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This study reports a comparative analysis of the polyphenolic composition and nutraceutical properties of different olive mill wastewater (OMWW) and corresponding extra virgin olive oil (EVOO) extracts. Specifically, four OMWWs and corresponding EVOOs from cultivars Frantoio (A) and Leccino (B) obtained from different crushing seasons (early-stage (A1 and B1) and later-stage (A2 and B2)) were analyzed. Employing HPLC-DAD and LC-MS methods, the primary polyphenol content was identified and quantified. Overall, OMWW extracts showed a greater polyphenolic content compared to corresponding EVOO extracts, with OMWW B1 displaying the highest levels of polyphenols. The antiradical properties of extracts towards radical species (DPPH, ABTS, O2−, and HOCl−) were demonstrated in vitro, revealing a correlation with polyphenolic content. In fact, OMWW B1 and B2 demonstrated the strongest antiradical activity. Exploring nutraceutical properties of OMWWs, the intestinal permeation of the main polyphenols in a co-culture model (Caco-2 and HT29-MTX cell lines) was assessed, with tyrosol achieving a permeation of almost 60%. Furthermore, the involvement in the inflammation process has been evaluated in cell studies on THP1-derived macrophages by immunocytochemistry, demonstrating that OMWW B1 may exert an anti-inflammatory effect by modulating specific phenotype expression on macrophages. In conclusion, this study provides evidence supporting the reuse of OMWWs as a source of polyphenols with nutraceutical properties.

The Mediterranean diet and consumption of EVOO are associated with multiple beneficial effects for human health, e.g. reduction in cardiovascular risk and mortality, improvement in the lipid profile, and the prevention of chronic diseases, such as cancers and neurodegenerative diseases. In EVOO, more than 30 different phenolic-derived compounds have been identified, representing one of the most promising bioactive classes in olive oil. This review explores the hepatoprotective properties of two of these compounds, tyrosol and oleocanthal, focusing on their mechanisms of action. Recent studies have shown that these compounds, which share a similar chemical structure with a hydroxyl group attached to an aromatic hydrocarbon ring, can potentially mitigate chronic liver diseases, such as MASLD and liver fibrosis, as well as their progression to liver cancer. Consequently, they deserve attention for future pharmacological drug development. In vitro and in vivo studies have suggested that these compounds exert these effects through the regulation of cellular pathways involved in antioxidant response, lipid metabolism, transcription factor activity, and NF-κB signaling. Understanding the mechanisms underlying the hepatoprotective properties of tyrosol and oleocanthal may provide valuable information for the development of therapeutic agents based on their chemical structures capable of targeting chronic liver diseases.

IntroductionThe pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the demise of motor neurons has been linked to excitotoxicity caused by excessive calcium influx via N-methyl-D-aspartate receptors (NMDARs), suggesting that uncompetitive NMDAR antagonism could be a strategy to attenuate motor neuron degeneration. REL-1017, the dextro-isomer of racemic methadone, is a low-affinity uncompetitive NMDAR antagonist. Importantly, in humans REL-1017 has shown excellent tolerability in clinical trials for major depression.MethodsHere, we tested if REL-1017 improves the disease phenotypes in the G93A SOD1 mouse, a well-established model of familial ALS, by examining survival and motor functions, as well as the expression of genes and proteins involved in neuroplasticity.ResultsWe found a sex-dependent effect of REL-1017 in G93A SOD1 mice. A delay of ALS symptom onset, assessed as 10%-decrease of body weight (p

Hepatocellular carcinoma (HCC), the most common primary liver cancer, accounts for 830,180 related deaths worldwide in 2020, according to GLOBOCAN, representing the fourth leading cause of cancer-related death, with a five-year survival rate of about 18% for advanced stage, and the second leading cause in men of cancer-related mortality worldwide [...]

Gender Medicine has had an enormous expansion over the last ten years. Autoimmune liver diseases include several conditions, i.e., autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and conditions involving the liver or biliary tree overlapping with AIH, as well as IgG4-related disease. However, little is known about the impact of sex in the pathogenesis and natural history of these conditions. The purpose of this review is to provide an update of the gender disparities among the autoimmune liver diseases by reviewing the data published from 1999 to 2023. The epidemiology of these diseases has been changing over the last years, due to the amelioration of knowledge in their diagnosis, pathogenesis, and treatment. The clinical data collected so far support the existence of sex differences in the natural history of autoimmune liver diseases. Notably, their history could be longer than that which is now known, with problems being initiated even at a pediatric age. Moreover, gender disparity has been observed during the onset of complications related to end-stage liver disease, including cancer incidence. However, there is still an important debate among researchers about the impact of sex and the pathogenesis of these conditions. With this review, we would like to emphasize the urgency of basic science and clinical research to increase our understanding of the sex differences in autoimmune liver diseases.

