Innate Immune Training of Granulopoiesis Promotes Anti-tumor Activity.

Trained innate immunity, induced via modulation of mature myeloid cells or their bone marrow progenitors, mediates sustained increased responsiveness to secondary challenges. Here, we investigated whether anti-tumor immunity can be enhanced through induction of trained immunity. Pre-treatment of mice with ß-glucan, a fungal-derived prototypical agonist of trained immunity, resulted in diminished tumor growth. The anti-tumor effect of ß-glucan-induced trained immunity was associated with transcriptomic and epigenetic rewiring of granulopoiesis and neutrophil reprogramming toward an anti-tumor phenotype; this process required type I interferon signaling irrespective of adaptive immunity in the host. Adoptive transfer of neutrophils from ß-glucan-trained mice to naive recipients suppressed tumor growth in the latter in a ROS-dependent manner. Moreover, the anti-tumor effect of ß-glucan-induced trained granulopoiesis was transmissible by bone marrow transplantation to recipient naive mice. Our findings identify a novel and therapeutically relevant anti-tumor facet of trained immunity involving appropriate rewiring of granulopoiesis.

Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity.

Trained innate immunity fosters a sustained favorable response of myeloid cells to a secondary challenge, despite their short lifespan in circulation. We thus hypothesized that trained immunity acts via modulation of hematopoietic stem and progenitor cells (HSPCs). Administration of ß-glucan (prototypical trained-immunity-inducing agonist) to mice induced expansion of progenitors of the myeloid lineage, which was associated with elevated signaling by innate immune mediators, such as IL-1ß and granulocyte-macrophage colony-stimulating factor (GM-CSF), and with adaptations in glucose metabolism and cholesterol biosynthesis. The trained-immunity-related increase in myelopoiesis resulted in a beneficial response to secondary LPS challenge and protection from chemotherapy-induced myelosuppression in mice. Therefore, modulation of myeloid progenitors in the bone marrow is an integral component of trained immunity, which to date, was considered to involve functional changes of mature myeloid cells in the periphery.

A self-sustained loop of inflammation-driven inhibition of beige adipogenesis in obesity.

In obesity, inflammation of white adipose tissue (AT) is associated with diminished generation of beige adipocytes ('beige adipogenesis'), a thermogenic and energy-dissipating function mediated by beige adipocytes that express the uncoupling protein UCP1. Here we delineated an inflammation-driven inhibitory mechanism of beige adipogenesis in obesity that required direct adhesive interactions between macrophages and adipocytes mediated by the integrin α4 and its counter-receptor VCAM-1, respectively; expression of the latter was upregulated in obesity. This adhesive interaction reciprocally and concomitantly modulated inflammatory activation of macrophages and downregulation of UCP1 expression dependent on the kinase Erk in adipocytes. Genetic or pharmacological inactivation of the integrin α4 in mice resulted in elevated expression of UCP1 and beige adipogenesis of subcutaneous AT in obesity. Our findings, established in both mouse systems and human systems, reveal a self-sustained cycle of inflammation-driven impairment of beige adipogenesis in obesity.

Liver endothelial cells in NAFLD and transition to NASH and HCC.

Non-alcoholic fatty liver disease (NAFLD) is considered as the hepatic manifestation of metabolic syndrome, which is characterised by obesity, insulin resistance, hypercholesterolemia and hypertension. NAFLD is the most frequent liver disease worldwide and more than 10% of NAFLD patients progress to the inflammatory and fibrotic stage of non-alcoholic steatohepatitis (NASH), which can lead to end-stage liver disease including hepatocellular carcinoma (HCC), the most frequent primary malignant liver tumor. Liver sinusoidal endothelial cells (LSEC) are strategically positioned at the interface between blood and hepatic parenchyma. LSECs are highly specialized cells, characterised by the presence of transcellular pores, called fenestrae, and exhibit anti-inflammatory and anti-fibrotic characteristics under physiological conditions. However, during NAFLD development they undergo capillarisation and acquire a phenotype similar to vascular endothelial cells, actively promoting all pathophysiological aspects of NAFLD, including steatosis, inflammation, and fibrosis. LSEC dysfunction is critical for the progression to NASH and HCC while restoring LSEC homeostasis appears to be a promising approach to prevent NAFLD progression and its complications and even reverse tissue damage. In this review we present current information on the role of LSEC throughout the progressive phases of NAFLD, summarising in vitro and in vivo experimental evidence and data from human studies.

Diagnostic Challenges during Inflammation and Cancer: Current Biomarkers and Future Perspectives in Navigating through the Minefield of Reactive versus Dysplastic and Cancerous Lesions in the Digestive System.

