The Effects of Probiotics,Prebiotics and Synbiotics in Non-Alcoholic Fat Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH): A Systematic Review

Modifications in the microbiota caused by environmental and genetic reasons can unbalance the intestinal homeostasis, deregulating the host’s metabolism and immune system, intensifying the risk factors for the development and aggravation of non-alcoholic fat liver disease (NAFLD). The use of probiotics, prebiotics and synbiotics have been considered a potential and promising strategy to regulate the gut microbiota and produce beneficial effects in patients with liver conditions. For this reason, this review aimed to evaluate the effectiveness of probiotics, prebiotics, and symbiotics in patients with NAFLD and NASH. Pubmed, Embase, and Cochrane databases were consulted, and PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines were followed. The clinical trials used in this study demonstrated that gut microbiota interventions could improve a wide range of markers of inflammation, glycemia, insulin resistance, dyslipidemia, obesity, liver injury (decrease of hepatic enzymes and steatosis and fibrosis). Although microbiota modulators do not play a healing role, they can work as an important adjunct therapy in pathological processes involving NAFLD and its spectrums, either by improving the intestinal barrier or by preventing the formation of toxic metabolites for the liver or by acting on the immune system.     

Plasma Levels of Homocysteine and Cysteine Increased in Pediatric NAFLD and Strongly Correlated with Severity of Liver Damage

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of metabolic abnormalities ranging from simple triglyceride accumulation in the hepatocytes to hepatic steatosis with inflammation, ballooning and fibrosis. It has been demonstrated that the pathogenesis of NAFLD involves increased oxidative stress, with consumption of the major cellular antioxidant, glutathione (GSH). Liver has a fundamental role in sulfur compound metabolism, although the data reported on plasma thiols status in NAFLD are conflicting. We recruited 63 NAFLD patients, and we analyzed all plasma thiols, such as homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly) and GSH, by high-performance liquid chromatography (HPLC) with fluorescence detection. Hcy, Cys and CysGly plasma levels increased in NAFLD patients (p < 0.0001); whereas GSH levels were decreased in NAFLD patients when compared to controls (p < 0.0001). On the contrary, patients with steatohepatitis exhibited lower levels of Hcy and Cys than subjects without. Furthermore, a positive correlation was found between Hcy and Cys and the presence of fibrosis in children with NAFLD. Taken together, these data demonstrated a defective hepatic sulfur metabolism in children with NAFLD, and that high levels of Hcy and Cys probably correlates with a pattern of more severe histological liver damage, due to mechanisms that require further studies.     

Role of Endocrine-Disrupting Chemicals in the Pathogenesis of Non-Alcoholic Fatty Liver Disease: A Comprehensive Review

Non-alcoholic fatty liver disease (NAFLD) is considered the most common liver disorder, affecting around 25% of the population worldwide. It is a complex disease spectrum, closely linked with other conditions such as obesity, insulin resistance, type 2 diabetes mellitus, and metabolic syndrome, which may increase liver-related mortality. In light of this, numerous efforts have been carried out in recent years in order to clarify its pathogenesis and create new prevention strategies. Currently, the essential role of environmental pollutants in NAFLD development is recognized. Particularly, endocrine-disrupting chemicals (EDCs) have a notable influence. EDCs can be classified as natural (phytoestrogens, genistein, and coumestrol) or synthetic, and the latter ones can be further subdivided into industrial (dioxins, polychlorinated biphenyls, and alkylphenols), agricultural (pesticides, insecticides, herbicides, and fungicides), residential (phthalates, polybrominated biphenyls, and bisphenol A), and pharmaceutical (parabens). Several experimental models have proposed a mechanism involving this group of substances with the disruption of hepatic metabolism, which promotes NAFLD. These include an imbalance between lipid influx/efflux in the liver, mitochondrial dysfunction, liver inflammation, and epigenetic reprogramming. It can be concluded that exposure to EDCs might play a crucial role in NAFLD initiation and evolution. However, further investigations supporting these effects in humans are required.     