Primary biliary cholangitis (PBC) is an autoimmune liver disease characterised by the immune-mediated destruction of small and medium intrahepatic bile ducts, with variable outcomes and progression. This review summarises the state of the art regarding the risk of neoplastic progression in PBC patients, with a particular focus on the molecular alterations present in PBC and in hepatocellular carcinoma (HCC), which is the most frequent liver cancer in these patients. Major risk factors are male gender, viral infections, e.g., HBV and HCV, non-response to UDCA, and high alcohol intake, as well as some metabolic-associated factors. Overall, HCC development is significantly more frequent in patients with advanced histological stages, being related to liver cirrhosis. It seems to be of fundamental importance to unravel eventual dysfunctional molecular pathways in PBC patients that may be used as biomarkers for HCC development. In the near future, this will possibly take advantage of artificial intelligence-designed algorithms.

The gut microbiota is a complex system, playing a peculiar role in regulating innate and systemic immunity. Increasing evidence links dysfunctional gut microbiota to metabolic dysfunction-associated steatotic liver disease (MASLD) due to the activation of multiple pathways in the gut and in the liver, including those mediated by Toll-like receptors (TLRs), that sustain hepatic inflammation. Thus, many efforts have been made to unravel the role of microbiota-associated dysfunction in MASLD, with the final aim of finding novel strategies to improve liver steatosis and function. Moreover, recent evidence underlines the role of adipose tissue in sustaining hepatic inflammation during MASLD development. In this review, we focus on the recently discovered strategies proposed to improve the alteration of gut microbiota observed in MASLD patients, with a particular insight into those known to modulate gut microbiota-associated dysfunction and to affect the complex crosstalk between the gut, the adipose tissue, and the liver.

Liver fibrosis is a common and reversible feature of liver damage associated with many chronic liver diseases, and its onset is influenced by sex. In this study, we investigated the mechanisms of liver fibrosis and regeneration, focusing on understanding the mechanistic gaps between females and males. We injected increasing doses of carbon tetrachloride into female and male mice and maintained them for a washout period of eight weeks to allow for liver regeneration. We found that male mice were more prone to developing severe liver fibrosis as a consequence of early chronic liver damage, supported by the recruitment of a large number of Ly6Chigh MoMφs and neutrophils. Although prolonged liver damage exacerbated the fibrosis in mice of both sexes, activated HSCs and Ly6Chigh MoMφs were more numerous and active in the livers of female mice than those of male mice. After eight weeks of washout, only fibrotic females reported no activated HSCs, and a phenotype switching of Ly6Chigh MoMφs to anti-fibrogenic Ly6Clow MoMφs. The early stages of liver fibrosis mostly affected males rather than females, while long-term chronic liver damage was not influenced by sex, at least for liver fibrosis. Liver repair and regeneration were more efficient in females than in males.

Liver fibrosis is the result of a chronic pathological condition caused by the activation of hepatic stellate cells (HSCs), which induces the excessive deposition of extracellular matrix. Fibrogenesis is sustained by an exaggerated production of reactive oxidative species (ROS) by NADPH oxidases (NOXs), which are overactivated in hepatic inflammation. In this study, we investigated the antifibrotic properties of two phenolic compounds of natural origin, tyrosol (Tyr) and hydroxytyrosol (HTyr), known for their antioxidant and anti-inflammatory effects. We assessed Tyr and HTyr antifibrotic and antioxidant activity both in vitro, by a co-culture of LX2, HepG2 and THP1-derived Mϕ macrophages, set up to simulate the hepatic microenvironment, and in vivo, in a mouse model of liver fibrosis obtained by carbon tetrachloride treatment. We evaluated the mRNA and protein expression of profibrotic and oxidative markers (α-SMA, COL1A1, NOX1/4) by qPCR and/or immunocytochemistry or immunohistochemistry. The expression of selected miRNAs in mouse livers were measured by qPCR. Tyr and HTyr reduces fibrogenesis in vitro and in vivo, by downregulating all fibrotic markers. Notably, they also modulated oxidative stress by restoring the physiological levels of NOX1 and NOX4. In vivo, this effect was accompanied by a transcriptional regulation of inflammatory genes and of 2 miRNAs involved in the control of oxidative stress damage (miR-181-5p and miR-29b-3p). In conclusion, Tyr and HTyr exert antifibrotic and anti-inflammatory effects in preclinical in vitro and in vivo models of liver fibrosis, by modulating hepatic oxidative stress, representing promising candidates for further development.

Liver cancer remains a global health challenge and its incidence is growing worldwide [...]

The electronic cigarettes mimic combustible cigarettes through a heating technology that vaporizes a refill liquid consisting of solvents, flavors, and nicotine. E-cigarettes are sometimes still used as a support for smoking cessation, even if in 2019 an acute lung injury outbreak occurred in the USA, affecting mainly adolescents and young adults, and was correlated to eCigs. Therefore, due to the lack of a definite knowledge about the mechanism(s) of refill liquid toxicity and considering that previous investigations gave controversial results, the aim of the present study was the cytotoxicity assessment of different refill liquids on human endothelial cells, evaluated by means of two different in vitro approaches, i.e. the resazurin and the LDH release assays. Our results clearly demonstrated that different refill liquids (6 samples) display different levels of cytotoxicity in our cellular model, although their cytotoxicity was always lower than that observed for the condensate obtained from traditional cigarettes (3 samples). These results suggest that accurate evaluations should be provided for refill liquids, in particular to correlate their toxicity to their chemical composition, with the final aim of obtaining useful information for the agencies involved in the regulation of their components.

