabolism by inhibiting insulin signal transduction and reducing hepatic fat accumulation. Moreover, the activation of GPR43 can inhibit the expression of interleukin 6 (IL-6, IL-1β and tumor necrosis factor alpha (TNF-α), exerting anti-inflammatory effects, suppressing the hyperglycemic-induced oxidative stress response and the activation of nuclear factor-κB (NF-κB), inhibiting the IL-6/JAK1/STAT3 signaling pathway, reducing liver damage and further developing MASLD-related HCC[5,29,30].

Propionate and butyrate act as signaling molecules by binding to GPR41, promoting gluconeogenesis. By activating the APN-AMPK-PPARα pathway, they activate AMPK phosphorylation in HepG2 cells, inhibit hepatic lipogenesis, increase fatty acid oxidation and reduce hepatic lipid accumulation, thereby inhibiting MASLDF. CD4+ T cells and innate lymphoid cells can produce IL-22 via GPR41 to maintain the integrity of the intestinal mucosa[15,31,32]. Once GPR41 is in action, propionate has the capacity to hold back the expression of IL-4, IL-5, and IL-17A. And butyrate can put a brake on the expression of inducible nitric oxide synthase (iNOS), TNF-α, IL-6, and monocyte chemoattractant protein - 1 (MCP-1)[33].

Butyrate can activate the GPR109A receptor and has been found to reduce the lipase activity in the fat level, plasma triglyceride level and free-fatty-acid level, decrease insulin secretion, improve glucose metabolism and adipose tissue inflammation, and exert anti - inflammatory effects together with GPR41 and GPR43 in the intestine[33]. During the stage of liver inflammatory response, β-hydroxybutyric acid (BHB)-GPR109A has an inhibitory effect on the activity of the NLRP3 inflammasome, promotes the secretion of anti-inflammatory cytokines by macrophages in the liver, reduces the production of IL-1β and IL-18 mediated by them, alleviates inflammation, and has been proven to have a mitigating effect on MASH caused by IR and obesity[27]. Among them, BHB is one of the recognized ligands of GPR109A, which is mainly produced in the liver. When the level of BHB increases, it can bind to GPR109A and activate it. Meanwhile, as a signal transduction, it regulates lipolysis and energy metabolism[34]. However, in colonic epithelial cells, the binding of acetate to GPR43 causes the efflux of K⁺ or the increase of Ca²⁺, thereby stimulating the initiation of the NLRP3 inflammasome, and the GPR109A-dependent induction by butyrate can both lead to an increase in the level of IL-18[35], which promotes the expression of tight junction proteins in epithelial cells, maintains the integrity of the intestinal barrier, and inhibits the translocation of endotoxins and harmful bacteria[36].

In the signal transduction of SCFA-involved AMPK, the expression of PGC-1α in adipose tissue is increased, the expression of PPARγ and p-ACC in the liver is increased, and these proteins are involved in the expression of the main lipolytic enzymes, such as hormone-sensitive lipase and adipose triglyceride lipase, thereby reducing the levels of triglycerides and fatty acids. In addition, AMPK can also inhibit the expression of SREBP-1c and the synthesis of fatty acids in the liver (Figure 2)[33].

Figure 2 The therapeutic effects of different gut microbiota on diseases through the production of short-chain fatty acids via different pathways and their binding to GPR43, GPR41 and GPR109A receptors. SCFA: Short-chain fatty acid. Created from BIOGDP.com[93].

