Interplay between intestinal permeability and metabolic dysfunction-associated steatotic liver disease: Could there be a role for extra virgin olive oil consumption?

Abstract

Metabolic dysfunction-associated steatotic liver disease is a multifaceted disease associated with obesity, insulin resistance (IR), type 2 diabetes mellitus - in a word, metabolic syndrome - which has been extensively studied because it is related to an alteration of the normal metabolism of glucose and lipids, ultimately leading to triglyceride accumulation within hepatocytes. This lipid overload triggers an inflammatory status, also influenced by gut-liver axis dysfunction, with gut dysbiosis, which alters intestinal permeability, causing inflammation and IR in a vicious circle. Several approaches have been attempted to treat this condition and stop its possible evolution towards increasingly serious stages, but the first step is always lifestyle modification. The Mediterranean diet seems to be the most reliable for affecting liver steatosis, probably thanks to extra virgin olive oil, a healthy food with a high content of monounsaturated fatty acids and variable concentrations of phenols (oleocanthal) and phenolic alcohols, such as hydroxytyrosol and tyrosol. This review investigates the mechanisms underlying the bidirectional and synergistic relationships among metabolic dysfunction–associated steatotic liver disease, IR, and the gut-liver axis, specifically focusing on the role of extra virgin olive oil as one of the main antioxidant components of the Mediterranean diet.

Key Words: Metabolic dysfunction-associated steatotic liver disease; Metabolic syndrome; Intestinal permeability; Gut-liver axis; Extra virgin olive oil

Core Tip: Several studies have proven the close relationship between eating behaviour, intestinal permeability, and its influence on the “leaky gut” and the development of metabolic dysfunction-associated steatotic liver disease. This review aims to focus on the possible role of extra virgin olive oil consumption, as a fundamental component of the Mediterranean diet, on the gut-liver axis, with particular attention to its role as an antioxidant and anti-inflammatory functional food.

INTRODUCTION

Liver steatosis is characterized by an accumulation of fat in more than 5% of the liver’s hepatocytes. In 1980, Ludwig et al[1] first reported a liver condition mimicking alcoholic hepatitis that could progress to cirrhosis in people who did not consume a significant amount of alcohol. For many years, this condition was defined as non-alcoholic fatty liver disease (NAFLD), a definition for which it was necessary to exclude an alcoholic aetiology[2]. Subsequently, in 1999, several authors reported the association of NAFLD with metabolic syndrome (MetS) and described the association of MetS with liver steatosis and its evolution towards hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (HCC)[3]. In those years, it was observed that NAFLD was associated with insulin resistance (IR), obesity, and type 2 diabetes mellitus (T2DM). However, in 2008, it was observed that NAFLD could also be present in lean people. Lean NAFLD was still associated with IR, but it had lower rates of the other components of MetS[4,5]. Recently, a multisociety Delphi consensus statement was published on the new fatty liver disease nomenclature, changing the definition of NAFLD into steatotic liver disease (SLD), an overarching term to encompass the various aetiologies of steatosis, which includes metabolic dysfunction-associated SLD (MASLD), a condition in which steatosis is associated with metabolic dysfunctions typical of MetS, T2DM, arterial hypertension, visceral obesity, hypertriglyceridemia and low high-density lipoprotein (HDL) levels - all of which are conditions related to IR[6].

With their definition of hepatic steatosis, the experts wanted to simultaneously include steatosis associated with metabolic disease, alcoholic steatosis, and other secondary forms of steatosis. Although the terms NAFLD, metabolic dysfunction-associated fatty liver disease (MAFLD), and MASLD are similar, there are key differences among them. Several studies, however, have indicated that the term MASLD is an indicator for defining patients with liver metabolic diseases, while the term MAFLD combines the concept of disease with alcohol-intake related aetiology[7]. MASLD is considered the most common liver disease in the world; recent meta-analyses estimate that it makes up 38% of all the causes of chronic liver disease (CLD) in adults, and between 7% and 14% in children[8,9]. As has been known for years, the SLD spectrum can evolve from simple steatosis (MASLD) to metabolic dysfunction-associated steatohepatitis (MASH) and then, with progressive fibrosis development, to liver cirrhosis (LC), which can degenerate into HCC[7].

In recent years, the global burden of CLD remains substantial, and the role of SLD and its associated spectrum of pathologies is becoming increasingly relevant, especially after a decline in hepatitis B and C virus infections in some regions. Therefore, it is becoming increasingly important to make a socio-economic commitment to recognizing and treating risk factors in time, and to understanding the complex physio-pathological mechanisms that determine the onset and evolution of this disease in order to be able to intervene with appropriate therapies[10]. Among the possible mechanisms involved, several studies are looking at “gut-liver axis” dysfunction and, more specifically, at altered intestinal permeability. The “intestinal barrier” is, in fact, a complex structure whose alteration has been evoked as a trigger and progression factor of liver damage in MASLD[11]. Extra virgin olive oil (EVOO), a functional food and fundamental component of the Mediterranean diet (MD), is rich in polyphenols, and its antioxidant properties have been studied among the factors that could reverse gut-impaired permeability, probably by downregulating oxidative stress-mediated zonulin activation[12]. This review summarizes the most recent data on the mechanisms underlying the bidirectional and synergistic relationships among MASLD, IR, and the gut-liver axis, specifically focusing on EVOO’s involvement, given that it is one of the primary contributors of antioxidants to the MD.