Significance of Circulating Cell-Free DNA Species in Non-Alcoholic Fatty Liver Disease

The pathogenetic mechanisms involved in the progression of non-alcoholic fatty liver disease (NAFLD) have not been completely elucidated, while the significance of circulating cell-free DNA (cf-DNA) species has been rarely evaluated in NAFLD. Herein, we assessed the serum levels of cf-DNA species in NAFLD patients and investigated their potential associations with patients’ characteristics and severity of liver disease. Forty-nine adult patients with NAFLD of any stage were included in this cohort study. Cf-DNA was isolated from patients’ sera and the levels of several distinct cf-DNA species including total cf-DNA, gene-coding cf-DNA, Alu repeat sequences, mitochondrial DNA copies and 5-methyl-2′-deoxycytidine were determined. Cirrhotic compared to non-cirrhotic patients had significantly lower serum levels of cf-DNA and RNAse P coding DNA as well as higher expression of 5-methyl-2′-deoxycytidine. After adjustment for the significant clinico-epidemiological factors, lower serum levels of cf-DNA or RNAse P were independently associated with the presence of cirrhosis. Serum levels of total and gene-coding DNA are associated with the presence of cirrhosis in NAFLD patients regardless of clinical or epidemiological parameters and may therefore be used as a screening tool for NAFLD progression.     

Lessons from Mouse Models of High-Fat Diet-Induced NAFLD

Nonalcoholic fatty liver disease (NAFLD) encompasses a clinicopathologic spectrum of diseases ranging from isolated hepatic steatosis to nonalcoholic steatohepatitis (NASH), the more aggressive form of fatty liver disease that may progress to cirrhosis and cirrhosis-related complications, including hepatocellular carcinoma. The prevalence of NAFLD, including NASH, is also increasing in parallel with the growing epidemics of obesity and diabetes. However, the causal relationships between obesity and/or diabetes and NASH or liver tumorigenesis have not yet been clearly elucidated. Animal models of NAFLD/NASH provide crucial information, not only for elucidating the pathogenesis of NAFLD/NASH, but also for examining therapeutic effects of various agents. A high-fat diet is widely used to produce hepatic steatosis and NASH in experimental animals. Several studies, including our own, have shown that long-term high-fat diet loading, which can induce obesity and insulin resistance, can also induce NASH and liver tumorigenesis in C57BL/6J mice. In this article, we discuss the pathophysiology of and treatment strategies for NAFLD and subsequent NAFLD-related complications such as NASH and liver tumorigenesis, mainly based on lessons learned from mouse models of high-fat diet-induced NAFLD/NASH.     

The Alterations of Mitochondrial Function during NAFLD Progression—An Independent Effect of Mitochondrial ROS Production

The progression of non-alcoholic fatty liver (NAFL) into non-alcoholic steatohepatitis implicates multiple mechanisms, chief of which is mitochondrial dysfunction. However, the sequence of events underlying mitochondrial failure are still poorly clarified. In this work, male C57BL/6J mice were fed with a high-fat plus high-sucrose diet for 16, 20, 22, and 24 weeks to induce NAFL. Up to the 20th week, an early mitochondrial remodeling with increased OXPHOS subunits levels and higher mitochondrial respiration occurred. Interestingly, a progressive loss of mitochondrial respiration along “Western diet” feeding was identified, accompanied by higher susceptibility to mitochondrial permeability transition pore opening. Importantly, our findings prove that mitochondrial alterations and subsequent impairment are independent of an excessive mitochondrial reactive oxygen species (ROS) generation, which was found to be progressively diminished along with disease progression. Instead, increased peroxisomal abundance and peroxisomal fatty acid oxidation-related pathway suggest that peroxisomes may contribute to hepatic ROS generation and oxidative damage, which may accelerate hepatic injury and disease progression. We show here for the first time the sequential events of mitochondrial alterations involved in non-alcoholic fatty liver disease (NAFLD) progression and demonstrate that mitochondrial ROS are not one of the first hits that cause NAFLD progression.     

The Dipeptidyl Peptidase-4 Inhibitor Teneligliptin Attenuates Hepatic Lipogenesis via AMPK Activation in Non-Alcoholic Fatty Liver Disease Model Mice

Non-alcoholic fatty liver disease (NAFLD), which is strongly associated with metabolic syndrome, is increasingly a major cause of hepatic disorder. Dipeptidyl peptidase (DPP)-4 inhibitors, anti-diabetic agents, are expected to be effective for the treatment of NAFLD. In the present study, we established a novel NAFLD model mouse using monosodium glutamate (MSG) and a high-fat diet (HFD) and investigated the effects of a DPP-4 inhibitor, teneligliptin, on the progression of NAFLD. Male MSG/HFD-treated mice were divided into two groups, one of which received teneligliptin in drinking water. Administration of MSG and HFD caused mice to develop severe fatty changes in the liver, but teneligliptin treatment improved hepatic steatosis and inflammation, as evaluated by the NAFLD activity score. Serum alanine aminotransferase and intrahepatic triglyceride levels were significantly decreased in teneligliptin-treated mice (p < 0.05). Hepatic mRNA levels of the genes involved in de novo lipogenesis were significantly downregulated by teneligliptin (p < 0.05). Moreover, teneligliptin increased hepatic expression levels of phosphorylated AMP-activated protein kinase (AMPK) protein. These findings suggest that teneligliptin attenuates lipogenesis in the liver by activating AMPK and downregulating the expression of genes involved in lipogenesis. DPP-4 inhibitors may be effective for the treatment of NAFLD and may be able to prevent its progression to non-alcoholic steatohepatitis.     

