Non-alcoholic fatty liver disease (NAFLD) is one of the most frequent causes of chronic liver disease in the Western world, probably due to the growing prevalence of obesity, metabolic diseases, and exposure to some environmental agents. In certain patients, simple hepatic steatosis can progress to non-alcoholic steatohepatitis (NASH), which can sometimes lead to liver cirrhosis and its complications including hepatocellular carcinoma. Understanding the mechanisms that cause the progression of NAFLD to NASH is crucial to be able to control the advancement of the disease. The main hypothesis considers that it is due to multiple factors that act together on genetically predisposed subjects to suffer from NAFLD including insulin resistance, nutritional factors, gut microbiota, and genetic and epigenetic factors. In this article, we will discuss the epidemiology of NAFLD, and we overview several topics that influence the development of the disease from simple steatosis to liver cirrhosis and its possible complications. NAFLD has become, in recent years, one of the most common liver diseases in the world. It is based on different liver disorders characterized by accumulation of fat (hepatic steatosis) in more than 5% of hepatocytes , primariThe term NAFLD has undergone an evolution throughout history, as knowledge about the disease and diagnostic methods have advanced . RecentlNAFLD can have different forms of presenting itself, from simple accumulation of fat that is a metabolic disorder that does not present symptoms ) to a symptomatic non-alcoholic steatohepatitis (NASH). NAFLD is asymptomatic in most cases and is associated with obesity and characteristics of metabolic syndrome (MS), as mentioned before ,10. NASHThe prevalence of NAFLD evolves in parallel with obesity and varies among countries and ethnicities. Globally, it has been estimated at around 25% in the general population , increasThe number of patients in the general population with NAFLD who develop NASH is unknown, but it is greater than 10% of the overall NAFLD population . PrimaryComplex interactions among environmental factors, metabolism and demography, genetic variants, and gut microbiota are involved in the pathogenesis of NAFLD . CurrentCurrently, different studies have shown a strong association between NAFLD and each of the risk factors associated with MS ,21,22, eObesity is considered the main risk factor for NAFLD, since body mass index (BMI) and waist circumference correlate positively with both the presence of NAFLD and disease progression . InteresNevertheless, a proportion of patients with NAFLD and normal body mass index (BMI) represent the already known lean or non-obese NAFLD. This condition lacks the complications associated with obesity, so it is expected to be less severe than in obese NAFLD , but datLipotoxicity and glucotoxicity play a crucial role in the development of simple steatosis in the liver and its progression to NASH. In this regard, a high fat and carbohydrate diet, which obese patients are more prone to follow, favors fat deposition in the liver by different mechanisms including mitochondrial defects and endoplasmic reticulum and oxidative stress, as mentioned ,33. ThenMS has multiple definitions, but some of its agreed manifestations are increased waist circumference, hyperglycemia, dyslipidemia, and systemic hypertension . MS inciIn MS patients, the capacity of insulin to inhibit glucose production is reduced, resulting in mild hyperglycemia, which in turn stimulates insulin secretion, causing a state of hyperinsulinemia. Insulin normally decreases the amount of VLDL, by suppressing its hepatic production, or by inhibiting lipolysis of adipose tissue . In patiT2DM has a strong relationship with the progression of NAFLD, in fact more than 50% of patients with T2DM have NAFLD ,44. DiabIR, on the other hand, is considered one of the critical cellular abnormality that causes the development of both NAFLD and T2DM, and has been recognized as an integral component of NAFLD pathogenesis , worseniIt has been increasingly recognized that inflammatory pathways are critically involved in IR development. However, is still unclear when the inflammatory processes begin ,54. MoreBacteroidetes and Firmicutes, while the predominant prokaryotic microorganisms are Euryarchaeota [The human gut microbiome is made up of 10–100 trillion microorganisms, mainly bacteria, with this number about ten times higher than the number of eukaryotic human cells , susceptrchaeota . The comRecent studies have shown that the gut microbiota affects hepatic carbohydrate and lipid metabolisms and also influences the balance between pro-inflammatory and anti-inflammatory effectors in the liver, which directly affects NAFLD and its progression to NASH. Several experiments based on manipulation of the microbiome provide the most important evidence on the role of the gut microbiome in obesity and NAFLD in mice and humans. In an interesting study, germ-free mice fed with a high-fat diet exhibited lower levels of liver lipids compared to conventionally housed mice . In anotProteobacteria [Bacteroides [Gammaproteobacteria (phylum Proteobacteria) and Prevotella (Bacteroides) compared with the microbiota of obese children without NAFLD [Proteobacteria and decrease in Firmicutes, suggesting that the gut microbiome may not be stable during disease progression [NAFLD and NASH in humans tend to coincide with the existence of obesity and poor dietary habits, which makes it difficult to differentiate the effects of diet from those produced by the altered microbiome, and the metabolic changes that accompany both in liver disease. The abundance of some bacterial species in humans such as bacteria , or Bactteroides  were assut NAFLD . It is ngression . Short-tgression . Similargression .Taken together, these studies show that there may be correlations between intestinal bacterial composition and NAFLD or NASH. However, these observations are limited by the lack of reproducibility of the studies and the absence of a mechanism to explain their effects on NAFLD and NASH. Furthermore, in most studies, the microbiome is sampled from stool samples, being the bacterial composition different from the communities present in the most proximal areas of the intestine .loci associated with the progression of the disease.The interaction between the genetic status and the environmental factors may explain part of the inter-individual variability observed in the manifestation of the phenotype and severity of NAFLD. Unbiased genetic and epidemiological studies show strong evidence for the heritability of characteristic traits of NAFLD and have identified gene Several clinical studies using family members demonstrate that first-degree relatives are at higher risk for NAFLD, suggesting a genetic predisposition to the disease ,70. PareEpidemiological studies describe inter-individual differences in the NAFLD prevalence depending on the ethnicity. Hispanic Americans have the highest prevalence of NAFLD, followed by Americans of European descent, and African Americans having the lowest prevalence ,77,78. IThe first genome-wide association study (GWAS) performed in NAFLD showed that the contribution of the ancestry to the observed differences in the accumulation of hepatic fat content and susceptibility to NAFLD can be partly explained by the genetic status of the individuals . Since tDifferent variants of genes implicated in the cellular metabolism of lipids in the liver define the genetic risk factors for NAFLD. As such, the most relevant loci affecting NAFLD are PNPLA3, TM6SF2, GCKR, MBOAT7, and HSD17B13 .This gene encodes an enzyme that is expressed mainly in adipose tissue, retina, and the liver ,88. WithPNPLA3 has been widely studied in NAFLD since the missense variant rs738409 C>G encoding for the I148M allele of PNPLA3 was first discovered in 2008 by GWAS and reported to explain most of the genetic contribution to the hepatic triglyceride accumulation and tendency to NAFLD in patients of different ethnicity . The PNPknockout (KO) mice revealed that lack of PNPLA3 protein did not affect the accumulation of fat in the liver, IR, or levels of liver enzymes [knock in and overexpression of the variant PNPLA3 I148M in the liver on high-fat diet reproduced the NAFLD phenotype observed in humans [Functional in vitro studies to characterize the biological relevance of the PNPLA3 I148M variant in NAFLD showed that PNPLA3 I148M is a gene variation that causes loss of function ,138. How enzymes . Interesn humans . Addition humans . For thin humans . From a n humans  in mice,n humans .TM6F2 is a protein that is mainly expressed in the liver and small intestine where it regulates the intracellular trafficking and secretion of VLDL and cholesterol ,95. The The relevance of this locus in NAFLD was stablished in 2014 by an exome-wide association study that identified the missense variant rs58542926 C>T that encodes for the mutant TM6FS2 E167K . In thisThis gene is expressed in the liver of vertebrates and encodes for a protein that acts as an allosteric inhibitor of glucokinase (GCK), an enzyme responsible for blood glucose homeostasis. GCK is activated by increased levels of glucose in the portal vein and catalyzes the beginning of the glycolytic pathway by phosphorylating the glucose that enters in the cell . GCK hasThe loss-of-function variant rs1260326 C>T SNP, which encodes for the P446L protein of GCKR, is related to steatosis in the liver and risk of NAFLD even in obese children and adolescents  and NASHMechanistical studies using recombinant human GCKR wild type and P446L proteins showed that physiological levels of F6P failed to activate P446L GCKR, resulting in increased activity of GCK . The conMBOAT7 gene expression yields the enzyme lysophosphatidylinositol (LPI) acyltransferase, an endomembrane protein involved in the metabolism of lipids in the liver  that catA GWAS first identified the MBOAT7 variant rs641738 C>T as a risk locus in the pathogenesis of cirrhosis driven by alcohol . Later sThis gene encodes for retinol dehydrogenase, which is involved in steroid hormone signaling as well as bile acids and lipid metabolism . HSD17B1Recently, it has been described that several polymorphisms including rs72613567, rs143404524, and rs62305723 have a protective role in liver injury as they have been associated with reduced serum hepatic ALT and AST levels, diminished risk for liver damage, progression toward HCC and liver-related mortality in alcoholic and nonalcoholic liver disease ,114,115.Although the causal role of certain loci and NAFLD pathogenesis is undeniable, it is becoming clear that the rising prevalence of NAFLD cannot be explained exclusively by the contribution of environmental and genetic factors. Epigenetics, which constitutes the reversible and heritable change in gene expression without modification of the underlying nucleotide sequence, serves as a mechanistic bridge in this phenomenon . As a maWe can find examples of epigenetics on the basis of NAFLD as early as in embryo development. Maternal obesity, diabetes, or Western diet consumption  lead to The relationship between DNA methylation and other metabolic diseases has been extensively studied . The