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Background: High risk HPVs (HR-HPVs) are DNA viruses considered as primary etiologic factors in malignancies of the low female genital tract. Their presence has also been documented in oropharyngeal and laryngeal cancers. However, HPV infection is considered a necessary but not sufficient cause of tumoral development; meantime, increasing evidences on the tumorigenic role of cancer stem cells (CSCs) have been documented in the literature. CSCs represent a small subpopulation of neoplastic cells with self-renewal potential, capable of maintaining tumor growth and cell differentiation, also involved in metastatic process, recurrence and resistance to chemotherapeutic agents. In the present study, performed on KB cell lines, we evaluated the tumor forming potential of CSCs, and their relationship with the HPV infection status. Methods: We started our study by identifying the most aggressive cell line on the minimal number of cells able of growth in vivo in a model of athymic nude mice (BALB/c nu/nu). We used an oral-derived KB cell-line separated in the KB-CD133+ and KB-CD133- populations, by using immunomagnetic beads and fluorescence-activated cell sorting (FACS). The separated populations were injected in athymic nude mice (BALB/c nu/nu). Xenograft tumors have been analysed for tumor size, CD133 expression by immunohistochemistry (IHC) and for DNA HR-HPV integration by in situ hybridization (ISH), comparing CD133 enriched xenograft tumors versus the CD133 non-enriched ones. Results: On standard conditions the KB cell line has a poor population of glycosylated CD133 marker (

Oral squamous cell carcinoma (OSCC) is one of the most aggressive and deadliest tumors worldwide. The aryl hydrocarbon receptor (AHR) is a nuclear transcription factor known as a dioxin receptor and mediates the toxic effects of industrial contaminants. In addition, AHR has been implicated in multiple cellular processes and its expression has been shown to play a critical role in tumorigenesis, including human oral cancer cell lines. In the present study, we evaluated the expression of AHR/HSP-90 in 25formalin‑fixed, paraffin-embedded human oral cancer specimens by IHC analysis. CYP1A1 expression was regarded as an AHR reporter gene. The data indicated a complete correlation between AHR expression and cancer grade enabling us to propose AHR as a prognostic marker of oral cancer. Moreover, in OSCC cell line CAL27, we observed the modulatory effect of polydatin, a widespread natural substance and direct precursor of resveratrol, on AHR expression. A computational approach was performed to predict the site of interaction of polydatin on the AHR surface. Our studies confirm the involvement of AHR signaling in the clinicopathological specimens of oral cancer and suggest the use of polydatin for oral cancer prevention.

Health promotion strategies and lifestyle changes are important in disease prevention. Oral health has received a large amount of attention previously as it is a fundamental component of general health and it contributes to the quality of life. Therefore, the study of associations between diet, health and the presence of bioactive compounds in food is receiving a substantial amount of attention. In the present review the effects and targets of a natural polyohenolic stilbenoid compound; resveratrol (3,5,4'-trihydroxystilbene; RSV) is assessed, and the future prospects for RSV in promoting oral health are considered. RSV is a phytoalexin, synthesized by a wide range of plants and abundantly extracted in grape skin, it has been purported to exert a multiplicity of anti-inflammatory, anti-viral, anti-microbial, estrogenic, anticancer, cardioprotective, neuroprotective and immunomodulatory functions. In this review, following an introduction documenting the biochemistry of RSV and RSV glucosides, the bioavailability and pharmacokinetics of RSV are described. Considering its multiple properties, the present review has focused on the potential benefits of RSV as an antioxidant and chemopreventive agent.

Health promotion strategies and lifestyle changes are important in disease prevention. Oral health has received a large amount of attention previously as it is a fundamental component of general health and it contributes to the quality of life. Therefore, the study of associations between diet, health and the presence of bioactive compounds in food is receiving a substantial amount of attention. In the present review the effects and targets of a natural polyohenolic stilbenoid compound; resveratrol (3,5,4'-trihydroxystilbene; RSV) is assessed, and the future prospects for RSV in promoting oral health are considered. RSV is a phytoalexin, synthesized by a wide range of plants and abundantly extracted in grape skin, it has been purported to exert a multiplicity of anti-inflammatory, anti-viral, anti-microbial, estrogenic, anticancer, cardioprotective, neuroprotective and immunomodulatory functions. In this review, following an introduction documenting the biochemistry of RSV and RSV glucosides, the bioavailability and pharmacokinetics of RSV are described. Considering its multiple properties, the present review has focused on the potential benefits of RSV as an antioxidant and chemopreventive agent.

