Gut microbial metabolites in MASLD: Implications of mitochondrial dysfunction in the pathogenesis and treatment

Abstract

With an increasing prevalence, metabolic dysfunction–associated steatotic liver disease (MASLD) has become a major global health problem. MASLD is well-known as a multifactorial disease. Mitochondrial dysfunction and alterations in the gut bacteria are 2 vital events in MASLD. Recent studies have highlighted the cross-talk between microbiota and mitochondria, and mitochondria are recognized as pivotal targets of the gut microbiota to modulate the host's physiological state. Mitochondrial dysfunction plays a vital role in MASLD and is associated with multiple pathological changes, including hepatocyte steatosis, oxidative stress, inflammation, and fibrosis. Metabolites are crucial mediators of the gut microbiota that influence extraintestinal organs. Additionally, regulation of the composition of gut bacteria may serve as a promising therapeutic strategy for MASLD. This study reviewed the potential roles of several common metabolites in MASLD, emphasizing their impact on mitochondrial function. Finally, we discuss the current treatments for MASLD, including probiotics, prebiotics, antibiotics, and fecal microbiota transplantation. These methods concentrate on restoring the gut microbiota to promote host health.

INTRODUCTION

Metabolic dysfunction–associated steatotic liver disease (MASLD) is characterized by excessive accumulation of fat in the liver tissue in the absence of reasons for secondary hepatic fat accumulation, such as genetic disorders, viral hepatitis, and excessive alcohol consumption.¹ Actually, the overload of fat in the liver reflects the hepatic manifestation of systemic lipid metabolism disorder; thus, the name of the disease has recently been redefined from NAFLD to MASLD.²,³ MASLD is currently the most common chronic liver disease worldwide.⁴ The prevalence of MASLD in Mainland China was ~30% in 2020,⁵ and the prevalence of MASLD ranged from 29.5% in 1999–2000 to 40.3% in 2015-2016 in the American adult population.⁶ Concerningly, with disease progression, a substantial portion of benign metabolic dysfunction–associated steatotic liver may evolve into metabolic dysfunction–associated steatohepatitis (MASH), cirrhosis, and hepatocellular cancer.⁷ Therefore, prior treatment and early prevention are vital in patients with MASLD. However, the underlying mechanism of MASLD remains unclear, and definitive treatments for MASLD and MASH are lacking.⁸

With advances in culture-independent techniques, intestinal bacteria have been found to play a crucial role in MASLD and are closely associated with the development of hepatic steatosis, fibrosis, and inflammation.⁹ Unfortunately, there is no conclusive mechanism that illustrates how the gut microbiota contributes to the pathogenesis of MASLD. Recently, the interdependence of the intestinal flora and mitochondria has received particular attention in many studies.¹⁰,¹¹,¹² According to the endosymbiotic theory, mitochondria in eukaryotic cells have been described as endosymbiotic organelles evolved from α-proteobacteria, sharing significant similarities in structural components and metabolic pathways with bacteria.¹³ Based on the metabolic reconstruction model Recon2, Thiele et al identified 437 mitochondria-related metabolites, 325 of which overlapped with gut microbiota metabolites, implying strong cross-talk between these 2 components.¹⁴ Additionally, metabolites of gut bacteria are reported to modulate energy metabolism, oxidative stress, and inflammation through mitochondrial metabolism–related pathways in animal models.¹² Accurately, mitochondrial effects have been proposed as a vital mechanism by which the gut flora can affect health and disease.¹⁵ Mitochondrial dysfunction has also emerged as a pivotal pathological feature in MASLD, and literature suggests that compromised hepatic mitochondrial fatty acid oxidation and mitochondrial turnover are intimately related to increasing MASLD severity in patients with obesity.¹⁶ This review will concentrate on the cross-talk between microbiota and mitochondria in MASLD and discuss treatments for MASLD associated with the manipulation of the gut microbiota.

THE ROLE OF MITOCHONDRIAL DYSFUNCTION IN MASLD

Each hepatocyte contains 1000–2000 mitochondria, and the liver is a mitochondrion-rich organ.¹⁷ Mitochondria are the most important sites for converting nutrients into energy. Thus, mitochondrial dysfunction plays a vital role in the pathogenesis of diseases related to metabolic syndrome.⁸,¹⁰,¹⁸ Additionally, there is accumulating evidence for the crucial role of mitochondrial dysfunction in the formation of multiple events, including hepatocyte steatosis, oxidative stress, inflammation, and fibrosis, during the progression of MASLD.¹⁸ Hence, MASLD is considered to be a mitochondrial disease.¹⁹ Nevertheless, mitochondria have complex and dynamic structures and functions, revealing their dysfunction in multiple ways, including deficient mitochondrial oxidative metabolism, oxidative stress, and mitochondrial quality control imbalance.

Defect in mitochondria fatty acid oxidation

Mitochondrial fatty acid oxidation (mtFAO) is an important method to reduce fat accumulation in the liver.²⁰ In the classical mtFAO pathway, free fatty acids (FFAs) are converted to acyl acyl-CoA in the cytoplasm, which can be gradually metabolized to water, carbon dioxide, and a large amount of ATP through β-oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS) in mitochondria (Figure 1).¹⁸ In the liver, fatty acids that are not metabolized in time are stored as triglycerides (TG) in lipid droplets. Even though a part of TG is packaged into VLDL for secretion from the liver to circulation, the effect size with regard to liver fat accumulation is extremely weak,²¹ and high serum VLDL levels have been considered an independent risk factor for cardiovascular disease.²² However, long and persistent accumulation of FFAs and chronic acyl-CoA by mtFAo defects separate the function of the tricarboxylic acid cycle from mitochondrial respiration, resulting in excess reactive oxygen species(ROS) production.²³,²⁴

FIGURE 1.

(A) MtFAO is a vital way for the liver to reduce fat accumulation. In the classical pathway of mtFAO, FFAs are converted to acyl-CoA in the cytoplasm, which can be gradually metabolized to water, CO₂, and a large number of ATP by way of β-oxidation, tricarboxylic acid cycle and OXPHOS in mitochondria. However, mtFAO function is defective in subjects with MASLD and animal models, which is manifested as decreased expression of β-oxidation and OXPHOS-related genes and protein, including CoA, CPTI, CPT2, and β-HAD, and mitochondrial complex (Ⅰ, Ⅱ, Ⅲ, Ⅳ, and Ⅴ). Deficient ETC function could result in the production of ROS, which could further impair mitochondrial function. (B) Excessive ROS can cause OS and play an important role in mitochondrial dysfunction, inflammation, and lipid peroxidation. ROS will generate oxidative damage to mtDNA and be associated with decreased MMP and increased MP. Increased MP enables mtDAMP release into the cytosol, promoting an inflammatory response. Apparently, ROS can directly activate inflammatory pathways, promoting MASLD. Additionally, OS can contribute to insulin resistance, facilitating fat accumulation in the liver by promoting lipolysis, DNL, and hepatic uptake of FFAs. Besides, elevated ROS levels will lead to increases in LP, which can produce toxic byproducts (such as 4-HNE and MDA), thereby inducing mitochondrial dysfunction, more ROS production and inflammation. In conclusion, mitochondrial dysfunction can lead to increased mitochondrial production of ROS, which causes a vicious cycle of mitochondrial dysfunction, leading to more ROS production. Abbreviations: β-HA, β-hydroxyacyl-CoA-dehydrogenase; CPT1, carnitine palmitoyl transferase 1; CPT2, carnitine palmitoyl transferase 2; DNL, de novo lipogenesis; ETC, electron transport chain; FFA, free fatty acid; 4-HNE, 4-hydroxynonenal; LP, lipid peroxidation; MDA, malondialdehyde; MASLD, metabolic dysfunction–associated steatotic liver disease; MMP, mitochondrial membrane potential; MP, mitochondrial permeability; mtDAMP, mitochondrial damage-associated molecular patterns; mtFAO, mitochondrial fatty acid oxidation; OS, oxidative stress; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species; TCA cycle, tricarboxylic acid cycle.