Hepatocellular carcinoma (HCC), the primary hepatic malignancy, represents the second-highest cause of cancer-related death worldwide. Many efforts have been devoted to finding novel biomarkers for predicting both patients’ survival and the outcome of pharmacological treatments, with a particular focus on immunotherapy. In this regard, recent studies have focused on unravelling the role of tumor mutational burden (TMB), i.e., the total number of mutations per coding area of a tumor genome, to ascertain whether it can be considered a reliable biomarker to be used either for the stratification of HCC patients in subgroups with different responsiveness to immunotherapy, or for the prediction of disease progression, particularly in relation to the different HCC etiologies. In this review, we summarize the recent advances on the study of TMB and TMB-related biomarkers in the HCC landscape, focusing on their feasibility as guides for therapy decisions and/or predictors of clinical outcome.

Liver fibrosis, which is the outcome of wound-healing response to chronic liver damage, represents an unmet clinical need. This study evaluated the anti-fibrotic and anti-inflammatory effects of the polyphenol oleocanthal (OC) extracted from extra virgin olive oil (EVOO) by an in vitro/in vivo approach. The hepatic cell lines LX2 and HepG2 were used as in vitro models. The mRNA expression of pro-fibrogenic markers, namely alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 chain (COL1A1), a panel of metalloproteinases (MMP1, MMP2, MMP3, MMP7, MMP9) and vascular endothelial growth factor A (VEGFA) as well as the pro-oxidant genes NADPH oxidases (NOXs) 1 and 4 were evaluated in TGF-β activated LX2 cells by qRT-PCR. α-SMA and COL1A1 protein expression was assessed by immunofluorescence coupled to confocal microscopy. VEGFA release from LX2 was measured by ELISA. We also evaluated the amount of reactive oxygen species (ROS) produced by H2O2 activated- HepG2 cells. In vivo, OC was administered daily by oral gavage to Balb/C mice with CCl4-induced liver fibrosis. In this model, we measured the mRNA hepatic expression of the three pro-inflammatory interleukins (IL) IL6, IL17, IL23, chemokines such as C-C Motif Chemokine Ligand 2 (CCL2) and C-X-C Motif Chemokine Ligand 12 (CXCL12), and selected miRNAs (miR-181-5p, miR-221-3p, miR-29b-3p and miR-101b-3p) by qRT-PCR. We demonstrated that OC significantly downregulated the gene/protein expression of α-SMA, COL1A1, MMP2, MMP3, MMP7 and VEGF as well as the oxidative enzymes NOX1 and 4 in TGFβ1-activated LX2 cells, and reduced the production of ROS by HepG2. In vivo OC, beside causing a significant reduction of fibrosis at histological assessment, counteracted the CCl4-induced upregulation of pro-fibrotic and inflammatory genes. Moreover, OC upregulated the anti-fibrotic miRNAs (miR-29b-3p and miR-101b-3p) reduced in fibrotic mice, while downregulated the pro-fibrotic miRNAs (miR-221-3p and miR-181-5p), which were dramatically upregulated in fibrotic mice. In conclusion, OC exerts a promising antifibrotic effect via a combined reduction of oxidative stress and inflammation involving putative miRNAs, which in turn reduces hepatic stellate cells activation and liver fibrosis.

Primary biliary cholangitis (PBC) is a rare autoimmune cholestatic liver disease that may progress to fibrosis and/or cirrhosis. Treatment options are currently limited. The first-line therapy for this disease is the drug ursodeoxycholic acid (UDCA), which has been proven to normalize serum markers of liver dysfunction, halt histologic disease progression, and lead to a prolongation of transplant-free survival. However, 30–40% of patients unfortunately do not respond to this first-line therapy. Obeticholic acid (OCA) is the only registered agent for second-line treatment in UDCA-non responders. In this review, we focus on the pharmacological features of OCA, describing its mechanism of action of and its tolerability and efficacy in PBC patients. We also highlight current perspectives on future therapies for this condition.

Acute and chronic hepatic damages are caused by xenobiotics or different diseases affecting the liver, characterized by different etiologies and pathological features. It has been demonstrated extensively that liver damage progresses differently in men and women, and some chronic liver diseases show a more favorable prognosis in women than in men. This review aims to update the most recent advances in the comprehension of the molecular basis of the sex difference observed in both acute and chronic liver damage. With this purpose, we report experimental studies on animal models and clinical observations investigating both acute liver failure, e.g., drug-induced liver injury (DILI), and chronic liver diseases, e.g., viral hepatitis, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), autoimmune liver diseases, and hepatocellular carcinoma (HCC).