In the setting of pronounced inflammation, changes in the epithelium may overlap with neoplasia, often rendering it impossible to establish a diagnosis with certainty in daily clinical practice. Here, we discuss the underlying molecular mechanisms driving tissue response during persistent inflammatory signaling along with the potential association with cancer in the gastrointestinal tract, pancreas, extrahepatic bile ducts, and liver. We highlight the histopathological challenges encountered in the diagnosis of chronic inflammation in routine practice and pinpoint tissue-based biomarkers that could complement morphology to differentiate reactive from dysplastic or cancerous lesions. We refer to the advantages and limitations of existing biomarkers employing immunohistochemistry and point to promising new markers, including the generation of novel antibodies targeting mutant proteins, miRNAs, and array assays. Advancements in experimental models, including mouse and 3D models, have improved our understanding of tissue response. The integration of digital pathology along with artificial intelligence may also complement routine visual inspections. Navigating through tissue responses in various chronic inflammatory contexts will help us develop novel and reliable biomarkers that will improve diagnostic decisions and ultimately patient treatment.

Lipidomic Analysis of Liver and Adipose Tissue in a High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease Mice Model Reveals Alterations in Lipid Metabolism by Weight Loss and Aerobic Exercise.

Detailed investigation of the lipidome remodeling upon normal weight conditions, obesity, or weight loss, as well as the influence of physical activity, can help to understand the mechanisms underlying dyslipidemia in metabolic conditions correlated to the emergence and progression of non-alcoholic fatty liver disease (NAFLD). C57BL/6 male mice were fed a normal diet (ND) or a high-fat diet (HFD) for 20 weeks. Subgroups within the high-fat diet (HFD) group underwent different interventions: some engaged in exercise (HFDex), others were subjected to weight loss (WL) by changing from the HFD to ND, and some underwent a combination of weight loss and exercise (WLex) during the final 8 weeks of the 20-week feeding period. To support our understanding, not only tissue-specific lipid remodeling mechanisms but also the cross-talk between different tissues and their impact on the systemic regulation of lipid metabolism are essential. Exercise and weight loss-induced specific adaptations in the liver and visceral adipose tissue lipidomes of mice were explored by the UPLC-TOF-MS/MS untargeted lipidomics methodology. Lipidomic signatures of ND and HFD-fed mice undergoing weight loss were compared with animals with and without physical exercise. Several lipid classes were identified as contributing factors in the discrimination of the groups by multivariate analysis models, such as glycerolipids, glycerophospholipids, sphingolipids, and fatty acids, with respect to liver samples, whereas triglycerides were the only lipid class identified in visceral adipose tissue. Lipids found to be dysregulated in HFD animals are related to well-established pathways involved in the biosynthesis of PC, PE, and TG metabolism. These show a reversing trend back to basic levels of ND when animals change to a normal diet after 12 weeks, whereas the impact of exercise, though in some cases it slightly enhances the reversing trend, is not clear.

Short term starvation potentiates the efficacy of chemotherapy in triple negative breast cancer via metabolic reprogramming.

BACKGROUND: Chemotherapy (CT) is central to the treatment of triple negative breast cancer (TNBC), but drug toxicity and resistance place strong restrictions on treatment regimes. Fasting sensitizes cancer cells to a range of chemotherapeutic agents and also ameliorates CT-associated adverse effects. However, the molecular mechanism(s) by which fasting, or short-term starvation (STS), improves the efficacy of CT is poorly characterized. METHODS: The differential responses of breast cancer or near normal cell lines to combined STS and CT were assessed by cellular viability and integrity assays (Hoechst and PI staining, MTT or H2DCFDA staining, immunofluorescence), metabolic profiling (Seahorse analysis, metabolomics), gene expression (quantitative real-time PCR) and iRNA-mediated silencing. The clinical significance of the in vitro data was evaluated by bioinformatical integration of transcriptomic data from patient data bases: The Cancer Genome Atlas (TCGA), European Genome-phenome Archive (EGA), Gene Expression Omnibus (GEO) and a TNBC cohort. We further examined the translatability of our findings in vivo by establishing a murine syngeneic orthotopic mammary tumor-bearing model. RESULTS: We provide mechanistic insights into how preconditioning with STS enhances the susceptibility of breast cancer cells to CT. We showed that combined STS and CT enhanced cell death and increased reactive oxygen species (ROS) levels, in association with higher levels of DNA damage and decreased mRNA levels for the NRF2 targets genes NQO1 and TXNRD1 in TNBC cells compared to near normal cells. ROS enhancement was associated with compromised mitochondrial respiration and changes in the metabolic profile, which have a significant clinical prognostic and predictive value. Furthermore, we validate the safety and efficacy of combined periodic hypocaloric diet and CT in a TNBC mouse model. CONCLUSIONS: Our in vitro, in vivo and clinical findings provide a robust rationale for clinical trials on the therapeutic benefit of short-term caloric restriction as an adjuvant to CT in triple breast cancer treatment.