Liver fat metabolism is highly dependent on mtFAO, and defects in mitochondrial respiration are important factors that can facilitate the development of MASLD.²⁵ β-hydroxyacyl-CoA-dehydrogenase is one of the most important enzymes involved in β-oxidation. In addition, impaired mitochondrial β oxidation also causes alternative peroxisomal and cytochrome oxidation of FFA, producing peroxide products and amounts of ROS.²⁶ Mary et al reported that β-hydroxyacyl-CoA-dehydrogenase decreased by 40%–50% in patients with MASH when compared with controls, suggesting impaired β-oxidation.²⁷ Interestingly, their study also identified that the degree of liver fibrosis was associated with a loss of OXPHOS complexes CⅡ, CⅢ, and CⅣ.²⁷ Carnitine palmitoyl transferase Ⅰ (CPT1) and Carnitine palmitoyl transferaseⅡ 2 (CPT2) are located in outer and inner mitochondrial membranes, respectively. Together with carnitine, they are required for the movement of long-chain fatty acyl-CoAs into the mitochondrial matrix for β-oxidation. Moreover, CPT1 and CPT2 are always used as indicators of the β-oxidation capacity.²⁸,²⁹,³⁰,³¹ In addition, multiple studies have revealed a reduction in CPT1 and CPT2 in high-fat diet (HFD)-induced animal models.²⁸,²⁹,³⁰,³¹ Correspondingly, the administration of syringic acid to MASLD mice exhibited anti-hepatic steatosis effects by upregulating CPT1 and CPT2 expression.³¹ CoA is another enzyme that catalyzes the conversion of LFAs into long-chain acyl-CoA. Additionally, CoA was decreased upon mtFAO defects in MASLD mice (Figure 1).³¹ Mitochondrial cristae are an important part of the inner mitochondrial membrane, including 5 major membrane protein complexes, the mitochondrial complexes I–V, which are responsible for OXPHOS and ATP synthesis (Figure 1).¹⁸ However, evidence has shown that the abundance of mitochondrial cristae is decreased in brown adipose tissue of obese mice, providing supportive evidence that mitochondrial respiration is impaired in metabolic diseases.³² Additionally, mitochondrial complex (I, II, III, and V) activity has also been reported to be decreased in MASLD mice.³⁰,³³ Notably, deficient mitochondrial complex function could contribute to the production of ROS, which in turn damages mitochondrial function by oxidizing a variety of macromolecules, including proteins, lipids, and DNA. AMP-activated protein kinase (AMPK) is a key regulator of fatty acid metabolism in the liver, and sirtuins (SIRTs) and proliferator-activated receptorγ coactivator (PGC)-1x are its downstream mediators. In MASLD, the expression of PGC-1α mRNA and sirt expression in the liver are reduced.³⁴ Studies have shown that the activation of the AMPK/PGC-1α or AMPK/Sirt3 signaling pathway in hepatocytes can regulate lipid metabolism, improve mitochondrial respiratory capacity and oxidation-reduction reaction homeostasis, reduce hepatic lipid accumulation and oxidative stress, and thus improve mitochondrial dysfunction.³⁵,³⁶

Based on the above discussion, mtFAO deficiency is an important factor in lipid accumulation in the liver. MtFAO is a complex metabolic process, and its defects are not only manifested by reduced β-oxidation, OXPHOS-related gene or protein expression, and ATP synthesis, but also by the massive production of ROS and the resulting oxidative damage (Figure 1). Therefore, enhancing the mtFAO capacity is a promising therapeutic strategy.

ROS and mitochondrial dysfunction

Physiologically, ROS are produced as secondary messengers in several important biological processes.³⁷ Meanwhile, excess ROS can be detoxified by antioxidant defense enzymes (ie, peroxiredoxins, glutathione peroxidases, catalase, and superoxide dismutases) to maintain redox homeostasis.³⁸ When high levels of ROS overwhelm the antioxidant defense system, they lead to a state of oxidative stress (OS) in vivo,³⁸ which is a well-known key causative factor in initiating inflammation, lipid peroxidation, and insulin resistance (IR) events. Certainly, besides a consequence of fat accumulation in the liver, IR is a vital facilitator of fat accumulation in the liver by promoting lipolysis, de novo lipogenesis in the liver, and hepatic uptake of FFAs.³⁹,⁴⁰ In addition to their ability to generate ROS, mitochondria are vulnerable to oxidative damage.¹⁸ As mitochondrial DNA (mtDNA) lacks protective histones and is close to the ROS source, mtDNA is susceptible to ROS-mediated oxidative damage, which affects mtDNA replication and transcription, causing further mitochondrial functional impairment. Oxidative damage to mtDNA has been demonstrated in animal models and patients with MASLD, accompanied by the inhibition of mitochondrial complex activity.⁴¹,⁴² Moreover, the increased level of ROS might contribute to a decrease in ΔΨm, which in turn increases mitochondrial membrane permeability, leading to the release of mitochondrial damage-associated molecular pattern (eg, mtDNA, cytochrome c, cardiolipin, and heat shock protein 60) into the cytoplasm, which are key stimulators of the inflammatory response pathway.⁴³,⁴⁴ Using a rodent model and primary mouse KCs, Pan et al found that the release of mtDNA can activate KCs and upregulate the expression of NLRP3, which has been proposed as a potential cause of progression from simple steatosis to MASH.⁴⁴ Noticeably, in MASLD rats, high levels of ROS can directly activate the NLRP3 inflammasome and promote the expression of pro-inflammatory cytokines (eg, TNFα, IL-6, and NF-kβ), contributing to liver inflammation and fibrosis progression.⁴⁵,⁴⁶ In human MASLD, another adverse effect of OS is the promotion of hepatic lipid peroxidation, which can produce toxic byproducts,³⁹ including 4-hydroxynonenal and malondialdehyde (MDA).⁴²,⁴⁷,⁴⁸ As a result, this can induce mitochondrial dysfunction, more ROS production, and inflammation. In addition, decreased antioxidant enzyme (such as superoxide dismutase [SOD] and glutathione peroxidase) activity is common in patients with MASLD, and animal models provide an opportunity for disease progression.⁴⁹,⁵⁰

Excessive ROS production is a hallmark of mitochondrial dysfunction. Oxidative stress caused by ROS can trigger IR, lipid peroxidation, inflammation, and mtDNA oxidative damage, thereby exacerbating mitochondrial function and liver damage during MASLD progression (Figure 1).

Aberrant mitochondrial quality control

Mitochondrial quality control (mitoQC) is a complex set of mechanisms that have evolved to maintain a healthy mitochondrial network and cellular homeostasis in dynamic host environments. These mechanisms include mitochondrial redox regulation, proteosis, mitophagy, mitochondrial dynamics, and biogenesis.⁵¹ The maintenance of normal mitochondrial morphology and volume is also dependent on mitoQC; however, in rodent models of MASLD, fragmented cristae, swelling, and outer member disruption of mitochondria are frequent pathological features,⁵²,⁵³,⁵⁴ indicating that aberrant mitoQC is involved in the pathogenesis of MASLD. In this chapter, we discuss how mitophagy, mitochondrial dynamics, and mitochondrial biogenesis contribute to MASLD development.

Mitophagy

A dynamic cellular process called mitophagy helps cells eliminate extraneous, damaged, or defective mitochondria. This is a type of specialized autophagy. “PINK/PARKIN” is referred to as a canonical mitophagy pathway.⁵⁵ Evidence shows that Pink and Parkin protein levels are lowered in MASLD animal models, along with apoptosis, mitophagy blockade, and altered mitochondrial architecture.⁵⁶,⁵⁷,⁵⁸ Liraglutide has a solid track record of its protective effects against MASLD.⁵⁹,⁶⁰ Yu et al found that liraglutide inhibited the production of ROS, exerted anti-inflammatory effects, reduced lipid accumulation, and inhibited the activation of NLRP3 inflammasome and cell pyroptosis by activating PINK expression, suggesting that liraglutide ameliorates MASLD by enhancing mitophagy.⁶¹ The Bcl-2 family member Bnip3 is connected in the control of mitophagy. Both patients with MASH and the MASLD mouse model had lower levels of Bnip3 expression,²⁷,⁶² and the liver mitochondria of BNIP3-deficient mice had a lower ∆Ψm value and reduced oxygen consumption.⁶³ Macrophage stimulating 1 (Mst1) is a regulatory factor that can affect the apoptosis of cancer cells by inhibiting mitochondrial phagocytosis, and BNIP3 plays an essential role in the induction of mitochondrial autophagy under Mst1 and Mst2-mediated mitochondrial stress.⁶⁴ In addition, Mst1 can regulate the expression of Parkin through the AMPK pathway, inhibit the phagocytosis of Parkin-related mitophagy, and restore the apoptosis of hepatocyte mitochondria, thus promoting the improvement of MASLD.⁶⁵

Together, these findings indicate that MASLD evolution is aided by poor mitophagy (Figure 2A).