PATHOGENESIS OF SLD

The histological definition of “fatty liver” involves the identification of intracellular triglycerides (TGs) in 5% or more of hepatocytes[2,6]. TGs build up in hepatocytes as fat globules, and steatosis is diagnosed when hepatic TG levels surpass the 95th percentile, which, for healthy, lean subjects, is > 55 mg per g of hepatic tissue. Hepatocytes convert fatty acids from the bloodstream into TGs; some TGs are synthesized not only from carbohydrate sources but also from alternative sources [de novo lipogenesis (DNL)]. These lipids, stored as lipid droplets, are released into the bloodstream as very low-density lipoproteins (VLDL). Normally, the systemic contribution of free fatty acids to the systemic circulation of DNL is 25%. In MASLD, due to IR, the increase in both DNL and the release of free fatty acids into the circulation by adipose tissue causes lipid accumulation in the liver and leads to steatosis[13,14]. The increased fat in the liver induces high oxidative stress with mitochondrial dysfunction, an increase in the production of mitochondrial reactive oxygen species (ROS), and the consequent apoptosis of hepatocytes. Cell death leads to the recruitment of inflammatory cells with a further increase in liver damage mediated by proinflammatory cytokines [tumor necrosis factor alpha (TNF-α), transforming growth factor beta, interleukin (IL)-6, IL-1beta]with the activation of hepatic stellate cells (HSCs). HSCs trigger the production of the extracellular matrix and fibrosis that, if increasingly severe, can evolve into LC; fibrosis and cirrhosis favour the onset of HCC[15-18].

The progression of MASLD into its evolutionary forms of MASH, LC, and HCC is a very complex phenomenon with multifactorial genesis. The two-hit hypothesis explains the evolution of LSD in two moments: The first hit is constituted by the accumulation of fat resulting from a high-fat diet, obesity, and IR, and the second is due to the intervention of inflammatory cytokines, adipokines, mitochondrial malfunction, and oxidative stress; but this theory has proven to be overly simplistic[19]. The more recent multiple-hits hypothesis, which suggests that different factors in genetically predisposed subjects may be responsible for the genesis and evolution of MASLD, appears more convincing[20-22]. These factors cause inflammation and stress in the liver environment, determining metabolic dysfunction. As reported above, intestinal barrier dysfunction and the alteration of the intestinal microbiota can lead to MASLD and its progression[23,24], although the mechanisms are still not fully understood[25] (Figure 1).

Figure 1 The natural history of metabolic dysfunction-associated steatotic liver disease. TJs: Tight junctions; LPS: Lipopolysaccharide; HSCs: Hepatic stellate cells; ROS: Reactive oxygen species; LC: Liver cirrhosis; MASH: Metabolic dysfunction-associated steatohepatitis; MASLD: Metabolic dysfunction-associated steatotic liver disease; HCC: Hepatocellular carcinoma.

GUT-LIVER AXIS

The gut-liver axis is the bidirectional relationship between the gut (including the microbiota) and the liver through intestinal blood drained by the portal vein and the bile duct, which creates continuous bidirectional “metabolic” cooperation through biliary acids, hormones, and products of digestion and absorption. It includes interactions between dietary, genetic, and environmental factors. For years, it has been considered to play a pivotal role in the genesis and evolution of CLD and, in particular, SLD[24,26] (Figure 2). Communication between the intestine and the liver happens through the portal vein, which allows for the arrival of intestinal products and feedback through bile acids (BAs) and antibodies. The intestinal barrier, made up of mucus and closely lined epithelial cells sealed by tight junctions (TJs), immune cells, and soluble mediators (e.g., immunoglobulin A, antimicrobial peptides), is part of the gut-liver axis, allowing effective bidirectional communication[23,24].

Figure 2 The gut-liver axis, a bidirectional “metabolic” cooperation through bile acids, hormones, and products of digestion and absorption, includes interactions between diet, genetic, and environmental factors, which, if altered, can contribute to the development of metabolic dysfunction-associated steatotic liver disease. TJs: Tight junctions; LPS: Lipopolysaccharide; MASLD: Metabolic dysfunction-associated steatotic liver disease; SCFAs: Short-chain fatty acids.

On one side, gut-derived products are transported across the intestine to the portal vein and on to the liver. On the other side, the liver secretes bile, especially BAs, and antibodies originate from the liver and flow across to the intestine. This is a very dynamic and resilient functional system, and the mechanisms maintaining the function of the intestinal barrier have major effects on metabolic balance in both health and disease conditions. For example, the gut microbiota undergoes continuous adaptations to lifestyle and foods and is responsible for the biotransformation of primary (hepatic) BA into secondary and tertiary (intestinal) BA, thus enriching the total BA pool in the body. The first physiological function of BA contributes to fat digestion and absorption and the stimulation of gut nuclear- and membrane-associated receptors[27]. In addition, BA controls the gut microbiome. Thus, the intestinal barrier function with the maintenance of gut homeostasis relies on the anatomical and functional integrity of the microbiome, mucus, enterocytes, immune system, and gut vascular barrier (GVB). Thus, an interruption of the intestinal barrier and subsequent increased intestinal permeability can both allow the translocation of bacteria or their products as well as food-derived toxins/antigens and promote the absorption of excess nutrients through the altered TJs. Increased intestinal permeability can be influenced by the Western diet with high fat and alcohol intake, the disruption of the gut microbiome, increased pro-inflammatory cytokine production, and the use of antibiotics[28,29].