SCFAs regulated by diet play a therapeutic role in MASLD: Dietary fiber is an important substrate for the fermentation of gut microbiota and can be decomposed by gut microbiota to produce SCFAs. For example, water-soluble dietary fiber such as inulin can be fermented to produce SCFAs like butyrate. In Beisner et al's study[37], it was found that inulin could promote the integrity and function of the mucosal barrier in mice and humans, alleviate intestinal barrier dysfunction, and also have a beneficial impact on bacteria translocation induced by a high-fructose diet, for example, it could prevent the increase in plasma LPS levels. Chambers et al[38] developed inulin-propionate ester. After clinical trials with volunteers, it was found that inulin could be released through microbial hydrolysis in the colon, increasing the delivery of propionate to the colon and reducing lipid accumulation in liver cells. It has certain therapeutic value for liver steatosis related to MASLD. Besides, pectin and guar gum are also common soluble dietary fibers, which can prevent the development of MASLD, induce the browning of adipose tissue, change the IM, and promote the increase in the relative abundance of SCFA-producing bacteria[39]. In another experiment by Hao et al[40], hawthorn polysaccharide CPP extracted from hawthorn fruits could increase the total SCFA content in the feces of MASLD mice, especially the levels of butyric acid and acetic acid, by regulating SCFA-producing bacteria, and then improve the development of MASLD. And polysaccharides with similar functions also include Poria cocos water-insoluble polysaccharide, Astragalus polysaccharide, and fructooligosaccharide, etc.[40,41]. In dietary treatment, a grape polyphenol rich in type B proanthocyanidins was also found. It could increase the metabolic consumption of butyrate, reduce the concentration of butyrate in feces, strengthen the tight junctions of intestinal epithelial cells, simultaneously reduce the portal vein delivery of lipogenic butyrate and sugar, prevent Western diet-induced obesity and liver steatosis, and reduce liver fat accumulation by decreasing the ratio of butyrate to propionate[42].

The gut microbiota is involved in the circulation and metabolism of BAs and contributes to MASLD

According to epidemiological survey statistics, cholecystectomy patients are at increased risk of developing colorectal cancer and MASLD. The function of the gallbladder lies in the secretion and storage of BAs. As antibacterial agents, BAs affect the homeostasis of the gut microbiota through a number of vital activities and are regulated by microorganisms[43]. Moreover, BAs affect BA receptors, regulate inflammatory factors and fat-metabolism pathways, and participate in the synthesis of new primary BAs by liver cells, playing a vital part in the onset and advancement of MASLD.

Cholesterol is converted into BAs through the classical pathway (CYP7A1-CYP8B1) and the alternative pathway (CYP27A1-CYP7B1): A large amount of saturated fatty acids and cholesterol are important factors influencing the development of MASLD into MASH and liver fibrosis. In a mouse experiment, high -dietary cholesterol led to IR, steatosis, steatohepatitis and fibrosis in mice and ultimately caused HCC. High cholesterol, a lipotoxic molecule of MASLD, affects the development of the disease, and the synthesis of BAs through two biological pathways by cholesterol has become an effective means of eliminating cholesterol[44].

In the liver, cholesterol is catalyzed by cholesterol-7α-hydroxylase (CYP7A1) to form 7α-hydroxy-cholesterol, and then the side chain is oxidatively cleaved by sterol-12α-hydroxylase (CYP8B1) to form primary BAs, namely, cholic acid (CA) and chenodeoxycholic acid (CDCA), which the main pathway by which cholesterol synthesizes BAs and is called the classical pathway. In the other alternative pathway, cholesterol is catalyzed by sterol-27-hydroxylase (CYP27A1) to generate 27-hydroxy-cholesterol, which is further catalyzed by oxysterol-7α-hydroxylase (CYP7B1) to generate CDCA[45]. Moreover, the high hydrophobicity of CA and CDCA leads to their strong cytotoxicity, and excessive accumulation activates the inflammatory response and oxidative stress response, resulting in the necrosis and apoptosis of liver cells[46].

Highly hydrophobic primary BAs can be conjugated with glycine or taurine to reduce their hydrophobicity and toxicity[47]. Moreover, they can emulsify lipids to promote the digestion and absorption of lipids and may inhibit the growth of Clostridium perfringens and the activity of extracellular cholylglycine hydrolase, thus inhibiting the growth of small -intestine bacteria[48,49]. After the BAs in the intestine complete the digestion of fat, most fat is reabsorbed back to the liver for continuous circulation, and a small portion is supplied for microbial metabolism, including dissociation and dihydroxylation, to form secondary BAs[50].