amoAlthough the effect seen in NAFLD is hypomethylation, several interesting examples of hypermethylated genes with reduced expression can be found. Insulin-like growth factor-binding protein (IGFBP)-2 is often repressed in patients with NAFLD and NASH via methylation . InteresAnother prominent example of a hypermethylated gene in NAFLD is Peroxisome Proliferator-Activated receptor gamma coactivator (PGC)-1α, a master regulator of various aspects of energy metabolism, especially fatty acid oxidation and mitochondrial biogenesis, which are features involved in the pathogenesis of fatty liver. Patients of NAFLD have decreased PGC1α expression due to promoter methylation, which correlates with mitochondrial defects and IR . To be nUnlike DNA methylation, histone modifications are less understood in the context of fatty liver disease . NeverthSirtuin 1 (SIRT1), on the other hand, is a histone deacetylase that has been traditionally related to hepatic metabolic regulation . As mattmiRNAs are known to regulate multiple biological pathways involved in the pathogenesis of NAFLD such as lipid uptake, de novo lipogenesis, lipid oxidation, and hepatic lipid export, apoptosis, cell proliferation, or fibrosis . In factmiR-33a and miR-33b, for example, negatively regulate the levels of ATP-binding cassette transporter 1 (ABCA1), which controls high-density lipoprotein biogenesis, thereby promoting high levels of circulating VLDL and triglycerides . In the NAFLD and aging are known to be strongly correlated, with increasing age being one of the strongest epidemiological factors for NAFLD, NASH, and fibrosis ,199,200.Unlike what happens with age, the influence of sex on the prevalence and course of NAFLD is not so clear. Available studies are divided between both sex, some have shown a female preponderance, while others have shown a male preponderance in the prevalence of NAFLD . In an iIn recent years, NAFLD has become a biological marker of social affluence and sedentary lifestyle ,24. As wNumerous studies have suggested that dietary composition may predispose people to NAFLD. In developed societies, human nutrition has undergone drastic changes in recent decades that, combined with a decrease in the population’s physical activity, has triggered a marked imbalance between energy consumption and energy expenditure . The risLeslie T. et al. reported in 2014  that NAFOne of the underlying mechanisms that could lead to the onset NAFLD from this particular scenario might be epigenetics, as mentioned before; the mechanism that could also be responsible for the IR that this population suffer ,217. IntSeveral recent studies have reported the relationship between smoking and the incidence of an accelerated disease progression and advanced fibrosis in NAFLD ,228,229.Zein et al.  demonstrAccording to some epidemiological studies, exposure to environmental particles is positively associated with increased morbidity and daily mortality caused by diseases closely related to life habits including ischemic heart disease  or T2DM 2.5 can affect NAFLD through different ways [2.5) may be an important risk factor for NAFLD progression. PM2.5 may also prompt the expression of proinflammatory factors in adipocytes [Long-term exposure to PMent ways . In 2007ent ways  publisheent ways  demonstripocytes ,243,244,ipocytes  and causipocytes .2.5 for more than 30 min showed an abnormal liver insulin signal, with an altered expression of endothelial NOS and protein kinase C. More recently, Yin et al. [2.5 exposure could inhibit the expression of PPARα and PPARγ, inducing hepatic steatosis, inflammation, and IR. According to numerous studies, oxidative stress is the main cause of damage of PM2.5 in cells, either through direct effects on structure and function of biological macromolecules like DNA, lipids, and proteins, or indirectly through the activation of intracellular oxidant pathways [In 2009, Sun et al.  found thn et al.  demonstrpathways ,250,251.Despite the results on the inflammatory and oxidative properties of air pollutants, the lack of data supports the need of more studies on the effects of environmental factors, notably air pollution, on NAFLD.NAFLD is a growing health problem worldwide. Recently, an international expert group has agreed to change the name of NAFLD to metabolic (dysfunction) associated fatty liver disease (MAFLD) ,253. Ove


Non-alcoholic fatty liver disease (NAFLD) is a fast-spreading epidemic across the globe and has serious implications far beyond that of a “benign” liver condition. It is usually an outcome of ectopic fat storage due to chronic positive energy balance leading to obesity and is associated with multiple health problems. While association with cardiovascular disease and hepatocellular cancer is well recognized, it is becoming clear the NAFLD carries with it an increased risk of cancers of extrahepatic tissues. Studies have reported a higher risk for cancers of the colon, breast, prostate, lung, and pancreas. Fatty liver is associated with increased mortality; there is an urgent need to understand that fatty liver is not always benign, and not always associated with obesity. It is, however, a reversible condition and early recognition and intervention can alter its natural history and associated complications. Non-alcoholic fatty liver disease (NAFLD) is one of the commonest indicators of ill-health in the modern era of obesogenic lifestyle, next to obesity itself ,2. It afThere are multiple causes for fatty liver, but the term NAFLD is conventionally restricted to that related to nutritional causes, obesity, and metabolic syndrome. Causes of fatty liver can be genetic  , drugs , surgeryEpidemiologyThe prevalence of NAFLD is disturbingly high across the world, varying between 25% and 35% globally. The highest prevalence is reported from South America (31%) and Middle East (32%), followed by Asia (27%) and the USA (24%), while the prevalence is lowest in Africa 14%) [% [14]. TRecent figures suggest that more than 50% of the Omani population is overweight, and 30% have a body mass index (BMI) of more than 30% . NeighboSurprisingly, in a study of HCC diagnosed from Oman between 2008 and 2015, of 284 patients, only two of 227 patients with cirrhosis (0.9%) were reported to have a cryptogenic etiology  , and NAFLD is a complex, “chicken and egg” situation. While the events that spark the inflammation that converts steatosis to steatohepatitis have been comprehensively reviewed , what caInsulin is an anabolic, fat-storage hormone, and hyperinsulinemia results in varying degrees of obesity depending on the fat storage capacity of the individual . The laIt is clear now that hyperinsulinemia is one of the earliest manifestations of modern-day chronic calorie excess/adiposity and appears much before the onset of insulin resistance and T2DM . The modThe hyperinsulinemic syndrome  has otheNAFLD and extrahepatic cancers (EHC)It was conventionally thought that the earliest manifestation of NAFLD, the fatty liver (NAFL), is a benign condition and becomes a medical problem only when it progresses to NASH, and later to fibrosis, cirrhosis with decompensation, and/or HCC. However, recent evidence suggests that the finding of hepatic steatosis is not benign and has significant health consequences . NAFLD iIn a prospective study from Rochester County, Minnesota, Allen et al.  followedIn a long-term population-based cohort study from Sweden of 8,892 patients of biopsy-proven NAFLD , EHC wasA meta-analysis by Mantovani et al.  of 10 coAnother meta-analysis of 26 studies  has conf A decade-long study from China  also shoWhat is interesting is that obesity is not an essential requirement for EHCs associated with NAFLD. Obesity-related cancers (ORCs) are a recognized entity , and theIn Allen’s study , NAFLD wAssociation is, of course, not causation and the larger question is, does fatty liver directly cause cancer? Causality is difficult to establish, and there could be another proximate cause for both fatty liver and cancer. Reversal of NAFLD leads to a drop in the incidence of cancers, the most effective way being bariatric surgery . It is kMechanisms of carcinogenesisThe molecular links between obesity and cancer have been speculated on ; recent The liver itself secretes a vast repertoire of hepatocytes ,70, someThe mechanism(s) is much more than of academic interest as blocking the proliferative/mutagenic signals should  uncouple NAFLD from its remote effects such as EHCs. This should be an area of research priority as it is unlikely that efforts to curb the incidence of NAFLD will bear fruit in near future. ImplicationsEffect on Natural HistoryIt is an intriguing question whether the presence of NAFLD alters the natural history of cancer. Some studies suggest that fatty liver increases the risk of liver metastasis (in lung cancer) , but othEffect on Drug ResponseAnother evolving issue is the altered dynamics of immune checkpoint inhibitors (ICIs), in patients with NAFLD. ICIs themselves have been reported to cause NAFLD  and alsoHigh-Risk WarningAt the general practitioner level, there should be increasing awareness that hepatic steatosis is not a benign condition; it should not be dismissed as an incidental finding in the ultrasound scan of the abdomen, as is done for renal or liver cysts. Firstly, about 20% of NAFL progress to NASH, and 40% of the latter go on to develop fibrosis; the risk of progression to NASH and fibrosis can be rapid, with NAFL progressing to fibrosis in an average of 14 years while NASH can in half that duration , as doctRecommendations for Screening?It has been suggested that patients with NAFLD should be candidates for targeted screening for EHCs . Three oManagementReversal by Weight LossReversal of NAFLD can either be by weight loss or by specifically targeting NAFLD. Weight loss through lifestyle modification (diet and exercise) is the most cost-effective intervention to reverse NAFLD ,89 but dTargeted ReversalSpecific treatment of NAFLD is in its nascent stages . It is cAn epidemiological study from China  gives inSeveral agents that directly reverse NAFLD such as controlled-release mitochondrial protonophore (CRMP) are being studied . Two ratFinally, research is essential to discover the mechanisms linking NAFLD to EHCs so as to enable intervention. It is unlikely that we will achieve any form of control over the burgeoning obesity and NAFLD rates in near future . The preChronic positive energy balance overwhelms safe fat storage depots in subcutaneous WAT, leading to ectopic fat storage in unsafe sites such as VAT and liver. This causes insulin resistance, metabolic syndromes, and, as is increasingly clear, EHC. Clinicians and their patients should be made aware that fatty liver is just the tip of an iceberg; NAFLD is an indicator of an underlying plethora of metabolic disturbances. Aggressive measures must be instituted early to reverse it. Obesity and NAFLD will continue to rise and will overwhelm the health care system in a not-too-distant future. Current advice on the control of obesity such as lifestyle changes is a clear failure. It would be prudent to investigate the causative link(s) between NAFLD and EHCs with the ultimate aim of discovering methods to uncouple them.


Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Its worldwide prevalence is rapidly increasing and is currently estimated at 24%. NAFLD is highly associated with many features of the metabolic syndrome, including obesity, insulin resistance, hyperlipidaemia, and hypertension. The pathogenesis of NAFLD is complex and not fully understood, but there is increasing evidence that the gut microbiota is strongly implicated in the development of NAFLD. In this review, we discuss the major factors that induce dysbiosis of the gut microbiota and disrupt intestinal permeability, as well as possible mechanisms leading to the development of NAFLD. We also discuss the most consistent NAFLD-associated gut microbiota signatures and immunological mechanisms involved in maintaining the gut barrier and liver tolerance to gut-derived factors. Gut-derived factors, including microbial, dietary, and host-derived factors involved in NAFLD pathogenesis, are discussed in detail. Finally, we review currently available diagnostic and prognostic methods, summarise latest knowledge on promising microbiota-based biomarkers, and discuss therapeutic strategies to manipulate the microbiota, including faecal microbiota transplantation, probiotics and prebiotics, deletions of individual strains with bacteriophages, and blocking the production of harmful metabolites. Non-alcoholic fatty liver disease (NAFLD) is characterised by an excessive intrahepatic fat accumulation, i.e., steatosis, without significant alcohol consumption. Liver steatosis is defined as fat accumulation, in >5% of hepatocytes . NAFLD mThe first stage of alcoholic liver disease (ALD) is also characterised by hepatic steatosis. However, unlike NAFLD, the primary trigger of ALD, i.e., excessive alcohol consumption, is known and the disease is preventable. Ethanol probably does not play a prominent role in NAFLD pathogenesis but is discussed as one of the possible contributing factors. A detailed discussion of the role of the gut microbiota in ALD pathogenesis is beyond the scope of this review and has been discussed elsewhere .NAFLD is closely associated with many features of metabolic syndrome, including obesity, insulin resistance, hyperlipidaemia, and hypertension ,4 and inNAFLD is the most common chronic liver condition in the USA and Europe. Its global prevalence is rapidly increasing and is currently estimated at 24%. The highest rates are reported from the Middle East (32%) and South America (31%) and the lowest from Africa (14%) . The estNAFLD pathogenesis is complex and not fully understood. The current understanding is that NAFLD is caused by a complex interplay of environmental factors mostly dietary, gut microbiota disturbances, and host factors.This review discusses the involvement of the gut microbiota in the pathogenesis of NAFLD, focusing on factors that modulate microbiota composition and intestinal permeability. In addition, NAFLD-associated microbiota signatures, immunological mechanisms behind liver tolerance to gut-derived antigens, and the gut–liver axis will be explained. Finally, we review advances in microbiota-based biomarkers and therapeutic interventions.Enterobacteriaceae family, a subgroup of Proteobacteria phylum, is frequently observed in many immune-mediated and metabolic diseases including NAFLD [Liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), cirrhosis, and hepatocellular carcinoma  are assong NAFLD ,12. The ng NAFLD .Diverse gut microbiota of each individual may endow the host with unique metabolic apparatus and the ability to adapt to changing environment and substrate availability. With decreasing microbial diversity during urbanisation/industrialisation this adaptability was partially lost as human gut microbiota gained new abilities aimed at sugar and xenobiotics processing ,15.Dysbiosis might be caused by host-derived factors such as genetic background, health status , and lifestyle habits or, even more importantly, by environmental factors such as diet , xenobiotics , or hygienic environment.Profound shifts in gut bacterial and fungal microbiota can be quickly achieved with shifts in macronutrients. These changes have significant physiological consequences, as diets rich in animal protein or simple sugars worsen the intestinal inflammation induced by dextran sulphate sodium. However, while the former increases proinflammatory tuning in gut monocytes the latter worsens the gut barrier function. In both cases, however, interactions between diet and microbiota are necessary for this deleterious effects as they fail to appear in germ-free condition or after transfer of the microbiota to naive mice ,17.The effect of food additives on gut microbiota has been long overlooked, but recently, several groups, including ours published data demonstrating that some human gut microbes are highly susceptible to food preservatives  and thatHost-derived factors modifying gut microbiota load and composition are bactericidal fluids produced by gastric glands and the liver, i.e., gastric acid and bile, and antimicrobial molecules, such as defensins, lysozymes, and antibacterial lectins (Reg3γ) produced by Paneth cells or SIgA produced by plasma cells .Dysbiotic microbiota can influence the host immune and metabolic systems and mucosal integrity via various mechanisms. Immune system-modulating mechanisms include the modulation of inflammasome signalling through microbial metabolites, the modulation of Toll-like receptor (TLR) and NOD-like receptor (NLR) signalling, the degradation of secretory IgA (SIgA), the shifting of the balance between regulatory and proinflammatory T cell subsets, direct mucolytic activity and others . MetabolWhether dysbiosis is a direct cause of NAFLD or merely reflects disease-associated changes in the host’s immune and metabolic systems remains unclear. However, there is accumulating evidence from both preclinical and clinical studies suggesting that gut microbiota dysbiosis plays a key role in the initiation of the disease and its maintenance.The gut microbiota alterations associated with NAFLD are dependent on the clinical stages of the disease . The mosEnterobacteriaceae (family), Escherichia, Bacteroides, Dorea, and Peptoniphilus (genus) and decreased Rikenellaceae, Ruminococcaceae (family), Faecalibacterium, Coprococcus, Anaerosporobacter, and Eubacterium (genera) , (genera) . The NAFirrhosis  as well irrhosis , T2DM [3irrhosis , or in iirrhosis  or IBS [irrhosis . F. prauoperties . Anotherfibrosis , is assofibrosis . Interestococcus , in the Candida overgrowth [Unlike patients with ALD who have fungal dysbiosis characterised by decreased diversity and ergrowth , patientStaphylococcus and Acinetobacter can be cultured from venous blood of cirrhosis patients [Evidence of viable liver or circulating microbiota in NAFLD patients is limited. Nevertheless, bacteria of genera patients . These cpatients .The gut–liver axis is a bidirectional communication through the biliary tract, portal vein, and systemic circulation. Liver-derived factors, such as bile acids, influence gut microbiota composition and function, and gut-derived products, either dietary or microbial, regulate bile acid synthesis and as well as glucose and lipid metabolism in the liver. The disruption of the gut–liver axis by, for example, environmental factors inducing gut dysbiosis and/or increased intestinal permeability leads to proinflammatory changes in the liver, and its failure to regulate gut microbiota results in further disease progression . A comprThe key function of the barrier is to protect tissues and organs from harmful luminal contents, such as parasites, microorganisms, MAMPs, microbial metabolites, dietary antigens, or toxins while preserving nutrient absorption. The intestinal barrier consists of several functional elements. The physical barrier consists of commensal bacteria, mucins secreted by goblet cells, and the intestinal epithelium sealed with tight junction proteins. The immunological barrier includes components of cellular and humoral immunity. Humoral factors, such as antimicrobial peptides and SIgA, control the load and composition of the microbiota in the lumen. The major antimicrobial peptides are defensins, cathelicidins, resistin-like molecules, and lectins produced by specialised epithelial cells and Paneth cells. SIgA is secreted by plasma cells of the lamina propria, activated by antigen-presenting cells, and transported into the intestinal lumen by epithelial cells after binding to the polymeric Ig receptor (pIgR) . SIgA biThere is no consensus as to which factors are major contributors to increased intestinal permeability, however, there is accumulating evidence that environmental factors, especially an unhealthy diet characterised by low fibre, high sugar and HFCS content, and some food additives play a significant role. For example, chronic fructose consumption is associated with tight junction disruption  and incrThe liver is evolutionarily programmed to tolerate low-level exposure to innocuous dietary and microbial antigens delivered via the portal vein. Liver tolerance is maintained by hepatic antigen-presenting cells (HAPCs), which include dendritic cells, liver sinusoidal endothelial cells (LSECs), Kupffer cells, and hepatic stellate cells ,57. AntiGut-derived factors involved in pathogenesis of NAFLD might originate in the diet, be a product of gut microbiota, or be host-derived. Dietary factors, such as ethanol, fructose, or choline, might act directly or after processing by microbiota. Microbiota-derived factors comprise microbial components, such as LPS, and products of microbiota metabolism. Host-derived factors are, for example, primary bile salts or mucin.The exposure of the liver to whole bacteria and/or their components is under normal/healthy conditions insignificant. However, if the gut barrier is disrupted due to the direct effects of dietary factors, such as ethanol or fructose, or indirectly due to gut dysbiosis, the liver is exposed to a significant microbial load. The increased exposure to microorganisms and their products, such as LPS, peptidoglycan, viral or bacterial DNA, or fungal beta-glucan, leads to the induction of proinflammatory changes. These components, collectively labelled microbe-associated molecular patterns (MAMPs), are then recognised by liver innate immune cells . The activation and long-term maintenance of inflammation leads to fibrosis, cirrhosis, or even HCC.The metabolites which are exclusively produced by gut microbiota were identified by comparing metabolomic profiles of germ-free or antibiotic-treated mice with conventional mice. The microbiota-derived metabolites implicated in NAFLD pathogenesis include the choline metabolite trimethylamine (TMA), the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), and SCFA ,68,69.Fructose is a monosaccharide that is naturally present in fruits and honey. However, fructose is also a major component of high-fructose corn syrup (HFCS), a ubiquitous sweetener made from corn starch, and sucrose, a glucose-fructose disaccharide. Human exposure to fructose-containing sugars was historically very low. Two hundred years ago, the yearly per capita sugar consumption in Europe and the United States was about 8 kg; by 1900, the consumption had increased to almost 40 kg, and the current consumption of fructose-containing sweeteners including HFCS is close to 70 kg per person per year [Abundant evidence both from preclinical and clinical studies suggests a major role for fructose in the pathogenesis of NAFLD and also NASH . FructosImportantly, fructose also has direct