Background: Seminal vesicle protein number 4 (SV-IV) is a small, basic, multifunctional, intrinsically disordered secretory protein synthesized in large amounts by rat seminal vesicle epithelium under androgen transcriptional control. SV-IV-immunorelated proteins occur in other rat tissues and in humans. Methods: The in vitro effect of SV-IV on human FcΕRI+ cells was investigated by standard immunologic, biochemical and molecular biology procedures. Results: SV-IV-induced histamine release from human basophils and lung mast cells without any influence on leukotriene C4 release and cell migration. The histamine release rate was slower compared with that induced by anti-IgE, the temperature dependence of the event being similar. SV-IV-induced histamine release was Ca2+-dependent, suggesting a physiological interaction of the protein with FcΕRI+ cells. SV-IV and anti-IgE acted synergistically on the histamine release. SV-IV did not induce de novo synthesis of cytokines and growth factors (transforming growth factor-β1, interleukin-10, interleukin-13, tumor necrosis factor-α, vascular endothelial growth factor A) in FcΕRI+ cells. Conclusions: SV-IV protein induces in human FcΕRI+ cells the release of histamine, a proinflammatory, antiapoptotic and immunosuppressive biogenic amine. These data: (1) are consistent with the antiapoptotic and immunosuppressive properties of SV-IV; (2) confirm a regulatory feature of SV-IV on mammal inflammatory reactivity by either inhibiting the arachidonate cascade pathway or stimulating proinflammatory cytokine release from lymphocyte/monocytes and histamine from FcΕRI+ cells; (3) raise the possibility of a protective role of SV-IV on implanting hemiallogenic blastocysts against maternal reactive oxygen species and immunological attacks at the uterine implantation site.

The enzymatic activities of purified horseradish peroxidase, selenium-dependent glutathione peroxidase, thyroid peroxidase and myeloperoxidase, but not that of lactoperoxidase, were markedly enhanced when added into a reaction mixture containing 5 μm native seminal vesicle protein 4, a major protein secreted from rat seminal vesicle epithelium. A further increase of horseradish peroxidase activity was obtained using Ser58-phosphorylated or acetylated seminal vesicle protein 4. The activating effect of native seminal vesicle protein 4 was highest (about 60-fold) on horseradish peroxidase when 4-chloro-1-naphtol was used as the electron donor substrate. The main kinetics parameters of the stimulatory effect on horseradish peroxidase were evaluated and the enzyme–electron donor substrate interaction was investigated by HPLC and electrospray-MS. A native seminal vesicle protein 4/4-chloro-1-naphtol noncovalent adduct was detected when the protein and 4-chloro-1-naphtol were present in the appropriate molar ratio in the horseradish peroxidase-catalyzed reaction. By contrast, no adducts were formed between native seminal vesicle protein 4 and horseradish peroxidase. This native seminal vesicle protein 4/4-chloro-1-naphtol interaction might underlie the native seminal vesicle protein 4-induced horseradish peroxidase stimulation. Furthermore, native seminal vesicle protein 4 was shown by spectrophotometric and electrospray-MS analysis to interact with NADPH, an electron donor substrate of the selenium-dependent glutathione peroxidase/glutathione reductase redox system, with formation of an adduct between them. Although further investigation is required to elucidate the mechanism of adduct formation, this interaction, probably by promoting the release of the NADPH electrons required for glutathione disulphide reduction, could explain the stimulatory effect of seminal vesicle protein 4 on mammalian peroxidases possibly involved in its physiological function on the selenium-dependent glutathione peroxidase/glutathione reductase system. The biological significance of these properties of native seminal vesicle protein 4 might be related to its ability to downregulate reactive oxygen species and oxidative stress-induced apoptosis.