Primary and secondary BAs work together to activate farnesol X receptor and TGR5 receptors: Conjugated BAs are dissociated by bile salt hydrolase (BSH) to remove glycine and taurine, forming free BAs such as free CA and CDCA. The activity of this enzyme is derived from the gut microbiota, including Firmicutes, Bacteroidetes, and Actinobacteria[51]. It is produced mainly by gram-positive lactic acid bacteria, Clostridium, Bifidobacterium, and Enterococcus, as well as by symbiotic gram-negative Bacteroides, Parabacteroides, and archaea (including Methanobrevibacter smithii and Methanosphera stadtmanae)[52,53]. With the help of the 7α-dehydroxylated gut microbiota, CA and CDCA are dehydroxylated to form secondary BAs such as lithocholic acid (LCA) and deoxycholic acid (DCA)[54]. The main bacteria involved are Lachnospiraceae, Ruminococcaceae, Blautia, Eubacterium, Clostridium, Lactobacillus, Bacteroides, and Clostridium, including Clostridium scindens, Clostridium marinum, Clostridium piliforme, etc.[55,56]. These primary BAs and secondary BAs formed through dissociation and dehydroxylation with the help of the gut microbiota jointly participate as signal molecules in the activation of farnesol X receptor (FXR) and the G-protein - coupled receptor BA receptor TRG5, affecting lipid metabolism and inflammatory responses.

CDCA, LCA, DCA, and CA are effective activators of FXR and TGR5. FXR activates the expression of PPARα, promoting fatty acid oxidation and reducing the accumulation of triglycerides[57]. FXR can affect the synthesis of BAs and regulate the BA concentration in the body by inducing small heterodimer partner to inhibit the transcription of the key rate-limiting enzymes CYP7A1 and CYP8B1 in the classical pathway[58,59]. When the concentration of BA is too low, disruption of the homeostasis of the gut microbiota, a decrease in BA 7α-dehydroxylation bacteria, and a transformation into toxic gram-negative bacteria can occur[60]. However, excessively high concentrations of BA cause cholestasis and damage the liver through proapoptotic and proinflammatory effects, including the enhancing effects of CDCA and DCA on the expression of inflammatory cytokines such as TNF-α, IL-1α, IL-1β, IL-6, and LPS receptor genes. In addition, the slightly toxic DCA and LCA can reduce the abundance of Firmicutes, especially the species Blautia and Ruminococcus, while increasing the abundance of proinflammatory pathogenic bacteria in Proteobacteria, especially Enterobacteriaceae[61]. Moreover, under specific conditions, LCA and DCA can bind to the surface receptors of HSCs, triggering a series of cascade reactions to activate the inflammasome and produce proinflammatory factors[46,57,62].

TGR5 is involved in the regulation of metabolism and energy expenditure as well as inflammation[54]. TGR5 is expressed in subcutaneous white adipose tissue and can promote the browning of white adipose tissue and induce the activation of PPARα and PGC-1α, thereby enhancing mitochondrial oxidative phosphorylation and energy metabolism. Moreover, the activation of TGR5 stimulates the secretion of GLP-1 by intestinal enteroendocrine L cells, increasing the insulin secretion of pancreatic β cells and improving insulin sensitivity[63]. The activation of FXR and TGR5 not only increases the expression of tight junction proteins, protects the integrity of the intestinal barrier and inhibits bacterial translocation[59] but also inhibits the inflammatory regulator nuclear factor NF-κB, thereby holding back inflammatory factors such as iNOS, COX-2, TNF-α and IL-6. These inflammatory factors can activate HSC, leading to liver fibrosis and inflammatory reactions (Figure 3).

Figure 3 In the enterohepatic circulation, cholesterol within the liver is metabolized into bile acids via two distinct pathways. Influenced by the gut microbiota, primary bile acids are further transformed into secondary bile acids, which include cholic acid (CA), chenodeoxycholic acid, lithocholic acid, and cholic acid. These secondary bile acids can activate the farnesoid X receptor and TGR5 receptor, thereby exerting a disease - alleviating effect. DCA: Deoxycholic acid; CDCA: Chenodeoxycholic acid; LCA: Lithocholic acid; CA: Cholic acid; BA: Bile acid; BSH: Bile salt hydrolase; HSC: Hepatic stellate cell. Created from BIOGDP.com[93].

Intestinal microbes produce LPS to accelerate the progression of MASLD

The LPS, which forms part of the outer membrane of gram-negative bacteria, is considered a major bacterial bioproduct in the pathogenesis of MASLD[64] and also an effective inducer of liver inflammation[65]. Multiple studies have shown that one of the characteristics of MASLD patients is an increased level of endotoxin circulation[66], and this change is closely related to the gut microbial flora.