THE GUT BARRIERS

The gut barrier is a complex structure made up of mechanical, chemical, immunological, and microbial barriers (Table 1). In the gut lumen, depending on the site, there is a mixture of intestinal secretions, bile, saprophytic and pathogenic bacteria that are in contact with the epithelial layer, made up of epithelial cells and mucus above them, with the epithelial cells connected by intercellular junctions. There is also a vascular barrier that allows water and macromolecules to be transported to the liver via portal circulation[23].

Table 1 Types of intestinal barriers, roles, and examples for each category.

Type of barrier	Description	Examples and functions
Mechanical barrier	Physical structures that prevent the entry of pathogens and unwanted substances	Intestinal epithelium: A layer of cells that lines the intestine; tight junctions: Connections between epithelial cells that prevent the passage of substances; mucosa: Production of mucus that traps pathogens and foreign particles
Vascular barrier	A vascular network that provides nutrients and oxygen and removes waste	Capillaries, lymphatics; a barrier that opposes the entry of bacteria from the intestinal lumen into the circulatory system; maintains the proper functioning of intestinal cells and the immune system
Chemical barrier	Chemical substances that inhibit the growth of pathogenic microbes and protect the intestine	Digestive enzymes, such as pepsin and lipase, which break down nutrients; gastric acid: Its low pH kills many pathogens; natural antibiotics, such as defensin, which acts against bacteria
Immunological barrier	Components of the immune system that monitor and defend the intestine from pathogens	Immune cells: Such as T and B lymphocytes, macrophages, and dendritic cells; immunoglobulins: In particular, IgA, which neutralize pathogens and toxins; gut-associated lymphoid tissue (GALT): Lymphoid structures that play a key role in intestinal immune response
Microbial barrier	Microbes that colonize the intestine and compete with pathogens for resources (microbiome)	Intestinal flora: Beneficial bacteria such as Lactobacillus and Bifidobacterium that maintain balance and inhibit the growth of pathogens; nutritional competition: Beneficial bacteria occupy ecological niches and resources, making it difficult for pathogens to colonize; production of antimicrobial substances: Some bacteria produce substances that inhibit the growth of other microorganisms

GALT: Gut-associated lymphoid tissue.

Mechanical barrier

The mechanical barrier is made up of the mucus layer, intestinal epithelial cells (IECs), intercellular TJs, and the lamina propria. The mucus layer is secreted by goblet cells. All together, these elements constitute a mechanical defence against germ aggression[30,31].

GVB

The GVB is located under the epithelial layer and constitutes another barrier that opposes the entry of bacteria from the intestinal lumen into the circulatory system[32].

Chemical barrier

The chemical barrier protects against the passage of bacteria or chemical substances into the circulatory system. It is made up of gastric juices, bile salts, pancreatic and intestinal secretions, glycoproteins, mucopolysaccharides, digestive enzymes, lysozymes, and antimicrobial peptides. Gastric juices with their acidic pH destroy some bacteria, bile salts, and BAs, in addition to regulating the absorption of lipids and carbohydrates, inactivate some pathogenic bacteria, and promote the growth of certain species of bacteria that have antimicrobial properties[33,34].

Immunological barrier

Immune cells in the intestinal barrier are essential for maintaining a balance between providing defence against pathogens and tolerating harmless substances, such as nutrients and beneficial microbes. IECs act as triggers for innate immunity, goblet cells secrete mucus but also respond to inflammatory stimuli, collaborate with IECs, phagocytes, lymphoid cells present in the gut, and B and T lymphocytes, which interact with the microbiota in establishing innate and adaptive immunity. Goblet cells also act as antigen-presenting cells against dendritic cells. The first line of defence against molecules, microbes, and antigens is the innate immune system. If pathogens manage to overcome this first obstacle, they are attacked by dendritic cells, macrophages, or Natural Killer cells. These cells, through receptors such as toll-like receptors and nucleotide-binding oligomerization domain receptors, recognize molecular patterns associated with pathogens released by bacteria, viruses, and fungi. There are also intestinally localized T lymphocytes that have an important role as an adaptive immune system[35-39]. Finally, it should be remembered that the intestinal immune defences include the gut lymphatic system, consisting of lymphocytes and lymphoid tissue in the intestinal wall, T helper, B and plasma cells, Peyer’s patches, intraepithelial lymphocytes, mucosa-associated lymphoid follicles, macrophages dispersed throughout the intestinal mucosa, and secretory immunoglobulin A[40].

Microbial barrier

The microbiota is a component of the intestinal barrier. It is made up of over one hundred trillion microorganisms (bacteria, fungi, protozoa, archaea, and viruses). This complex ecosystem lives in symbiosis with the host, cooperating in various functions like maintaining the host’s immune homeostasis (through interactions with mucosal cells), defence against the invasion of pathogens (by creating a real biological barrier), stimulation of the immune system, and production of enzymes[39-43]. It also produces vitamins, promotes the fermentation of polysaccharides, transformation of complex carbohydrates into digestible ones, conversion of bile salts, formation of short-chain fatty acids (SCFAs), and absorption of salts (iron, calcium). Dietary changes, drug intake, and colonization by other germs favour changes in the microbiome (dysbiosis) and alter those functions that it performs in physiological conditions. Dysbiosis is defined as the excessive proliferation of some bacterial species and the reduction of colonies of commensal bacteria, which is frequently associated with the pathogenesis of various inflammatory diseases and potential infections[44,45]. Dysbiosis is considered one of the causes of MASLD and, probably, of its progression to MASH[46].

