Gut microbiome and nonalcoholic fatty liver disease

Meng-Yuan Wu ,Jian-Gao Fan

a Xiangya School of Medicine, Central South University, Changsha 410013, China

b Department of Gastroenterology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 20 0 092, China

c Shanghai Key Lab of Pediatric Gastroenterology and Nutrition, Shanghai 20 0 092, China

Keywords: Nonalcoholic fatty liver disease Gut microbiome Dysbiosis Intestinal permeability

ABSTRACT Nonalcoholic fatty liver disease (NAFLD) has become the most prevalent chronic liver disease globally and imposed a heavy economic burden on society and individuals. To date,the pathological process of NAFLD is not yet fully elucidated. Compelling evidences have demonstrated the pivotal role of gut microbiota in the pathogenesis of NAFLD,and gut dysbiosis has been commonly observed in patients with NAFLD. Gut dysbiosis impairs gut permeability,allowing the translocation of bacterial products such as lipopolysaccharides (LPS),short-chain fatty acids (SCFAs),and ethanol to the liver via portal blood flow. This review aimed to shed light on the underlying mechanisms by which gut microbiota influences the development and progression of NAFLD. In addition,the potential application of gut microbiome as a non-invasive diagnostic tool and a novel therapeutical target was reviewed.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease that affects approximately 30% of the global adult population. A rising proportion of nonalcoholic steatohepatitis (NASH),the progressive form of NAFLD,has resulted in an alarming increase in liver cirrhosis and hepatocellular carcinoma (HCC) [1] .NAFLD-related liver failure imposes a substantial economic burden on society and has become the second leading indication for liver transplantation in the Western world [2] .

NAFLD has a complex phenotype shaped by both genetic and environmental factors. Only a proportion of individuals with NASH develop further inflammation and fibrosis,resulting in the ultimate forms: cirrhosis and HCC. Moreover,a series of histological changes are observed in NAFLD progression,including hepatic steatosis,lobular inflammation,hepatocyte ballooning,Mallory-Denk bodies,and ultimately liver fibrosis from peri–central region to periportal areas [3] . A multiple-hit hypothesis has been proposed for NAFLD progression. The parallel factors comprise genetic background,metabolic disorders,gut-liver axis dysfunction,inflammatory response,and dietary habits [4] . Epidemiological data indicate substantial inter-patient variation in the susceptibility and severity of NAFLD and liver-related complications. A genome-wide association study validated interethnic differences in the prevalence and severity of NAFLD between Hispanics,European American,and African American patients in the USA urban population. Hispanic individuals have the highest prevalence of hepatic steatosis and are more likely to progress to hepatic inflammation [5] . A more recent study showed that Hispanics of Mexican origin are at a higher risk of steatosis than those from Puerto Rico and Dominican descent when metabolic risk factors were controlled [6] . This ethnic variability is partly attributed to genetic variation in Palatin-like phospholipase domain-containing 3 (PNPLA3),which is linked to serum aspartate aminotransferase (AST) levels [7] . Using magnetic resonance imaging and magnetic resonance elastography,Loomba et al. [8] performed a prospective twin study which showed that only monozygotic twins with NAFLD showed a robust correlation in both hepatic steatosis and fibrosis. The heritability of hepatic fat fraction was estimated to be 52% of individuals while heritable components accounted for 50% of liver fibrosis. These results fit well with the former study that genetic factors play a vital role in the progression of NAFLD. Insulin resistance is another key factor involved in the pathogenesis of NAFLD. The defective response to insulin results in increased release of free fatty acids (FFAs)from adipocytes [9] . The circulating FFAs can activate inflammatory pathways,thereby promoting liver fibrosis [10] . Insulin resistance also disturbs lipid metabolism in the liver. In the condition of insulin resistance,hepaticdenovolipogenesis (DNL) is enhanced as a result of increased sterol-regulatory binding protein 1c (SREBP-1c) [ 11 ]. Additionally,defects in insulin signaling pathways cause the inhibition ofβ-oxidation of FFAs,thus promoting lipid accumulation [ 12 ].