On the one hand, as a membrane component of Gram-negative bacteria, changes in the concentration of LPS are associated with changes in the quantity of relevant bacteria that produce LPS, especially Proteobacteria, including Shigella and Escherichia[67]. For example, in mice with relatively high levels of LPS, Enterobacter cloacae B29, Klebsiella pneumoniae A7 and Escherichia coli (E. coli) PY102 in Enterobacteriaceae, which are strains with proinflammatory endotoxins, were isolated. Furthermore, in MASLD mice, the abundance of Bacteroides, Bifidobacterium, and Eubacterium decreased. Bacteroides and Bifidobacterium have endotoxin activity that is 1000 times lower than that of Enterobacteriaceae and cannot induce immune activation[68]. Similar to the results of another experiment, the serum LPS concentration was positively correlated with the numbers of E. coli and Enterococcus but negatively correlated with the numbers of Lactobacillus, Bifidobacterium and Bacteroides[64]. High levels of Prevotella and Helicobacter pylori in the intestines of patients with this disease were also found to result in increased levels of LPS[14].

On the other hand, gut dysbiosis or the increase in intestinal permeability caused by the lack of related metabolites such as SCFAs and BAs and their related receptors enables LPS to pass through the intestinal barrier into the bloodstream and reach the liver, resulting in an elevated serum LPS concentration and triggering endotoxemia. Many studies have demonstrated that the amount of LPS in MASLD patients is up to five times the level in the healthy control group in the serum[66]. However, in Loguercio et al's study[69], it was believed that all MASLD patients did not have endotoxemia. The reason for the contradictory results may lie in the relatively short half-life of LPS. Coupled with the efficient recognition and degradation by Kupffer cells in the liver, it is not easy to detect endotoxemia in the blood unless under the conditions of infection or inflammation, which requires more sensitive tests for confirmation[70]. Before LPS in the blood is recognized by Kupffer cells, it needs to first bind with lipopolysaccharide-binding protein (LBP) to form an LPS-LBP complex. This complex is more stable than LPS and is usually used as a detection indicator of endotoxemia instead of LPS. Compared with that in MASLD patients, the LBP concentration in MASH patients is significantly greater and is related to hepatic steatosis and ballooning degeneration[70], further indicating that the inflammatory phenomenon in MASLD patients is related to increased LPS levels. Xue's team reported that the levels of LPS and TLR4 expression were negatively correlated with the ratio of Bifidobacterium/Enterobacter. The elevated expression of TLR4 contributed to the development of MASLD, and the degree of liver injury in MASH mice in which the TLR4 gene was knocked out was significantly reduced[61]. LPS are recognized by LBP and CD14 and then transported to the cell surface to bind with TLR4 and MD-2. Subsequently, the MyD88-dependent pathway and NF-κB are activated causing Kupffer cells to release proinflammatory factors such as TNF-α, IL-1, and IL-6[71,72], thus leading to liver inflammation and local intestinal mucosal hypoxia - ischemia and further increasing intestinal permeability. TLR4 can activate stellate cells and produce TNF-α. TNF-α disrupts the insulin signaling pathway, increases IR leading to steatosis and mitochondrial dysfunction, boosts the generation of ROS and lipid peroxidation, thereby indirectly leading to liver injury[14,64,71].

The proinflammatory effect of LPS and TLR4 on MASLD can be alleviated by intestinal probiotics. Lactobacillus can significantly weaken the TLR4 signaling pathway and reduce the activity of PPAPγ to reduce steatosis in hepatocytes previously exposed to LPS and regulate the NOD-NF-κB pathway to induce the release of anti-inflammatory cytokines[71]. In an experiment, Bifidobacterium adolescentis (B. adolescentis) effectively maintained the intestinal barrier, reduced intestinal permeability, reduced the amount of lipopolysaccharide produced by the gut microbiota, inhibited the TLR4/NF-κB pathway, and relieved hepatic steatosis and inflammation (Figure 4).

Figure 4 In the intestine, lipopolysaccharide-producing microorganisms spread through the intestinal leakage into the portal vein circulation and then reach the liver, where they release lipopolysaccharide. By binding to the TLR4-MD2 receptor, this process exacerbates the occurrence of metabolic dysfunction-associated steatotic liver disease. LPS: Lipopolysaccharide; LBP: Lipopolysaccharide-binding protein; IR: Insulin resistance; MASLD: Metabolic dysfunction-associated steatotic liver disease. Created from BIOGDP.com[93].