The liver is always interacting with gut-derived bacteria and microbial products in a distinctive local immune environment, often hesitating in tolerance since an intact intestinal epithelial barrier can protect the liver from an excess of gut bacteria and their metabolites[47]. Bacterial products derived from the gut, collectively known as pathogen-associated molecular patterns and metabolism-associated molecular patterns, reach the liver via portal circulation and systemic circulation through the mesenteric lymph nodes and may induce an inflammatory response by activating toll-like receptors.

INTESTINAL PERMEABILITY ALTERATIONS: THE “LEAKY GUT”

Intestinal permeability has been conceptualized and defined rather broadly to include both a transcellular route, which involves membrane channels and transporters, as well as endocytic mechanisms, and an altered paracellular route controlled by TJs. A number of years ago, the “leaky gut” concept, also called increased intestinal permeability, was described, and today it is experiencing a surge in interest in both scientific literature and the media because of its possible association with a variety of conditions that are not necessarily involved with the gastrointestinal tract, including diabetes, Alzheimer’s disease, and asthma.

The idea of the “leaky gut” theory is that increased intestinal permeability can be influenced by both exogenous (alcohol intake, diet, etc.) and endogenous (intestinal inflammation, psychological stress, etc.) factors. Pathogenic or commensal bacteria, bacterial components, and food antigens manage to penetrate into the lamina propria and, thus, into the systemic circulation, where they can provoke the type of systemic inflammation characteristic of various disease conditions. Most research on this topic has concentrated on assessing the intestinal permeability of the paracellular route, even though there are other transepithelial transport routes that could be more relevant in disorders of the gastrointestinal system[47].

RELATIONSHIP BETWEEN THE GUT-LIVER AXIS AND MASLD

The relationship between intestinal barriers and MASLD involves alterations of the intestinal barrier, including the vascular changes, dysbiosis, and alteration of the immunological barrier[38].

Intestinal barrier alterations

Several studies have shown that, in MAFLD, the intestinal epithelial barrier is damaged, with the rupture of TJs and lower expression of zonula occludens resulting in increased intestinal permeability[46]. Studies on animal models have found that mice treated with high-fat diets have a lower expression of junctional adhesion molecules and an intestinal inflammatory pattern characterized by reduced T regulatory cells, increased interferon-gamma-producing Th1 and CD8+ T cells, and increased IL-17-producing T cells in the lamina propria that favour the onset of MASLD[48-51]. However, while there is consensus regarding the role of altered intestinal permeability, steatosis, and the severity of steatosis, not all studies report an association with hepatic fibrosis and inflammation[24]. Another observation that appears to be confirmed by several studies is that the disruption of the vascular barrier favours the passage of bacteria and proinflammatory molecules into the portal circulation and then into the liver[23].

The gut microbiome in MASLD and MASH

Data regarding the role of various bacterial species in the onset of MASLD are conflicting. Some studies have found a relationship with a higher prevalence of Gram-negative bacteria; in particular, the increase of Bacteroides, Escherichia, and Enterobacteriaceae has been considered to be associated with severe fibrosis[52-55]. Others have reported that the presence of Bacteroides in patients with MASH is lower than in controls and that their presence is not correlated with diet[56]. These conflicting results may likely depend on many variables: Age, characteristics of the study population, and geographical area. Therefore, further studies - and not only on guinea pigs - must be conducted to clarify this issue. Nevertheless, considering the whole microbiome, and thus not dividing it into single bacterial species, the role of dysbiosis in the onset of MASLD/MASH is more univocal. In fact, dysbiosis that, on the one hand, damages the intestinal epithelial barrier (leaky gut), on the other, can determine an alteration of the homeostasis of the immune system. Microbiome components or their metabolites, such as SCFAs or lipopolysaccharide (LPS), translocate into the portal circulation, reach the liver, and trigger a cascade of cytokines and inflammation mediators that, in turn, favour mechanisms that lead to the progression of steatosis in MASH and fibrosis[57-59].

Some studies on faecal transplantation in animal models have highlighted the role of microbiota: It has been demonstrated that transferring faeces from obese mice with hepatic steatosis to germ-free mice can reproduce NAFLD features. Accordingly, an improvement in intestinal permeability and a consequent therapeutic effect on NAFLD has been noted after faecal microbiota transplantation or after probiotics supplementation[60]. A recent report showed that NAFLD patients with intestinal barrier damage and increased intestinal permeability were characterized by more severe disease status, such as worse liver dysfunction, hyperlipidaemia, liver fat deposition, and IR. In particular, they found a positive relation between serum D-lactate (a marker of increased intestinal permeability) and markers of hepatocyte necrosis, cholestasis, and TG metabolism[61,62]. This is probably the result of the translocation of bacterial components, particularly LPS, into the portal vein and thus into the liver, resulting in liver inflammation and injury. Moreover, LPS levels seem to be related to fibrosis in NAFLD patients.