Extracellular Vesicles: A New Frontier in Biomarker Discovery for Non-Alcoholic Fatty Liver Disease

In recent years, the global burden of obesity and diabetes has seen a parallel rise in other metabolic complications, such as non-alcoholic fatty liver disease (NAFLD). This condition, once thought to be a benign accumulation of hepatic fat, is now recognized as a serious and prevalent disorder that is conducive to inflammation and fibrosis. Despite the rising incidence of NAFLD, there is currently no reliable method for its diagnosis or staging besides the highly invasive tissue biopsy. This limitation has resulted in the study of novel circulating markers as potential candidates, one of the most popular being extracellular vesicles (EVs). These submicron membrane-bound structures are secreted from stressed and activated cells, or are formed during apoptosis, and are known to be involved in intercellular communication. The cargo of EVs depends upon the parent cell and has been shown to be changed in disease, as is their abundance in the circulation. The role of EVs in immunity and epigenetic regulation is widely attested, and studies showing a correlation with disease severity have made these structures a favorable target for diagnostic as well as therapeutic purposes. This review will highlight the research that is available on EVs in the context of NAFLD, the current limitations, and projections for their future utility in a clinical setting.     

Contribution of Dysregulated DNA Methylation to Autoimmunity

Epigenetic mechanisms, such as DNA methylation, histone modifications, and non-coding RNAs are known regulators of gene expression and genomic stability in cell growth, development, and differentiation. Because epigenetic mechanisms can regulate several immune system elements, epigenetic alterations have been found in several autoimmune diseases. The purpose of this review is to discuss the epigenetic modifications, mainly DNA methylation, involved in autoimmune diseases in which T cells play a significant role. For example, Rheumatoid Arthritis and Systemic Lupus Erythematosus display differential gene methylation, mostly hypomethylated 5′-C-phosphate-G-3′ (CpG) sites that may associate with disease activity. However, a clear association between DNA methylation, gene expression, and disease pathogenesis must be demonstrated. A better understanding of the impact of epigenetic modifications on the onset of autoimmunity will contribute to the design of novel therapeutic approaches for these diseases.     

Molecular Mechanisms and New Treatment Strategies for Non-Alcoholic Steatohepatitis (NASH)

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD), in which most patients exhibit non-progressive, non-alcoholic fatty liver (NAFL) attributable to simple steatosis. Multiple hits, including genetic differences, fat accumulation, insulin resistance and intestinal microbiota changes, account for the progression of NASH. NAFLD is strongly associated with obesity, which induces adipokine secretion, endoplasmic reticulum (ER) and oxidative stress at the cellular level, which in turn induces hepatic steatosis, inflammation and fibrosis. Among these factors, gut microbiota are acknowledged as having an important role in initiating this multifactorial disease. Oxidative stress is considered to be a key contributor in the progression from NAFL to NASH. Macrophage infiltration is apparent in NAFL and NASH, while T-cell infiltration is apparent in NASH. Although several clinical trials have shown that antioxidative therapy with vitamin E can effectively control hepatitis pathology in the short term, the long-term effects remain obscure and have often proved to be ineffective in many other diseases. Several long-term antioxidant protocols have failed to reduce mortality. New treatment modalities that incorporate current understanding of NAFLD molecular pathogenesis must be considered.     

Epigenetic Mechanisms in Penile Carcinoma

Penile carcinoma (PeCa) represents an important public health problem in poor and developing countries. Despite its unpredictable behavior and aggressive treatment, there have only been a few reports regarding its molecular data, especially epigenetic mechanisms. The functional diversity in different cell types is acquired by chromatin modifications, which are established by epigenetic regulatory mechanisms involving DNA methylation, histone acetylation, and miRNAs. Recent evidence indicates that the dysregulation in these processes can result in the development of several diseases, including cancer. Epigenetic alterations, such as the methylation of CpGs islands, may reveal candidates for the development of specific markers for cancer detection, diagnosis and prognosis. There are a few reports on the epigenetic alterations in PeCa, and most of these studies have only focused on alterations in specific genes in a limited number of cases. This review aims to provide an overview of the current knowledge of the epigenetic alterations in PeCa and the promising results in this field. The identification of epigenetically altered genes in PeCa is an important step in understanding the mechanisms involved in this unexplored disease.     