FIGURE 2.

(A) The roles of mitophagy, mitochondrial dynamics, and mitochondrial biogenesis play in MASLD progression. (B) The gut-liver axis is altered in the development of MASLD. When patients with MASLD were compared with healthy subjects, the differences in BA profiles persisted, which can be mainly reflected in the production of both primary and secondary bile acids. Meanwhile, according to the changes in the content of different BA species, it can be inferred that FXR signaling is inhibited in MASLD. Additionally, intestinal epithelial barrier damage is also one of the key factors promoting MASLD, which can result in an increased risk of metabolites of bacterial origin and microbial translocation from the gut to the periphery. (C) The intestinal flora is rich in metabolites, including both beneficial and unfavorable components. From the bacteria and mitochondria interaction perspective, LPS, EE, and TMAO can exert dramatic detrimental effects on mitochondrial function, including inhibiting mitochondrial respiration, increasing oxidative stress, and impairing mitochondrial quality control. Conversely, mitochondrial dysfunction was beneficially impacted by I3A, NAM, and SCFAs. However, their expression was reduced in patients with MASLD. Notably, decreased mitochondrial respiratory function in the gut epithelium affects the structure of the gut microbiota, generating an increase in harmful metabolites, including TMAO. (D) Gut microbiota-involved biosynthesis and metabolism of bile acids. Primary BAs are synthesized in hepatocytes from cholesterolviaclassical or alternative pathways. In the classical pathway, cholesterol is initiated by CYP7A1 and converted into 2 primary BAs, CA, and CDCA. The alternative pathway is initiated by CYP27A1 and forms mostly CDCA. They are further conjugated with taurine (in mice) or glycine (in humans) and transformed into conjugated BAs (eg, TCA, GCA, TCDCA, and GCDCA). Part of BAs is converted to secondary bile acids(eg, UDCA, DCA, LCA, TUDCA, TDCA, TLCA, GUDCA, GDCA, and GLCA) by various organisms by intestinal flora. FXR is activated by unconjugated BAs, thus stimulating the expression of SHP, which further binds to LRH1 to inhibit CYP7A1 transcription. In addition, FGF19/FGF15 released by intestinal epithelial cells also induces JNK1/2 and ERK1/2 signaling by binding to FGFR4/β-klotho complexes. Abbreviations: BA, bile acid; DCA, deoxycholic acid; EE, endogenous ethanol; FGFR4, FGF receptor 4; FXR, farnesoid X-activated receptor; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; I3A, indol-3-acetic acid; LCA, lithocholic acid; LPS, lipopolysaccharide; LRH1, liver receptor homolog 1; MASLD, metabolic dysfunction–associated steatotic liver disease; NAM, nicotinamide; SCFAs, short-chain fatty acids; SHP, small heterodimer partner; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TMAO, trimethylamine N-oxide; UDCA, ursodeoxycholic acid.

Mitochondrial biogenesis

Mitochondrial biogenesis is an important mediator of mitoQC because of its ability to synthesize new mitochondria, which helps cells to adapt to their energy requirements. PGC-1α is a pivotal regulator of mitochondrial fatty acid oxidation and biogenesis.⁶⁶ A low level of PGC-1 has been reported to be associated with decreased antioxidant mechanisms in a mouse model of diet-induced MASLD.⁴¹ Besides, In an in vitro study, Zhang et al showed that Lycium barbarum polysaccharides increase PGC-1-dependent mitochondrial biogenesis in an MASLD cell model, which contributes to improving liver energy metabolism and attenuating intracellular lipid accumulation.⁶⁷ Likewise, a 2020 study in mice revealed that supplementation with neohesperidin could mitigate diet-induced insulin resistance and hepatic steatosis, which are associated with upregulated mitochondrial biogenesis.⁶⁸ Collectively, mitochondrial biosynthesis was inhibited during MASLD development (Figure 2A).

Mitochondrial dynamics

Mitochondrial dynamics encompasses 2 complementary processes: mitochondrial fission and fusion. Mitochondrial fission is mainly mediated by dynamin-related protein 1 (Drp1), which can seriously damage mitochondria in healthy mitochondrial networks and function in mitophagy to eliminate mitochondrial garbage. In addition, mitochondrial fusion can promote the exchange of mitochondrial components (eg, proteins, lipids, and genomes) to maintain mitochondrial health, and optic atrophy 1 and mitofusins promote fusion of the mitochondrial inner and outer membranes, respectively.⁵¹ Indeed, mitochondrial fission has been shown to be excessive in mice fed a choline-deficient, L-amini-acid-defined diet. The same study revealed that mdivi1, a chemical mitochondrial fission inhibitor, is capable of rebalancing mitochondrial dynamics and attenuating fibrosis and inflammation in the livers of MASLD mice.⁵² Similarly, several ongoing studies have shown an elevation of Drp1 and downregulation of Mfn2 and Opa1 in the MASLD disease model induced by HFD feeding, suggesting that disrupted mitochondrial fusion-fission balance is involved in MASLD progression.⁵³,⁵⁴ In addition, Han et al revealed that excessive mitochondrial fission seems to be associated with AMPK pathway inhibition.⁵³

THE GUT-LIVER AXIS AND MASLD

Both the liver and gut are involved in the digestion and absorption of food. Due to the existence of portal circulation and the biliary tree, there are many connections between these 2 organs, including metabolism, neuroendocrine, and immune. The gut provides ~70% of the blood for the liver through the portal vein, enterogenic substances such as gut microbial products, exogenous toxins, and fragments of digested food enter the liver through the portal vein.⁶⁹,⁷⁰ The liver responds to these intestinal signals by excreting bile acids, cytokines, and other bioactive substances into the gut, a 2-way link often referred to as the “gut-liver axis” (Figure 2B).⁷⁰

Association of MASLD pathogenesis and gut dysbiosis

Gut microbiome dysbiosis has attracted the attention of researchers in metabolic disorders. The human gastrointestinal tract is colonized by a complex microbial community, gut microbiota (GM), consisting of bacteria, archaea, protists, fungi, and viruses.⁷¹ GM is characterized by highly dynamic changes that vary with age, gender, diet, hormonal changes, and medication.⁷² In addition to absorbing host energy, GM actively participates in human physiological activities, including digestion, immunity, neurotrophy, and inflammation. Gut bacteria have a powerful metabolic capacity. They ferment hard-to-digest dietary fiber and produce short-chain fatty acids (SCFAs), which provide the body with an energy source. Endogenous substances, such as bile acids (BAs) and vitamins, are also metabolized by gut bacteria.⁷³ The composition and abundance of GM is accepted as a barometer of overall health.¹⁶,⁷⁰ Obvious changes in the composition of the fecal microbiota have been reported in patients with MASLD. For example, 2 studies⁷⁴,⁷⁵ have recently reported an upregulation of Firmicutes/Bacteroidetes ratio in obese youths with MASLD and adults with MASLD, respectively, indicating that the Firmicutes/Bacteroidetes ratio is associated with steatosis and obesity. Apart from this, the gut microbiome is an essential component of the immune barrier. Host bacteria attach to the surface of intestinal epithelial cells to form the intestinal barrier. They can also secrete antimicrobial substances such as microproteins or lower local pH by releasing metabolites to prevent pathogenic bacteria from colonizing them.⁷⁶ Moreover, Metabolites produced by specific microbes in the gut microbiome, such as SCFAs, tryptophan, and BAs, can help regulate host immune responses and homeostasis.⁴,⁷⁷