Recently, some preclinical and clinical studies have demonstrated the ability of brown seaweeds in reducing the risk factors for metabolic syndrome. Here, we analyzed the beneficial effect of a nutraceutical formulation containing a phytocomplex extracted from seaweeds and chromium picolinate in animal models of liver steatosis of differing severities (rats with non-alcoholic fatty liver disease (NAFLD) and its complication, non-alcoholic steatohepatitis (NASH)). This treatment led to a significant drop in hepatic fat deposition in both models (p < 0.01 vs. untreated animals), accompanied by a reduction in plasma inflammatory cytokines, such as interleukin 6, tumor necrosis factor α, and C reactive protein, and myeloperoxidase expression in liver tissue. Furthermore, a modulation of the molecular pathways involved in lipid metabolism and storage was demonstrated, since we observed the significant reduction of the mRNA levels of fatty acid synthase, diacylglycerol acyltransferases, the sterol-binding protein SREBP-1, and the lipid transporter perilipin-2, in both treated NAFLD and NASH rats in comparison to untreated ones. In conclusion, this nutraceutical product was effective in reducing liver steatosis and showed further beneficial effects on hepatic inflammation and glycemic control, which were particularly evident in rats characterized by a more severe condition, thus representing a therapeutic option for the treatment of NAFLD and NASH patients.

A small library of new angelicin derivatives was designed and synthesized with the aim of bypassing the side effects of trimethylangelicin (TMA), a promising agent for the treatment of cystic fibrosis. To prevent photoreactions with DNA, hindered substituents were inserted at the 4 and/or 6 positions. Unlike the parent TMA, none of the new derivatives exhibited significant cytotoxicity or mutagenic effects. Among the synthesized compounds, the 4-phenylderivative 12 and the 6-phenylderivative 25 exerted a promising F508del CFTR rescue ability. On these compounds, preliminary in vivo pharmacokinetic (PK) studies were carried out, evidencing a favorable PK profile per se or after incorporation into lipid formulations. Therefore, the selected compounds are good candidates for future extensive investigation to evaluate and develop novel CFTR correctors based on the angelicin structure.

4,6,4′-trimethylangelicin (TMA) is a promising pharmacological option for the treatment of cystic fibrosis (CF) due to its triple-acting behavior toward the function of the CF transmembrane conductance regulator. It is a poorly water-soluble drug, and thus it is a candidate for developing a self-emulsifying formulation (SEDDS). This study aimed to develop a SEDDS to improve the oral bioavailability of TMA. Excipients were selected on the basis of solubility studies. Polyoxyl-35 castor oil (Cremophor® EL) was proposed as surfactant, diethylene glycol-monoethyl ether (Transcutol® HP) as cosolvent, and a mixture of long-chainmono-,di-, and triglycerides (Maisine® CC) or medium-chain triglycerides (LabrafacTM lipophile) as oil phases. Different mixtures were prepared and characterized by measuring the emulsification time, drop size, and polydispersity index to identify the most promising formulation. Two formulations containing 50% surfactant (w/w), 40% cosolvent (w/w), and 10% oil (w/w) (Maisine® CC or LabrafacTM lipophile) were selected. The results showed that both formulations were able to self-emulsify, producing nanoemulsions with a drop size range of 20–25 nm, and in vivo pharmacokinetic studies demonstrated that they were able to significantly increase the oral bioavailability of TMA. In conclusion, SEEDS are useful tools to ameliorate the pharmacokinetic profile of TMA and could represent a strategy to improve the therapeutic management of CF.

Ursodeoxycholic acid (UDCA) is the first-line therapy used for the treatment of PBC. In recent years, new pharmacological agents have been proposed for PBC therapy to cure UDCA-non-responders. Obeticholic acid (OCA) is registered in many countries for PBC, and fibrates also seem to be effective in ameliorating biochemistry alteration and symptoms typical of PBC. Moreover, a variety of new agents, acting with different mechanisms of action, are under clinical evaluation for PBC treatment, including PPAR agonists, anti-NOX agents, immunomodulators, and mesenchymal stem cell transplantation. Since an insufficient amount of data is currently available about the effect of these novel approaches on robust clinical endpoints, such as transplant-free survival, their clinical approval needs to be supported by the consistent improvement of these parameters. The intensive research in this field will hopefully lead to a novel treatment landscape for PBC in the near future, with innovative therapies based on the combination of multiple agents acting on different pathogenetic mechanisms.

Background: Berberine is a natural alkaloid with hypoglycemic properties. However, its therapeutic use is limited by a very low oral bioavailability. Here we developed a new oral formulation of berberine based on Sucrosomial® technology and tested its effect on insulin resistance. Methods: Sucrosomial® berberine was first tested in vitro in the hepatoma cell line Huh7 to assess its effect on proteins involved in glucose homeostasis and insulin resistance. The pharmacokinetics and efficacy on insulin resistance were then studied in C57BL/6 mice fed with standard (SD) and high-fat diet (HFD) for 16 weeks and treated daily during the last 8 weeks with oral gavage of Sucrosomial® berberine or berberine. Results: Sucrosomial® berberine did not affect Huh7 cell viability at concentrations up to 40 µM. Incubation of Huh7 with 20 µM of Sucrosomial® and control berberine induced glucokinase (GK) and the phosphorylation of 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK), both known targets for the control of insulin resistance. In vivo, we observed an 8-fold higher plasma concentration after 3 weeks of oral administration of 50 mg/kg/day of Sucrosomial® formulation compared to berberine. HFD, compared to SD, induced insulin resistance in mice as determined by oral glucose tolerance test (OGTT). The treatment with a 6.25 mg/kg/daily dose of Sucrosomial® berberine significantly reduced the area under the curve (AUC) of OGTT (73,103 ± 8645 vs. 58,830 ± 5597 mg/dL × min), while control berberine produced the same effects at 50 mg/Kg/day (51518 ± 1984 mg/dL × min). Under these conditions, the two formulations resulted in similar berberine plasma concentration in mice. Nevertheless, a different tissue distribution of metabolites was observed with a significant accumulation of reduced, demethylated and glucuronide berberine in the brain after the oral administration of the Sucrosomial® form. Glucuronide berberine plasma concentration was higher with Sucrosomial® berberine compared to normal berberine. Finally, we observed similar increases of AMPK phosphorylation in the liver in response to the treatment with Sucrosomial® berberine and berberine. Conclusions: The Sucrosomial® formulation is an innovative and effective technology to improve berberine gastrointestinal (GI) absorption with proven in vitro and in vivo activity on insulin resistance.