Polydatin, a hydroxystilbene derived from the rhizome of Polygonum cuspidatum, elicits hepatoprotective and neuroprotective effects through its anti-oxidant properties. The present study aimed to determine the effects of oral administration of polydatin in alcoholic patients in order to improve liver biochemical parameters, serum oxidative stress and mental state. We enrolled 20 chronic alcoholic patients hospitalized for rehabilitative therapy. The patients were divided into two groups receiving the following treatment regimes for two weeks: administration of an anti-oxidant nutritional supplement containing glutathione and vitamin C (group 1), or glutathione, vitamin C and polydatin (group 2).The results of the present study show that elevated plasma aspartate aminotransferase and alanine aminotransferase levels in patients after two weeks of alcohol withdrawal were significantly reduced by polydatin (group 2), when compared to group 1. Polydatin also significantly reduced lipid peroxidation levels. Finally, our preliminary data resulting from the analysis of the Mini-Mental Status suggest that polydatin improves cognitive performance.Daily dietary administration of polydatin should be considered for prevention and treatment of liver disease and cognitive impairment in alcoholic patients.

Previous data showing an increase of receptor binding activity of [R16]VIP, a vasoactive intestinal peptide (VIP) structural analogue containing arginine at the position 16 of its amino acid sequence, have pointed out the importance of a positive charge at this site. Here, the functional characterization of three VIP polyaminated adducts (VIPDap, VIPSpd, and VIPSpm), obtained by a transglutaminase-catalysed reaction between the VIP Gln16 residue and 1,3-diaminopropane (Dap), spermidine (Spd), or spermine (Spm), is reported. Appropriate binding assays and adenylate cyclase enzymatic determinations have shown that these VIP adducts act as structural VIP agonists, both in vitro and in vivo. In particular, their IC50 and EC50 values of human and rat VIP/pituitary adenylate cyclase activating peptide (PACAP)1 and VIP/PACAP2 receptors indicate that VIPDap is a VIP agonist, with an affinity and a potency higher than that of VIP, while VIPSpd and VIPSpm are also agonists but with affinities lower than that of VIP. These findings suggest that the difference in adduct agonist activity reflects the differences in the positive charge and carbon chain length of the polyamine covalently linked with the VIP Gln16 residue. In addition, the data obtained strongly suggest that the length of polyamine carbon chain could be critical for the interaction of the agonist with its receptor, even though possible hydrophobic interaction cannot be ruled out. In vivo experiments on murine J774 macrophage cell cultures have shown the ability of these compounds to stimulate the inducible nitric oxide synthase activity at the transcriptional level.

Introduction. Although altered regulation of the Wnt pathway via beta-catenin is a frequent event in several human cancers, its potential implications in oral/oropharyngeal squamous cell carcinomas (OSCC/OPSCC) are largely unexplored. Work purpose was to define association between beta-catenin expression and clinical-pathological parameters in 374 OSCCs/OP-SCCs by immunohistochemistry (IHC).Materials and Methods. Association between IHC detected patterns of protein expression and clinical-pathological parameters was assessed by statistical analysis and survival rates by Kaplan-Meier curves. Beta-catenin expression was also investigated in OSCC cell lines by Real-Time PCR. An additional analysis of the DNA content was performed on 22 representative OSCCs/OPSCCs by DNA-image-cytometric analysis.Results and Discussion. All carcinomas exhibited significant alterations of beta-catenin expression (P<0.05). Beta-catenin protein was mainly detected in the cytoplasm of cancerous cells and only focal nuclear positivity was observed. Higher cytoplasmic expression correlated significantly with poor histological differentiation, advanced stage, and worst patient outcome (P<0.05). By Real-Time PCR significant increase of beta-catenin mRNA was detected in OSCC cell lines and in 45% of surgical specimens. DNA ploidy study demonstrated high levels of aneuploidy in beta-catenin overexpressing carcinomas.Conclusions. This is the largest study reporting significant association between beta-catenin expression and clinical-pathological factors in patients with OSCCs/OPSCCs.