Gut microbiome and NAFLD

There are trillions of commensal microbes in the human gut,including different groups of bacteria,resident archaea,fungi,and viruses,which are distributed in various sites of the gastrointestinal tract and interact with the host to maintain intestinal homeostasis. The composition of the gut microbiota changes with age throughout human life. Before the time of delivery,the early infant microbiome in utero environment is transferred via the placental,amniotic fluid,and the umbilical cord [13–15] . The microbial contact in utero could affect the fetal immune system as the expression of Toll-like receptor (TLR)-related genes change in both the placenta and the fetal intestine with maternal probiotic supplementation [16] . During the first year of the infant period,the microbiota develops into a more complex and dissimilar pattern,with more similarities to their mother’s gut microbiota [14] .As solid foods are introduced,an increase in Bacteroidetes is observed [17] . Until the age of 3 years old,infant gut composition gradually gains an adult-like structure [18] . In addition to genetic background,several pivotal factors induce shifts in the species and composition of intestinal colonization,including the mode of delivery,and gestational age [ 19,20 ]. Infant antibiotic treatment and feeding styles as well as wider environment exposure also have been associated with the microbial profile [ 14,21,22 ]. The microbial composition and abundance vary along the alimentary tract.The caecum and colon are colonized with the highest abundance of microorganisms (1011and 1012,respectively) and the level decreases from the small intestine (104–107) to the stomach and duodenum (10–103). Bacteroidota and Bacillota play a predominant role in intestinal homeostasis,which account for over 90% of bacterial phyla colonizing the healthy human gut,while the other three major phyla of the gut microbiota (Proteobacteria,Actinobacteria,and Fusobacteria) represent only a small percentage [23] . The gut microbiota is functionally diverse and actively takes part in various activities of the host,including energy intake,glucose and lipid metabolism,and immune modulation. Additionally,it can recognize immune regulators and promote the differentiation of immune cells,thereby modulating autoimmune and adaptive immune responses [24] . A number of intrinsic and extrinsic factors are associated with the composition and abundance of gut microbiota.Mutations in nucleotide-binding oligomerization domain 2 (NOD2)result in decreasing bacterial diversity and dysbiosis in the microbiota. Dietary factors,lifestyle,use of drugs,and environmental stimuli also cause quantitative and qualitative alterations in the intestinal microflora. As a consequence,the gut microbiome is highly heterogeneous among individuals.

The liver and the gastrointestinal tract both originate from endoderm during fetal development and interact with each other bidirectionally. This tight connection depends on two functional structures: vascular circulation and the gut mucosal barrier. The portal circulation drained from the gastrointestinal tract transports nutrients and microbial metabolites to the liver where inflammatory cells are activated in response to gut-derived signals.In turn,the liver releases bile acids and secretes many bioactive mediators into the biliary tract and systemic circulation. Portal blood flow comprises proinflammatory microbial components like lipopolysaccharides (LPS),which can be recognized by pattern recognition receptors (PRRs) of hepatocytes and induces inflammation cascades [25] . The past decade has witnessed an explosion of studies focusing on microbial communities residing within the human body,especially the gut microbiome with their host in the pathogenesis of metabolic disorders including obesity,diabetes,and NAFLD. Indeed,Asian patients with NAFLD have lower total bacterial diversity and richness than healthy individuals [26] . It has been shown using 16S rRNA sequencing thatBacteroideswere substantially increased with the severity of liver lesions,while a reduction inPrevotellawas observed in patients with NASH [27] .The high level ofBacteroideswas paralleled by an increase in fecal deoxycholic acid,which was reported to induce apoptosis in rat liver [ 27,28 ]. Schwimmer et al. [29] used 16S rRNA amplicon sequencing and metagenomic shotgun sequencing to access fecal microbiota composition in children with NAFLD. The abundance ofαdiversity was decreased with the severity of NAFLD and the high abundance ofPrevotellacopriwas associated with more severe fibrosis. Germ-free (GF) animal models,which lack all microorganisms,have been established to study the association of gut microbes with the host. It was found that GF mice are resistant to high-fat diet (HFD)-induced NASH and this effect is linked to intestinal blood vessel permeability. The disruption of the gut vascular barrier (GVB) occurs following epithelial barrier impairment,allowing the translocation of bacteria to the liver. Transplanting gut microbiota from HFD-fed mice to GF mice leads to the increase of epididymal adipose tissue even on a standard diet,indicating that dysbiosis is involved in NASH progression [30] .