In addition to reduced richness and diversity of the microbiome, the breakdown of the intestinal barrier can also lead to intestinal dysbiosis.⁷⁸ The intestinal barrier plays a vital role in the gut-liver axis, including physical, immune, and biochemical components.⁷⁹ Intestinal blood vessels and a single layer of epithelial cells, connected by tight junction proteins, together with the mucus layer and microorganisms, form a physical barrier. Biochemical barriers are maintained and mediated by molecules with antimicrobial properties, such as BAs and antimicrobial proteins. The main components of the immune barrier include secreted Ig A and lymphoid follicles containing a variety of immune cells.⁸⁰ A complex relationship exists between the gut microbiota and the immune system. Most gut microbiome-mediated diseases are associated with impaired immune responses.⁸¹ Certainly, the intestinal barrier constitutes the body’s first line of defense for immunity, intact intestinal barrier prevents noxiousness from entering the circulation by permselective and active transport in a healthy state.⁶⁹ However, under pathological conditions, the stimuli (eg, dysbiosis, gut inflammation, and toxins) can disrupt the intestinal barrier and increase permeability, leading to the translocation of intestinal bacteria and endotoxin entry into the portal vein system,⁸² allowing more bacteria and bacteria-derived components and metabolites to escape to the liver, stimulating the activation of immune cells in the liver, the release of inflammatory cytokines, and the progression of fibrosis,⁸³ thus causing an imbalance in enterohepatic homeostasis, which is a pathological basis for various chronic liver diseases.⁸⁴ Repairing the intestinal microbiota may be an effective strategy for MASLD treatment. It is well known that unhealthy diet is an important driver of MASLD. An animal study found that energy-dense diets could successfully induce MASLD, which is simultaneously accompanied by elevated gut permeability.⁸⁵ Another study found that Lactobacillus plantarum NA136 treatment could regulate gut microbial dysbiosis, thus strengthening the intestinal microbiota and reducing inflammation in the liver.⁸⁶

The liver has the function of fine-tuning inflammation and immune effector cells and can control the inflammatory response of the GM and its degradation products, which reach the liver through portal vein blood.⁸⁷,⁸⁸ The liver provides a second line of defense against disease-causing agents that evade the intestinal mucosal immune defense.⁸⁹ Excessive accumulation of FFA can lead to the transfer of lipopolysaccharide (LPS) to the liver and interaction with Toll-like receptors (TLRs) (mainly TLR4-CD14 complexes) on macrophages and stellate cells, thereby enabling hepatocytes, KCS, and other pro-inflammatory cells to activate downstream MAPK and NF-κB signaling pathways. Ultimately, it promotes the release of pro-inflammatory cytokines.⁹⁰ TNF, IL-6, and IL-1β have been found to be highly expressed in MASLD.⁹¹ TNF is one of the earliest cytokines to be studied. In addition, IL-1 family members have been shown to affect insulin-glucose metabolism and regulate metabolic dysfunction in patients with MASLD. Moreover, IL-1β is one of the key cytokines leading to the induction and persistence of liver inflammation.⁹² IL-6 is also expressed at increased concentrations in the liver and adipose tissue of patients with MASLD.⁹³ Loss of IL-17 can promote MASLD/MASH progression, and recent evidence has demonstrated that IL-17 can exert a gut microbiota-mediated restoring function of the gut barrier, thereby inhibiting hepatocyte damage.⁹⁴ Furthermore, the role of inflammasomes and TLRs in MASLD has been reported several times. NLRP3 and various TLRS affect key pathways involved in MASLD, including inflammatory infiltration of the liver and adipose tissue and regulation of insulin sensitivity.⁹⁵ Actually, regulating the gut microbiome through the activity of certain TLRs or inflammatory mediators may lead to alterations in microbiome-related inflammation, which in turn may worsen liver inflammation and MASLD⁹⁶ (Figure 3).

FIGURE 3.

Schematic diagram of liver immune changes in MASLD. After excessive accumulation of FFAs, hepatic steatosis causes the release of DAMPs and other substances and stimulates TLRs, which enables hepatocytes, KCS, and other pro-inflammatory cells to activate downstream MAPK and NF-κB signaling pathways, thereby activating NLRP3 and ultimately promotes the release of pro-inflammatory cytokines (including IL-β, TNF-α, IL-6, IL-8, TGF-β, etc.). Immune activation and hepatocyte injury will affect HSC activation by upregulating pro-fibrotic and pro-inflammatory cytokines such as TGF-β1 and IL-β, further aggravating MASLD. In addition, activated DC coordinates the T-cell immune response. Moreover, the increased level of ROS might contribute to the release of mtDAMP release, which can activate KCs and upregulate the NLRP3 expression. Abbreviations: BA, bile acid; DC, dendritic cell; FFA, free fatty acid; MASLD, metabolic dysfunction–associated steatotic liver disease; TLR, toll-like receptors.

Collectively, the gut-liver axis is a pivotal player in MASLD pathogenesis, and alterations in the gut-liver axis primarily result from gut bacterial dysbiosis (Figure 2B). Thus, a full understanding of the structural characteristics of the gut microbiota in MASLD is required to develop effective treatment strategies for improving MASLD.

THE LINK BETWEEN MICROBIAL METABOLITES AND MITOCHONDRIAL DYSFUNCTION IN MASLD

Multiple lines of evidence suggest that the adverse effects of the gut microbiota on many nondigestive systems are achieved through various bacterial metabolites. Changes in microbial metabolites may reflect compositional alterations in the gut microbiota during MASLD development. Recently, the interaction between mitochondria and microbiota has attracted particular attention, and evidence has shown that damaging factors resulting from gut-flora-induced mitochondrial dysfunction act as vital regulators of lipid accumulation in the liver.⁹⁷,⁹⁸,⁹⁹,¹⁰⁰ A review in this chapter aims to summarize several microbial metabolites linked to MASLD as well as their impact on mitochondrial dysfunction (Figure 2C).

Bile acids

Bile acids (BAs) act as vital mediators for communication between the gut and the liver.¹⁰¹ The gut microbiota can biotransform and modify BAs, substantially influencing the synthesis and metabolism of BAs.¹⁰² Catalyzed by cholesterol 7α-monooxygenase or sterol 27-hydroxylase, cholesterol can be converted to primary BA in pericentral hepatocytes that can be further conjugated to taurine or glycine to produce primary conjugated BAs, which are stored in the gallbladder and spilled into the small intestine after feeding the host.¹⁰³ However, 95% of BAs are actively reabsorbed and transported back to the liver through the portal vein; the remaining BAs are converted into secondary BAs by bacterial-dependent modifications and lost into feces (Figure 2D).¹⁰⁴ Furthermore, gut microbiota can not only directly biotransform primary BA into secondary BA, but also control the BA synthesis by regulating the bile acids synthetase through dehydroxyisomerization, oxidative desulfurization, and esterification, including deconjugation, 7α-Dehydroxyisomerization, oxidation, isomerization, desulfurization, and esterification.¹⁰⁵,¹⁰⁶ In turn, in addition to promoting the absorption of lipids and lipid-soluble vitamins, BAs can also regulate diverse immunological and metabolic pathways by activating bile acid receptors, including farnesoid X-activated receptor (FXR) and G protein-coupled BA receptor 1, and play a beneficial role in maintaining gut microbiota homeostasis and intestinal microbiota integrity.⁶⁹ Therefore, gut microbiota disorder may lead to BA synthesis and metabolism disorder, which may affect lipid metabolism, hepatobiliary function, and intestinal health. The imbalance in BA homeostasis plays a critical role in MASLD progression. Clinical cohort studies have shown the plasma BA profiles differ between patients with MASLD/MASH and healthy controls. For example, Jiao et al found an elevated production of both primary and secondary BAs in patients with MASLD.¹⁰⁷ Nevertheless, the same study revealed that deoxycholic acid (DCA), an FXR antagonist, was increased, whereas chenodeoxycholic acid, an FXR agonist, was decreased in MASLD, suggesting that FXR signaling is inhibited in MASLD, which could promote obesity and steatosis in the liver (Figure 2D).¹⁰⁷ In another study, researchers proved that the alteration of circulating BA composition was associated with the histological features of MASH.¹⁰⁸ In addition, BA has an effect on the antioxidant capacity of mitochondria. In liver conditions with biliary stasis, the antioxidant capacity of mitochondria is reduced.¹⁰⁹ Studies have found that ursodeoxycholic acid, taurine deoxycholic acid, and lipophilic BA, such as DCA, chenodeoxycholic acid, and lithocholic acid, can inhibit electron transport chain in isolated mitochondria of rat liver.¹¹⁰ In addition, recent studies have proved that CA and DCA can reduce mitochondrial potential, and the levels of OXPHOS complexes I, II and III also inhibit mitochondrial biogenesis and affect mitochondrial function, thus increasing ROS production.¹¹¹

Overall, as a major mediator of enterohepatic circulation, BAs, play a pivotal role in nutrient absorption and signal transduction by regulating the gut microbiota and activating different BA receptors, and it is of great interest to understand the effect of changes in bile acid metabolism in the gut and mitochondrial cross-talk in MASLD.