Analysis of the Role of Stellate Cell VCAM-1 in NASH Models in Mice.

Non-alcoholic fatty liver disease (NAFLD) can progress to non-alcoholic steatohepatitis (NASH), characterized by inflammation and fibrosis. Fibrosis is mediated by hepatic stellate cells (HSC) and their differentiation into activated myofibroblasts; the latter process is also promoted by inflammation. Here we studied the role of the pro-inflammatory adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) in HSCs in NASH. VCAM-1 expression was upregulated in the liver upon NASH induction, and VCAM-1 was found to be present on activated HSCs. We therefore utilized HSC-specific VCAM-1-deficient and appropriate control mice to explore the role of VCAM-1 on HSCs in NASH. However, HSC-specific VCAM-1-deficient mice, as compared to control mice, did not show a difference with regards to steatosis, inflammation and fibrosis in two different models of NASH. Hence, VCAM-1 on HSCs is dispensable for NASH development and progression in mice.

Genetic and Diet-Induced Animal Models for Non-Alcoholic Fatty Liver Disease (NAFLD) Research.

A rapidly increasing incidence of non-alcoholic fatty liver disease (NAFLD) is noted worldwide due to the adoption of western-type lifestyles and eating habits. This makes the understanding of the molecular mechanisms that drive the pathogenesis of this chronic disease and the development of newly approved treatments of utmost necessity. Animal models are indispensable tools for achieving these ends. Although the ideal mouse model for human NAFLD does not exist yet, several models have arisen with the combination of dietary interventions, genetic manipulations and/or administration of chemical substances. Herein, we present the most common mouse models used in the research of NAFLD, either for the whole disease spectrum or for a particular disease stage (e.g., non-alcoholic steatohepatitis). We also discuss the advantages and disadvantages of each model, along with the challenges facing the researchers who aim to develop and use animal models for translational research in NAFLD. Based on these characteristics and the specific study aims/needs, researchers should select the most appropriate model with caution when translating results from animal to human.

Estrogen Receptor Subtypes Elicit a Distinct Gene Expression Profile of Endothelial-Derived Factors Implicated in Atherosclerotic Plaque Vulnerability.

In the presence of established atherosclerosis, estrogens are potentially harmful. MMP-2 and MMP-9, their inhibitors (TIMP-2 and TIMP-1), RANK, RANKL, OPG, MCP-1, lysyl oxidase (LOX), PDGF-ß, and ADAMTS-4 play critical roles in plaque instability/rupture. We aimed to investigate (i) the effect of estradiol on the expression of the abovementioned molecules in endothelial cells, (ii) which type(s) of estrogen receptors mediate these effects, and (iii) the role of p21 in the estrogen-mediated regulation of the aforementioned factors. Human aortic endothelial cells (HAECs) were cultured with estradiol in the presence or absence of TNF-α. The expression of the aforementioned molecules was assessed by qRT-PCR and ELISA. Zymography was also performed. The experiments were repeated in either ERα- or ERß-transfected HAECs and after silencing p21. HAECs expressed only the GPR-30 estrogen receptor. Estradiol, at low concentrations, decreased MMP-2 activity by 15-fold, increased LOX expression by 2-fold via GPR-30, and reduced MCP-1 expression by 3.5-fold via ERß. The overexpression of ERα increased MCP-1 mRNA expression by 2.5-fold. In a low-grade inflammation state, lower concentrations of estradiol induced the mRNA expression of MCP-1 (3.4-fold) and MMP-9 (7.5-fold) and increased the activity of MMP-2 (1.7-fold) via GPR-30. Moreover, p21 silencing resulted in equivocal effects on the expression of the abovementioned molecules. Estradiol induced different effects regarding atherogenic plaque instability through different ERs. The balance of the expression of the various ER subtypes may play an important role in the paradoxical characterization of estrogens as both beneficial and harmful.

Circulating cell-free DNA species affect the risk of hepatocellular carcinoma in treated chronic hepatitis B patients.

Hepatocellular carcinoma (HCC) may still develop in chronic hepatitis B (CHB) patients even under effective long-term oral antiviral therapy, but its pathogenesis in the setting of long-standing inhibition of viral replication has not been completely elucidated. We investigated whether species of circulating cell-free DNA (cfDNA) may be involved in the process of hepatocarcinogenesis in treated CHB patients. Serum samples were obtained from HBeAg-negative CHB patients with (HCC cases, n = 37) or without HCC development during the first 5 years of oral antiviral therapy (controls, n = 74). HCC cases and controls were matched 1:2 for age, sex and platelets. Determination of different circulating cfDNA species (before HCC diagnosis in HCC cases) including total cfDNA quantity, levels of Alu repeat DNA and RNase P coding DNA, copies of mitochondrial DNA and levels of 5-methyl-2'-deoxycytidine as an indicator of DNA methylation was performed. HCC cases compared with controls had higher median levels of Alu247 (123 vs 69 genomic equivalent, p = .042) and RNase P coding DNA (68 vs 15 genomic equivalent, p < .001). In contrast, median cfDNA concentration, Alu115 levels, Alu247/Alu115 ratio as an index of DNA integrity and mitochondrial DNA copies did not differ significantly between HCC cases and controls. Receiver operating characteristic curve analysis showed that levels RNase P coding DNA offered good prediction of subsequent HCC development (c-statistic: 0.80, p < .001). In conclusion, serum levels of RNase P coding DNA are increased years before HCC diagnosis and could be potentially helpful in the prediction of the HCC risk in treated HBeAg-negative CHB patients.