and harmful effects on the liver. The unique metabolism of fructose which is distinct from glucose metabolism leads to ATP depletion ,74,75, tFor humans and many animals, choline is an essential nutrient with many functions. It is required for the synthesis of phosphatidylcholine, which in turn is required for the synthesis of cellular membranes and VLDL, which are responsible for the transport of triglycerides out of the liver . CholineHowever, under certain conditions, choline may play a negative role in NAFLD pathogenesis because specific subsets of gut bacteria can convert it into trimethylamine (TMA) ,86 whichCholine conversion by microbiota seems to be involved in NAFLD pathogenesis by at least two mechanisms. Firstly, it decreases choline liver bioavailability, which leads to the inefficient export of VLDL particles out of the liver, lipid accumulation, and liver inflammation . SecondlSCFAs are defined as fatty acids with fewer than six carbons and the most abundant SCFAs are acetate, propionate, and butyrate. They are generated by gut microbiota mainly from dietary nondigestible starch and fibre in the colon and both composition of individual’s microbiota and source of nondigestible fibre determine which SCFAs are produced . They plOn the other hand, butyrate is a major source of energy for colonocytes, and the supplementation of tributyrin, a butyrate prodrug, as well as acetate and propionate, was shown to protect against diet-induced obesity, hepatic steatosis, and insulin resistance ,95.Published data on the immunomodulatory effects of SCFAs are mostly positive, i.e., favouring the induction of anti-inflammatory T cells. However, the evidence that SCFAs might, under certain conditions, induce proinflammatory T cells, such as Th1 and Th17 exists ,97. InteObviously, further research is needed to determine how significant the contribution of SCFA in NAFLD pathogenesis is and to discriminate whether the net effect is positive or negative. Additionally, it is important to discriminate between circulating and faecal SCFA levels as circulating SCFA are more directly linked to metabolic health .Klebsiella pneumoniae, are significant alcohol producers and that the NAFLD phenotype is transferable to experimental mice using FMT [Even healthy subjects with no alcohol consumption have low levels of blood ethanol , and gutsing FMT .NAFLD has been associated with increased luminal and serum levels of ethanol and its metabolites, acetate, and acetaldehyde. Additionally, children with an inflammatory form of NAFLD, NASH, have increased serum levels of ethanol compared to healthy children . HoweverIncreased levels of endogenous ethanol and its metabolites fuel the progression of the disease by several mechanisms. In the gut, ethanol and its metabolite, acetaldehyde, increase intestinal permeability by stimulating the production of inflammatory cytokines, decreasing the production of AMPs, and disrupting tight junctions. This leads to gut barrier dysfunction, the translocation of microbiota-derived molecules into the liver resulting in increased production of inflammatory cytokines and the induction of lipogenesis. In the liver, ethanol disrupts lipid metabolism by inducing fatty acid uptake and de novo lipogenesis, impairing fatty acid oxidation and inhibiting VLDL export . ContinuInterestingly, gut microbiota can also metabolise ethanol and protect the liver against alcohol-induced liver injury. This was well documented by a study in germ-free mice showing that the absence of microbiota leads to increased liver exposure to ethanol, increased expression of ethanol metabolising enzymes, and exacerbation of hepatic steatosis . TherefoThe primary bile acids (BAs), i.e., cholic and chenodeoxycholic (CDCA), are organic molecules synthesised in a multistep process from cholesterol in the pericentral hepatocytes and secreted in the form of glycine/taurine conjugated bile salts via the bile duct together with other bile components such as bilirubin phospholipid, cholesterol, amino acids, porphyrins, and xenobiotics, into the duodenum . The BAsSeveral preclinical and clinical studies associate the disturbance of bile acid metabolism with NAFLD. There are two well-studied mechanistic pathways implicated in the NAFLD pathogenesis. Firstly, a decreased signalling via the bile acid receptor farnesoid X receptor (FXR) due to an increased ratio of secondary to primary BAs (DCA/CDCA) leads to dysregulation of lipid and glucose metabolism ,108. AddThe primary source of SIgA in the intestine is intestinal plasma cells. However, plasma cells capable of producing microbiota-specific SIgA are also present in the liver . The livThe most common diagnostic and prognostic methods for NAFLD are blood tests, imaging methods, and liver stiffness assessment methods. Blood tests are used to detect elevated liver enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Such laboratory abnormalities are often the first sign of liver dysfunction. The most common tests used to visualise the liver include ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI). MRI is the most sensitive test for imaging hepatic steatosis and can be used to calculate the amount of fat in the liver. Liver stiffness or elasticity could be measured with ultrasound or magnetic resonance elastography (MRE). The low frequency vibrations are visualised to create a map, an elastogram, that shows the stiffness and elasticity over the liver. Elastography methods can detect the early stages of liver fibrosis and may eliminate the need for an invasive test, i.e., liver biopsy ,115.An increased knowledge of gut microbiota, MAMPs, and microbiota-derived metabolites’ involvement in the NAFLD pathogenesis could be exploited in novel diagnostic and therapeutic approaches. NAFLD is a heterogeneous group of conditions, which consist of several subtypes driven by various combinations of the above-mentioned factors, and this knowledge should be reflected in both diagnosis and therapy.Veillonellaceae with liver fibrosis in non-obese NAFLD patients and suggest Veillonellaceae as a diagnostic marker [Currently, a liver biopsy is still required for the diagnosis and monitoring of disease progression. Therefore, noninvasive and reliable markers for NAFLD evaluation are urgently needed. Recent progress in gut microbiota research suggests that some microbiota members and metabolites might be useful as diagnostic and prognostic markers. For example, Loomba et al. found that a panel of gut microbiota consisting of 37 bacterial strains can be used to accurately diagnose advanced fibrosis in patients with NAFLD  and Lee c marker .Bacteroidaceae and Prevotella [There are several microbiota-derived metabolites that could be used as NAFLD biomarkers. Some of the most promising are succinate, phenylacetic acid, and 3-(4-hydroxyphenyl) lactate. Interestingly, NAFLD patients have often a decreased microbial gene richness resulting in an alteration in aromatic amino acid and branched-chain amino acid metabolism. For example, the above mentioned 3-(4-hydroxyphenyl) lactate which is associated with liver fibrosis, is a product of aromatic amino acid metabolism . Serum levotella , was fouevotella .The deep knowledge of gut microbiota could be exploited for therapeutic purposes on several levels. Firstly, the whole microbiota communities might be restored using FMT. Secondly, single strains or collections of beneficial strains (probiotics) could be introduced to gut microbiota to deliver missing functionality while harmful or undesirable strains might be deleted using antibiotics, antimycotics, or bacteriophages. Lastly, microbial metabolic pathways could be targeted to decrease or block the production of harmful metabolites or to stimulate the production of those which are beneficial.Data on the efficacy of FMT in the treatment of NAFLD are limited. To date, FMT has been used successfully in cirrhotic patients with hepatic encephalopathy  and alcoProbiotics, prebiotics, and synbiotics have also been used in NAFLD treatment. Probiotics are nonpathogenic live microorganisms that, when consumed, can have a positive effect on host health . The mosE. faecalis, using a bacteriophage was shown to be effective in ethanol-induced liver disease in humanised mice [The therapeutic strategy of targeting single strain, specifically cytolytic sed mice .The therapeutic approaches aimed at manipulating microbiota to increase the production of protective metabolites or to block/decrease the production of harmful metabolites are also promising. For example, the conversion of dietary choline into TMA by microbial TMA lyases can be blocked by 3,3-dimethyl-1-butanol, which is a structural analogue of choline . MicrobiIncreasing evidence suggests that the gut microbiota plays a critical role in the pathogenesis of NAFLD. Metabolites derived from the gut, whether beneficial or harmful, are transported via the portal vein directly to the liver for further processing, including energy production, detoxification, and synthesis. Problems arise when the liver becomes overloaded with harmful, toxic or proinflammatory molecules due to unhealthy dietary habits, dysbiosis of the gut microbiota and/or increased gut permeability. These molecules originate from either the diet  or the microbiota  and negatively affect liver physiology. The dysfunctional liver cannot effectively control the gut microbiota via bile acids and other microbiome-regulating factors, resulting in gut dysbiosis and gut barrier dysfunction. This reinforcing loop has deleterious effects on the liver, gut, and human health in general.The major disruptors of the gut microbiota are environmental factors, particularly diet and medications. Genetic factors provide the playing field for NAFLD pathogenesis but may not be responsible for the steady increase in NAFLD incidence due to their relatively stable nature.Several clinical studies have uncovered promising NAFLD-associated microbiome signatures that could be used for noninvasive NAFLD diagnosis or monitoring of disease progression. However, the usefulness of the signatures needs to be further validated in well-designed large cohort longitudinal studies that take into account important confounding factors such as comorbidities, including T2DM and obesity, ethnic background, medication and diet. The diagnostic and prognostic value of microbiota signatures could be further enhanced by combining them with microbiota-derived metabolites detected in blood/plasma, urine, or faeces. Such an approach will allow more accurate differentiation of NAFLD subtypes and more precise evaluation of the efficacy of therapy.A comprehensive understanding of the interactions between microbiota and liver will enable us to develop effective microbiota-based therapies. There will be two main approaches that can be used in combination. The first will be direct manipulation of the gut microbiota by removing unwanted strains, introducing beneficial strains  or replacing the dysbiotic microbiota with FMT. The second approach will be based on exploiting microbial metabolites by either inducing or blocking their production.In summary, a growing body of evidence suggests that NAFLD is triggered and driven by a gut microbiota distorted by environmental factors. A deep knowledge of the interactions between microbiota and liver will allow us to introduce new noninvasive diagnostic and prognostic methods based on NAFLD-specific microbiota signatures and metabolites, and also to develop effective microbiota-based therapies, such as probiotic interventions, FMT, or metabolite manipulations.