Mechanisms

During the past few years,a host of studies have revealed the mechanisms linking gut microbiota and NAFLD progression,ranging from increased intestinal permeability to the alterations in gut metabolites ( Fig. 1 ). These molecular mechanisms are discussed below.

Fig. 1. Mechanisms contributing to microbiome regulation of NAFLD. Microbiota dysbiosis induces increased gut permeability,allowing the translocation of bacterial products into the portal vein. NAFLD: nonalcoholic fatty liver disease; SCFAs: short-chain fatty acids; LPS: lipopolysaccharide; TMA: trimethylamine; TLRs: Toll-like receptors.

Gut barrier dysfunction

As the first line of protection against pathogens,the intestinal gut barrier comprises more than one physical barricade (epithelial cell tight junctional complexes and the mucosal surface covering the intestinal epithelial cell). The epithelium,including enterocytes,Paneth cells,and goblet cells,produces antimicrobial peptides and chloride to form a chemical barrier that separates the microbiota from the tissues. In addition,the intestinal barrier participates in immune responses with innate and adaptive immune cells secreting cytokines,chemokines,and antimicrobial peptides(AMPs) in the lamina propria and gut-associated lymphoid tissue.Therefore,apart from nutrient absorption,a functional gut barrier is highly dynamic and responsive to microbial invaders and harmful stimuli by affecting commensal bacteria composition and regulating immune defense. In contrast,the microbiota can trigger an immune response,thereby influencing intestinal barrier integrity.Therefore,the maintenance of intestinal homeostasis relies on the complex bidirectional communication between the microbiota and the intestinal barrier. Aberrant alterations in the crosstalk among gut microbiota,intestinal epithelial cells,and gut immune system can increase the risk of a wide array of gastrointestinal diseases,and there is a growing number of studies revealing the role of gut barrier dysfunction or altered gut permeability in NAFLD. Miele et al. [31] first reported an increased gut permeability correlated with the severity of steatosis in patients with biopsy-proven NAFLD. And this abnormality is associated with the high prevalence of small intestinal bacterial overgrowth (SIBO) in these patients. Based on glucose breath test,approximately 60% of the NAFLD patients have SIBO,compared with only 20.8% of healthy controls. These differences are linked to the disruption of intercellular tight junctions,as the expression of zonula occludens-1 (ZO-1),a marker for assessing tight junction integrity,was significantly decreased in NAFLD individuals. However,the causal relationship between NAFLD/NASH and disruption of the gut epithelial barrier has not been demonstrated. In diet-induced mouse models of NASH,enhanced intestinal permeability was observed after initial liver injury and inflammation [30] . Therefore,whether the change in gut permeability is a causal factor or a consequence of NAFLD still needs further investigation. Additionally,gut barrier dysfunction contributes to the development of NAFLD. The leaky tight junctions allow microbial components,such as LPS,to cross the intestinal wall and enter to the portal vein. LPS binds to TLR-4 and consequently induces hepatic inflammation,cell death,and fibrosis [32] . In NAFLD biopsies,higher LPS hepatocyte localization was detected compared with normal livers,consistent with increased expression of TLR4 in NAFLD patients.Invivoexperiments showed that inhibition of TLR reduces lobular inflammation and poorly absorbable antibiotics inhibit LPS-TLR4 signaling,thereby attenuating the liver fibrosis development in NASH. A higher number of TLR4 + macrophages and TLR4 + platelets observed in liver biopsies may act as possible mediators in LPS-induced liver damage [33] .Another mediator is the NOD-like receptor protein 3 (NLRP3) inflammasome,which mediates the generation of active caspase-1 by pro-caspase-1. Caspase-1 facilitates the production of inflammatory cytokines,such as IL-1βand IL-18,thereby mediating pyroptosis,inflammation,and fibrosis. Gut-derived LPS provides the first signal of triggering NLRP3 inflammasome formation while specific factors such as mitochondrial reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress act as the second signal [34] . Gaul et al. [35] reported extracellular NLRP3 inflammasome particles released from primary human hepatocytes can be internalized by hepatic stellate cells. NLRP3 blockade reduces hepatic expression of pro-IL-1βand hepatic infiltration with macrophages and neutrophils. Furthermore,NLRP3 inhibition attenuates liver fibrosis with significantly reduced pro-fibrotic markers.