Short-chain fatty acids

Short-chain fatty acids (SCFAs) are bacterial metabolites derived from indigestible dietary fibers and saccharides.¹⁰⁰ The ample evidence has demonstrated that SCFAs exert beneficial effects on the systemic glycolipid metabolism and intestinal mucosal barrier function.¹⁶,¹⁸,¹¹² Clearly, SCFAs have been accepted as beneficial regulators for mitochondrial metabolism.¹⁰ However, patients with MASLD show a depletion of SCFA-producing bacteria in stool samples, including Prevotella-9, Lachnospiraceae, and Ruminococcus.¹¹³ Interestingly, in vivo studies of HFD-fed mice revealed that sodium butyrate supplementation could mitigate hepatic steatosis and inflammation by upregulating the abundance of SCFA-producing bacteria, although the pathogenic mechanism remains unclear.¹¹⁴ Based on this observation, Maria et al found that butyrate reduced the lipid content and OS levels in the liver of an obese mouse model.⁹⁹ Notably, these effects were mainly related to improved mitochondrial respiration through the activation of the AMPK-acetyl-CoA carboxylase pathway.⁹⁹ In another study, a significant increase in propionic acid and isobutyric acid concentrations was found in the feces of Methylation-controlled J protein knockout mice fed CDA-HFD.¹¹⁵ In the hyperinsulin-induced dysfunction of HepG2 cells, sodium butyrate can significantly increase the mitochondrial DNA content of HepG2 cells, increase the membrane potential function, and enhance the activities of SOD and glutathione peroxidase. It depletes levels of pro-oxidant NADPH oxidase 2, ROS, and MDA, thereby improving mitochondrial.¹¹⁶ In addition to these beneficial effects, butyrate was shown to elevate the expression of Mfn1, Mfn2, and Opa1 protein mRNAs in the liver, promoting mitochondrial fusion.⁹⁹ Interestingly, sodium acetate has been reported to prevent HSC activation and alleviate liver fibrosis by activating the AMPK/PPARγ pathway.¹¹⁷ However, the association between sodium acetate and the mitochondrial function of HSC requires further study. Additionally, at the cellular level, the addition of propionate to FFA-treated calf hepatocytes reversed mitochondrial dysfunction, OS, and apoptosis, which was associated with the upregulation of PGC-1α, which is specific for propionate.¹¹⁸ Similarly, Deng et al demonstrated that SCFAs, particularly butyrate, can prevent mitochondrial dysfunction by lowering ROS production and caspase-3 protein expression in THLE-2 Cells after exposure to hippuric acid.¹¹⁹ Besides, studies in obese rodent models have revealed that butyrate can lead to the upregulation of CPT-1b and cytochrome c oxidase I gene expression, as well as the downregulation of histone deacetylase activity in skeletal muscle cells. Changes in these mechanisms can improve mitochondrial function and contribute to increased energy expenditure and insulin sensitivity.¹²⁰ Notably, histone deacetylases have been associated with mitochondrial energy metabolism and quality control.¹²¹,¹²² SCFA treatment exhibits beneficial effects by altering histone deacetylase mRNA expression and attenuating systemic inflammation in obese patients.¹²³

Collectively, SCFA therapy has been shown to have antioxidant effects by downregulating pro-inflammatory mediators, maintaining the integrity of the intestinal microbiota, and increasing mitochondrial respiratory capacity.¹²⁴ These data indicated that SCFAs exert a protective role in MASLD by optimizing mitochondrial function (Figure 4).

FIGURE 4.

Microbiota-derived metabolites impact mitochondrial dysfunction in MASLD. Gut microbiota and its metabolites affect the structure and function of hepatocyte mitochondria through enterohepatic circulation, and the interaction between microbiota and mitochondria completes the progression of gut microbiota to promote MASLD. Abbreviations: ADH, alcohol dehydrogenase; AhR, aryl hydrocarbon receptor; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AMPK, AMP-activated protein kinase; BA, bile acids; CPT1, carnitine palmitoyl transferase Ⅰ; CPT2, carnitine palmitoyl transferase Ⅱ; Drp1, dynamin-related protein 1; EE, endogenous ethanol; Fndc5, Fibronectin type III domain-containing protein 5; FXR, farnesoid X-activated receptor; GSH, glutathione; I3A, indol-3-acetic acid; LPS, lipopolysaccharide; MASLD, metabolic dysfunction–associated steatotic liver disease; MCJ, methylation-controlled J protein; MDA, malondialdehyde; NAM, nicotinamide; OXPHOS, oxidative phosphorylation; PGC-1α, proliferator-activated receptorγ coactivator-1α; SCFAs, short-chain fatty acids; SDHB, succinate dehydrogenase complex subunit B; SH-SY5Y, human neuroblastoma cell line; SIRTs, sirtuins; SOD2, superoxide dismutase; TG, triglycerides; TLR4, toll-like receptors 4; TMAO, trimethylamine N-oxide.

Endogenous ethanol

Endogenous ethanol (EE) is produced by gut bacteria through carbohydrate metabolism.¹²⁵ Several studies have reported that ethanol-producing bacteria (including Clostridium, Escherichia, Klebsiella pneumoniae, and Proteobacteria) in MASLD have a higher representation, contributing to the high serum level of ethanol in the absence of alcohol ingestion.⁹⁸,¹²⁶,¹²⁷ Thus, some researchers have described MASLD as an endogenous alcohol-associated fatty liver disease.¹²⁸ Additionally, MASLD and alcohol-associated liver disease share similar disease phenotypes, including hepatic steatosis, inflammation, and fibrosis, as well as a common mechanistic background, such as mitochondrial dysfunction.¹²⁵ Ethanol may cause liver damage by inducing intestinal cytochrome P450 2E1 and iNOS to increase oxidative stress and steatosis, and eventually dysregulate tight junction proteins and damage the intestinal microbiota.¹²⁹,¹³⁰ Liver mitochondria play an important role in ethanol metabolism. Following the conversion of ethanol to acetaldehyde by cytosolic alcohol dehydrogenase in hepatocytes, mitochondrial acetaldehyde dehydrogenase, and cytochrome P450 2E1 catalyze the conversion of hepatocytes into acetate.¹³¹ Apparently, acetaldehyde, a potentially toxic molecule, was reported to upregulate the levels of ROS and Ca2+ in SH-SY5Y cells, thereby promoting excessive Drp1 phosphorylation and mitochondrial fragmentation.¹³² In addition, FLD has been directly induced in mice by the inadministration of Klebsiella pneumoniae with high-yield alcohol, showing upregulation of genes associated with cytochrome P450 2E1 and lipogenesis, suggesting that endogenous alcohol produced by the intestinal bacterium HiAlc-Kpn may be sufficient to cause the liver pathogenesis observed in mice.⁹⁸ Additionally, SIRT has been demonstrated to promote mitochondrial fatty acid oxidation and induce stress tolerance.¹³³ Nevertheless, SIRT1 and SIRT3 expression was downregulated and functionally insufficient in a mouse model of alcohol-induced fatty liver, along with reduced mitochondrial content and inadequate ATP production.¹³⁴ Moreover, similar results were reported in a study by Fan et al, in which alcohol-dependent mitochondrial dysfunction was associated with downregulation of SIRT1.¹³⁵ A preclinical study has shown that endogenous ethanol produced by gut bacteria can induce mitochondrial damage in liver cells. Yuan and his colleagues considered Klebsiella pneumoniae as a contributor to MASLD.⁹⁸ Their study further verified the induction of MASLD by Klebsiella pneumoniae in mice and the effect of EE on the liver in vivo, and the results showed that the content of EE in blood was positively correlated with the levels of TG and MDA in liver tissue, and the activity of AST and ALT in serum. Moreover, EE induced by Klebsiella pneumoniae could impair mitochondrial integrity, decrease ATP content, and increase mitochondrial ROS accumulation and DNA damage.⁹⁷,⁹⁸ Furthermore, it should be noted that their study also demonstrated that EE could reduce the expression of Mfn1 but upregulate the expression of Drp1 and Fis1, generating mitochondrial disintegration.⁹⁸

Collectively, although the underlying molecular mechanisms of endogenous ethanol in MASLD have rarely been specifically studied, there is no denying that EE is probably produced by the microbiota in the colon and is largely involved in the highly complex pathogenesis of MASLD, leading to liver damage and steatosis. However, the combination and interaction of these factors remain to be clarified, and the association between EE and mitochondrial dysfunction in MASLD is also worth further exploration.