Nonalcoholic fatty liver disease (NAFLD), the most common cause of chronic liver disease, ranges from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH), which is a more aggressive form characterized by hepatocyte injury, inflammation, and fibrosis. Increasing evidence suggests that NASH is a risk factor for hepatocellular carcinoma (HCC), which is the fifth most common cancer worldwide and the second most common cause of cancer-related death. Recent studies support a strong mechanistic link between the NASH microenvironment and HCC development. The liver has a large capacity to remove circulating pathogens and gut-derived microbial compounds. Thus, the liver is a central player in immunoregulation. Altered immune responses are tightly associated with the development of NASH and HCC. The objective of this study was to differentiate the roles of specific immune cell subsets in NASH and HCC pathogenesis. Clarifying the role of specific cells in the immune system in the transition from non-alcoholic fatty liver disease (NAFLD) to liver cancer will help to understand disease progression and may open avenues towards new preventive and therapeutic strategies. NAFLD is the most common chronic liver disease. Growing evidence suggests that its most aggressive form, non-alcoholic steatohepatitis (NASH), can promote the development of liver cancer, the second most common cause of cancer deaths worldwide. Chang-Woo Lee and colleagues at Sungkyunkwan University, Suwon, South Korea review the immunological distinction between NASH and liver cancer, focusing on the levels and activities of six key types of immune system cells. Chronic inflammation mediated by the immune system can create conditions for NAFLD, NASH and liver cancer to develop and worsen. HCC is the sixth leading cause of cancer-related deaths globally and is expected to become the third leading cause of liver cancer-related deaths by 20302. Such changes in HCC incidence are affected by obesity, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD), which is the most common liver disease3. Although NAFLD has a spectrum of liver pathologies similar to those of alcohol-induced fatty liver damage, NAFLD can occur in patients even in the absence of alcohol abuse4. NAFLD is characterized by a steatosis or the accumulation of triglycerides in lipid droplets inside hepatocytes (hepatic steatosis)5. Such accumulation of lipids is closely associated with metabolic syndromes such as obesity, type 2 diabetes, hypertension, and dyslipidemia6. NAFLD is highly prevalent on every continent. The global prevalence of NAFLD was ~25%. The Middle East has the highest prevalence rate of 32%, followed by South America (31%). Africa has the lowest prevalence at 14%7. NAFLD can progress to a more severe form called nonalcoholic steatohepatitis (NASH). NASH is marked by abnormal fat accumulation in the liver and immune cell infiltration into the liver due to chronic hepatitis and inflammation. In addition, it seems that most NASH patients develop progressive fibrosis7. NASH can cause liver diseases such as cirrhosis and HCC and is also associated with an increased risk of cardiovascular disease8.Hepatocellular carcinoma (HCC) is the most common type of liver cancer and accounts for 70–85% of all liver cancer cases9. NASH is the fastest increasing cause of HCC in the United States10. As such, the incidences of NAFLD and NASH increase each year. Patients with these disorders are highly likely to have more than one metabolic syndrome. These individuals are at high risk of developing HCC12. The incidence of NAFLD/NASH-released HCC has continuously increased in many ethnic groups, including in the United States13 Europe16, South Korea17, and Japan18, over the past decades. A study released in 2010 stated that NAFLD/NASH (59%) was the most common etiological risk factor in the United States, followed by diabetes (36%) and hepatitis C virus (22%)19. Given recent advances in anti-hepatitis C virus (HCV) therapy, NASH is highly likely to become a major cause of progressive liver disease within the next three decades.The prevalence of NASH among NAFLD patients in the United States has been estimated to be 21% . The prevalence of NASH in the United States accounts for ~3–4% of the entire population20. To overcome this growing burden of NASH and NAFLD/NASH-HCC, it is crucial to understand the factors associated with NASH and HCC to develop preventive and therapeutic strategies.Thus, the epidemiology of NASH-associated HCC is continuously changing as the number of patients with metabolic syndrome surges yearly. Compared to patients with other causative factors, patients with NASH-associated HCC are more prone to complications such as diabetes, obesity, dyslipidemia, and hypertension. These factors can exacerbate the clinical complexity of patients and eventually result in a difficult situation for clinical management. Additionally, although patients with lesions caused by HCV or HBV can be partially treated because of the development of treatments, effective treatment is currently unavailable for NASH-associated HCC patients22. In pathological liver injury, these cells are part of a complex proinflammatory and fibrogenic background, and hepatocyte death occurs, promoting disease progression. Various pathobiological factors, including proinflammatory cytokines (such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α), leptin, hyperinsulinemia, the gut microbiota, bile acid, and free fatty acid, can interact with components in the liver microenvironment. These factors may cause inflammation, fibrosis, and lipotoxicity as a result of interactions with the liver microenvironment. In the long term, the interactions of these factors with the liver microenvironment may lead to the progression to NASH and increase the possibility of HCC development21.Recent studies have shown that the liver microenvironment may play a crucial role in NAFLD/NASH and HCC progression. The liver provides a unique proinflammatory microenvironment that is composed of a variety of immunologically active cells, including Kupffer cells (KCs), T cells, antigen-presenting cells (APCs), and hepatic stellate cells (HSCs)23. NASH may trigger the death of liver cells by inducing metabolic stress in hepatocytes. The generation of damage-associated molecular patterns (DAMPs) occurs after an influx of activated immune cells and is called sterile chronic inflammation. HCC is an inflammation-related cancer. A chronic inflammatory state can trigger the initiation and development of cancer24. Altered immune responses in NASH with chronic inflammation are also associated with the development of HCC27.A proinflammatory microenvironment created by toxic lipid-induced hepatocyte injury (lipotoxicity), which may occur under NASH conditions, has a significant effect on the deterioration of NASH28. Thus, it is crucial to understand that the microenvironment of the liver plays a key role in the pathogenesis of NAFLD, NASH, and HCC. The pathogenesis of NASH and HCC is affected by various factors within the microenvironment. However, as inflammation is the most prominent feature of NASH and HCC, the present study reviews the function of immune cells in the respective microenvironments of NASH and HCC.Various factors and microenvironmental changes have been reported in HCC patients. In particular, cancer-related microenvironmental components such as immune cells, fibroblasts, endothelial cells, and extracellular matrix (ECM) can facilitate tumor cell differentiation, proliferation, and invasion29. NK cells play crucial roles in NAFLD and NASH -γ and the formation of a proinflammatory environment35.Natural killer (NK) cells are a group of innate immune cells that show cytolytic activity against cells under stress, such as tumor cells and virus-infected cellsASH Fig. . The num36. In addition, it seems that NK cell function is decreased by myeloid-derived suppressor cells (MDSCs) during the development of HCC  cells and CD8+ cytotoxic T cells. Th cells can be classified into Th1 and Th2 cells. Th1 cells produce IL-2 and IFN-γ and are involved in cellular immunity by regulating macrophages  to regulate neutrophils39  diet can trigger the loss of CD4+T lymphocytes and promote the development of HCC in liver-specific MYC oncogenic transgenic mice. In the same context, obesity-induced lipid accumulation can result in the selective loss of CD4+ T lymphocytes and promote disease progression from NAFLD to hepatocellular carcinoma43. These CD4+ T cells are biased to Th1 and Th17 subtypes in NASH conditions44. Th1 cells can promote the differentiation of macrophages into M1 macrophages that play a proinflammatory role by secreting IFN-γ and TNF-α46. Th17 cells secrete IL-17, which exacerbates hepatic steatosis and inflammation and induces the transition from simple steatosis to hepatic steatosis48. Th17 cells also produce neutrophil-attracting chemokines to recruit neutrophils and lymphocytes in NASH49. Fibrosis progression is further induced through HSCs50. In contrast, the number of hepatic regulatory T cell (Tregs) is decreased in NASH51. In mice with HFD-induced steatosis, Treg apoptosis can be induced by increased oxidative stress, whereas liver inflammation is markedly reduced by adoptive transfer of Tregs52. In fact, the ratio of Th17 cells/Tregs is higher in NASH patients than in NAFLD patients and normal controls, indicating that the ratio of Th17/Tregs may play a crucial role in the NASH environment53.The selective loss of CD454. The number of Tregs is significantly increased in HCC patients55. An increase in Tregs is mainly observed around tumor sites. Tregs extracted from the tumor site contribute to uncontrolled growth of HCC cells by inhibiting the proliferation of CD8+ cytotoxic T cells  and play a cytolytic role , and GrB production might be interrupted by the increased numbers of Tregs in tumors60  cells are unconventional cells that express NK and T cell surface markers and secrete various cytokines, such as IFN-γ and IL-4, to control innate and adaptive immunity. NKT cells account for 30% of hepatic nonparenchymal cellsr64 Fig. . The nums64 Fig. . Additio 65 Fig. . Moreove66. Based on differences in T cell receptor (TCR) usage, these NKT cells can be classified into two types: Type I and Type II. Type I NKT cells express invariant TCRα chains that are readily detectable by α-galactosylceramide -loaded CD1d tetramers, while type II NKT cells express a broader TCR repertoire68. However, studies on Type II NKT cells in the liver are insufficient because of the absence of a specific marker. Thus, Type I NKT cells called invariant NKT (iNKT) cells have been mostly studied in patient tumors  and inflammatory cytokines such as IL-1β, TNF, and IL-6. They participate in Th1 responses and mediate resistance to intracellular parasites and tumors. In contrast, M2 cells are characterized by an IL-12loIL-23loIL-10hiTGF-βhi phenotype in vitro. M2 cells participate in polarized Th2 responses, allergies, parasite clearance, dampening of inflammation, tissue remodeling, angiogenesis, immunoregulation, and tumor promotion73. In the liver, hepatic resident macrophages are called KCs and are involved in inflammation signaling and metabolic changes.Macrophages that circulate in the blood or exist in various organs and tissues are the first barriers to any disease. These cells play the most important role in innate and acquired immunity. Macrophages are generally divided into two phenotypes: classically activated (M1) macrophages and alternatively activated (M2) macrophages74. The level of KCs decreases in NASH, while the infiltration of Ly6C+ monocyte-derived macrophages is increased in the early stage of NASH in an MCD diet model75. In NASH, toll-like receptor (TLR) 4 in KCs is more highly expressed than other TLRs76. When LPS binds to TLR4, KCs are activated, and the production of proinflammatory cytokines such as TNF-α, IL-1β, IL-2, IL-6, IL-10, and IFN-γ is enhanced, triggering the recruitment of lymphocytes and other leukocytes77 and promoting the activation of NF-κB, MAPK, ERK1, p38, JNK, and IRF378. Additionally, the expression of hepatic inflammatory genes is increased by oxidized low-density lipoprotein (LDL) trapped in KC lysosomes in a NASH model79. Recent studies have shown an increase in endolysosomal lipid accumulation in KCs during the progression from NAFLD to NASH80, indicating that KCs play a crucial role in the progression of NASH. Along with KCs, infiltrated Ly6C+ macrophages produce cytokines such as TNF-α and IL-1β, thus promoting inflammation and activating HSCs81.In NASH, various metabolic syndromes