Discovering Biomarkers for Non-Alcoholic Steatohepatitis Patients with and without Hepatocellular Carcinoma Using Fecal Metaproteomics

High-calorie diets lead to hepatic steatosis and to the development of non-alcoholic fatty liver disease (NAFLD), which can evolve over many years into the inflammatory form of non-alcoholic steatohepatitis (NASH), posing a risk for the development of hepatocellular carcinoma (HCC). Due to diet and liver alteration, the axis between liver and gut is disturbed, resulting in gut microbiome alterations. Consequently, detecting these gut microbiome alterations represents a promising strategy for early NASH and HCC detection. We analyzed medical parameters and the fecal metaproteome of 19 healthy controls, 32 NASH patients, and 29 HCC patients, targeting the discovery of diagnostic biomarkers. Here, NASH and HCC resulted in increased inflammation status and shifts within the composition of the gut microbiome. An increased abundance of kielin/chordin, E3 ubiquitin ligase, and nucleophosmin 1 represented valuable fecal biomarkers, indicating disease-related changes in the liver. Although a single biomarker failed to separate NASH and HCC, machine learning-based classification algorithms provided an 86% accuracy in distinguishing between controls, NASH, and HCC. Fecal metaproteomics enables early detection of NASH and HCC by providing single biomarkers and machine learning-based metaprotein panels.     

The New Therapeutic Approaches in the Treatment of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease which is characterized by extremely complex pathogenetic mechanisms and multifactorial etiology. Some of the many pathophysiological mechanisms involved in the development of NAFLD include oxidative stress, impaired mitochondrial metabolism, inflammation, gut microbiota, and interaction between the brain-liver-axis and the regulation of hepatic lipid metabolism. The new therapeutic approaches in the treatment of NAFLD are targeting some of these milestones along the pathophysiological pathway and include drugs like agonists of peroxisome proliferator-activated receptors (PPARs), glucagon-like peptide-1 (GLP-1) agonists, sodium/glucose transport protein 2 (SGLT2) inhibitors, farnesoid X receptor (FXR) agonists, probiotics, and symbiotics. Further efforts in biomedical sciences should focus on the investigation of the relationship between the microbiome, liver metabolism, and response to inflammation, systemic consequences of metabolic syndrome.     

MAFLD and CKD: An Updated Narrative Review

Accumulating evidence now indicates that non-alcoholic fatty liver disease (NAFLD), which is the most common chronic liver disease observed in clinical practice worldwide, is independently associated with an increased risk of incident chronic kidney disease (CKD). Given that NAFLD is linked to insulin resistance, obesity and type 2 diabetes mellitus, an international panel of experts have recently proposed a name change from NAFLD to metabolic associated fatty liver disease (MAFLD). Since the diagnostic criteria for NAFLD and MAFLD are different, observational studies assessing the potential concordance (or even superiority) of MAFLD, compared with NAFLD, in detecting patients at increased risk of hepatic and extra-hepatic complications (including CKD) are required. Hence, in the last two years, some observational studies have investigated the potential relationship between MAFLD and CKD. The result is that, at present, evidence regarding the concordance or even superiority of MAFLD, compared with NAFLD, in detecting patients at higher risk of CKD is still preliminary, although some data indicate that MAFLD identifies patients with CKD as accurately as NAFLD. In this narrative review, we will discuss: (a) the epidemiological evidence assessing the association between NAFLD and risk of incident CKD, (b) the epidemiological data investigating the association between MAFLD and risk of CKD and (c) the biological mechanisms underlying the association between NAFLD/MAFLD and CKD.     

Cannabinoids and Chronic Liver Diseases

Nonalcoholic fatty liver disease (NAFLD), alcohol-induced liver disease (ALD), and viral hepatitis are the main causes of morbidity and mortality related to chronic liver diseases (CLDs) worldwide. New therapeutic approaches to prevent or reverse these liver disorders are thus emerging. Although their etiologies differ, these CLDs all have in common a significant dysregulation of liver metabolism that is closely linked to the perturbation of the hepatic endocannabinoid system (eCBS) and inflammatory pathways. Therefore, targeting the hepatic eCBS might have promising therapeutic potential to overcome CLDs. Experimental models of CLDs and observational studies in humans suggest that cannabis and its derivatives may exert hepatoprotective effects against CLDs through diverse pathways. However, these promising therapeutic benefits are not yet fully validated, as the few completed clinical trials on phytocannabinoids, which are thought to hold the most promising therapeutic potential (cannabidiol or tetrahydrocannabivarin), remained inconclusive. Therefore, expanding research on less studied phytocannabinoids and their derivatives, with a focus on their mode of action on liver metabolism, might provide promising advances in the development of new and original therapeutics for the management of CLDs, such as NAFLD, ALD, or even hepatitis C-induced liver disorders.     