Short-chain fatty acids

Breakdown and absorption of nutrients are mainly carried out in the small intestine,where there are various enzymes including amylase,lipase,and protease. In normal conditions,most carbohydrates are converted into sugar molecules allowing their absorption by the intestine. However complex carbohydrates,such as dietary fiber and resistant starch will escape digestion in the small intestine and reach the colon due to the lack of the corresponding enzymes. The colon is colonized with the largest gut bacterial species and these abundant microbes convert the nondigestive carbohydrates into various short-chain fatty acids (SCFAs): butyrate,acetate,and propionate. Lachnospiraceae and Ruminococcaceae are the major common bacterial families that produce SCFAs [36] . Most SCFAs produced by gut microbiota not only provide energy sources for enterocytes and colonocytes but also act as signaling molecules by activation of G protein-coupled receptors (GPR41 and GPR43) expressed at the surface of the enteroendocrine L -cells. GRP43 knockout mice display impaired SCFA-induced GLP-1 and PYY secretion [37] . Moreover,butyrate plays a role in microbial environment regulation by communicating with colonocytes. The epithelium hypoxic environment is an essential requirement for anaerobic bacteria. Butyrate controls the amount of oxygen by activating the nuclear receptor proliferatoractivated receptor gamma (PPAR-γ) in colonic cells,thereby influencing anaerobic conditions. PPAR-γactivates mitochondrialβoxidation,an oxygen consumption process,leading to the limited diffusion of oxygen from the colonocytes to the gut lumen. In turn,epithelial hypoxia allows anaerobiosis in the luminal part,resulting in a dominance of obligate anaerobes,which release SCFAs [38] . Importantly,butyrate and,to a lesser extent,propionate inhibit histone deacetylases (HDACs) and function as potent anti-inflammatory agents. SCFAs can regulate cytokine expression in T cells and regulatory T cells (Tregs) differentiation via HDAC inhibition [ 36,39 ]. Several studies point out the fact that SCFAs are associated with the pathogenesis of NAFLD. SCFAs regulate lipid metabolism via a cascade event in which the downregulation of PPARγactivates UCP2-AMPK-ACC pathway. The activation of AMP-activated protein kinase (AMPK) promotes energy expenditure by shifting metabolism in liver tissue from lipogenesis to fatty-acid oxidation.Invivoexperiment showed that SCFA supplementation increases hepatic lipid oxidation capacity and therefore reduces hepatic triglycerides [40] . Another mechanism involves the activation of GPR43 which influences the energy balance by inhibiting fat deposition and increasing fat consumption. SCFAs reduction under GF conditions or treated with antibiotics abolished differences in the body weight and insulin resistance regardless of GPR43 being knocked out [41] . Additionally,SCFAs are involved in NAFLD progression via inflammatory signals. SCFAs stimulate GPR43 which attenuates inflammation in colitis,arthritis,and asthma [42] . SCFAs supplementation greatly alleviated macrophage aggregations and proinflammatory responses (TNF-α,JNK p46,and NF-κB p65) in mice with methionine-choline deficient (MCD) dietinduced NASH [43] . However,the effect of SCFAs is quite complex regardless of many investigations focused on their roles in NAFLD pathogenesis. A higher concentration of SCFAs in the stool was observed in patients with NAFLD and/or NASH compared with healthy individuals,consistent with an increased abundance of SFCAs-producing bacterial groups [44] . In some studies,dietary soluble fiber indeed provides positive metabolic effects. However,incorporating soluble fiber inulin induces icteric HCC,which was microbiota-dependent [45] . Thus,using dietary supplementation of fiber as a treatment for NAFLD might need to be extremely careful and personalized,and further investigations are now needed to disentangle the association between SCFAs and NAFLD.