and insulin resistance promote the accumulation of free fatty acids (FFAs) in hepatocytes and blood, triggering the innate immune response by stimulating lipotoxin and LPS. In a healthy liver, the KCs primarily function as the body’s frontline defense against phagocytosis and pathogens from the portal vein and arterial circulation82. Indeed, KCs can secrete profibrogenic cytokines such as transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF)78. In other words, KCs contribute to inflammation and fibrosis progression through various processes, exacerbating NASH 84.KCs act as a major mediator in fibrosis Fig. . In vivoASH Fig. . In addi85 -induced HCC model, hepatocellular carcinogenesis was attenuated when the activation of KCs was mitigated by deletion of the proinflammatory myeloid cell surface receptor triggering receptor expressed on myeloid cell-1 (TREM-1), which is expressed by KCs. It has been revealed that the activation of KCs is crucial for tumor development in the early stage of chemical-induced carcinogenesis86. Furthermore, IL-1a secreted either by oncogene-induced senescent hepatocytes or by apoptotic hepatocytes in the DEN model can induce the production of IL-6 in KCs and trigger compensatory proliferation and tumor progression, which are essential for HCC development22. Once a primary tumor is established, liver-infiltrated macrophages play a more prominent role than KCs. Macrophages infiltrate near tumors. Macrophages present in the TME are called tumor-associated macrophages (TAMs)87. A tumor with highly infiltrated TAMs leads to a worse prognosis in HCC88  of HCC have low expression of costimulatory molecules but high expression of coinhibitory molecules. Moreover, due to the presence of MDSCs, the antitumor function of KCs is suppressed85 Fig. . Recentl91. Macrophages that are transformed into the M2 phenotype can exacerbate HCC. M2-polarized macrophages enable epithelial–mesenchymal transition (EMT) and the migration of HCC cells via the TLR4/signal transducer and activator of transcription (STAT) 3 signaling pathway92. Moreover, increased stability of HIF-1α in hypoxic conditions in HCC increases the secretion of IL-1β and promotes EMT and metastasis of tumor cells93. TAMs can also secrete IL-6 and promote the expansion of cancer stem cells and tumor formation through IL-6/STAT3 signaling94 , a TNF superfamily member, is secreted by adipocytes. BAFF regulates the maturation and development of B cells101. NASH patients have higher levels of BAFF than control patients with simple steatosis. BAFF is associated with the degree of ballooning hepatocytes and hepatic fibrosis102. In BAFF-knockout mice, the number of mature B cells is decreased. Recent studies have shown that deletion of BAFF in the NAFLD model attenuates hepatic fat accumulation, inhibits inflammation and fibrosis in VAT, improves insulin resistance, and weakens liver steatosis. These results indicate that BAFF plays a role in exacerbating NAFLD and NASH103. BAFF-knockout mice show reduced systemic inflammation104. In response to LPS stimulation, hepatic B cells produce more IFN-γ, IL-6 and tumor necrosis factor (TNF)-α but less IL-10 than those from secondary lymphoid tissue, indicating that hepatic B cells promote inflammatory responses105. Increased TGF-β levels in NASH patients106 trigger the transformation of IgM-expressing B cells into IgA-expressing B cells with regulatory activity. The level of serum IgA is increased in NASH patients. A high level of IgA is associated with the state of fibrosis108. Additionally, the accumulation of liver-resident IgA+PDL1+ cells that secrete IL-10 concurrent in NASH can promote tumor growth by inhibiting the function of CD8+ T cells107.B cells are immune cells that play a role in antibody secretion, antigen presentation, T cell costimulation, and cytokine secretion. B cells play an immunomodulatory role and contribute to autoimmunity and disease pathogenesis109. An increased level of tumor-infiltrating B cells can lead to better clinical outcomes. However, intratumor infiltration of B cells is impaired during the progression of HCC110. B cells secrete immunoglobulin with a direct antitumor effect that is beneficial for HCC patients111  is also associated with hepatic cholesterol accumulation, inflammation, and fibrosis. MPO enhances macrophage cytotoxicity and induces neutrophil activation116. Additionally, neutrophil-derived human neutrophil peptide (HNP)-1 induces hepatic fibrosis through HSC proliferation and exacerbates NASH117 . These cells serve as mediators of inflammation119) expelled by neutrophils are increased in sera samples from NASH patients. NETs play a key role in the formation of chronic inflammatory liver microenvironment in NASH that promotes the progression to HCC. The formation of NETs has been observed in livers of STAM mice , followed by an influx of monocyte-derived macrophages, the formation of inflammatory cytokines, and progression to HCC120.Neutrophils also play a crucial role in the transition from NASH to HCC Fig. . The lev122. Increased levels of IL-17 in HCC promote the secretion of CXC chemokines to recruit neutrophils around the peritumoral stromal regions  and promote the growth and metastasis of malignant cells that are activated by GM-CSF, which is secreted by hepatoma cells in the HCC microenvironment125. Taken together, these findings indicate that neutrophils play a protumorigenic role in HCC progression, growth, and metastasis.Neutrophils are enriched mainly around the peritumoral stromal region in HCC. The frequency of neutrophil infiltration in the liver is an indicator of poor survival in HCC126. Since DCs induce the adaptive immune response, the importance of DCs during hepatic inflammation has emerged. The hepatic DC population becomes expanded and mature in the NASH liver127. When DCs are depleted, intrahepatic fibrosis-associated inflammation is markedly exacerbated. Hence, in NASH, DCs can limit CD8+ T cell expansion and restrict Toll-like receptor expression and cytokine production in innate immune effector cells, including KCs, neutrophils, and inflammatory monocytes127. DCs can capture HCC-related antigens, process antigens, and activate antigen-specific T cells to remove tumors. However, in advanced HCC, the functions of DCs and IL-12 production are impaired. Thus, antigen-specific T cells cannot be activated properly128.Other myeloid cells also have various roles in NASH and HCC pathogenesis. Particularly, dendritic cells (DCs) are the most powerful APCs and can induce adaptive immune responses and generate tolerance to self-antigensintLy6Chi monocytes appear to be the predominant source of TNFα production at a later time point in MCD diet-induced disease, leading to NASH progression129. These infiltrated monocytes have potent proinflammatory activities. In addition, together with KC, infiltrated monocytes can propagate lipid accumulation induced by the MCD diet mainly through the production of TNFα129. In HCC patients, PD-L1+ monocytes are highly enriched in the peritumoral stroma. These monocytes are activated to suppress tumor-specific T cell immunity. In fact, high infiltration of these monocytes is associated with poor survival of HCC patients130.Monocytes play a pivotal role in inflammation and metabolic stresses. The infiltration of monocytes is an important feature of NASH. CD11b24. As mentioned above, NAFLD and NASH are major causes of the increased prevalence of HCC. Additionally, NASH-related HCC mainly occurs in the context of cirrhosis. Although NASH-related HCC cases occur mainly in cirrhotic patients, an increasing number of HCC cases have been reported in NAFLD and NASH patients with little or no cirrhosis131. While many studies on the respective microenvironments of NASH and HCC have been conducted, such studies on changes in the progression from NASH to HCC are insufficient. Since various immune cells have different functions that can affect the development of NASH and HCC, it is critical to develop an experimental model to identify the transition from NASH to HCC.Acute inflammation is a useful reaction to achieve tissue recovery by promoting regeneration. Conversely, chronic inflammation is maladaptive and provides an environment that is conducive to the development of NASH and HCC. Chronic injury triggers the secretion of significant amounts of proinflammatory molecules, including IL-1, IL-6, TNF-α, and lymphotoxin-β, that can facilitate HCC development132. Thus, patients with NASH are at increased risk of adverse liver-related outcomes, with the degree of fibrosis contributing most significantly to this increased risk. However, medications have not yet been approved by the Federal Drug Administration (FDA) or European Medicines Agency (EMA) for the treatment of NASH. Many agents are currently being studied in clinical trials Phase 2 clinical trials have been for the TLR4 antagonist JKB-121 , which targets cells expressing TLRs, such as KCs, and solithromycin , which inhibits TNF-α/CXCL8 production and MMP9 activity in monocytic cells. Obeticholic acid (OCA) is a derivative of the primary human bile acid chenodeoxycholic acid (CDCA) and functions as an agonist of farnesoid X receptor (FXR). There was a randomized placebo-controlled, phase IIb trial and a large phase III trial to evaluate the safety and efficacy of OCA for the treatment of NASH and fibrosis133. Elafibranor is an agent currently in a phase III trial for the treatment of NASH . Elafibranor, a dual peroxisome proliferator-activated receptor (PPAR)-α/δ agonist133, and cenicriviroc (CVC), a dual CCR2/CCR5 chemokine receptor antagonist that has been shown to play key roles in hepatic inflammation and fibrosis134, are being investigated in current phase III clinical trials in patients with NASH and fibrosis, respectively . Additionally, the immune checkpoint inhibitors nivolumab , pembrolizumab , and tremelimumab  are undergoing phase III clinical trials in patients with HCC.NASH with advanced cirrhosis is currently the primary etiology for liver transplantation and is estimated to become the leading indication for liver transplantation135. Furthermore, instead of using only checkpoint inhibitors, combined therapies targeting immune cells involved in the progression of HCC or targeting cells participating in angiogenesis with checkpoint inhibitors are now presenting promising outcomes137. However, combinational therapy that controls steatosis, chronic inflammation, and fibrosis could be the most efficient therapeutic strategy for NASH treatment. In addition, overcoming the hypocellular hepatic microenvironment is the most important condition for inducing efficient drug effects. Thus, developing effective drug candidates that can reverse the functions of immune cells in the respective microenvironments of NASH and HCC is expected to become a novel therapeutic strategy.Various mechanisms and microenvironmental changes are involved in the onset and progression of NASH and HCC, raising a fundamental question on whether one targeted treatment would be effective. Indeed, most of the abovementioned drug candidates did not show expected therapeutic efficacies in phase II or phase III clinical trials. Importantly, a combination therapy that uses more than two types of targeted drugs to provide additional and/or synergistic effects could be a more effective strategy for NASH and HCC treatment. For instance, antidiabetic drugs or antifibrotic drugs with an FXR agonist, a well-known drug for NASH, have shown notable effects in clinical trials


