1,8-Cineole is a plant-derived monoterpene and a major constituent of Eucalyptus essential oil. Previously, we demonstrated that 1,8-cineole inhibited hepatocellular carcinoma (HCC) HepG2 cell ...growth. However, the underlying mechanisms remain unknown. Here, we evaluated the mechanisms of action of 1,8-cineole and the potential benefits of its combination with anticancer compounds harboring “anti-senescence” properties in HepG2 cells.
Cell viability was determined by the MTT assay. Cell cycle was assessed through flow cytometry (FC) and western blot (WB). Senescence was determined by the SA-β-galactosidase assay, and apoptosis by caspase-3 activity, WB, and TUNEL. MAPKs (ERK, JNK, and p38), AMPK, and Akt/mTOR were analyzed by WB. Reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were evaluated by FC and fluorescence microscopy.
1,8-Cineole inhibited cell proliferation by promoting G0/G1 arrest. While 1,8-cineole was unable to trigger apoptosis, it induced cellular senescence. 1,8-Cineole promoted ROS production, ΔΨm depolarization, AMPK, ERK, and p38 activation and mTOR inhibition. Antioxidants, like N-acetyl-L-cysteine and vitamins, prevented HepG2 cell growth inhibition and senescence induced by 1,8-cineole. Pre-incubation with 1,8-cineole sensitized HepG2 cells to the anti-senescence compounds, quercetin, simvastatin, U0126, and SB202190. Combinations of 1,8-cineole and each compound synergistically inhibited cell viability, and combined treatment with 1,8-cineole and simvastatin induced apoptosis.
1,8-Cineole induces G0/G1 arrest and senescence in HepG2 cells through oxidative stress and MAPK, AMPK, and Akt/mTOR pathways, and sensitizes cells to anti-senescence drugs, suggesting that 1,8-cineole has potential as an antineoplastic and adjuvant compound in combination with anti-senescence drugs in HCC therapy.
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•1,8-Cineole (CN) promotes G0/G1 cell cycle arrest and senescence in HepG2 cells.•CN induces AMPK, ERK and p38 kinases and suppresses mTOR/p70S6K pathway.•CN induces ROS which contribute to cell growth inhibition and senescence.•CN sensitizes cells to anticancer drugs harboring anti-senescence properties.
Alcohol dependence and alcoholic liver disease represent a major public health problem with substantial morbidity and mortality. By yet incompletely understood mechanisms, chronic alcohol abuse is ...associated with increased intestinal permeability and alterations of the gut microbiota composition, allowing bacterial components, bacteria, and metabolites to reach the portal and the systemic circulation. These gut-derived bacterial products are recognized by immune cells circulating in the blood or residing in remote organs such as the liver leading to the release of pro-inflammatory cytokines which are considered important mediators of the liver-gut-brain communication. Although circulating cytokines are likely not the sole factors involved, they can induce liver inflammation/damage and reach the central nervous system where they favor neuroinflammation which is associated with change in mood, cognition, and drinking behavior. In this review, the authors focus on the current evidence describing the changes that occur in the intestinal microbiota with chronic alcohol consumption in conjunction with intestinal barrier breakdown and inflammatory changes sustaining the concept of a gut-liver-brain axis in the pathophysiology of alcohol dependence and alcoholic liver disease.
Preclinical studies have largely supported that alcohol-consumption induces the development of an important neuro-inflammation and this neuro-inflammation contributes to alcohol-drinking behaviors, ...notably through TLR4 and LPS related mechanisms. The neuro-inflammation originates from a direct interaction of ethanol with the neuronal and immune brain cells, but also from the generation of an inflammation at the periphery. Ethanol in particular interacts with the intestine to develop a gut dysbiosis and an increase in gut permeability, that allows the liberation of bacterial fragments to the systemic circulation and induces a pro-inflammatory response in the systemic circulation and peripheral organs, and in particular the liver. Peripheral cytokines or activated peripheral cells may cross the blood-brain barrier and activate neuro-inflammation. In humans, peripheral inflammation and intestinal dysbiosis are related to symptoms of alcohol use disorders (AUD), such as depression, anxiety and alcohol-craving, However, the dysbiosis, could also participate in a different manner to the symptomatology of the addiction, possibly by interacting with the stress system, by interfering with the sleep processes and altering the abilities for social interactions. The role of the gut suggests that interventions with probiotics or prebiotics might in the future be of interest for the treatment of the addiction.
This article is part of the Special Issue entitled “Alcoholism”.
•A review on peripheral sources of inflammation in AUD is proposed.•The intestine appears as a major source of peripheral inflammation.•Other peripheral sources of inflammation like the gut-wall or the liver are also suggested.•Mechanisms for transduction of peripheral inflammation to the CNS are evoked.•Hypotheses regarding the relation between peripheral inflammation and the symptoms of AUD is are described.