Nicotinamide and nicotinamide adenine dinucleotide

The gut microbiota-derived metabolite nicotinamide (NAM) serves as a precursor of NAD+.¹³⁶ NAD⁺ is a classic coenzyme that exerts its biological role through REDOX and non-redox reactions.¹³⁷ In mammals, NAD⁺ is synthesized through 3 main pathways: the de novo synthesis pathway of tryptophan, the recovery pathway of NAM, and the Preiss-Handler pathway of niacin (NA).¹³⁸ In addition, the role of gut microbiota in NAD⁺ synthesis has received significant attention in recent years. Evidence shows that Bacteroidetes, Clostridium, Proteobacteria, Actinobacteria, and Firmicutes are capable of synthesizing niacin in the healthy human intestines.¹³⁹ Using 16S rRNA sequencing technology, significant differences were discovered in the phenotypes of amino acid metabolism of gut microbiota between the MASLD and healthy control groups.¹⁴⁰ Studies have shown that normal gut microbiota transplantation can not only restore gut microbiota imbalance but also induce an increase in serum NMN levels and enhance the biosynthesis of NAD⁺ in the pancreas.¹⁴¹ In addition, Shats et al demonstrated the importance of gut microbiota in promoting NAD⁺ with oral nicotinamide riboside (NR) supplementation, and found that partially labeled NR in normal mice was converted to NAM in the colonic lumen at a rate 3 times greater than that of NR in germ-free mice, suggesting that the conversion of NR to NAM is mainly done by the gut microbiota. Furthermore, this study also found that conventional mice treated with oral pyridine-15NNR had significantly increased levels of NA and NAR in portal vein blood, and the increase in NAM in germ-free mice was also significantly diminished, suggesting that both amidated and deamidated NAD⁺ precursors produced by the gut microbiota from NR reach the circulatory system.¹⁴²

Notably, the gut microbiome has a unique NAD⁺ metabolic pathway, which may help improve the metabolic flexibility of the host.¹⁴³ In contrast to the liver, the gut microbiota, such as Clostridium, Firmicutes, and Proteus, which regulate key enzymes NAMNAT and NAD⁺, synthesize NAD⁺ precursors into NAD⁺ through a specific de novo synthesis pathway. At the same time, some Firmicutes convert quinolinic acid to NAMN and synthesize NAD⁺ through NMNS and NMNAT. In addition, gut microbiota supplementation of NAD⁺ precursors NAM, NA, and NR also plays a crucial role in the synthesis of NAD⁺. Only Actinomyces, Firmicutes, and Proteobacteria can absorb NAM and NA, while the salvage of NR is only found in Firmicutes and Proteobacteria.¹³⁹ It has also been reported that there is another important pathway in the gut microbiota, namely the catalytic conversion of NAM to NA through PncA11, which combines the recycling and Preiss-handler pathways, which is known as deamidation.¹⁴² Moreover, Kim et al also confirmed the pathway whereby orally isotopically labeled NMN in rats was partially deaminated to NAMN by intestinal flora before absorption. Although the extent was significantly lower than that of the amidation metabolites NMN and NAD⁺, the level of nicotinate adenine dinucleotide in the liver was increased.¹⁴⁴ The deamidation process is considered to be an important step in the regulation of NAM and NR-enhanced NAD⁺ levels by intestinal flora.¹⁴⁵ Shen et al reported a liver-protective effect of nicotinamide; that is, nicotinamide pretreatment protects HepG2 cells from palmitate-induced lipotoxicity and cell death through SIRT1-mediated induction of autophagy.¹⁴⁶ Indeed, mitochondria harbor a large number of oxidoreduction reactions in which NAD⁺ is a vital player acting as an electron transfer carrier.¹⁴⁷ NAD⁺ not only plays a key role in energy metabolism and promotes mitochondrial function but is also a precursor to NADPH, an important component of the antioxidant defense system.¹⁴⁸ NAD⁺ can accept electrons for reduction to NADH, which can further transfer electrons into the respiratory chain and oxidize to NAD⁺.¹⁴⁹ Principally, NAD⁺ deletion results in mitochondrial dysfunction, which is an established cause of metabolic disorders.¹⁵⁰ Furthermore, the concentration of NAD⁺ within the mitochondria was significantly decreased in animal models of MASLD and obesity.³²,⁵⁰ Of note, a study in mice revealed that oral administration of NR, a precursor of NAD⁺, could rescue the MASLD phenotype (eg, fatty liver, low insulin sensitivity, fibrosis, and inflammation) through the increase of mitochondrial respiratory and content.³² Besides, it should also be noted that the effect of NR on mitochondrial function is mediated through SIRT1-dependent and SIRT3-dependent mitochondrial unfolded proteins.³² Similarly, in obese mice, Carles et al found that supplementation with NR reversed mitochondrial dysfunction in obese mice, thereby enhancing the oxidation capacity in skeletal muscle and brown adipose tissue, which could contribute to weight loss in obese mice.⁵⁰ Moreover, mitochondrial SOD2 is an important antioxidant enzyme, and NAD⁺ has been reported to facilitate the deacetylation of SOD2 by triggering SIRT3 activity to increase its antioxidant properties.⁵⁰ Interestingly, NAD⁺-boosting therapy has been reported to increase mitochondrial biogenesis and autophagy by modulating SIRT2-mediated Fndc5 deacetylation and deubiquitination.¹⁵¹ The above evidence demonstrates that the therapeutic potential of NAM in MASLD models is related to mitochondrial dysfunction (Figure 4).

Lipopolysaccharide

LPS, also known as an endotoxin, is a potent inducer of inflammation.¹⁵² LPS expression was found to be increased in liver tissue and serum in patients with MASLD as well as in an HFD model.¹⁵³ Moreover, LPS has been demonstrated to increase with MASLD disease severity¹⁵⁴ and is proposed to be a potential serum biomarker for MASLD and MASH.¹⁵⁵ An interesting animal study found that fatty liver formation was associated with high serum endotoxin levels triggered by gut flora disturbances.¹⁵⁶ Inflammation has been associated with oxidative stress, and the treatment of THLE-2 cells with LPS can successfully induce oxidative stress.¹⁵⁷ Using this cellular model, Li et al found that LPS contributed to the expression of MCJ, an endogenous inhibitor of mitochondrial complex I and mitochondrial membrane potential, increased ROS levels, and reduced mitochondrial membrane potential.¹⁵⁷ Notably, Wei et al revealed that LPS treatment could promote hepatocyte apoptosis by stimulating the release of cytochrome C from the mitochondria into the cytosol.¹⁵⁸ Moreover, LPS significantly reduced the copy number of mtDNA in the liver and further affected the activity of mitochondrial complexes IV and V, ultimately generating impaired mitochondrial OXPHOS and decreasing ATP production.¹⁵⁹ An additional study reported that LPS could induce sepsis in C57BL/6 mice and damage the liver, leading to increased SOD2 acetylation, SIRT3 depletion, mitochondrial morphological changes, and upregulation of heat shock protein 60 in the liver.¹⁶⁰ Collectively, LPS is a vital player in MASLD, which can impair mitochondrial function and lead to hepatocyte apoptosis, OS, and inflammation (Figure 4).