Treatments for NAFLD: State of Art

Non-alcoholic fatty liver disease (NAFLD) is to date the most common chronic liver disease in clinical practice and, consequently, a major health problem worldwide. It affects approximately 30% of adults in the general population and up to 70% of patients with type 2 diabetes (T2DM). Despite the current knowledge of the epidemiology, pathogenesis, and natural history of NAFLD, no specific pharmacological therapies are until now approved for this disease and, consequently, general strategies have been proposed to manage it. They include: (a) lifestyle change in order to promote weight loss by diet and physical activity, (b) control of the main cardiometabolic risk factors, (c) correction of all modifiable risk factors leading the development and progression of advanced forms of NAFLD, and (d) prevention of hepatic and extra-hepatic complications. In the last decade, several potential agents have been widely investigated for the treatment of NAFLD and its advanced forms—shedding some light but casting a few shadows. They include some glucose-lowering drugs (such as pioglitazone, glucagon-like peptide-1 (GLP-1) receptor agonists, sodium-glucose co-transporter-2 (SGLT-2) inhibitors), antioxidants (such as vitamin E), statins or other lipid lowering agents, bile and non-bile acid farnesoid X activated receptor (FXR) agonists, and others. This narrative review discusses in detail the different available approaches with the potential to prevent and treat NAFLD and its advanced forms.     

Methylated DNA and microRNA in Body Fluids as Biomarkers for Cancer Detection

Epigenetic alterations including DNA methylation and microRNAs (miRNAs) play important roles in the initiation and progression of human cancers. As the extensively studied epigenetic changes in tumors, DNA methylation and miRNAs are the most potential epigenetic biomarkers for cancer diagnosis. After the identification of circulating cell-free nuclear acids, increasing evidence demonstrated great potential of cell-free epigenetic biomarkers in the blood or other body fluids for cancer detection.     

Cancer Development,Progression, and Therapy: An Epigenetic Overview

Carcinogenesis involves uncontrolled cell growth, which follows the activation of oncogenes and/or the deactivation of tumor suppression genes. Metastasis requires down-regulation of cell adhesion receptors necessary for tissue-specific, cell–cell attachment, as well as up-regulation of receptors that enhance cell motility. Epigenetic changes, including histone modifications, DNA methylation, and DNA hydroxymethylation, can modify these characteristics. Targets for these epigenetic changes include signaling pathways that regulate apoptosis and autophagy, as well as microRNA. We propose that predisposed normal cells convert to cancer progenitor cells that, after growing, undergo an epithelial-mesenchymal transition. This process, which is partially under epigenetic control, can create a metastatic form of both progenitor and full-fledged cancer cells, after which metastasis to a distant location may occur. Identification of epigenetic regulatory mechanisms has provided potential therapeutic avenues. In particular, epigenetic drugs appear to potentiate the action of traditional therapeutics, often by demethylating and re-expressing tumor suppressor genes to inhibit tumorigenesis. Epigenetic drugs may inhibit both the formation and growth of cancer progenitor cells, thus reducing the recurrence of cancer. Adopting epigenetic alteration as a new hallmark of cancer is a logical and necessary step that will further encourage the development of novel epigenetic biomarkers and therapeutics.     

Role of Insulin Resistance in MAFLD

Many studies have reported that metabolic dysfunction is closely involved in the complex mechanism underlying the development of non-alcoholic fatty liver disease (NAFLD), which has prompted a movement to consider renaming NAFLD as metabolic dysfunction-associated fatty liver disease (MAFLD). Metabolic dysfunction in this context encompasses obesity, type 2 diabetes mellitus, hypertension, dyslipidemia, and metabolic syndrome, with insulin resistance as the common underlying pathophysiology. Imbalance between energy intake and expenditure results in insulin resistance in various tissues and alteration of the gut microbiota, resulting in fat accumulation in the liver. The role of genetics has also been revealed in hepatic fat accumulation and fibrosis. In the process of fat accumulation in the liver, intracellular damage as well as hepatic insulin resistance further potentiates inflammation, fibrosis, and carcinogenesis. Increased lipogenic substrate supply from other tissues, hepatic zonation of Irs1, and other factors, including ER stress, play crucial roles in increased hepatic de novo lipogenesis in MAFLD with hepatic insulin resistance. Herein, we provide an overview of the factors contributing to and the role of systemic and local insulin resistance in the development and progression of MAFLD.