Non-alcoholic fatty liver disease (NAFLD) is a leading cause of liver cirrhosis and hepatocellular carcinoma. NAFLD is associated with metabolic disorders such as obesity, insulin resistance, dyslipidemia, steatohepatitis, and liver fibrosis. Liver-resident (Kupffer cells) and recruited macrophages contribute to low-grade chronic inflammation in various tissues by modulating macrophage polarization, which is implicated in the pathogenesis of metabolic diseases. Abnormalities in the intestinal environment, such as the gut microbiota, metabolites, and immune system, are also involved in the pathogenesis and development of NAFLD. Hepatic macrophage activation is induced by the permeation of antigens, endotoxins, and other proinflammatory substances into the bloodstream as a result of increased intestinal permeability. Therefore, it is important to understand the role of the gut–liver axis in influencing macrophage activity, which is central to the pathogenesis of NAFLD and nonalcoholic steatohepatitis (NASH). Not only probiotics but also biogenics (heat-killed lactic acid bacteria) are effective in ameliorating the progression of NASH. Here we review the effect of hepatic macrophages/Kupffer cells, other immune cells, intestinal permeability, and immunity on NAFLD and NASH and the impact of probiotics, prebiotics, and biogenesis on those diseases. Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver disorders worldwide and its prevalence is increasing ,2. NAFLDSeveral cross-sectional clinical studies have focused on the pathogenesis of NASH. In addition, mouse models of NASH mimic the pathogenesis of diet-induced obesity and its resulting metabolic disturbances, including NAFLD and NASH ,7,8,9,10Innate immune responses of NAFLD and NASH involve resident Kupffer cells, recruited macrophages derived from bone marrow cells, neutrophils, and natural killer T cells. These cells contribute to the progression of NASH by inducing inflammation, by promoting the production of cytokines, chemokines, eicosanoids, nitric oxide, and ROS. In addition, excessive free fatty acids (FFAs) and cholesterol cause hepatic lipotoxicity and stimulate macrophage activation, and production of proinflammatory cytokines . PalmitaLiver macrophages comprise several populations and play a key role in liver immune homeostasis and the pathogenesis of liver disease . Kupfferhi) inflammatory blood monocytes. The latter differentiate to CD11b+F4/80+ inflammatory macrophages (M1-type), which have phagocytic activity and secrete proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, and ROS [lowF4/80+ macrophages (M2-type) with an immunosuppressive, pro-fibrogenic phenotype is observed in the reparative phase of NAFLD and NASH. M2-type macrophages secrete high levels of IL-13 and transforming growth factor-β1 (TGFβ1), resulting in progressive fibrosis [Kupffer cells and recruited macrophages regulate liver immune homeostasis and the development of liver diseases. Kupffer cells recruit additional immune cells, including neutrophils and lymphocyte antigen 6C-high  and include CCR2 + Ly6Chi and CX3CR1 + Ly6C- monocytes. Under inflammatory conditions, CCR2 + Ly6Chi monocytes transmigrate and differentiate into M1 macrophages. In steady state, CX3CR1 + Ly6C- monocytes differentiate into anti-inflammatory M2 macrophages and mediate tissue repair . In highThe increased prevalence of NAFLD may be linked to increased energy intake caused by dietary changes, such as increased intake of carbohydrate , fat, and fructose. Moreover, increased use of corn syrup or fructose as sweeteners and sucralose as a non-caloric artificial sweetener may affect the development of NAFLD ,62,63. TThe liver and gut are impacted by nutrients and the microbiome via the biliary tract, portal vein, and systemic mediators. Liver damage caused by disruption of the gut microbiome, its derived metabolites, and the gut immune system is implicated in the pathogenesis of obesity-induced insulin resistance and NAFLD. The liver is exposed to portal system products, such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) and is strongly influenced by diet-induced dysbiosis. PAMPs and DAMPs induce an inflammatory response in hepatocytes, Kupffer cells, and hepatic stellate cells (HSCs) by a Toll-like receptor (TLR) cascade, enhancing release of cytokines and chemokines , resulting in liver damage. Mice fed a HF or choline-deficient diet and patients with NAFLD have increased intestinal permeability ,65,66, tEscherichia coli and Bacteroides vulgatus and advanced fibrosis in patients with NAFLD [Escherichia abundance was higher in obese children with NASH compared to those with only obesity [Bacteroides and Ruminococcus were significantly increased, and Prevotella decreased, in patients with NASH (stage ≥ 2 fibrosis) compared to those without NASH, shown by 16S amplicon sequencing [Bacteroides and Prevotella are competitors in the gut microbiota, depending on dietary composition [Several clinical studies have suggested a link between the gut microbiota  and the pathogenesis of NAFLD, but causality has not been established . Shotgunth NAFLD . Escheri obesity . Moreovequencing . This fiposition .+ and TCR− IELs exhibit different subtypes depending on the developmental conditions: TCR+ IELs are induced after antigens are encountered, natural TCRαβ+ IELs undergo thymic agonist selection, TCRγδ+ IELs differentiate either intrathymically or extrathymically, and the development of TCR− IELs is similar to that of peripheral innate lymphocytes [+), NK T lymphocytes, dendritic cells, macrophages, ILCs, IgA+ plasma cells, IgG+ and IgM+ plasma cells, and B lymphocytes [+ T cells in the LP comprised primarily T helper (Th) 17 cells and regulatory T (Treg) cells. Th17 cells release IL-17A, IL-17F, and IL-22, preventing bacterial dissemination by inducing the expression and secretion of antimicrobial peptides [Intestinal immune cells contribute to the establishment of the intestinal mucosal barrier. These cells are classified as intraepithelial and lamina propria cells. Intraepithelial cells include intestinal intraepithelial lymphocytes (IELs), encompassing several T cell receptor (TCR)-positive and -negative subsets. TCRphocytes ,75. IELsphocytes ,77. CD4+peptides . IL-17A peptides . The redpeptides . By contpeptides . Notablypeptides ,81. Intepeptides . The effMany pharmacotherapeutic strategies have been used for NASH, which can progress to cirrhosis. Insulin sensitizers, such as metformin or pioglitazone, have been studied for the treatment of NASH. In addition, vitamin E, a food ingredient with antioxidant properties, has also been studied for its therapeutic effect on NASH ,84. In tLactobacillus, Streptococcus, Lactococcus, Enterococcus, Bifidobacterium, Bacillus, and Clostridium. These probiotics promote an anti-inflammatory environment and intestinal epithelial growth and survival and counteract pathogenic bacteria by modulating immunity. Prebiotics were defined by Gibson et al. in 1995 as, “A non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health” [Studies have investigated the ability of functional foods, such as lactic acid bacteria, to improve the gut microbiome in NASH and NAFLD. Functional foods are classified as probiotics, prebiotics, and biogenics based on their mechanisms of action . Probiot health” .Prebiotics include oligosaccharides and dietary fibers. Oligosaccharides encompass fructooligosaccharides and galactoses, such as lactulose, which have highly selective availability to bifidobacteria and promote their growth. The term “synbiotic” refers to a combination of probiotics and prebiotics, as defined by Gibson et al.Biogenics was proposed by Mitsuoka et al., and applies to food ingredients that contain biologically active peptides and immunopotentiators  produced directly or indirectly by modulation of the intestinal microflora . BiogeniLactobacillus, Bifidobacterium, and Streptococcus, or Lactobacillus alone. VSL#3 protected against insulin resistance and NAFLD by inhibiting inflammatory signaling, such as c-Jun N-terminal kinase (JNK) and nuclear factor-κB (NF-κB) and restoring the reduced number of hepatic natural killer T cells caused by a HF diet [Lactobacillus plantarum NA136 isolated from fermented food suppressed the body weight gain and decreased the mass of fat tissues of HF diet and fructose-fed mice (NAFLD model).; lipids, AST, and ALT levels were also reduced L. plantarum NA136 decreased de novo lipogenesis and increased fatty acid oxidation by activating the AMPK pathway to phosphorylate ACC and suppress SREBP-1/FAS signaling in a NASH model. Furthermore, L. plantarum NA136 reduced oxidative stress in the liver by activating the AMPK/NF-E2-related factor 2 (Nrf2) pathway in a NAFLD model. These effects resulted in L. plantarum NA136 attenuating NAFLD [Lactobacillus paracasei decreased the expression of TLR-4, CCL2 and TNF-α and attenuated hepatic steatosis. L. paracasei decreased the proportion of M1 Kupffer cells and increased that of M2, leading to M2-dominant shift in the liver of a NASH animal model [TM, containing Lactobacillus acidophilus NCIMB 30175, Lactobacillus plantarum NCIMB 30173, Lactobacillus rhamnosus NCIMB 30174, and Enterococcus faecium NCIMB 30176) on the composition of human intestinal microbiota was studied using the M-SHIME® system on an in vitro human intestinal model. Three probiotics showed colonization and growth in the luminal and mucosal compartments of the proximal and distal colon, and growth in the luminal proximal colon. This increased the proximal and distal colonic lactate concentrations. Lactate stimulated growth of lactate-consuming bacteria, resulting in increased short-chain fatty acid (SCFA) production, especially butyrate. Additionally, the probiotics exerted immunomodulatory effects, such as increased production of anti-inflammatory cytokines (IL-10 and IL-6) and decreased production of proinflammatory chemokines  [Several animal model studies and clinical trials have reported evidence of benefits. For example, VSL#3 and modified VSL#3 are a mixture of several probiotic bacteria of the genera  HF diet ,93. By c HF diet . Other a HF diet ,96. Lactng NAFLD . Moreoveal model . The effd MCP-1) . A meta-d MCP-1) .Faecalibacterium prausnitzii and Bifidobacterium and reduced the plasma endotoxin level by increasing GLP-1 secretion as well as the GLP-2 trophic effect on gut barrier integrity [Prebiotics may have a beneficial effect on NAFLD and NASH. In animal models, prebiotics altered the gut microbiota composition and increased the plasma glucagon-like peptide-2 (GLP-2) level, improving gut barrier function. Moreover, prebiotics reduced liver inflammation and improved metabolic disorders in obesity and diabetes . Furtherntegrity . In humantegrity . Moreoventegrity .Lactobacillus reuteri GMNL-263 (Lr263) reduced fibrosis in the liver and heart by TGF-β suppression in HF diet-fed mice [Lactobacillus plantarum L-137 (HK L-137), which is isolated from fermented fish and rice dishes, attenuated adipose tissue and hepatic inflammation in DahlS. fa/LeprfaZ-Lepr rats as a model of metabolic syndrome [Lactobacillus pentosus strain S-PT84, isolated from Kyoto pickles (shibazuke), reportedly enhances splenic natural killer activity and interferon-γ production in mice [Lactobacillus pentosaceus LP28 (LP28), isolated from longan fruit (Euphoria longana), showed reduced body weight gain, liver triglyceride and cholesterol in HF diet-fed mice. However, heat-killed LP28 did not prevent metabolic syndrome [Heat-killed lactic acid bacteria in biogenics, which are easier to handle compared with live lactic acid bacteria, have been used in studies of NAFLD and NASH. Heat-killed fed mice . Similarfed mice . Moreovesyndrome . Live an in mice ,109. Hea in mice . By contsyndrome . Heat-kiLactobacillus plantarum 299 v for at least 1 week preoperatively during the postoperative period in elective surgical patients did not influence bacterial translocation, gastric colonization, or the incidence of postoperative septic morbidity [Probiotic, prebiotic, and biogenic treatment of NAFLD and NASH is new and under development, and these agents regulate the gut microbiota and immunity. By contrast, probiotics do not regulate the intestinal environment or improve the symptoms of acute pancreatitis or Crohn’s disease. For instance, administration of orbidity ,113. A morbidity ,115. HowNAFLD is a common chronic liver disease worldwide, and its prevalence is increasing. Moreover, its association with obesity, type 2 diabetes mellitus, insulin resistance, metabolic syndrome, and progression to cirrhosis and hepatic carcinoma increase its clinical importance. The pathogenesis of NASH and NAFLD is complex, involving not only the hepatic immune system (monocyte or macrophage polarization) mechanisms but also adipokines produced by adipose tissue, and microbiome. Moreover, an altered gut microbiota composition and intestinal immunity are related to liver disease and are important in progression from NAFLD to NASH. In human and animal studies of NAFLD and NASH, probiotics, prebiotics and biogenics reduced serum levels of liver aminotransferases, inflammatory cytokines, and chemokines, and ameliorated insulin resistance and hepatic steatosis . Probiot