PROBIOTICS HAVE A THERAPEUTIC EFFECT ON MASLD BY REDUCING THE EXPRESSION OF PROINFLAMMATORY FACTORS AND REGULATING THE INTESTINAL FLORA

Probiotics are described by the World Health Organization as "live microorganisms that are beneficial to health when consumed in sufficient quantities" and are safe and nontoxic bacteria that is capable of enhancing the micro-ecological equilibrium of the host and exert beneficial effects on the host's health[73]. Among them, the most common traditional probiotics are lactic acid bacteria and bifidobacteria, which are isolated mainly from fermented dairy products and feces[74]. These two types of strains also play important roles in regulating the intestinal barrier, lipid metabolism and gut microbiota, and are considered traditional probiotics for effectively preventing and alleviating MASLD. In addition, some species, such as Akkermansia, F. prausnitzii, Bacteroides acidifaciens, and Christensenellaceae, are also regarded as part of the new generation of probiotics, with the potential to be more effective, more precise and disease specific (Table 2).

Table 2 Probiotics regulate the gut microbiota and related disease factors.

Probiotics	Phylum (Gram negative/positive)	Changes	Function
Bacillus subtilis + Enterococcus faecium[78]	Firmicutes (G+)	Firmicutes/Bacteroidetes↓	IL-1β, IL-6, TNF-α↓
		Akkermansia, Ruminococcus_UCG-014, Eubacterium_coprostanoligenes_group↑	TLR4, NF-κB↓
		Lachnoclostridium, Oscillibacter, Family_XIII_AD3011_group, Anaerovorax, Acetatifactor, Coriobacterium_UCG-002, Negativibacillus, Angelakisella, Ruminococcus, Harryflintia, Escherichia-Shigella↑	LPS↓
Lactobacillus acidophilus KLDS1.0901[77]	Firmicutes (G+)	Roseburia, Lachnospiraceae UCG-006, Bacteroides, Enterorhabdus↓	IL-1β, IL-6, TNF-α, LPS, Lactic acid↓
		Blautia, Alistipes, Oscillibacter, Faecalibaculum, Ruminiclostridium, Lactobacillus, Ruminococcaceae UCG-009↑	IL-10↑
Lactobacillus plantarum NCU116[76]	Firmicutes (G+)	Allobaculum, Lactobacillus, Bifidobacterium↑	LPS↓
			Intestinal barrier intact
Bifidobacterium longum MG723, MG731[76]	Actinobacteria (G+)		F4/80, MCP-1, TGF-β, IL-1β, IL-18↓
			BAs↑
Bifidobacterium lactis, Bifidobacterium.longum DD98[76]	Actinobacteria (G+)		IL-6, IL-1β, TNF-α↓
Bifidobacterium longum R0175[76]	Actinobacteria (G+)	Alloprevotella spp.↑	IL-1, TNF-α, Chemokines↓
		Acetatifactor muris, Butyricimonas spp., Oscillibacter spp.↓
Bifidobacterium longum LC67[76]	Actinobacteria (G+)	Firmicutes, Proteobacteria, Tyzzerella, Escherichia-Shigella, Nestiinimonas, Osillibacter, Ruminiclostridium↓
		Faecalibacterium, Lactobacillus↑
Bifidobacterium bifidum[76]	Actinobacteria (G+)	Tyzzerella, Escherichia-Shigella, Nestiinimonas, Osillibacter↓	Propionate, butyrate↑
		Faecalibacterium, Ruminococcus↑
Lactobacillus paracasei Jlus66[98]	Actinobacteria (G+)	Firmicutes↑	IL-6, TNF-α, LPS↓
		Proteobacteria, Clostridia↓	IL-10↑
		Clostridiaceae, Oscillospiraceae, Bifidobacteriaceae↑
		Lactobacillus, Bifidobacterium↑
Lactiplantibacillus plantarum ZDY2013[75]	Actinobacteria (G+)	Lachnospirillaceae, Muribaculaceae, Lactobacillus spp., Alloprevotella and Blautia↑	LPS/NF-κB↓
		Fusobacteria, Verrucomicrobia↓	Tight binding protein, IR↑
Lactobacillus sakei MJM60958[79]	Actinobacteria (G+)	Salmonella gallinarum KCTC 2931, E. coli O1 KCTC 2441, Salmonella cholerae KCTC 2932, Pseudomonas aeruginosa KCCM 11802↓	Acetic acid↑
Akkermansia mucophila[23]	Verrucomicrobiota (G-)		γδT17, NF-κB, LPS↓
			Fat oxidation, Acetate, IL-10, Mucin (intestinal barrier integrity)↑
F. prausnitzii[84,86,87]	Firmicutes (G-)	Erysipelatoclostridium, Faecalibaculum, Blautia, Akkermansia, Bifidobacterium, Lactobacillus, Duboria↑	TNF-α, IL-1β, IL-6, TLR4↓
		Tyzzerella↓	Butyrate, intestinal barrier↑
Christensenella minuta[88]	Firmicutes (G-)		Acetate, butyrate↑
Bifidobacterium adolescentis[72]	Actinobacteria (G+)		Intestinal barrier↑
			TLR4/NF-κB, IL-6, IL-1β, TNF-α↓
Bacteroides ovatus[92]	Bacteroidetes (G-)	Lachnospiraceae_NK4A136_group, norank_f_Oscillospiraceae, Colidextribacterder↑	SCFA, intestinal barrier↑
		F/B, Ruminococcus_torques_group, Ruminococcus_gauvreauii_group, Erysipelothrix rhusiopathiae↓	LPS, CD163, IL-1β, TNF-α↓