Brain-derived neurotrophic factor (BDNF), a neurotrophin widely expressed in the central nervous system, exhibits important effects on neural plasticity. BDNF has been implicated in the mechanism of action of ketamine, a N-methyl-d-aspartic acid receptor (NMDAR) antagonist with rapid anti-depressant effects in humans. REL-1017 (esmethadone), the d-optical isomer of the racemic mixture d-l-methadone, is devoid of clinically relevant opioid activity at doses expected to exert therapeutic NMDAR antagonistic activity in humans. The present study was conducted to ascertain the effects of oral administration of 25 mg of REL-1017 for 10 days on plasma BDNF in healthy subjects confined to an inpatient unit for a phase 1 clinical trial. We observed an increase in post-treatment BDNF plasma levels compared to pre-treatment levels. Post-treatment, Day 10 BDNF plasma levels ranged from 2 to 17 times pre-treatment levels in the 25 mg REL-1017 treatment group, whereas in the placebo group, BDNF plasma levels remained unchanged (p = 0.028). Diastolic blood pressure decreased significantly in subjects treated with REL-1017, while no effect could be observed in the placebo group. In conclusion, the administration of 25 mg REL-1017 significantly increased BDNF plasma levels and significantly decreased diastolic blood pressure in healthy subjects confined to an inpatient unit for a phase 1 clinical trial.

The effect of liver steatosis on drug metabolism has been investigated in both preclinical and clinical settings, but the findings of these studies are still controversial. We here evaluated the pharmacokinetic profile of the main sofosbuvir metabolite GS-331007 in healthy animals and rats with non-alcoholic fatty liver disease (NAFLD) after the oral administration of a single 400 mg/kg dose of sofosbuvir. The plasma concentration of GS-331007 was evaluated by HPLC-MS. The expression of the two enzymes uridine monophosphate-cytidine monophosphate kinase 1 (UMP-CMPK1), and nucleoside diphosphate kinase (ND-PK), responsible for the formation of the active metabolite GS-331007-TP, were measured by qRT-PCR and Western Blot. We demonstrated that in rats with steatosis, the area under the plasma concentration-vs-time curve (AUC) and the peak plasma concentration (Cmax) of GS-331007 increased significantly whereas the expression of UMP-CMPK was significantly lower than that of healthy animals. The reduction of UMP-CMPK expression suggests an impairment of sofosbuvir activation to GS-331007-TP, giving a possible explanation for the reduction of sofosbuvir efficacy in patients affected by genotype 3 Hepatitis C virus (HCV), which is often associated with liver steatosis. Furthermore, since GS-331007 plasma concentration is altered by steatosis, it can be suggested that the plasma concentration of this metabolite may not be a reliable indicator for exposure-response analysis in patients with NAFLD.

Pregnane X receptor (PXR), a nuclear receptor known for modulating the transcription of drug metabolizing enzymes and transporters (DMETs), such as cytochrome P450 3A4 and P-glycoprotein, is functionally involved in chronic liver diseases of different etiologies. Furthermore, PXR activity relates to that of other NRs, such as constitutive androstane receptor (CAR), through a crosstalk that in turn orchestrates a complex network of responses. Thus, besides regulating DMETs, PXR signaling is involved in both liver damage progression and repair and in the neoplastic transition to hepatocellular carcinoma. We here summarize the present knowledge about PXR expression and function in chronic liver diseases characterized by different etiologies and clinical outcome, focusing on the molecular pathways involved in PXR activity. Although many molecular details of these finely tuned networks still need to be fully understood, we conclude that PXR and its modulation could represent a promising pharmacological target for the identification of novel therapeutical approaches to chronic liver diseases.

A peculiar role for oxidative stress in non-alcoholic fatty liver disease (NAFLD) and its transition to the inflammatory complication non-alcoholic steatohepatitis (NASH), as well as in its threatening evolution to hepatocellular carcinoma (HCC), is supported by numerous experimental and clinical studies. NADPH oxidases (NOXs) are enzymes producing reactive oxygen species (ROS), whose abundance in liver cells is closely related to inflammation and immune responses. Here, we reviewed recent findings regarding this topic, focusing on the role of NOXs in the different stages of fatty liver disease and describing the current knowledge about their mechanisms of action. We conclude that, although there is a consensus that NOX-produced ROS are toxic in non-neoplastic conditions due to their role in the inflammatory vicious cycle sustaining the transition of NAFLD to NASH, their effect is controversial in the neoplastic transition towards HCC. In this regard, there are indications of a differential effect of NOX isoforms, since NOX1 and NOX2 play a detrimental role, whereas increased NOX4 expression appears to be correlated with better HCC prognosis in some studies. Further studies are needed to fully unravel the mechanisms of action of NOXs and their relationships with the signaling pathways modulating steatosis and liver cancer development.