Background Human colon adenocarcinoma cells are resistant to chemotherapeutic agents, such as anthracyclines, that induce death by increasing the reactive oxygen species. A number of studies have been focused on chemo-preventive use of resveratrol as antioxidant against cardiovascular diseases, aging and cancer. While resveratrol cytotoxic action was due to its pro-oxidant properties. In this study, we investigate whether the Resveratrol (trans-3,5,49-trihydroxystilbene) and its natural precursor Polydatin (resveratrol-3-O-b-mono- D-glucoside, the glycoside form of resveratrol) combination, might have a cooperative antitumor effect on either growing or differentiated human adenocarcinoma colon cancer cells. Methods The polydatin and resveratrol pharmacological interaction was evaluated in vitro on growing and differentiated Caco-2 cell lines by median drug effect analysis calculating a combination index with CalcuSyn software. We have selected a synergistic combination and we have evaluated its effect on the biological and molecular mechanisms of cell death. Results Simultaneous exposure to polydatin and resveratrol produced synergistic antiproliferative effects compared with single compound treatment. We demonstrated that polydatin alone or in combination with resveratrol at 3:1 molar ratio synergistically modulated oxidative stress, cell cycle, differentiation and apoptosis. Worthy of note treatment with polydatin induced a nuclear localization and decreased expression of heat shock protein 27, and vimentin redistributed within the cell. Conclusions From morphological, and biochemical outcome we obtained evidences that polydatin induced a transition from a proliferative morphology to cell-specific differentiated structures and caused human CaCo-2 cell death by induction of apoptosis. Our data suggest the potential use of polydatin in combination chemotherapy for human colon cancer.

The statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) have been proven to be effective in lowering cholesterol and as anti-lipid agents against cardiovascular disease. Recent reports demonstrate an anticancer effect induced by the statins through inhibition of cell proliferation. Probably, these effects are due to suppression of the mevalonate pathway leading to the depletion of various downstream products that play an essential role in cell cycle progression, cell signaling and membrane integrity. To date, although many hypotheses have been proposed, the exact mechanism at the basis of cancer cell growth arrest induced by statins is not known. In this study, we have demonstrated that simvastatin, at a dose of 20 mu M for 24-72 h, induced in cancer cells but not in normal cells precise features of apoptosis including increased DNA fragmentation while, at the molecular level simvastatin induced overexpression of the pro-apoptotic gene Bax together with an inhibition of BCL-2, the gene that has the well-known function of protecting cells from apoptosis. The simvastatin-mediated induction of apoptosis in similar cancer cells but not in normal cells is very interesting and may be at the basis of cancer therapy using statins, usually in combination with chemotherapy or to be used as a cancer protective drug. Simvastatin may, thus, play a dual prophylactic role as a lipid-lowering drug for the prevention of heart disease and as an anticancer agent to prevent certain types of cancers.

Survivin (SVV) is a protein that belongs to the inhibitor of apoptosis proteins (IAP) family and is involved in the G2/M phase progression of the cell cycle as a spindle-associated molecule. The biological features of this protein are well documented and its activity appears to be involved in mitochondria-dependent and -independent antiapoptotic pathways. Overexpression of SVV at the transcriptional and translational level has been associated with cancer, a multifactorial disorder in which the occurrence of a -31G to C polymorphism in the promoter region may significantly contribute to the development of this pathology. To verify this hypothesis, the occurrence of a single nucleotide polymorphism (SNP) in cis-acting cell cycle-dependent elements (CDEs) and in cell cycle homology regions (CHRs) of the survivin TATA-less promoter was investigated. A total of 23 oral squamous cell carcinoma (OSCC) cell lines and normal epithelium-derived normal human epidermal keratinocyte (NHEK) cell lines were analyzed by RFLP and direct DNA sequencing of their promoter region. Furthermore, survivin expression at the transcriptional and translational levels was evaluated in these cells by RT-PCR and Western blotting, respectively. The findings indicate that the presence of a G or C allele is not directly correlated to survivin expression, at the mRNA or at the protein level, at least in the OSCC lines analyzed in this study.

Purpose: Survivin, an inhibitor of apoptosis protein and a cell cycle regulator, has been detected in the majority of human cancers. Five splice variants (survivin, survivin-2α, survivin-2B, survivin-3B, and survivin-ΔEx3) have been identified; their expressions have been investigated here. Methods: By means of RT real-time PCR and immunohistochemistry, we have evaluated survivin isoform expressions at both mRNA and protein levels in human normal oral tissue, precancerous lesions, and oral squamous cell carcinoma (OSCC). Their correlations with the pathological findings have also been analyzed. Results: Expression levels of all survivin transcript variants were markedly elevated in OSCC when compared to normal tissues. One-way analysis of variance (ANOVA) revealed highly significant up-regulation of survivin (P = 0.001), survivin-ΔEx3 (P = 0.001) and survivin-2B (P = 0.004), whereas survivin-3B showed a minor increase in OSCC compared to normal mucosa. Conclusions: Our findings suggest that survivin isoforms deregulation may have significant implications in tumor aggressiveness and prognosis. © 2008 Springer-Verlag.