Bile acids

Bile acid (BA) synthesis begins in the liver via two different pathways. Complex biosynthesis involves a series of biotransformative reactions catalyzed by a group of enzymes. A study reported a reduction in expression levels of several of these enzymes,including CYP7A1,CYP7B1,and CYP27A1 in conventionally raised(CONV-R) mice compared with GF mice,indicating gut microbiota altered the expression profile of genes involved in bile acid synthesis [46] . Primary BAs synthesized by the liver are stored in the gallbladder and released into the duodenum after meals. Approximately 95% of bile acids excreted from the biliary system are reabsorbed during the digestive process,predominantly in the brush border membrane of the terminal ileum by the apical sodiumdependent bile acid transporter (ASBT,also known as IBAT),and then recirculated back into the liver via the portal vein. This cyclic process is known as enterohepatic circulation and takes place in humans about six times per day. In the gut,BAs are converted into secondary BAs by the gut microbiota via deconjugation,oxidation,and epimerization.Bacteroides,Clostridium,Lactobacillus,Bi-fidobacterium,andListeriaare identified as the main bacterial genera of the gut microbiota involved in BA deconjugation [47–50] .BAs are not only involved in dietary absorption and cholesterol homeostasis but also can activate multiple signaling pathways with systemic endocrine functions. As endogenous ligands for the nuclear receptor (farnesoid X receptor,FXR),BAs activate FXR signaling and influence hepatic lipid metabolism. The activation of FXR leads to a series of NAFLD-relevant changes such as reduced levels of hepatic and plasma triacylglycerols,reduced inflammation,and increased insulin sensitivity. Patients with NASH had significantly higher BA concentrations in liver tissue,serum,and urine than healthy individuals [51] . Alterations in BA synthesis from the classical to the alterative pathway were observed in both NASH patients and mouse models of NASH,and the shift of the BA efflux pathway from the bile to systemic blood may be involved in liver fibrosis by activating hepatic stellate cells (HSCs) [ 52,53 ]. These findings indicate that changes in BA composition and efflux pathway are linked to NAFLD progression. As BA and gut microbiota can cross-talk and influence each other,and changes in intestinal flora modify BA metabolism,it is hypothesized that gut flora influences lipid and glucose metabolism through communication with BA. Notably,treatment with obeticholic acid,a more potent ligand to FXR than chenodeoxycholic acid,results in an improvement in the key features of NASH [54] . Thus,regulating BAs by targeting gut microbiota may evolve as a promising avenue for the treatment of NAFLD,but much more research is needed.

Choline

Diet lacking choline is widely used to establish mouse models of NASH and a reduced level of choline in diet leads to increased liver fat with changes in the composition of gut bacteria. Choline is an important vitamin-like nutrient found in food.About 70% choline in the human body is derived from diet and the remainder is obtained fromdenovoproduction in the body [55] .Choline can be used to make phospholipids or involved in methylation as a major source for methyl groups. Choline is stored in the liver,where it is mainly used in endogenous formation of phosphatidylcholine. The lack of phosphatidylcholine results in insufficient very low-density lipoprotein (VLDL) as the packaging and export of triglycerides in VLDL is hindered [56] . In mouse models,deletion of genes involved in endogenous choline biosynthesis (Pemt) or methylation-dependent biosynthesis as a donor of methyl groups leads to NAFLD [ 57,58 ]. This observation was translated to humans,where NAFLD patients have a higher rate of loss of function polymorphism inPemt[59] . Several mechanisms are proposed to explain the relationship between choline and NAFLD.One mechanism involves mitochondrial dysfunction,a central role in NAFLD development. As an important component of mitochondrial membrane,low choline affects fatty acid beta-oxidation with decreased ATP production by mitochondria in mice [ 60,61 ]. Another possible mechanism by which choline affects NAFLD progression is by cellular stress. Choline deprivation leads to robust phosphorylation of eIF2,a stress signal associated with hepatic steatosis [62] . In the gut,choline is metabolized by commensal bacteria into methylamines including trimethylamine (TMA),and catalyzed by flavin-monooxygenase-3 (FMO3) in the liver. TMA can be further converted into trimethylamine N-oxide (TMAO),an early biomarker of metabolic syndrome. Only a small fraction of the microorganisms including the genusClostridiumXIVaand a specificEubacteriumcan utilize choline to produce TMA,and the modification in the intestinal bacteria strongly influences the levels of TMA [ 55,63 ]. Circulating level of TMAO positively correlates with the presence and severity of NAFLD [64] . As demonstrated in mice,TMAO induces lipogenesis and aggravates liver steatosis and injury via the suppression of FXR signaling [65] .