Although selective tumor delivery of anticancer drugs has been sought by exploiting either passive targeting or by ligand-mediated targeting, a selective anticancer therapy remains an unmet medical need. Despite the advances which have been achieved by nanomedicines, nanosystems such as polymer-drug conjugates still miss the goal of clinical efficacy. In this study, we demonstrated that polymer-drug conjugates require a thoroughly chemical design and the right targeting agent/polymer ratio to be selective and effective towards cancer cells. In particular, two PEG conjugates carrying paclitaxel and targeted with different folic acid (FA)/PEG ratios (one or three) were investigated. The cytotoxicity study in positive (HT-29) and negative (HCT-15) FA receptor (FR)-cell lines demonstrated that the conjugates with one or three FAs were 4- or 28-fold more active in HT-29 cells, respectively. The higher activity of the 3-FA conjugate was confirmed by its strong impact on cell cycle arrest. Furthermore, FA targeting had a clear effect on migration and invasiveness of HT-29 cells, which were significantly reduced by both conjugates. Interestingly, the 3-FA conjugate showed also an improved pharmacokinetic profile in mice. The results of this study indicate that thorough investigations are needed to optimize and tune drug delivery and achieve the desired selectivity and activity towards cancer cells.

Metabolic syndrome is characterized by the coexistence of different metabolic disorders which increase the risk of developing type 2 diabetes mellitus and cardiovascular diseases. Therefore, metabolic syndrome leads to a reduction in patients’ quality of life as well as to an increase in morbidity and mortality. In the last few decades, it has been demonstrated that seaweeds exert multiple beneficial effects by virtue of their micro- and macronutrient content, which could help in the management of cardiovascular and metabolic diseases. This review aims to provide an updated overview on the potential of brown seaweeds for the prevention and management of metabolic syndrome and its associated diseases, based on the most recent evidence obtained from in vitro and in vivo preclinical and clinical studies. Owing to their great potential for health benefits, brown seaweeds are successfully used in some nutraceuticals and functional foods for treating metabolic syndrome comorbidities. However, some issues still need to be tackled and deepened to improve the knowledge of their ADME/Tox profile in humans, in particular by finding validated indexes of their absorption and obtaining reliable information on their efficacy and long-term safety.

Non-alcoholic fatty liver disease (NAFLD) and its complication non-alcoholic steatohepatitis (NASH) are important causes of liver disease worldwide. Recently, a significant association between these hepatic diseases and different central nervous system (CNS) disorders has been observed in an increasing number of patients. NAFLD-related CNS dysfunctions include cognitive impairment, hippocampal-dependent memory impairment, and mood imbalances (in particular, depression and anxiety). This review aims at summarizing the main correlations observed between NAFLD development and these CNS dysfunctions, focusing on the studies investigating the mechanism(s) involved in this association. Growing evidences point at cerebrovascular alteration, neuroinflammation, and brain insulin resistance as NAFLD/NASH-related CNS manifestations. Since the pharmacological options available for the management of these conditions are still limited, further studies are needed to unravel the mechanism(s) of NAFLD/NASH and their central manifestations and identify effective pharmacological targets.

Tumor-associated macrophages (TAMs), primarily the M2 phenotype, are involved in the progression and metastasis of colorectal cancer (CRC). Cuban brown propolis (Cp) and its main component Nemorosone (Nem) displays an antiproliferative effect on different cancer cells, including CRC cell lines. However, whether Cp and Nem could exploit its effect on CRC cells by targeting their relationship with TAMs remains to be elucidated. In this study, we differentiated the human monocytic THP-1 cells to M2 macrophages and confirmed this transition by immunofluorescence (IF) staining, qRT-PCR and zymography. An MTT assay was performed to determine the effect of Cp and Nem on the viability of CRC HT-29 cells co-cultured with M2 macrophages. Furthermore, the migration and invasion abilities of HT-29 cells were determined by Transwell assays and the expression levels of epithelial–mesenchymal transition (EMT) markers were analyzed by IF staining. We demonstrated that Cp and Nem reduced the viability of M2 macrophages and, accordingly, the activity of the MMP-9 metalloprotein. Moreover, we demonstrated that M2 macrophages produce soluble factors that positively regulate HT-29 cell growth, migration and invasion. These M2-mediated effects were counteracted by Cp and Nem treatments, which also played a role in regulating the expression of the EMT markers E-cadherin and vimentin. Taken together, our results indicate that Nem contained in Cp interferes in the crosstalk between CRC cells and TAMs, by targeting M2 macrophages.