F. prausnitzii: Faecalibacterium prausnitzii; E. coli: Escherichia coli; LPS: Lipopolysaccharide; BA: Bile acid; IR: Insulin resistance; SCFA: Short-chain fatty acid.

Lactobacillus plantarum (L. plantarum; including L. plantarum Zdy2013[75], L. plantarum NCU116, and L. plantarum NA136[76]) can reduce fat accumulation and inflammatory responses by regulating the gut microbiota, inhibiting the production of inflammation-stimulating factors and the LPS/NF-κB pathway, and regulating lipid metabolism. Among them, L. plantarum NA136 can protect the integrity of the intestinal barrier by augmenting the abundance of beneficial bacteria such as Lactobacillus and Bifidobacterium[76].

In different studies, the combination of Bacillus subtilis and Enterococcus faecium (LCBE) bacteria and Lactobacillus acidophilus KLDS1.0901 significantly reduced the expression levels of IL-6, TNF-α and IL-1β in high-fat diet (HFD)-fed mice, prevented increases in the levels of TLR4 and NF-κB, and reduced the expression of LPS[77,78]. Moreover, LCBE promoted the proliferation of beneficial bacteria such as Akkermansia and Ruminococcus_UCG - 014; reduced the abundances of harmful bacteria such as Ruminococcus, Lachnospiraceae, and E. Shigella; and regulated the balance of the gut microbiota[78]. L. acidophilus KLDS1.0901 restricted the growth of high-abundance Roseburia, Lachnospiraceae UCG - 006, Bacteroides and Enterorhabdus in high-fat-diet-fed mice and increased the abundances of bacteria such as Faecalibaculum, Ruminiclostridium, Alistipes, Oscillibacter, Blautia, Lactobacillus and Ruminococcaceae UCG - 009[77].

In an experiment by Nguyen et al[79], the lipid accumulation in oleic-acid-stimulated HepG2 cells was significantly inhibited by sakei MJM60958 (MJM60958) and cholesterol (OA-C), suppressed the growth of harmful bacteria such as Salmonella gallinarum KCTC 2931, E. coli O1 KCTC 2441, Salmonella choleraesuis KCTC 2932 and Pseudomonas aeruginosa KCCM 11802, and increased the acetic acid concentration in HFD-fed mice.

Bifidobacterium longum is a type of Bifidobacterium. B. longum MG723 and B. longum MG731, B. longum R0175, B. longum DD98, etc., can reduce the production of inflammatory factors. The former two can also promote the expression of BA synthesis genes and affect BA production and metabolism. B. longum R0175 and B. longum LC67 has the potential to elevate the quantity of beneficial bacteria like Prevotella, Lactobacillus and Faecalibacterium, and reduce the relative abundances of Acetatifactor muris, Butyricimonas spp., Tyzzerella, E. Shigella, Nestiinimonas, Osillibacter and Ruminiclostridium[76].