Indole-3-acetic acid

Over the past few years, the potential preventive role of indol-3-acetic acid (I3A) in metabolic diseases has attracted considerable attention.¹⁶¹ I3A is a tryptophan metabolite that has been repeatedly reported as an aryl hydrocarbon receptor (AhR) ligand,¹⁶²,¹⁶³ the AhR agonist in a variety of cells, including hepatocytes.¹⁶⁴,¹⁶⁵,¹⁶⁶ Moreover, the effect of I3A on AhR target genes is mediated by ligand activation of nuclear receptors.¹⁶⁷ This AhR can sense a wide range of gut signals and maintain homeostasis between the gut microbiota and the host.¹⁶⁸,¹⁶⁹ In addition, I3A can alleviate cytokine-mediated lipogenesis in hepatocytes by activating AhR.¹⁶⁷ Therefore, I3A can maintain intestinal homeostasis through AHR ligands and regulate the gut-liver axis.¹⁷⁰ However, the abundance of I3A-producing bacteria, such as Bcteroides, Alistipes, and Prevotellaceae, decreased in HFD-induced mouse models. Correspondingly, the expression of indole-related metabolites decreased in the feces of patients with MASLD and model mice, as confirmed by metabolic studies.¹¹³ Notably, in HFD-induced MASLD mice, oral administration of I3A alleviated the MASLD phenotype, mainly driven by the repair of mitochondrial respiration defects.¹⁵⁹ This effect was related to the increased expression of beta-oxidation and mitochondrial complex genes and decreased expression of lipogenesis genes in the liver. Moreover, the present study found that I3A treatment restored the level of ATP5A1, a component of ATP synthase, in an MASLD mouse model.¹⁷¹ Additionally, I3A has the ability to scavenge free radicals.¹⁷² It has been reported that macrophages pretreated with I3A were successively exposed to palmitic acid and LPS, which significantly reduced the mRNA levels of TNF-α, IL-1β, and McP-1, suggesting that I3A can reduce the expression of pro-inflammatory cytokines in macrophages and induce liver synthesis of FFAs. The study also demonstrated that I3A significantly reduced macrophage migration in response to MCP-1 in a dose-dependent manner. In addition, the study also demonstrated that I3A acts on hepatocytes to attenuate the upregulation of cell kinematics during adipogenesis, thereby attenuating free fatty acid-induced adipogenesis of hepatocytes.¹⁶⁷ Furthermore, I3A is reported to mitigate the upregulation of lipogenic genes involved in Srebf1, Scd1, PPARγ, Acaca, and Gpam, thereby reducing the accumulation of liver triglycerides after HFD induction in mice. In addition, it alleviates the increase in F4/80, MCP-1, and TNF-α expression and inhibits the infiltration of macrophages and reducing liver inflammation. The study also measured the levels of ROS and lipid peroxidation products, and the results showed that I3A can increase the expression of antioxidant enzymes (ie, SOD and GSH), reduce ROS levels, and protect the liver from oxidative stress.¹⁷¹ It has also been reported that I3A treatment increases the expression of Ahr and decreases the expression of Tlr4 mRNA that recognizes and activates LPS, thereby inhibiting macrophage activation and NF-κB signaling pathway, and ultimately improving the inflammatory response.¹⁷⁰ Melatonin, an indoleamine small molecular substance inside the human body, is not only a microbiota metabolite in the gut but also an endocrine secretion produced by the pineal gland, retina, skin, and others.¹⁷³ Type 2 diabetes is characterized by low serum melatonin serum levels, which may be associated with gut microbiota-mediated melatonin signaling.¹⁷³ Melatonin is a powerful antioxidant molecule that declines with age. Evidence shows that melatonin treatment can increase the expression of Mfn2, Opa1, and Drp1 in aging mice, thereby recovering mitochondrial dynamics altered by aging and maintaining mitochondrial morphology and number, which contributes to maintaining cardiac function.¹⁷⁴ In addition, in a mouse model of ochratoxin A-induced liver inflammation, melatonin treatment was found to regulate mitophagy, ameliorate oxidative stress, regulate gut microbiota disturbances, and repair intestinal microbiota functions.¹⁷⁵ These animal data provide evidence for the protective effect of I3A on MASLD under the protection of mitochondrial function (Figure 4), and targeting I3A may be an effective therapy for MASLD.

Trimethylamine N-oxide

A positive correlation between elevated serum trimethylamine N-oxide (TMAO) level and body weight, Visceral Adiposity Index, and Fatty Liver Index has been well described in overweight or obese subjects.¹⁷⁶ The importance of choline in maintaining hepatic lipid homeostasis has been recognized for several years.¹⁷⁷ However, under MASLD-associated dysbiosis, choline is overconsumed or metabolized to trimethylamine by anaerobic bacteria, followed by conversion of TMA to TMAO in the liver by choline-TMA lyase.¹⁷⁸ A recent study suggested that TMAO aggravates liver steatosis by suppressing BA-mediated hepatic FXR signaling.¹⁷⁹ Besides, it has been shown that long-term administration of 1.5% TMAO in drinking water induces oxidative stress in liver tissues.¹⁸⁰ Facultative anaerobic Enterobacteriaceae is closely associated with a pathological increase in TMAO in the gut.¹⁷⁸ Normally, the colonic epithelium depends on mitochondria to consume sufficient oxygen to maintain physiological hypoxia, forming an anoxic condition in the intestine, which restricts the growth of facultative anaerobic bacteria.¹⁸¹ However, a prolonged high-fat diet is reported to impair the host control mechanism, causing an increased number of respiratory electron acceptors, such as oxygen and nitrate, from the mucosal surface, supporting E. coli respiration and choline catabolism.¹⁷⁸ Succinate dehydrogenase, located on the inner membrane of mitochondria, is involved in OXPHOS and is closely linked to mitochondrial ROS production.¹⁸²Wu et al found that TMAO was capable of increasing succinate dehydrogenase complex subunit B expression and could lead to mitochondrial damage and high levels of ROS, resulting in endothelial cell pyroptosis and the progression of atherosclerotic lesions.¹⁸³ Another study demonstrated that TMAO reduces both pyruvate and fatty acid oxidation in cardiac mitochondria.¹⁸⁴ Based on these data, it is clear that TMAO negatively affects mitochondrial function (Figure 4). However, the association between TMAO and mitochondrial metabolism in hepatocytes requires further investigation.

TREATMENT OPTIONS FOR MASLD BASED ON MODULATION OF GUT MICROBIOTA

Optimizing the composition of the intestinal flora has been considered a vital factor in preventing or reversing the progression of MASLD in recent years because dysbiosis of the gut microbiota plays an essential role in the disease. Appropriate measures based on gut microbiota have been used in the clinical treatment of MASLD, such as the administration of probiotics, prebiotics, and antibiotics as well as the use of fecal microbiota transplantation (FMT). The beneficial effects of these measures have been validated in several animal and clinical studies, offering promising prospects for the treatment of MASLD. In addition to targeting the gut microbiota, these therapeutic strategies play a role in improving mitochondrial function.

Probiotics

Probiotics are a range of microbial food supplements that confer substantial health benefits to the host. It is reported that lactic acid bacteria cause Firmicutes/Bacteroidetes ratio and SCFA content changes in MASLD mice and, therefore, have an impact on energy and lipid metabolism and restrict the development of MASLD.¹⁸⁵ Notably, lactic acid bacteria fermentation enhanced the antioxidant capacity of black tea, and the lactic acid bacteria-fermented BT sample maintained mitochondrial membrane potential and inhibited ROS production in H₂O₂-treated CCD841 cells.¹⁸⁶ Additionally, a mouse model induced by HFD treated with probiotics containing Limosilactobacillus fermentum MG4231 and MG4244 exhibited enhanced lipid metabolism and reduced synthesis and liver inflammation, and these changes were associated with increased phosphorylation of AMPK and acetyl-CoA carboxylase, which aid mtFAO.¹⁸⁷ In another study, Bifidobacterium animalis subsp. lactis A6 conferred a relieving effect on obesity development in mice by regulating the gut bacterial community, normalizing the serum LPS level, improving mitochondrial biosynthesis, and upregulating the expression of uncoupling protein-1, which could increase the uncoupling mitochondrial OXPHOS to generate more heat.⁸,¹⁸⁸ Likewise, Dong et al found that Lactobacillus plantarum fermented barley extract restricts the development of obesity by repairing mitochondrial function.¹⁸⁹,¹⁹⁰,¹⁹¹ Overall, these preclinical studies provide evidence for the potential anti-MASLD effects of probiotics. Notably, various probiotic strains have been developed and used in the food industry, making it difficult to choose appropriate probiotics for patients with MASLD. In addition, the effectiveness of probiotics in MASLD has been inconsistent in clinical studies. For example, several recent clinical studies have demonstrated the therapeutic effect of probiotics in patients with MASLD, including the reduction of liver fat and inflammatory cytokine levels.¹⁹²,¹⁹³ In contrast, a 6-month study found that probiotics failed to alter important parameters of MASLD, such as blood lipid metabolism, hepatic steatosis, and fibrosis levels in patients with MASLD, but improved intestinal mucosal barrier function.¹⁹⁴ Therefore, the real benefits of probiotics in MASLD need to be explored in adequately designed clinical trials.