Non-alcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease, exposing to the risk of liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Angio-genesis is a complex process leading to the development of new vessels from pre-existing vessels. Angiogenesis is triggered by hypoxia and inflammation and is driven by the action of proangiogenic cytokines, mainly vascular endothelial growth factor (VEGF). In this review, we focus on liver angiogenesis associated with NAFLD and analyze the evidence of liver angiogenesis in animal models of NAFLD and in NAFLD patients. We also report the data explaining the role of angiogenesis in the progression of NAFLD and discuss the potential of targeting angiogenesis, notably VEGF, to treat NAFLD. Non-alcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease worldwide . NAFLD iAngiogenesis is a complex process leading to the development of new vessels from pre-existing vessels. Angiogenesis occurs under physiological conditions during normal wound healing and also in pathological contexts, such as tumorigenesis, so that antiangiogenic molecules  monoclonal antibody) are used in the treatment of different cancers, including HCC, according to recent guidelines . MoleculAngiogenesis also takes place during chronic liver diseases. Indeed, liver fibrosis progression is accompanied by angiogenesis, regardless of the etiology of the liver disease ,5. In thIn the context of chronic liver disease, angiogenesis leads to quantitative changes of liver vessels with the emergence of new vessels but also consists in qualitative changes of vessels (both pre-existing and new vessels), resulting in a process known as vascular remodeling. Such qualitative vascular changes include dedifferentiation of liver sinusoidal endothelial cells (LSECs), also called capillarization, defined by the loss of their fenestrae and the acquisition of a basement membrane . In the This review focuses on angiogenesis associated with NAFLD. First, we report the manifestations of angiogenesis in animal models and in patients with NAFLD. Second, we address the role of angiogenesis in the progression of NAFLD. Finally, we discuss the potential of targeting angiogenesis to treat patients with NAFLD.Liver angiogenesis can be assessed either directly, typically by showing an increase in the number of hepatic vessels, or indirectly, by measuring the expression of angiogenesis markers, such as markers of endothelial cells or proangiogenic cytokines. Because indirect methods are the most easily accessible, they are commonly used, but one should keep in mind their limitations. Indeed, the expression of endothelial cell markers does not necessarily correlate with the number of endothelial cells because some endothelial markers, such as CD105, are upregulated during the activation of endothelial cells associated with angiogenesis .Many rodent models have been used to study NAFLD: each of them mimicking one or several features of human NAFLD, i.e., steatosis, NASH, fibrosis and HCC . SeveralSpecific techniques of imaging were used to analyze the global vasculature of mouse liver with NAFLD. Using scanned electronic microscopy of vascular corrosion casts of the liver, Coulon et al. showed that NAFLD was associated with a global alteration of the hepatic vascular architecture that consisted not only in an increased number of vessels but also in a clearly different phenotype of vessels, which displayed an enlarged diameter and a disrupted organization . The samCompared to studies in animal models, data related to liver angiogenesis in patients with NAFLD are more limited. We showed that the liver from patients with NAFLD displayed an increased expression of the endothelial marker, vWF especially in patients with advanced fibrosis . FurtherNAFLD patients display increased serum levels of angiogenic markers such as VEGF, soluble VEGFR-1 (sVEGFR1) and sVEGFR2 . HoweverEvidence of liver angiogenesis in animal models of NAFLD and in NAFLD patients suggests a role of liver angiogenesis in NAFLD pathogenesis. During NAFLD, angiogenesis drives inflammation and fibrosis, as reviewed elsewhere . Liver aThe molecular pathways of angiogenesis are intermingled with those of NAFLD. Proangiogenic cytokines have an impact on NAFLD. Indeed, VEGF is also involved in lipogenesis so that anti-VEGFR2 treatment can induce changes in the expression of lipogenesis genes, as shown in MCD mice . HypoxiaIncreasing evidence indicates that macrophages play a critical role in NAFLD development and progression ,33. Yet,One of the mechanisms that promote angiogenesis is the secretion of extracellular vesicles. Extracellular vesicles are submicron membrane-bound structures secreted from different cell types. They contain a wide variety of molecules and exert important functions in cell-to-cell communication . Many stCirrhosis is associated with the risk of developing HCC in all chronic liver diseases including NAFLD. However, non-cirrhotic patients with NASH have a higher risk of HCC compared to patients with other types of liver disease . HCC is Ultimately, the best way to demonstrate the role of angiogenesis in the pathogenesis of NAFLD is to assess the effect of antiangiogenic treatments in NAFLD.A few antiangiogenic molecules have been tested in animal models of NAFLD. Coulon et al. showed that treatment with anti-VEGFR2 decreased steatosis and inflammation in MCD mice diet  and reduPlacental growth factor (PlGF) is a proangiogenic cytokine associated selectively with pathological angiogenesis. A previous study has shown that inhibition of PlGF reduced angiogenesis, inflammation and fibrosis in a non-NAFLD animal model of liver disease , whereasBesides anti-VEGFR2, another antiangiogenic agent has been assessed in NASH, i.e., the peptibody L1-10 which inhibits the interaction of Ang-2 with its receptor Tie2 . AdminisThe inhibition of formation of extracellular vesicles (by genetic inhibition of caspase 3) has been shown to limit the production of hepatocyte-derived proangiogenic extracellular vesicles and to protect mice fed with MCD diet from angiogenesis and fibrosis, independent of steatosis and inflammation .The treatment of CDAA rats with angiotensin II type 1 receptor blocker has been shown to decrease neovascularization and liver fibrosis . These rOne should note that the antifibrotic effects of the antiangiogenic treatments are not specific to NAFLD and have also been reported in other models of chronic liver diseases ,52,53,54In conclusion, many studies have provided convincing evidence of early and intensive liver angiogenesis in NAFLD pathogenesis both in animal models and in patients. Angiogenesis promotes the development of NAFLD, the progression of fibrosis and the emergence of HCC. Antiangiogenic treatment can reduce NASH and prevent HCC formation in different animal models. Antiangiogenic molecules approved to treat advanced hepatocellular carcinoma, notably Bevacizumab , could h


Obesity has become a worldwide health concern among the pediatric population. The prevalence of non-alcoholic fatty liver disease (NAFLD) is growing rapidly, alongside the high prevalence of obesity. NAFLD refers to a multifactorial disorder that includes simple steatosis to non-alcoholic steatohepatitis (NASH) with or devoid of fibrosis. NAFLD is regarded as a systemic disorder that influences glucose, lipid, and energy metabolism with hepatic manifestations. A sedentary lifestyle and poor choice of food remain the major contributors to the disease. Prompt and timely diagnosis of NAFLD among overweight children is crucial to prevent the progression of the condition. Yet, there has been no approved pharmacological treatment for NAFLD in adults or children. As indicated by clinical evidence, lifestyle modification plays a vital role as a primary form of therapy for managing and treating NAFLD. Emphasis is on the significance of caloric restriction, particularly macronutrients  in altering the disease consequences. A growing number of studies are now focusing on establishing a link between vitamins and NAFLD. Different types of vitamin supplements have been shown to be effective in treating NAFLD. In this review, we elaborate on the potential role of vitamin E with a high content of tocotrienol as a therapeutic alternative in treating NAFLD in obese children. The primary reason for chronic liver disease among children can be attributed to non-alcoholic fatty liver disease . NAFLD eAccording to the National Health and Morbidity Survey (NHMS) 2015, the prevalence of obesity among children aged 10–14 years in Malaysia was 14.4% . While iUntil today, there is no FDA approved drug in managing NAFLD among children. The safer choice of treatment to tackle this disease was probiotics or antioxidant agents . The ratThe prevalence of NAFLD was closely related to the increased prevalence of obesity in adults and children . The occThe prevalence of NAFLD increases with age, with a greater percentage of NAFLD among adolescents (15–19 years old) at 17.3% as compared to toddlers (2–4 years old) at 0.7% . NAFLD iPNPLA3) and transmembrane six superfamily 2 (TM6SF2) that affect the severity of liver damage as reported in adult patients with NAFLD  with metabolic syndrome were shown to have a significant increase in their ALT levels , and gamma-glutamyl transferase (GGT)  was noted in the patatin-like phospholipase domain containing three genes (th NAFLD . In a PAof NAFLD . The PNPT levels . A meta-se (GGT) . In an aHispanic . On the . The differences in storing fat capability in different adipose tissue compartments differed significantly between children with and without NAFLD . Ye. Ye. Theut NAFLD . In termut NAFLD . It was ut NAFLD . On the ut NAFLD .de novo lipogenesis in the liver. For instance, the conversion of fructose to fat in the liver further drives the accumulation of liver fat  plus spironolactone (25 mg once daily) for 52 weeks showed a significant decrease in liver fat score and homeostasis model of assessment-insulin resistance (HOMA-IR) in women with NAFLD , β (beta), δ (delta), and γ (gamma). Unlike tocopherol, tocotrienol is an unsaturated form that features an isoprenoid side chain, making it easier to absorb and penetrate better into tissues with saturated fatty layers, including the liver and the brain . TocotriIn vitro research revealed that α-tocotrienol considerably decreased the total cholesterol (TC) compared to untreated SH-SY5Y cells and cells treated with α-tocopherol (d-δ-tocotrienol inhibited the differentiation of murine 3T3-F442A preadipocytes through the downregulation of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation (Tocotrienol has been shown to have hypolipidemic properties in cells. The hypolipidemic property of tocotrienol is mediated through reduction of HMG-CoA (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase), a key enzyme for the cholesterol biosynthesis , 99. In copherol . A studycopherol . Tocotricopherol . Adipogecopherol . Zhao etcopherol . The stucopherol , 104. Bacopherol . Neverthcopherol . These rntiation . Shen etntiation , 108. Thntiation . The actntiation . Additiontiation  in LPS-intiation .Similar ntiation . In humantiation , 113.In vivo analysis of TRF supplementation with royal jelly in calorie restricted diet-fed rats indicated significant weight reduction and reduced expression of inflammatory cytokines (i.e. TNF-a and MCP1) as compared to TRF or royal jelly intervention alone (CPT1A), and cytochrome P450 family 7 subfamily A member 1 (CYP7A1)  mRNA, a pro-inflammatory marker was observed (Mcp1), Nlrp3, Tnfα, Il-1β, and cdc11  . High-pu7a1 gene . Since Nobserved . The autobserved . Furtherobserved . Shen etobserved . Likewisnd cdc11 .n = 200) of subjects with or without NAFLD, Ezaizi et al. reported a significant association between high-sensitivity C-reactive protein (hs-CRP) with NAFLD in Asian Indians , which are supposed to reduce cardiovascular-related, hepatic morbidity in NASH patients. Nevertheless, patients often found that participating in physical activity and changing dietary habits are tough to achieve and maintain. Therefore, the characteristics of tocotrienol, which are easily found and exceptional acceptability to people, have made it a practical treatment choice for NAFLD patients. NAFLD clinical experiments showed a moderate improvement in histology and liver biochemistry induced by tocotrienol treatment. However, further monotherapy clinical trials and pharmacological assessments are still required to explain the fundamental molecular mechanisms of treatment. Also, possible unpleasant consequences of tocotrienol to identify the best daily intake needed for pediatric NAFLD. In conclusion, tocotrienol is recommended as a functional diet to be used in combination with existing diabetes and obesity regulation strategies for the treatment of pediatric NAFLD.NM and ZA contributed to the conception of the idea for the manuscript. FA-B and AI performed literature search and drafted the manuscript. RR, NM, ZA, and KM critically revised the work. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.