Background and Aims
The progression of alcohol‐associated liver disease (ALD) in its early precirrhotic stages can be a silent process. Serum keratin 18 levels (K18‐M65) predict severe events and ...mortality in advanced stages of ALD, but data on this biomarker in early stages are scarce. We evaluated the diagnostic accuracy of K18‐M65 levels in identifying early forms of ALD.
Methods
We prospectively evaluated two cohorts of actively drinking patients with alcohol use disorder (AUD) following a rehabilitation program (training (n = 162) and validation (n = 78)) and matched healthy controls (n = 21). Clinical, laboratory, and imaging data were used to distinguish AUD patients with simple steatosis (minimal ALD) and steatohepatitis/fibrosis (early ALD). We measured serum K18‐M65 levels and assessed their ability to predict early ALD.
Results
High levels of K18‐M65 characterized AUD patients with early ALD, while levels in the minimal ALD group were similar to those in healthy controls. K18‐M65 levels distinguished minimal liver disease from early ALD (AUROC = 0.8704; p < 0.0001) with an optimal cutoff at 265.9 U/L. K18‐M65 levels strongly correlated with transaminases and predicted early ALD (OR 25.81; 95% CI 3.166–336.1; p < 0.0001), controlled attenuation parameter, and liver stiffness independently from transaminases and other potential confounders. K18‐M65 levels did not discriminate between fibrosis and steatohepatitis but correlated with histological signs of hepatocellular injury and inflammation (all p < 0.05). The K18‐M65 cutoff detected early ALD in the validation cohort with high accuracy (sensitivity 86.67%, specificity 96.67%) and a very high positive likelihood ratio (28.6; 95% CI 4.14–197.73).
Conclusions
Serum K18‐M65 levels can be used as a biomarker to detect early ALD stages with excellent predictive value.
The study prospectively evaluated and validated in two cohorts of actively drinking alcohol use disorder (AUD) patients the diagnostic value of keratin 18 fragment M65 (K18‐M65) for identifying early forms of alcohol‐associated liver disease (ALD). K18‐M65 detected with high diagnostic accuracy ALD in pre‐cirrhotic stages outperforming serum transaminases in identifying early ALD. K18‐M65 primarily correlated with hepatocellular injury and inflammation on histology. K18‐M65 could be used as a non‐invasive screening tool for detection of early forms of ALD in AUD patients.
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•Fecal levels of C. albicans and ECE1 are increased in patients with alcoholic hepatitis.•Candidalysin enhances ethanol-induced liver disease and is associated with higher mortality ...in mice.•Candidalysin damages hepatocytes in a dose-dependent manner.•Candidalysin is associated with the severity of liver disease and mortality in patients with alcoholic hepatitis.
Alcohol-associated liver disease is a leading indication for liver transplantation and a leading cause of mortality. Alterations to the gut microbiota contribute to the pathogenesis of alcohol-associated liver disease. Patients with alcohol-associated liver disease have increased proportions of Candida spp. in the fecal mycobiome, yet little is known about the effect of intestinal Candida on the disease. Herein, we evaluated the contributions of Candida albicans and its exotoxin candidalysin in alcohol-associated liver disease.
C. albicans and the extent of cell elongation 1 (ECE1) were analyzed in fecal samples from controls, patients with alcohol use disorder and those with alcoholic hepatitis. Mice colonized with different and genetically manipulated C. albicans strains were subjected to the chronic-plus-binge ethanol diet model. Primary hepatocytes were isolated and incubated with candidalysin.
The percentages of individuals carrying ECE1 were 0%, 4.76% and 30.77% in non-alcoholic controls, patients with alcohol use disorder and patients with alcoholic hepatitis, respectively. Candidalysin exacerbates ethanol-induced liver disease and is associated with increased mortality in mice. Candidalysin enhances ethanol-induced liver disease independently of the β-glucan receptor C-type lectin domain family 7 member A (CLEC7A) on bone marrow-derived cells, and candidalysin does not alter gut barrier function. Candidalysin can damage primary hepatocytes in a dose-dependent manner in vitro and is associated with liver disease severity and mortality in patients with alcoholic hepatitis.
Candidalysin is associated with the progression of ethanol-induced liver disease in preclinical models and worse clinical outcomes in patients with alcoholic hepatitis.
Candidalysin is a peptide toxin secreted by the commensal gut fungus Candida albicans. Candidalysin enhances alcohol-associated liver disease independently of the β-glucan receptor CLEC7A on bone marrow-derived cells in mice without affecting intestinal permeability. Candidalysin is cytotoxic to primary hepatocytes, indicating a direct role of candidalysin on ethanol-induced liver disease. Candidalysin might be an effective target for therapy in patients with alcohol-associated liver disease.