Prebiotics

Prebiotics can be fermented by gut microbiota to provide health benefits to the host.¹⁹⁵ Polyphenols are known for their prebiotic effects and are ubiquitous in plants. Li et al revealed that blueberry leaves contain 8 polyphenols, chlorogenic acid, D-catechin, L-epicatechin, rutin, isoquercitrin, cyanidin-3-O-glucoside, iridin, and quercetin.¹⁹⁶ The administration of polyphenols in blueberry leaves enhances antioxidant capacity in the liver and prevents the development of MASLD in mice by decreasing ROS and MDA levels and upregulating SOD2 expression in the liver. Polyphenols in blueberry leaves also repair mitochondrial dysfunction by increasing MMP, augmenting cellular ATP content, and reversing the decrease in mtDNA in the liver.¹⁹⁶ Rafiei et al found that several plant polyphenols that protect against steatosis were mediated by various mitochondria-related mechanisms in a cell model of MASLD. For instance, while cyanidin improves mitochondrial function primarily by restoring MMP levels to normal, kuromanin improves mitochondrial function primarily by increasing the mRNA levels of CPT1 and deacetylated PGC-1 and inhibiting the upregulation of FAS.¹⁹⁷ Likewise, evidence has shown that the polyphenol-rich foods Taurisolo and cocoa polyphenols may also attenuate the development of MASLD by restoring optimal mitochondrial function.¹⁹⁸,¹⁹⁹ Moreover, using a zebrafish model and human hepatocytes, Zhang et al showed that Lycii fructus polysaccharides restore redox balance and resist mitochondrial-mediated apoptosis, and therefore affect the development of MASLD.²⁰⁰ Resistant dextrin is another form of a prebiotic. A recent study showed that the anti-MASLD capability of resistant dextrin contributes to endothelial function improvement and intestinal flora structure optimization, in addition to maintaining mitochondrial integrity and function.²⁰¹ Taken together, the protective effect of prebiotics on mitochondria is crucial for preventing MASLD progression.

The beneficial effects of probiotics on MASLD have been supported by clinical studies. The combined use of camelina sativa oil and resistant dextrin could help patients with MASLD with metabolic risk factors and mental wellness.²⁰² Additionally, a study on obese individuals found that inulin-propionate ester administered to patients at doses of 10 mg daily for 24 weeks decreased weight gain, abdominal fat distribution, and liver fat content.²⁰³ It is noteworthy that a different investigation carried out by the same research group found that oral administration of inulin-propionate ester (20 mg/day) for 6 weeks did not alter intrahepatocellular lipid levels.²⁰⁴

Antibiotics

In a published article, Yuan et al described an intriguing case of MASH with auto-brewing syndrome and attributed the morbidity of the patient to the high level of endogenous alcohol induced by gut microbial dysbiosis, and the blood alcohol concentration and severity of MASH were alleviated after dietary changes and antibiotic treatment.⁹⁸ In mice fed with HFD, neomycin has been shown to reduce intestinal inflammation, hepatic steatosis, and atrophic intestinal lining.²⁰⁵ Additionally, rifaximin is poorly absorbed in the intestine and is recommended for the treatment of functional gastrointestinal disorders and hepatic encephalopathy.²⁰⁶ Its reported that the administration of rifaximin for 4 weeks could mitigate the disease phenotype of MASH in mice, accompanied by alterations in the intestinal microecology and decreased DCA in the ileum.²⁰⁷ In addition, a recent small clinical study, which focused on the effects of rifaximin on MASH, also found that rifaximin had positive effects on liver function, LPS, TLR4, IL-6, and MASLD-liver fat score in patients with MASH but was ineffective in lowering TG and BMI.²⁰⁶ Nevertheless, a prospective clinical study of 15 subjects found no effect of rifaximin therapy of 6-week duration on either hepatic inflammation, hepatic lipid content, or hepatic and peripheral insulin sensitivity in patients with MASH.²⁰⁸ Notably, another interesting study reported that inulin supplementation for 12 weeks after brief metronidazole therapy for 1 week could enhance the beneficial metabolic effects of short-term, very-low-calorie diets for 4 weeks among patients with MASLD.²⁰⁹

Overall, antibiotics may have a limited ability to treat MASLD, and it is important to consider any potential negative consequences they may have on human health, including the resistance of microorganisms to antibiotics and the reduction of beneficial bacteria by antibiotics, along with liver and kidney damage to the host.²⁰⁷,²¹⁰ The total impact of antibiotics on MASLD should be the focus of future studies.

Fecal microbiota transplantation

Fecal microbiota transplantation (FMT) aims to reconstitute a healthy gut microbiota with a “physiological” microbiome obtained from fecal material from a healthy donor.²¹¹ FMT has been clinically applied to treat a variety of metabolic syndromes, including MASLD, obesity, and diabetes.²¹²,²¹³ Six-week FMT treatment has been reported to promote beta cell regeneration, lower endotoxin burden, and promote pathological damage in the intestine and liver in a mouse model of type 2 diabetes mellitus.²¹⁴ Another interesting study showed that transplanting microbiota from healthy people could reduce body weight and liver triglyceride levels in HFD-fed mice compared with transplanting microbiota from patients with MASLD.²¹⁵ Similarly, a randomized clinical study conducted by Xue et al²¹⁶ revealed that FMT treatment delayed the progression of MASLD by altering the gut microecology. Additionally, the authors claimed that the effect of FMT was more pronounced in lean patients with MASLD than in patients with obesity. Conversely, another study reported that FMT reduced small intestinal permeability but failed to improve IR and hepatic proton density fat fraction in patients with MASLD.²¹⁷ Siew et al reported that a combination of lifestyle intervention and FMT alleviated hyperlipidemia and liver stiffness in patients with obesity with type 2 diabetes mellitus.²¹⁸

CONCLUSIONS AND FUTURE PERSPECTIVES

The gut microbiota of patients with MASLD shows an obvious alteration in composition and functionality, and in recent years, researchers have been exploring how the gut microbiota alters host homeostasis. In this review, the summarized data demonstrate a clear causal relationship between the gut microbiota and mitochondrial dysfunction during the development of MASLD. Oxygen consumption in gut epithelial mitochondria modulates the structure of the gut microbiota. Furthermore, altered gut microbiota and their metabolites affect the structure and function of hepatocyte mitochondria through enterohepatic circulation, which appears to be a key mechanism responsible for hepatocyte steatosis and imbalance in homeostasis. In conclusion, the interaction between the microbiota and mitochondria completes the storyline of the gut microbiota, promoting MASLD progression. Metabolites of the gut microbiota are diverse and can play either beneficial or detrimental roles in human physiological functions. Nevertheless, in this review, we summarized only 7 metabolites of gut microbiota, including BAs, SCFAs, ethanol, NAM, LPS, I3A, and TMAO, which may not be sufficient to generalize the overall impact of gut microbiota on mitochondrial function.Other metabolites (eg, H2S, NO, and p-cresols) should also be taken into consideration. In future studies, the correlation between gut microbiota and mitochondria warrants comprehensive and in-depth study. “Supplement the good ones, reduce the bad ones” appears to be the core strategy for therapeutic approaches targeting the modulation of the gut microbiota. In addition, probiotics, prebiotics, antibiotics, and FMT have been used to regulate the composition of the gut microbiota in patients with MASLD and to inhibit disease development. These treatments have been shown to be effective for treating MASLD in experimental animal models. However, the safety and efficacy of these therapeutic strategies should be demonstrated in future large-scale, randomized clinical studies. A better understanding of these mechanisms will aid in the development of potent therapeutic approaches to prevent MASLD.