In addition, BA metabolism could be involved in MASLD pathogenesis. Yang et al[63] have demonstrated that elevated BA levels are associated with NAFLD. These alterations may result from a high-fat diet that modifies the microbiome (dysbiosis) and, consequently, BA levels, but the altered microbiome can also modify BAs and promote steatosis through interaction with the farnesoid-X-receptor (FXR)[63]. BAs are synthesized in the hepatocytes as primary BAs (cholic acid and chenodeoxycholic acid) and transported from the canalicular side of the hepatocyte to undergo enterohepatic circulation as bile salts[46]. Absorbed by the enterocytes of the terminal ileum, they promote the synthesis of fibroblast growth factor-19 through the stimulation of the FXR. Once fibroblast growth factor-19 reaches the hepatocytes, it inhibits the synthesis of BAs through the activation of c-Jun N-terminal kinase and downregulates the gene encoding cholesterol 7α-hydroxylase[46,63]. BAs have other metabolic actions and have been recognized as signalling molecules in the body through FXR and Takeda G protein-coupled receptor 5, playing a key role in the control of hepatic DNL, VLDL, and plasma TG turnover[46,64-66]. Studies on mouse models have shown that gut FXR inhibition was related to the development of NAFLD. In particular, a very recent paper showed that salidroside (a major active constituent of a traditional Chinese herb) effectively alleviated lipid accumulation and inflammatory injury in NASH mice, altering the gut microbiome BA profile by decreasing the levels of conjugated BAs and tauro alpha/beta-muricholic acid and activating downstream FXR and Takeda G protein-coupled receptor 5[67].

LPS and its role in MASLD

LPS is the main component of the outer membrane of most Gram-negative bacteria, and its increased presence has been related to dysbiosis. LPS can activate NAPH oxidase, promoting an increase in oxidative stress that favours the onset of MASLD. Furthermore, LPS binds to TLR4 on Kupffer cells, promoting the release of TNF-α and IL-6. This results in the activation of HSCs that promote oxidative stress with the activation of nicotinamide adenine dinucleotide phosphate oxidase and the production of ROS and fibrosis. Therefore, dysbiosis via LPS is a source of oxidative stress that can induce the progression of MASLD to MASH and severe fibrosis[67,68]. Reducing plasma LPS levels can improve hepatic steatosis, suggesting that chronic low-grade inflammation induced by LPS is an important factor in MASLD progression[67-69]. Clinical studies have shown an increase in LPS in high-fat diets, and studies in mice have found that increased endotoxemia is associated with diabetes and obesity[70,71].

Metabolites of the microbiome and MAFLD/MASH

SCFAs: SCFAs are unsaturated fatty acids derived from the fermentation of dietary fibre by metabolites of some species of the microbiome, such as the anaerobic Gram-negative and Gram-positive bacteria (respectively) Akkermansia muciniphila and Faecalibacterium prausnitzii. The most frequent SCFAs in the intestine are butyrate, propionate, and acetate[72]. SCFAs are absorbed by the portal circulation and influence hepatic lipid metabolism via adenosine monophosphate kinase, which also increases energy consumption by oxidizing fats. In addition, SCFAs reduce the migration of immune cells, thus reducing inflammation mechanisms[73-75]. Experimental studies have shown that butyrate-producing probiotics correct enterohepatic immune disorders and MASH caused by a high-fat diet. It would therefore appear that these metabolites of a benign flora would be protective against MASLD/MASH[76-78]. Acetate promotes the release of glucagon-like peptide-1 (GLP-1), which reduces liver inflammation through mechanisms that are not only consequent to weight loss, but also by reducing IR, oxidative stress, and inflammatory processes. It is also known that clinical studies have demonstrated how GLP-1 receptor agonists (liraglutide, semaglutide, exenatide, and dulaglutide) improve MASLD and MASH[79-81].

Choline: Choline is a phospholipid partly absorbed from the intestine and partly produced by the liver. Choline deficiency causes hepatic steatosis because it leads to the accumulation of TG in the liver due to reduced synthesis of VLDL[82]. Dysbiosis may favour an excessive metabolization of choline to trimethylamine (TMA), which, in turn, is transformed into trimethylamine N-oxide (TMAO), with a consequent reduction of phosphatidylcholine mimicking the choline deficiency status and the consequent accumulation of TGs in the liver[83]. TMAO also promotes IR with the metabolic consequences of increased blood glucose and inflammation. Increased levels of TMAO have been found in patients with MASLD[84]. Increased TMAO may be due to dysbiosis linked to an increased Firmicutes/Bacteroidetes ratio[85].

Endogenous ethanol: It seems that dysbiosis can cause an increase in endogenous production of ethanol by the microbiome, which could be linked to the consequent onset of MASLD[86]. However, on this topic, the results are conflicting and require further studies[69].