Pseudomonas longum, another species in the Bifidobacterium genus, is an acetate-producing bacterium. It not only inhibits tumor formation and cancer cell proliferation and induces cancer cell death by producing acetate, thus impeding the development from MASLD to HCC but also enriches probiotic bacteria mainly including Bifidobacterium villosum, Bifidobacterium animalis and Oscillibacter PEA192, and has an exhausting effect on some harmful bacteria, such as Paeniclostridium sordellii and Enterobacter hormaechei[29].

Multiple studies have shown that Akkermansia muciniphila (L. plantarum) has therapeutic potential for MASLD. In an HFD-fed mouse experiment by Schneeberger et al[80], a positive relationship was noted between the level of A. muciniphila and the levels of fatty acid oxidation and fat browning; however, a negative relationship was detected between its level and plasma markers including inflammatory markers, lipid-synthesis-relevant markers, and markers associated with IR. This result was similar to that of another study in which the diameter of adipocytes in white adipose tissue was negatively correlated with the abundance of A. muciniphila, and adipocyte hypertrophy was associated with an increased risk of chronic proinflammatory cytokine secretion and IR[81]. Moreover, the production of fatty acids has a regulatory effect on A. muciniphila. For example, mice fed with lard showed a decreasing trend in the abundance of A. muciniphila, while the opposite was true for the fish oil-fed group[80]. A. muciniphila inhibits the development of MASH by downregulating TLR2 signaling to reduce the level of γδT17 cells, preventing γδT17 cells from promoting the polarization of macrophages to proinflammatory cells through IL-17 and inhibiting the accumulation of γδT cells and the release of cytokines and LPS[82]. In addition, A. muciniphila can prevent the infection of various pathogenic bacteria. A study revealed that repeatedly administering A. muciniphila to mice could protect the mice from infections by Clostridium difficile, Listeria monocytogenes, Fusobacterium nucleatum, Porphyromonas spp., and Salmonella typhimurium[83].

F. prausnitzii can reduce MASH-induced hepatic lipid accumulation and the resulting damage and ameliorate damage to the intestinal barrier. It is also an important producer of SCFAs, especially butyrate[84]. In the research of Shin et al[85], EB-FPDK9, EB-FPDK11 and EB-FPYYK1 in F. prausnitzii significantly improved the thickness of the mucosa and outer muscular layer and significantly reduced the mRNA expression of TLR4 compared with those in the MASH group. Moreover, they inhibited lipid accumulation by regulating the expression of genes that are involved in hepatic steatosis. This finding is similar to the research results of Hu and his team. They reported that five kinds of F. prausnitzii, namely A2-165, LB8, ZF21, PL45, and LC49, improved adipose tissue dysfunction, steatosis, inflammation and other phenomena in MASLD mice. Among them, LB8 and LC49 significantly reversed the abundances of Faecalibaculum, Tyzzerella, Erysipelatoclostridium, Blautia and Acetatifactor in MASLD mice, and enriched the abundances of salutary bacteria such as Bifidobacterium, Lactobacillus, Dubosia, Faecalibacterium and Akkermansia[86], regulating the disordered gut microbiota. In addition, with the exception of A2-165, the other four species can diminish the expression of TNF-α, IL-1β, and IL-6 and reduce hepatic oxidative stress and inflammatory reactions[84,87].

Christensenellaceae belongs to a family in the phylum Firmicutes and is regarded as a candidate next-generation probiotics, showing a negative correlation with MASLD. In SUN's experiment, several representative species within Christensenellaceae were studied. Among them, Christensenella minuta can reduce lipid accumulation and decrease diet-induced increases in body weight and blood glucose; a species within Christensenellaceae, Luoshenia tenuis, can regulate the levels of the hunger hormones PYY and GLP-1 to mitigate weight gain. All strains of Christensenellaceae can produce SCFAs, mainly acetic acid and butyric acid, and most of these strains can modify primary BAs[88], because Christensenellaceae can simultaneously exhibit the activities of BSH and 7α-dehydrogenase.