Ethanol

Microbial fermentation in the gut can convert dietary sugars into endogenous ethanol which then enters the circulation. In patients with NAFLD,there is a higher ethanol level in portal vein in comparison with individuals without steatosis,and the differences are related to the increased ethanol production by gut bacteria [66] .Klebsiellapneumoniaewas reported to be associated with endogenous ethanol production and mice fed with high ethanolproducing strains ofK.pneumoniaefrom a patient with NASH displayed immune responses related to hepatic steatosis. Furthermore,transferring strains into GF mice induced liver injury,indicating endogenous ethanol aggravates NASH pathogenesis [67] .Ethanol is involved in lipid metabolism and inflammation in the liver. Ethanol is catalyzed to highly toxic acetaldehyde by microsomal ethanol oxidase (EO) in the liver [68] . Acetaldehyde triggers alterations in mitochondrial structure and impairs mitochondrial functions,including the production of ROS. ROS mediates oxidative stress,causing DNA damage,lipid peroxidation,and neoantigens generation. Acetaldehyde and ROS can activate HSCs and enhancethe production of pro-fibrogenic mediators from immune cells,further promoting liver fibrogenesis [69] . Furthermore,acetaldehyde can be converted to acetate,which is then metabolized to acetyl-CoA,thereby contributing to fatty acid synthesis. However,acetate produced in the liver quickly enters the bloodstream,therefore its effect on fatty acid synthesis needs further validation.

Microbiota-based therapy for NAFLD

As a number of studies have stressed the role of gut microbiota in NAFLD,targeting gut microbiota for NAFLD diagnosis and treatment has gained growing interest. During the past few years,the progress of metagenomics and metabolomics has contributed enormously to the research as a much greater diversity of microbial communities has been identified. A study including both a discovery and a validation cohort reported a strong association between 3-(4-hydroxyphenyl) lactate with hepatic fibrosis after adjustment for age,sex,obesity,and Hispanic ethnicity [70] . Similarly,Hoyles et al. [71] demonstrated that plasma phenylacetate (PAA) was significantly correlated with steatosis,consistent with the increased microbial capacity for the metabolism of PAA. These results indicate that microbial metabolites might serve as better non-invasive diagnostic markers than conventional diagnostics. Moreover,the abundance of specific species of fecal gut microbiota by metagenomic sequencing is able to identify the severity of NAFLD. The high diagnostic accuracy of hepatic fibrosis suggests that gut microbiota holds promise as a future diagnostic marker for liverrelated complications [72] . Although several studies have set the stage to explore the potential role of microbiota as a diagnostic tool ( Table 1 ) [73–83],some limitations cannot be ignored including limited cohort size and the uncertain causality between microbiome and severity of NAFLD,and further investigations are needed to corroborate the clinical utility.

Table 1Clinical trials targeting dysbiosis in NAFLD.

Fecal microbiota transplantation (FMT)

A huge number of studies have explored the strategies to manipulate gut microbiota,thereby can be used to treat NAFLD.One solution to alter the composition of gut microbiota is FMT,which involves the transfer of fecal microbiota communities from a healthy donor to restore the balance of intestinal microbiota in the recipient. Xue et al. [73] showed that NAFLD patients had decreased fat accumulation with an improved abundance of gut microbiota after the FMT. Another study in cirrhotic patients revealed that oral capsular FMT decreases the incidence of infection and subsequent episodes of hepatic encephalopathy [84] . Furthermore,the intestinal barrier displayed a better immunoinflammatory state with reduced duodenal IL-6 expression,which was likely associated with the changed microbial composition after FMT. To date,limited FMT clinical trials have been performed in individuals with NAFLD. Although there are no significant improvements in hepatic steatosis following FMT,reduced toxic plasma metabolites derived from gut microbiota were detected,in line with decreased liver necro-inflammation in liver biopsy [85] . Notably,the safety of FMT remains an important concern as a therapeutic strategy. FMT allows the transmission of drug-resistant bacteremia among individuals,which likely causes severe infectious complications [86] . Gut barrier dysfunction,a vital factor in liver disease progression allows easier bacterial translocation in NAFLD patients,thus increasing the risk of infection. Therefore,improved donor screening is needed to prevent the transmission of microorganisms that induce infectious diseases.