EVOO

To date, in the absence of effective pharmacological treatments for MASLD/MASH and awaiting regulatory authority procedures for the use of new drugs, interventions related to nutrition, lifestyle, and regular exercise have been considered the best therapeutic options[87-89]. The MD, considered to belong to the Cultural Heritage of Humanity by UNESCO since 2010, is the cornerstone of the dietary interventions used in the prevention and treatment of MASLD. This diet has a high content of monounsaturated fats, low saturated fats, high consumption of cereals, vegetables, and fruit, and moderate intake of wine; EVOO is the main source of fat in the MD[90]. Epidemiological data have repeatedly demonstrated the association of higher olive oil intake with lower risks of several chronic illnesses. The results of a number of important studies have indicated the general benefits of olive oil to overall health, in particular regarding endothelial functioning, inflammation, cardiovascular disease, T2DM, blood pressure, haemostasis, body-mass composition, and cancer[91,92].

MD has also been proven to have beneficial effects on MASLD. These data are supported by several meta-analyses. Hassani Zadeh et al[93] showed that MD, compared to a Western diet, resulted in reduced cardiovascular risk. Similarly, Kawaguchi et al[94], analysing six studies, found that EVOO reduced the Fatty Liver Index and IR assessed according to the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR). These studies, however, may underestimate the possible role of confounder factors (e.g., other MD components). The beneficial effects on MAFLD would seem to be attributed to EVOO’s positive effect on the metabolic pathways of MetS, reducing cardiovascular risk and, in general, both the morbidity and mortality of these patients[95-99].

Some studies suggest that the effectiveness of the MD is specifically related to the intake of high amounts of EVOO[100]. According to the International Olive Council, EVOO is made up of 98%-99% TGs, and its monounsaturated fatty acids (MUFA), which range from between 55% and 83% of the overall quantity of fatty acids, consists primarily of omega-9 oleic acid (C18:1). Other, less prevalent MUFAs in EVOO are omega-7 palmitoleic acid (C16:1), heptadecenoic acid (C17:1) and galdoleic/9-eicosenoic acid (C20:1, -11). Besides MUFAs, EVOO contains polyunsaturated fatty acids (PUFA), including linoleic (C18:2, omega-6) and α-linolenic (C18:3, omega-3) acids with a high ratio of omega-6/omega-3. In addition, EVOO has a low proportion of saturated fatty acids, including margaric (C17:0), stearic (C18:0), arachidonic (C20:0), behenic (C22:0), lignoceric (C24:0), palmitic (C16:0), and myristic (C14:0) acids. EVOO also contains several, quantitatively less prevalent, minor substances, including soluble vitamins (beta-carotene, tocopherols), squalene, pigments (chlorophyll, carotenes), phytosterols, alcoholic triterpenes, and polyphenols. The polyphenols are made up of more than 30 different molecules: Secoiridoids, phenolic alcohols (mainly hydroxytyrosol and tyrosol), phenolic acids, lignans, and flavones. The “minor” compounds determine various properties: Colour, odour, aroma, taste, and aftertaste[101]. EVOO’s lipid profile and high omega-6/omega-3 ratio have been linked to its protective effects on cardiovascular, autoimmune, and inflammatory disorders, but also its anti-thrombotic and blood pressure regulatory qualities, and ensuring oxidative stability for a long shelf life[102]. Much evidence indicates that EVOO’s phenolic compounds perform biological activities due to their antioxidant, anti-inflammatory, and chemopreventive properties[103,104].

EVOO AND OXIDATIVE STRESS

A number of studies have concentrated on the ways that polyphenols in EVOO can diminish oxidative stress. Some have shown that LDLs are bound by polyphenols, thus preventing free radicals from oxidizing them[105]. Despite the lack of a complete definition of the mechanism behind the antioxidant activity of EVOO, some researchers have surmised that it may modulate nuclear factor-erythroid 2-related factor 2 expression, resulting in a greater expression of antioxidant molecules[106]. Such action could explain greater glutathione turnover following a meal rich in EVOO, with higher glutathione reductase and peroxidase activity, along with lower post-meal levels of protein carbonyl, lipid peroxide, and plasma hydrogen in the blood, perhaps due to a reduction of nicotinamide adenine dinucleotide phosphate oxidase activity. In addition, oleocanthal and hydroxytyrosol appear to impede copper-induced LDL oxidation by scavenging free radicals and chelating metals[107]. The elevated MUFA content of EVOO significantly lowers LDL-cholesterol and total cholesterol (TC) concentrations, reducing the ratios of TC/HDL and LDL/HDL[108]. In this situation, polyphenols and MUFAs act in synergy, resulting in the inhibition of pancreatic lipases, which delays the post-meal lipemic spike[109], as well as rapid lipid clearance[110]. EVOO additionally effects anti-inflammatory activity by altering pro-inflammatory gene activation and reducing the expression of pro-inflammatory cytokines.

Among the downregulated pro-inflammatory molecules that have been studied in humans are TNF-α, IL-6, IL-1 beta, visfatin, interferon-gamma, and cyclooxygenase-2[111]. In a randomized placebo-controlled crossover trial carried out at the Cardiology Department of the Hospital del Mar and the Municipal Institute of Medical Research, subjects consumed 50 mL of EVOO and refined olive oil (ROO) daily over two 3-week periods following 2-week washout periods. IL-6 (P < 0.002) and C-reactive protein (P = 0.024) were reduced after consumption of EVOO, with no observed changes in soluble intercellular and vascular adhesion molecules or lipid and glucose profiles. EVOO consumption may impart beneficial effects on stable heart disease patients as a treatment added to pharmacological therapies. Research concentrating specifically on MetS patients found that EVOO significantly reduced C-reactive protein values, plasma levels of IL-6, IL-7, and IL-18, and gene expression of pro-inflammatory molecules[111]. The modulation by EVOO of nuclear factor κB (NF-κB), a transcription factor regulating gene transcription in chemokines, inflammatory proteins, cytokines, adhesion molecules, and cyclooxygenase-2 (among others), has an important impact. NF-κB also participates in inflammatory processes linked to atherogenesis. EVOO, specifically its phenols, may alter NF-κB expression, diminishing the inflammatory cascade[112].