Alterations in the gut microbiome have been associated with the severity of nonalcoholic fatty liver disease (NAFLD). Previous studies focused exclusively on the bacteria in the microbiome; we ...investigated changes in the viral microbiome (virome) in patients with NAFLD.
In a prospective, cross-sectional, observational study, we extracted RNA and DNA virus-like particles from fecal samples from 73 patients with NAFLD: 29 patients had an NAFLD Activity Score (NAS) of 0–4, 44 patients had an NAS of 5–8 or liver cirrhosis (LCI), 37 patients had F0–F1 fibrosis, and 36 patients had F2–F4 fibrosis. As controls, 9 individuals without liver disease and 13 patients with mild primary biliary cholangitis were included in the analysis. We performed shotgun metagenomic sequencing of virus-like particles.
Patients with NAFLD and NAS 5–8/LCI had a significant decrease in intestinal viral diversity compared with patients with NAFLD and NAS 0–4 or control individuals. The presence of more advanced NAFLD was associated with a significant reduction in the proportion of bacteriophages compared with other intestinal viruses. Using multivariate logistic regression analysis with leave-1-out cross validation, we developed a model, including a viral diversity index and simple clinical variables, that identified patients with NAS 5–8/LCI with an area under the curve of 0.95 (95% confidence interval, 0.91–0.99) and F2–F4 fibrosis with an area under the curve of 0.88 (95% confidence interval, 0.80–0.95). Addition of data on viral diversity significantly improved multivariate models, including those based on only clinical parameters or bacterial diversity.
In a study of fecal viromes from patients with NAFLD and control individuals, we associated histologic markers of NAFLD severity with significant decreases in viral diversity and proportion of bacteriophages. We developed a model based on fecal viral diversity and clinical data that identifies patients with severe NAFLD and fibrosis more accurately than models based only on clinical or bacterial data.
Studies investigating the gut-liver axis have largely focused on bacteria, whereas little is known about commensal fungi. We characterized fecal fungi in patients with non-alcoholic fatty liver ...disease (NAFLD) and investigated their role in a fecal microbiome-humanized mouse model of Western diet-induced steatohepatitis.
We performed fungal internal transcribed spacer 2 sequencing using fecal samples from 78 patients with NAFLD, 16 controls and 73 patients with alcohol use disorder. Anti-Candida albicans (C. albicans) IgG was measured in blood samples from 17 controls and 79 patients with NAFLD. Songbird, a novel multinominal regression tool, was used to investigate mycobiome changes. Germ-free mice were colonized with feces from patients with non-alcoholic steatohepatitis (NASH), fed a Western diet for 20 weeks and treated with the antifungal amphotericin B.
The presence of non-obese NASH or F2-F4 fibrosis was associated with a distinct fecal mycobiome signature. Changes were characterized by an increased log-ratio for Mucor sp./Saccharomyces cerevisiae (S. cerevisiae) in patients with NASH and F2-F4 fibrosis. The C. albicans/S. cerevisiae log-ratio was significantly higher in non-obese patients with NASH when compared with non-obese patients with NAFL or controls. We observed a different fecal mycobiome composition in patients with NAFLD and advanced fibrosis compared to those with alcohol use disorder and advanced fibrosis. Plasma anti-C. albicans IgG was increased in patients with NAFLD and advanced fibrosis. Gnotobiotic mice, colonized with human NASH feces and treated with amphotericin B were protected from Western diet-induced steatohepatitis.
Non-obese patients with NAFLD and more advanced disease have a different fecal mycobiome composition to those with mild disease. Antifungal treatment ameliorates diet-induced steatohepatitis in mice. Intestinal fungi could be an attractive target to attenuate NASH.
Non-alcoholic fatty liver disease is one of the most common chronic liver diseases and is associated with changes in the fecal bacterial microbiome. We show that patients with non-alcoholic fatty liver disease and more severe disease stages have a specific composition of fecal fungi and an increased systemic immune response to Candida albicans. In a fecal microbiome-humanized mouse model of Western diet-induced steatohepatitis, we show that treatment with antifungals reduces liver damage.
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•More advanced disease severity in non-alcoholic fatty liver disease is associated with a specific composition of fecal fungi.•Changes are particularly present in non-obese patients.•Systemic antibodies against Candida albicans are increased in patients with non-alcoholic fatty liver disease and advanced fibrosis.•Antifungals protect germ-free fecal microbiome-humanized mice from Western diet-induced steatohepatitis.