The majority of deaths related to colorectal cancer (CRC) are associated with the metastatic process. Alternative therapeutic strategies, such as traditional folk remedies, deserve attention for their potential ability to attenuate the invasiveness of CRC cells. The aim of this study is to investigate the biological activity of brown Cuban propolis (CP) and its main component nemorosone (NEM) and to describe the molecular mechanism(s) by which they inhibit proliferation and metastatic potential of 2 CRC cell lines, i.e., HT-29 and LoVo. Our results show that CP and NEM significantly decreased cell viability and inhibited clonogenic capacity of CRC cells in a dose and time-dependent manner, by arresting the cell cycle in the G0/G1 phase and inducing apoptosis. Furthermore, CP and NEM downregulated BCL2 gene expression and upregulated the expression of the proapoptotic genes TP53 and BAX, with a consequent activation of caspase 3/7. They also attenuated cell migration and invasion by inhibiting MMP9 activity, increasing E-cadherin and decreasing β-catenin and vimentin expression, proteins involved in the epithelial−mesenchymal transition (EMT). In conclusion NEM, besides displaying antiproliferative activity on CRC cells, is able to decrease their metastatic potential by modulating EMT-related molecules. These finding provide new insight about the mechanism(s) of the antitumoral properties of CP, due to NEM content.

The Asian coastal communities have used the brown seaweeds Fucus vesiculosus and Ascophyllum nodosum since ancient times. Recently, some in vitro and in vivo studies have demonstrated their abilities in reducing risk factors for metabolic syndrome. Here, we analyzed the protective effect of a phytocomplex extracted from these seaweeds on the deposition of fat in the liver after the administration of a high-fat diet (HFD) to rats for five weeks. The administration of F. vesiculosus and A. nodosum led to significant reductions in microvescicular steatosis and plasma biochemical and lipid parameters, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total and conjugated bilirubin, and triglycerides. Furthermore, the postprandial glycemic peak was delayed and significantly reduced (p < 0.01) by the algal extract administration. In conclusion, this extract is effective in reducing microvescicular steatosis and improving glycemic control, thereby lowering the risk of nonalcoholic fatty liver disease, obesity, and diabetes, diseases related to the consumption of fat and sugar-enriched diets.

Glucocorticoids (GCs) are currently used for the therapeutic management of cholestatic diseases, but their use and molecular mechanism remain controversial. The aims of this study were 1) to assess the therapeutic effect of a 2-week treatment with the GC dexamethasone on hepatic damage in bile duct-ligated rats; 2) to investigate its effect on the activation of the nuclear receptors (NRs) pregnane X receptor (PXR), constitutive androstane receptor (CAR) and GC receptor (GR), and NF-kB, as well as on oxidative stress and bile acid (BA) hepatic composition. Cholestasis was induced by ligation of bile duct (BDL animals) in 16 male Wistar-Kyoto rats, and eight of them were daily treated by oral gavage with 0.125 mg/ml/kg DEX for 14 days. Eight Sham-operated rats were used as controls. Severity of cholestasis was assessed histologically and on plasma biochemical parameters. The nuclear expression of NF-kB (p65), GR, PXR and CAR was measured in hepatic tissue by Western Blot. Oxidative stress was evaluated by measuring malondialdehyde, carbonylated proteins, GHS and ROS content in rat livers. LC-MS was used to measure the plasma and liver concentration of 7 BAs. Histological findings and a significant drop in several markers of inflammation (p65 nuclear translocation, mRNA expressions of TNF-α, IL-1β, IL-6) showed that DEX treatment reversed cholestasis-induced inflammation, and similar results have been obtained with oxidative stress markers. The nuclear expression of p65 and CAR were inversely correlated, with the latter increasing significantly after DEX treatment (p

Since nutrition might have a significant impact on liver function, we analyzed the early effect of Western-type diet on hepatic tissue and lipid and drug metabolism in Wistar−Kyoto rats (n = 8); eight rats fed with a standard diet were used as controls. Histological analysis of liver tissue was performed, and plasma biochemical parameters were measured. Plasma concentration of six bile acids was determined by ultra-liquid chromatography-tandem mass spectrometry UHPLC-MS/MS. Hepatic gene expressions of enzymes involved in drug and lipid metabolism were assessed by means of real-time reverse transcription (qRT)-PCR. Liver of rats fed with a Western diet did not show macroscopic histological alterations, but number and diameter of lipid droplets increased, as well as DGAT1, GPAT4, SCD, FASN and SREBP2 expression. Furthermore, Western diet-fed animals showed an increase in the activation of hepatic stellate cells and macrophage number in liver tissue, as well as a significant increase in AST and bilirubin levels (p < 0.01), and in the LDL:HDL cholesterol ratio (p < 0.001). Plasma chenodeoxycholic acid concentration increased significantly, whereas cholic acid decreased (p < 0.05), and cytochrome P450 genes were generally downregulated. Significant changes in hepatic lipid and drug metabolism are early induced by the Western diet, prior to steatosis development. Such changes are associated with a peculiar alteration in circulating bile acids, which could represent an early marker of non-alcoholic fatty liver disease (NAFLD) development.