The enzymatic activities of purified horseradish peroxidase, selenium-dependent glutathione peroxidase, thyroid peroxidase and myeloperoxidase, but not that of lactoperoxidase, were markedly enhanced when added into a reaction mixture containing 5 mum native seminal vesicle protein 4, a major protein secreted from rat seminal vesicle epithelium. A further increase of horseradish peroxidase activity was obtained using Ser58-phosphorylated or acetylated seminal vesicle protein 4. The activating effect of native seminal vesicle protein 4 was highest (about 60-fold) on horseradish peroxidase when 4-chloro-1-naphtol was used as the electron donor substrate. The main kinetics parameters of the stimulatory effect on horseradish peroxidase were evaluated and the enzyme-electron donor substrate interaction was investigated by HPLC and electrospray-MS. A native seminal vesicle protein 4/4-chloro-1-naphtol noncovalent adduct was detected when the protein and 4-chloro-1-naphtol were present in the appropriate molar ratio in the horseradish peroxidase-catalyzed reaction. By contrast, no adducts were formed between native seminal vesicle protein 4 and horseradish peroxidase. This native seminal vesicle protein 4/4-chloro-1-naphtol interaction might underlie the native seminal vesicle protein 4-induced horseradish peroxidase stimulation. Furthermore, native seminal vesicle protein 4 was shown by spectrophotometric and electrospray-MS analysis to interact with NADPH, an electron donor substrate of the selenium-dependent glutathione peroxidase/glutathione reductase redox system, with formation of an adduct between them. Although further investigation is required to elucidate the mechanism of adduct formation, this interaction, probably by promoting the release of the NADPH electrons required for glutathione disulphide reduction, could explain the stimulatory effect of seminal vesicle protein 4 on mammalian peroxidases possibly involved in its physiological function on the selenium-dependent glutathione peroxidase/glutathione reductase system. The biological significance of these properties of native seminal vesicle protein 4 might be related to its ability to downregulate reactive oxygen species and oxidative stress-induced apoptosis.

Increase of VPAC receptor s binding to the (16)gamma-glutamyl diaminopropane vasoactive intestinal peptide (VIP-DAP) agonist, a vasoactive intestinal polypeptide (VIP) structural analogue containing a positive charge at position 16, has confirmed the importance of a positive charge at this site. By investigating the effect of distance from the peptide backbone Calpha of a positive charge in position 16, data are reported here concerning: (i) a novel chemical method used for the synthesis of a new family of (16)gamma-glutamyl diamine VIP derivatives differing among them for single carbon atoms and including diaminoethane (VIP-DAE2), diaminopropane (VIP-DAP3), diaminobutane (VIP-DAB4), diaminopentane (VIP-DAP5), and diaminohexane (VIP-DAH6); (ii) functional characterization of these compounds on human VPAC1 and VPAC2 receptors. In more detail, the EC50 and IC50 values, when measured as a function of the alkylic chain length, show in more detail, that the use of VIP-DAB4 derivative changes the IC50 but not the EC50, thus indicating on hVPAC2 receptor an unexpected relationship between binding and activity that differs from that obtained on hVPAC1.

Rat seminal vesicle protein 4 (RSV4) is a member of the seminal vesicle protein family present in rats, 90 residues long. When secreted, it is involved in many functions related to the reproduction ranging from semen coagulation to sperm capacitation, but its fragments have also shown in vitro pharmacological properties such as anti-inflammatory and pro-coagulant activity, important in the cancer development. However, no three-dimensional model of this protein is yet available probably because of the presence of intrinsic disorder in the structure. In this article we report structural studies in solution of RSV4 by SEC (size exclusion chromatography) and CD (circular dichroism). In solution the monomer is highly flexible and poorly organized with the presence of highly fluctuating helical segments and, as also suggested by SEC, is classifiable as NU-PMG (natively unfolded pre-molten globule). The lack of a cooperative sigmoidal structural transition induced by thermal and GdmCl perturbation in monomer supports the poor structural organization expected for a NU-PMG conformational model. The structure of RSV4 monomer was modeled computationally and subjected to molecular dynamics simulations to study its conformational changes and energetic stability.