EVOO, INTESTINAL PERMEABILITY, AND MASLD

Lifestyle choices can influence intestinal permeability, particularly in relation to energy-rich foods, physical activity, environmental factors, and medication use[113]. In this way, the MD is capable of altering the makeup and variety of the gut microbiome. In a study published in 2000, Manna et al[114] analysed the transepithelial transport and metabolism of 3,4-dihydroxyphenylethanol (DPE) in Caco-2 cells, a model of the human intestinal epithelium. The aim of this paper was to elucidate the kinetics of[114] DPE intestinal transport and its metabolism, using differentiated Caco-2 cell monolayers as the model system of the human intestinal epithelium. As a matter of fact, these cells, which closely mimic in vitro the food-intestinal tract interaction, have been successfully utilized to demonstrate DPE’s protective effect against ROS-mediated cytotoxicity. In conclusion, DPE has high bioavailability, confirming its potential beneficial role in the body. The nutritional benefits of olive oil are also linked to the presence of DPE and oleuropein.

Another subsequent study in an animal model aiming to assess the impact of EVOO on the gut microbiome, mucosal immunity, barrier integrity and metabolic health showed that, in C57BL/6 J mice who were exposed to a low-fat, lard [high fat (HF)], HF-EVOO, or HF-flaxseed oil diet for 10 weeks, the consumption of EVOO or flaxseed oil could beneficially impact the gut microbiome, enhance gut immunity, and assist in the preservation of metabolic health[115]. Several studies have focused on EVOO and its compounds, highlighting numerous beneficial effects on the various components of MetS. These include a quantitative reduction of body weight[116], a quantitative reduction of adipose tissue and positive metabolic modulation[104], a reduction of the lipid profile of TC, TC/HDL ratio, and LDL oxidation, and an increase in HDL concentrations[117], a reduction of glycaemic metabolism in the incidence of T2DM, fasting glucose levels, advanced glycation end-products (including glycated haemoglobin), and the activation of the GLP-1 incretin pathway[118].

Various metabolic pathways have been suggested to account for EVOO’s beneficial effects. These include: (1) Increased nuclear factor-erythroid 2-related factor 2 expression, leading to the enhancement of antioxidant molecules[129]; (2) Diminished activation of NF-kB, resulting in the adhesion molecules, chemokines, cytokines and enzymes involved in inflammation and oxidative stress being downregulated[120]; (3) Activation of the phosphoinositide 3-kinase/protein kinase B and mitogen-activated protein kinases (extracellular signal-regulated kinase and p38) signalling pathways, inducing endothelial nitric oxide synthase phosphorylation and modulating NO levels[121,122]; (4) Increased absorption of MUFAs and PUFAs, leading to a reduction in TC and LDL, as well as a decrease in the ratios of LDL/HDL and TC/HDL[112]; and (5) Changes to the composition of the gut microbiome and metabolic alterations, influencing intestinal permeability, antigen exposure, immune cell response, inflammation and oxidative stress[123] (Figure 3).

Figure 3 Metabolic pathways involved in the potential role of extra virgin olive oil in the restoration of normal intestinal permeability and hepatoprotection. Nrf2: Nuclear factor erythroid-2-related factor 2; NK cell: Natural Killer cell; IFNγ: Interferon γ.

Furthermore, mechanistic hypotheses linking the interconnected roles of EVOO, the intestinal microbiota, the enterohepatic circulation of BAs, and MASLD can be made in light of some of the literature data[46]. The well-known close correlation between diet-induced obesity, liver steatosis, and associated changes in the microbiota has also been explained by their effects on the BA profile and altered FXR signalling. In fact, the gut microbiota affects BA metabolism and, vice versa, BA can directly modulate bacterial growth in the gut by promoting FXR-induced expression of genes producing antimicrobial agents. In this perspective, Parséus et al[124] have suggested that FXR may contribute to increased adiposity by shifting the gut microbiota to a more obesogenic configuration. An EVOO-enriched diet, with its tendency to modify the intestinal microbial profile with a relative increase in the taxa within the Bacteroides phylum, could thus contribute to a reversion to the “lean” phenotype. Several research studies have suggested that EVOO may help reduce liver fat accumulation, reducing hepatic TG accumulation via DNL inhibition[13]. It also reduces oxidation and inflammation, potentially preventing or reversing MASLD and MASH, but so far, there is no conclusive evidence from the literature on these aspects.

In fact, a recent meta-analysis of seven clinical studies involving 529 participants (60.9% men) examined the effects of EVOO on various biochemical markers and body mass index, providing new insights into its potential benefits for liver health. The study assessed the connection between the consumption of olive oil and NAFLD, specifically its impact on liver enzymes such as aspartate aminotransferase. Subsequent tests examined biochemical and anthropometric parameters. The results showed that olive oil intake did not significantly improve biochemical markers (lipid metabolism, transaminase levels, cholestasis) or liver fat accumulation more than control diets, though this was probably due to an insufficient intervention duration. However, it did result in a significant reduction in body mass index (-0.57 kg/m²; 95% confidence interval: -1.08 to -0.06 kg/m²; P = 0.03).