B. acidifaciens belongs to the phylum Bacteroidetes. It can activate fat oxidation through the BA-TGR5-PPARα axis, improve energy metabolism, and reduce fat accumulation. BAs promote GLP-1 activation through TGR5 to improve IR[89]. B. acidifaciens also contributes to the production of acetate and hinders the development of MASH[90]. In a treatment experiment on mice with colitis, it was found that B. acidifaciens increased the relative abundances of Bacteroides, Parabacteroides, and Prevotella, while reducing the relative abundances of Bacteroides fragilis, Parabacteroides, and Streptococcus[91]. Although these bacteria are all bacteria associated with changes in MASLD, there are currently no targeted experiments to prove that the regulatory effect of B. acidifaciens on these microbiota is equally effective in MASLD patients.

A study of B. adolescentis revealed that B. adolescentis can maintain the intestinal barrier and inhibit the TLR4/NF-κB pathway by suppressing the production of lipopolysaccharide by bacteria, thereby increasing the sensitivity of FGF21. FGF21 can maintain glucose homeostasis through the regulation of hepatic insulin and, at the same time, reduce the manifestation of pro-inflammatory mediators such as IL-6, IL-1β, and TNF-α, regulate the activation of hepatocytes, and reduce liver damage and oxidative stress levels[72].

Bacteroides ovatus (B. ovatus) is a gram-negative anaerobic bacterium. Research has shown that B. ovatus can improve the gut microbiota of mice, reduce the F/B ratio in the intestines of HFD-fed mice; and decrease the abundances of Ruminococcus_torques_group, Ruminococcus_gauvreauii_group, Clostridium erysipelatos, etc. Meanwhile, the abundances of Lachnospiraceae_NK4A136_group, norank_f_Oscillospiraceae, and Colidextribacter in feces significantly increased. In addition, it increased the amounts of acetic acid, propionic acid, and butyric acid, decreased the expression of LPS and inflammatory mediators, downregulated the expression of de novo fat - producing genes such as Srebfl, Acaca, Scd1, and Fasn, and upregulated the expression of genes involved in the process of fatty acid oxidation. These changes resulted in weight loss and helped prevent steatohepatitis and liver damage[92].

CONCLUSION

The gut microbiota seems to play an important role in both the occurrence and development of MASLD. The influencing factors exist in multiple aspects, including affecting intestinal permeability, producing related endotoxins, inhibiting the reproduction of bacteria that produce beneficial metabolites, and producing pro-inflammatory factors. And these factors may, in turn, strengthen the impact on the gut microbiota. Comparing the variations in the gut microbiota between the affected population and the healthy population as well as patients at different stages of the disease (different disease-development states such as MAFL, MASH and different degrees of liver fibrosis) is helpful for using the gut microbiota as markers to reveal the disease progression and providing personalized microbiota regulation and metabolite regulation treatment plans for the disease reactions caused by the abnormal proliferation of different gut microbiota. Perhaps the treatment methods directly aimed at restoring the homeostasis of the gut microbiota will become an effective means to relieve and treat MASLD. In the future, more emphasis can be placed on multiple means such as prebiotics, probiotics, and antibiotics to restore the internal environment of the gut and formulate targeted regulation methods for MASLD patients with different microbiota compositions. Exploring how to screen and identify more efficient SCFA-producing microorganisms, develop new microbial preparations (such as probiotics, synbiotics, etc.) for the treatment of MASLD, precisely regulate the composition of the BAs pool and relevant metabolic trajectories through gut microbiota, and use multi - omics technology for joint analysis to find more accurate and stable microbial marker combinations for the development of MASLD have become potential research hotspots in the treatment of MASLD using gut microbiota. However, the microbial population is huge and the connections between different microbiota are close. The increase of one microbiota may lead to the decrease of multiple microbiota. How to identify the inter-restrictive and mutually promoting connections between microbiota and restore the homeostasis of a certain microbiota without disrupting the balance of other microbiota still requires greater efforts from us. This review also has certain limitations. Most studies on microbiota analysis rely on fecal samples, which cannot fully represent the microbiota state in the patient's intestines and the microbiota state of specific sites. Moreover, the disease development, microbiota composition, and metabolite state reflected by the samples are affected by regional and individual differences, which may have an impact on the generality and accuracy of the outcome. The data in this review are selected from representative high - abundance microbiota changes, and there are certain limitations for low - abundance microbiota due to the limitations of technological development and the complexity of the microbial community. In the future, we will conduct more in-depth research on the mechanism of the role that gut microbiota play in physiological activities like metabolism and immune regulation.