Previous data showing an increase of receptor binding activity of [R16]VIP, a vasoactive intestinal peptide (VIP) structural analogue containing arginine at the position 16 of its amino acid sequence, have pointed out the importance of a positive charge at this site. Here, the functional characterization of three VIP polyaminated adducts (VIPDap, VIPSpd, and VIPSpm), obtained by a transglutaminase-catalysed reaction between the VIP Gln16 residue and 1,3-diaminopropane (Dap), spermidine (Spd), or spermine (Spm), is reported. Appropriate binding assays and adenylate cyclase enzymatic determinations have shown that these VIP adducts act as structural VIP agonists, both in vitro and in vivo. In particular, their IC50 and EC50 values of human and rat VIP/pituitary adenylate cyclase activating peptide (PACAP)1 and VIP/PACAP2 receptors indicate that VIPDap is a VIP agonist, with an affinity and a potency higher than that of VIP, while VIPSpd and VIPSpm are also agonists but with affinities lower than that of VIP. These findings suggest that the difference in adduct agonist activity reflects the differences in the positive charge and carbon chain length of the polyamine covalently linked with the VIP Gln16 residue. In addition, the data obtained strongly suggest that the length of polyamine carbon chain could be critical for the interaction of the agonist with its receptor, even though possible hydrophobic interaction cannot be ruled out. In vivo experiments on murine J774 macrophage cell cultures have shown the ability of these compounds to stimulate the inducible nitric oxide synthase activity at the transcriptional level.

We compare the binding properties of [125I-VIP] and [125I]-Ro 25 1553 to VPAC1 receptors, expressed in stably transfected CHO cells. [125I]-VIP labelled two VPAC1 receptor states, while [125I]-Ro 25 1553 labelled selectively a limited number of high-affinity receptors. This high-affinity state probably corresponds to an agonist-receptor-Gs ternary complex as its properties (guanyl nucleotides, EC50 values and maximal effect) were affected by cholera toxin pre-treatment. Both high- and low-affinity receptors participated in the adenylate cyclase activation. This suggested that agonists activate not only low-affinity uncoupled receptors by facilitating the ternary complex formation, but also activated the high-affinity ternary complex by accelerating the GTP binding to emptied, receptor-bound G proteins.

Ochratoxin A (OTA) is a mycotoxin produced by fungal of Aspergillus species absorbed in human through contaminate food in gastrointestinal tract. OTA has been demonstrated to be teratogenic in a number of species including mice and potentially human. Mice exposed in uterus to OTA develop craniofacial abnormalities such as exencephaly, microencephaly, microphthalmia and facial clefts. An important role in differentiation of maxillofacial are exerted by the Hox related genes Dlx and Msx. In the present investigation we have confirmed that 2.75 mg/kg body weight OTA, given at gestational day 7.5, induces significant developmental craniofacial anomalies in mice and we have demonstrated the down expression of Dlx5, a member of Dlx gene family, that seems to be responsible of the observed deformities. These results support the hypothesis that Dlx5 is a target for ochratoxin and the inhibition of its function, directly or indirectly, could be at origin of the observed differentiation defects.

Aim To examine the impact of gut microbiota on non alcoholic fatty liver disease (NAFLD) pathogenesis. Data synthesis Emerging evidence suggests a strong interaction between gut microbiota and liver. Receiving approximately 70% of its blood supply from the intestine, the liver represents the first line of defence against gut-derived antigens. Intestinal bacteria play a key role in the maintenance of gut–liver axis health. Disturbances in the homeostasis between bacteria- and host-derived signals at the epithelial level lead to a break in intestinal barrier function and may foster “bacterial translocation”, defined as the migration of bacteria or bacterial products from the intestinal lumen to mesenteric lymph nodes or other extraintestinal organs and sites. While the full repertoire of gut-derived microbial products that reach the liver in health and disease has yet to be explored, the levels of bacterial lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria, are increased in the portal and/or systemic circulation in several types of chronic liver diseases. Derangement of the gut flora, particularly small intestinal bacterial overgrowth, occurs in a large percentage (20–75%) of patients with chronic liver disease. In addition, evidence implicating the gut–liver axis in the pathogenesis of metabolic liver disorders has accumulated over the past ten years. Conclusions Complex metabolic diseases are the product of multiple perturbations under the influence of triggering factors such as gut microbiota and diet, thus, modulation of the gut microbiota may represent a new way to treat or prevent NAFLD.