Chronic liver disease with cirrhosis is the 12th leading cause of death in the United States, and alcoholic liver disease accounts for approximately half of all cirrhosis deaths. Chronic alcohol ...consumption is associated with intestinal bacterial dysbiosis, yet we understand little about the contribution of intestinal fungi, or mycobiota, to alcoholic liver disease. Here we have demonstrated that chronic alcohol administration increases mycobiota populations and translocation of fungal β-glucan into systemic circulation in mice. Treating mice with antifungal agents reduced intestinal fungal overgrowth, decreased β-glucan translocation, and ameliorated ethanol-induced liver disease. Using bone marrow chimeric mice, we found that β-glucan induces liver inflammation via the C-type lectin-like receptor CLEC7A on Kupffer cells and possibly other bone marrow-derived cells. Subsequent increases in IL-1β expression and secretion contributed to hepatocyte damage and promoted development of ethanol-induced liver disease. We observed that alcohol-dependent patients displayed reduced intestinal fungal diversity and Candida overgrowth. Compared with healthy individuals and patients with non-alcohol-related cirrhosis, alcoholic cirrhosis patients had increased systemic exposure and immune response to mycobiota. Moreover, the levels of extraintestinal exposure and immune response correlated with mortality. Thus, chronic alcohol consumption is associated with an altered mycobiota and translocation of fungal products. Manipulating the intestinal mycobiome might be an effective strategy for attenuating alcohol-related liver disease.
Linalool is a plant-derived monoterpene with anticancer activity, however its mechanisms of action remain poorly understood. The aim of this work was to elucidate the anticancer mechanisms of action ...of linalool in hepatocellular carcinoma (HCC) HepG2 cells.
Cell viability and proliferation were determined by WST-1 assay and BrdU incorporation, respectively. Cell cycle analysis was assessed through flow cytometry (FC) and western blot (WB). Apoptosis was determined by caspase-3 activity, TUNEL assay and WB. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were analyzed by FC and fluorescence microscopy. Expression of Ras, MAPKs (ERK, JNK and p38) and Akt/mTOR pathways were evaluated by WB.
Linalool (0–2.5 mM) dose-dependently inhibited cell proliferation by inducing G0/G1 cell cycle arrest, through Cdk4 and cyclin A downregulation, p21 and p27 upregulation, and apoptosis, characterized by MMP loss, caspase-3 activation, PARP cleavage and DNA fragmentation. Low concentrations of linalool (1.0 mM) reduced membrane-bound Ras and Akt activity whereas higher amounts (2.0 mM) triggered mTOR inhibition and ROS generation, in correlation with MAPKs activation and Akt phosphorylation. ROS scavenger N-acetyl-L-cysteine partially rescued HepG2 cell growth and prevented MPP depolarization, ERK and JNK activation. Moreover, specific ERK and Akt phosphorylation inhibitors potentiated linalool anti-cancer activity, pointing Akt and ERK activation as pro-survival mechanisms in response to higher concentrations of linalool.
This report reveals that linalool induces G0/G1 arrest and apoptosis in HepG2 cells involving Ras, MAPKs and Akt/mTOR pathways and suggests that linalool is a promising anticancer agent for HCC therapy.
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The gastrointestinal tract is the natural habitat for a huge community of microorganisms, comprising bacteria, viruses, fungi and yeast. This microbial ecosystem codevelops with the host throughout ...life and is subject to a complex interplay that depends on multiple factors including host genetics, nutrition, life-style, stress, diseases and antibiotics use. The gut microbiota, that refers to intestinal bacteria, has profound influence on the host immune system, metabolism and nervous system. Indeed, intestinal bacteria supply the host with essential nutrients such as vitamins, metabolize bile acids and undigested compounds, defend against pathogen invasion, participate to the development of the intestinal architecture and the intestinal immune system and play an important role in the maintenance of the gut barrier function. More recently, the gut microbiota has been shown to influence brain functions, such as myelin synthesis, the blood-brain barrier permeability and neuroinflammatory responses but also mood and behavior. The cross-talk between microbes and the host implicates a vast array of signaling pathways that involve many different classes of molecules like metabolites produced by the bacteria from dietary or endogenous sources of carbohydrates and proteins (i.e. short-chain fatty acids (SCFAs), indole), neurotransmitters and inflammatory cytokines. This review will focus on the involvement of the gut microbiota in the pathophysiological aspects of alcohol dependence related to the gut barrier function, liver damage and psychological disturbances. We will also discuss the possibility to create new and realistic humanized animal models of alcohol dependence by the use of fecal transplantation.
•The gut microbiota is a dynamic ecosystem that influences host heath, including brain functions and behavior.•Alcohol abuse has a detrimental effect on the gut barrier and on intestinal microbiota.•Fecal transplantation is a new tool to analyze the causal role of gut microbiota in brain diseases.•Probiotics and prebiotics might represent a promising therapeutic approach in alcohol-use disorder.
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