Despite these findings, several factors need to be considered. The randomized clinical trials included in the analysis showed a high degree of heterogeneity, with variations in the quantity (effective clinical dosage range of 20-50 mg/day, polyphenol threshold > 300 mg/kg) and method of olive oil administration (capsules, addition to the diet, etc.), different dietary strategies, and various comparison substances. Moreover, the study generally considered olive oil without specifically focusing on EVOO, a crucial distinction given its unique composition that distinguishes it from regular olive oil[125]. The data reported in this meta-analysis led us to the hypothesis that benefits to liver function and potential reduction of steatosis would only be likely to come from olive oil with particular chemical characteristics, that is, EVOO. But despite limiting our database research to the most prominent in vitro, animal, and human studies that focused on analysing the impact of EVOO or its constituents on NAFLD/MASLD, it remains challenging to determine a definitive answer regarding EVOO’s effects on liver steatosis.

Indeed, although most of the studies concluded that there was EVOO conferred substantial benefits in terms of fat deposition in the liver as well as both general and liver-specific oxidative and inflammatory metabolism, the studies varied so widely in their design and methodological approaches that it is difficult to effectively compare them. Notably, the vast majority of the experiments were conducted using animal models, with very few using human subjects. This was a subgroup analysis nested within a multicentre, randomized, parallel-group clinical trial, PREvención con DIeta MEDiterránea (PREDIMED trial: No. ISRCTN35739639), aimed at assessing the effect of the MD on the primary prevention of cardiovascular disease. One hundred men and women (mean age: 64 ± 6 years) at high cardiovascular risk (62% with type 2 diabetes) from the Bellvitge-PREDIMED centre were randomly assigned to an MD supplemented with EVOO, an MD supplemented with mixed nuts, or a control diet (advice to reduce all dietary fat). No recommendations to lose weight or increase physical activity were given. The primary measurements were the percentage of liver fat and the diagnosis of steatosis, which were determined by magnetic resonance imaging. The association of diet with liver fat content was analysed by bivariate analysis after a median follow-up of 3 years. An energy-unrestricted MD supplemented with EVOO, a food with potent antioxidant and anti-inflammatory properties, is associated with a reduced prevalence of hepatic steatosis in older individuals at high cardiovascular risk[125].

Other experiments carried out with human subjects have demonstrated an inverse correlation between levels of EVOO components found in the liver, for example, sitosterol, and the extent of steatosis and lobular inflammation revealed by liver biopsy[126]. The specific elements of EVOO providing benefits to the liver comprise chlorophyll compounds, carotenoid pigments, phenols, tocopherols, and phytosterols. Their protective benefits to NAFLD may stem from their protection of mitochondria, preventing the reduction of levels of nitrated fatty acids induced by high dietary fat intake[127]. The phenols found in EVOO are directly responsible for this protective role, and it is believed that olive oil can act to lower liver fat deposition by itself, possibly providing a boost to the oxidation of fatty acids[128]. In addition to EVOO’s impact on reducing the oxidation of LDL, studies have proposed that improvements in NAFLD may stem from diminished activation of NF-κB and benefits to IR[129]. Moreover, experiments have demonstrated that several constituents of the MD have the ability to benefit NAFLD by affecting the transcription of genes related to metabolism.

The previously cited meta-analysis conducted by Tsamos et al[123] suggests that incorporating olive oil into the diet may bring about moderate, yet significant, benefits to body mass and composition. The particular characteristics of olive oil, specifically its central role in the MD and well-known benefits to cardiovascular as well as general health and increased life span, make it a highly recommended food for everyday dietary intake for all ages[123]. The apparent systemic benefits of EVOO derive from its ability to affect inflammation and reduce tissue damage from oxidative stress[130], and it has the same impact on the liver, possibly diminishing damage from inflammation and decreasing the accumulation of extracellular matrix proteins, thereby reducing inflammation and hepatic fibrosis. EVOO seems capable of directly acting on the metabolism of glucose and lipids, lowering the overall risk of cardiovascular disease and improving long-term mortality risk[131]. Improvements in the metabolism of glucose relate to insulin production, acting in synergy in the pancreas with GLP-1[132], as well as the insulin sensitivity of peripheral tissues[133]. Benefits to fat metabolism seem to be connected to higher MUFA and PUFA consumption, which consequently reduces TC, TG, LDL, and oxidized LDL, increases HDL, and reduces belly fat, along with improving its metabolism[134].

CONCLUSION

Current evidence indicates that intestinal barrier functions and composition can be regulated in general by components of the MD, and EVOO polyphenols in particular, by alleviating inflammation and, consequently, liver fat accumulation. However, many studies are difficult to compare because they only analyse animal models, and these models utilize different species, induce liver damage in varying ways, administer different amounts of EVOO, and use different approaches to assess hepatic damage. In addition, the precise quantity of EVOO consumed, its specific composition, and other factors could not able to be determined, rendering a broader analysis more arduous. However, given the preliminary results and the strong physio-pathological basis, prospective trials are needed to delve deeper and resolve the issue.