Probiotics, prebiotics, and synbiotics

Probiotics comprise live microorganisms to reverse dysbiosis,and it is considered a potential therapeutic strategy for NAFLD. In fatty liver mice models,compound probiotics could reduce hepatic fat accumulation and liver damage by modulating gut microbiota.The reduced serum inflammatory factors,such as TNF-α,IL-1β,and IL-18 showed the benefits of compound probiotics in the progression of NAFLD [87] . Moreover,probiotics can also influence gut permeability and mitochondrial function to prevent diet-induced NAFLD in mouse models [88] . Several clinical trials also validated the protective effect of probiotics on liver function. In a doubleblinded clinical trial,72 patients with NAFLD were enrolled and treated with probiotic yogurt containingLactobacillusacidophilusLa5andBifidobacteriumlactisBb12. After 8 weeks,the authors observed significantly reduced serum alanine aminotransferase (ALT)and AST in patients treated with probiotic yogurt compared with the placebo group [75] . Similarly,supplementation of probiotics containing six different microorganisms ofLactobacillusandBifidobacteriumstrains improved the function of the intestinal gut barrier,with an increased expression of CD8 + T lymphocytes and ZO-1,compared with the placebo group [76] . In addition to probiotics,prebiotics can also be used to modify the gut microbiota.Prebiotics refer to specific carbohydrate polymers that are poorly digested or absorbed. It can be fermented by bacteria and favor the production of SFCAs from bacteria,therefore helping to maintain gut homeostasis [89] . In animal models,it was revealed that the use of Fructo-oligosaccharides (FOS) alleviated hepatic steatosis in the regulation of fatty acid oxidation and cholesterol synthesis [90] . Additionally,several studies applied mixed probiotics and prebiotics,called synbiotics in NAFLD treatment. Malaguarnera et al. [81] utilized the synbiotics containingBifidobacteriumlongumand FOS plus lifestyle as an intervention for 24 weeks. The treated group showed improvement in serum AST levels,as well as reduced NASH activity. The positive effects are likely attributed to the reduced exposure to microbial metabolites that induce the activation of hepatic macrophages.

Conclusions and perspectives

Given the limited effective strategies for NAFLD treatment,numerous studies focusing on gut microbiota have provided novel approaches as well as challenges in NAFLD invention. However,some discrepancies among gut microbial signatures were observed across studies. One important factor is the type of method used in microbiota sequencing. 16S rRNA sequencing has been widely applied to identify NAFLD-related gut microbiota. Nevertheless,only a single region can be sequenced by 16S rRNA sequencing,which greatly limits its accuracy [91] . Therefore,it is imperative to apply more granular metagenomics sequencing to collect extended information that allows us to predict the function of unknown species.In addition,a number of experiments were carried out in mouse models,which develop limited human-specific histological alterations of NAFLD. Besides,mice and humans have substantially different dominant microbiota since some genera found in humans cannot colonize mice [92] . The difference in digestive tract architectures between rodents and humans also affects gut microbiota composition.

Since the composition of gut microbiota varies greatly among individuals,precision medicine is needed based on different microbiome signatures. Of note,probiotic supplementation under disruptive antibiotic conditions results in adverse effects with significantly delayed gut mucosal reconstitution and prolonged dysbiosis [93] . Given the effects of the probiotic,future studies are necessary to elucidate the molecular mechanisms of these gut microbiome-based medicine in both animal and human studies,and the methods to modulate gut microbiota for treatment must be carefully determined based on enough information about a patient’s microbiome.

Taken together,tremendous studies in gut microbiota have paved a path toward a deeper understanding of the pathogenesis and treatment of NAFLD. Despite the current progress in understanding the relationship between intestinal bacteria and humans,it is imperative to investigate the interactions between other microbes,such as viruses,fungi,archaea,and host in the progression of NAFLD.

Acknowledgments

We thank https://www.biorender.com/ for the help of figure.

CRediT authorship contribution statement

Meng-YuanWu:Writing – original draft.Jian-GaoFan:Conceptualization,Funding acquisition,Supervision,Writing – review& editing.

Funding

This study was supported by a grant from the National Natural Science Foundation of China ( 82170593 ).

Ethical approval

Not needed.

Competing interest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.