Principal component analysis (PCA) multivariate analysis was applied to study the cytotoxic activity of essential oils from various species of the Pistacia genus on human tumor cell lines. In particular, the cytotoxic activity of essential oils obtained from P. lentiscus, P. lentiscus var. chia (mastic gum), P. terebinthus, P. vera, and P. integerrima, was screened on three human adenocarcinoma cell lines: MCF-7 (breast), 2008 (ovarian), and LoVo (colon). The results indicate that all the Pistacia phytocomplexes, with the exception of mastic gum oil, induce cytotoxic effects on one or more of the three cell lines. PCA highlighted the presence of different cooperating clusters of bioactive molecules. Cluster variability among species, and even within the same species, could explain some of the differences seen among samples suggesting the presence of both common and species-specific mechanisms. Single molecules from one of the most significant clusters were tested, but only bornyl-acetate presented cytotoxic activity, although at much higher concentrations (IC50 = 138.5 µg/mL) than those present in the essential oils, indicating that understanding of the full biological effect requires a holistic vision of the phytocomplexes with all its constituents.

Edible seaweeds have been consumed by Asian coastal communities since ancient times. Fucus vesiculosus and Ascophyllum nodosum extracts have been traditionally used for the treatment of obesity and several gastrointestinal diseases. We evaluated the ability of extracts obtained from these algae to inhibit the digestive enzymes α-amylase and α-glucosidase in vitro, and control postprandial plasma glucose levels in a mouse model of non-alcoholic steatohepatitis (NASH); a liver disease often preceding the development of Type 2 diabetes (T2DM). This model was obtained by the administration of a high-fat diet. Our results demonstrate that these algae only delayed and reduced the peak of blood glucose (p < 0.05) in mice fed with normal diet, without changing the area under the blood glucose curve (AUC). In the model of NASH, the phytocomplex was able to reduce both the postprandial glycaemic peak, and the AUC. The administration of the extract in a diet particularly rich in fat is associated with a delay in carbohydrate digestion, but also with a decrease in its assimilation. In conclusion, our results indicate that this algal extract may be useful in the control of carbohydrate digestion and absorption. This effect may be therapeutically exploited to prevent the transition of NASH to T2DM.

Adrenomedullin (AM) is a multifunctional peptide endowed with various biological actions mediated by the interaction with the calcitonin receptor-like receptor (CLR), which couples to the receptor activity-modifying proteins 2 or 3 (RAMP2 or RAMP3) to form the functional plasma membrane receptors AM1 and AM2, respectively. In this study, we investigated for the first time the expression and localization of AM, CLR, RAMP2 and RAMP3 in human thymic tissue from newborns and in primary cultures of thymic epithelial cells (TECs) and thymocytes. Immunohistochemical analysis of thymic tissue showed that both AM and RAMP2 are abundantly expressed in the epithelial cells of medulla and cortex, blood vessels and mastocytes. In contrast, RAMP3 could not be detected. In cultured TECs, double immunofluorescence coupled to confocal microscopy revealed that AM is present in the cytoplasmic compartment, whereas RAMP2 could be detected in the cytoplasm and nucleus, but not in the cell membrane. At variance with RAMP2, CLR was not only present in the nucleus and cytoplasm of TECs, but could also be detected in the cell membrane. The nuclear and cytoplasmic localizations of RAMP2 and CLR and the absence of RAMP2 in the cell membrane were confirmed by western-blot analysis performed on cell fractions. AM, RAMP2 and CLR could also be detected in thymocytes by means of double immunofluorescence coupled to confocal microscopy, although these proteins were not present in the whole thymocyte population. In these cells, AM and RAMP2 were detected in the cytoplasm, whereas CLR could be observed in the cytoplasm and the plasma membrane. In conclusion, our results show that the AM system is widely expressed in human thymus from newborns and suggest that both AM1 receptor components CLR and RAMP2 are not associated with the plasma membrane of TECs and thymocytes but are located intracellularly, notably in the nucleus.

Although the induction of cytochrome P450 (CYP) has long been investigated in patients with cirrhosis, the question whether liver dysfunction impairs the response to CYP inducers still remains unresolved. Moreover, the mechanism underlying the possible effect of cirrhosis on induction has not been investigated. Since ethical constraints do not permit methodologically rigorous studies in humans, this question was addressed by investigating the effect of the prototypical inducer benzo[a]pyrene (BP) on CYP1A1 and CYP1A2 in cirrhotic rats stratified according to the severity of liver dysfunction. We simultaneously assessed mRNA level, protein expression and enzymatic activity of the CYP1A enzymes, as well as mRNA and protein expressions of the aryl hydrocarbon receptor (AhR), which mediates the BP effect. Basal mRNA and protein expressions of CYP1A1 were virtually absent in both healthy and cirrhotic rats. On the contrary, CYP1A2 mRNA, protein and enzyme activity were constitutively present in healthy rats and decreased significantly as liver function worsened. BP treatment markedly increased the concentrations of mRNA and immunodetectable protein, and the enzymatic activities of both CYP1A enzymes to similar levels in healthy and non-ascitic cirrhotic rats. Induced mRNA levels, protein expressions and enzymatic activities of both CYPs were much lower in ascitic rats and were proportionally reduced. Both constitutive and induced protein expressions of AhR were significantly lower in ascitic than in healthy rats. These results indicate that the inducibility of CYP1A enzymes is well preserved in compensated cirrhosis, whereas it is markedly reduced when liver dysfunction becomes severe. Induction appears to be impaired at the transcriptional level, due to the reduced expression of AhR, which controls the transcription of CYP1A genes.