The Role of Senescence in the Development of Nonalcoholic Fatty Liver Disease and Progression to Nonalcoholic Steatohepatitis.

In recent years, cellular senescence has generated a lot of interest among researchers because of its involvement in both the normal aging process and common human diseases. During senescence, cells undergo alterations that include telomere shortening, nuclear area enlargement, and genomic and mitochondrial DNA damage, leading to irreversible cell cycle arrest, and secretion of proinflammatory cytokines. Evidence suggests that the complex process of senescence is involved in the development of a plethora of chronic diseases including metabolic and inflammatory disorders and tumorigenesis. Recently, several human and animal studies have emphasized the involvement of senescence in the pathogenesis and development of liver steatosis including the progression to nonalcoholic steatohepatitis (NASH) as characterized by the additional emergence of inflammation, hepatocyte ballooning, and liver fibrosis. The development of nonalcoholic fatty liver disease (NAFLD) and its progression to NASH are commonly accompanied by several pathophysiological events including metabolic dysregulation and inflammatory phenomena occurring within the liver that may contribute to or derive from cellular senescence, implying that the latter may be both a stimulus and a consequence of the disease. Conclusion: In this review, we summarize the current literature on the impact of cellular senescence in NAFLD/NASH and discuss the effectiveness and safety of novel senolytic drugs and therapeutic options available to delay or treat the disease. Finally, we identify the open questions and issues to be addressed in the near future.

Robo4-mediated pancreatic endothelial integrity decreases inflammation and islet destruction in autoimmune diabetes.

In Type 1 Diabetes Mellitus (T1DM), leukocyte infiltration of the pancreatic islets and the resulting immune-mediated destruction of beta cells precede hyperglycemia and clinical disease symptoms. In this context, the role of the pancreatic endothelium as a barrier for autoimmunity- and inflammation-related destruction of the islets is not well studied. Here, we identified Robo4, expressed on endothelial cells, as a regulator of pancreatic vascular endothelial permeability during autoimmune diabetes. Circulating levels of Robo4 were upregulated in mice subjected to the Multiple Low-Dose Streptozotocin (MLDS) model of diabetes. Upon MLDS induction, Robo4-deficiency resulted in increased pancreatic vascular permeability, leukocyte infiltration to the islets and islet apoptosis, associated with reduced insulin levels and faster diabetes development. On the contrary, in vivo administration of Slit2 in mice modestly delayed the emergence of hyperglycaemia and ameliorated islet inflammation in MLDS-induced diabetes. Thus, Robo4-mediated endothelial barrier integrity reduces insulitis and islet destruction in autoimmune diabetes. Our findings highlight the importance of the endothelium as gatekeeper of pancreatic inflammation during T1DM development and may pave the way for novel Robo4-related therapeutic approaches for autoimmune diabetes.

Optimizing mechanical stretching protocols for hypertrophic and anti-apoptotic responses in cardiomyocyte-like H9C2 cells.

Cardiomyocytes possess the ability to respond to mechanical stimuli by reprogramming their gene expression. This study investigated the effects of different loading protocols on signaling and expression responses of myogenic, anabolic, inflammatory, atrophy and pro-apoptotic genes in cardiomyocyte-like H9C2 cells. Differentiated H9C2 cells underwent various stretching protocols by altering their elongation, frequency and duration, utilizing an in vitro cell tension system. The loading-induced expression changes of MyoD, Myogenin, MRF4, IGF-1 isoforms, Atrogin-1, Foxo1, Fuca and IL-6 were measured by Real Time-PCR. The stretching-induced activation of Akt and Erk 1/2 was also evaluated by Western blot analysis. Low strain (2.7% elongation), low frequency (0.25 Hz) and intermediate duration (12 h) stretching protocol was overall the most effective in inducing beneficial responses, i.e., protein synthesis along with the suppression of apoptosis, inflammation and atrophy, in the differentiated cardiomyocytes. These findings demonstrated that varying the characteristics of mechanical loading applied on H9C2 cells in vitro can regulate their anabolic/survival program.

The impact of sodium glucose co-transporter 2 inhibitors on non-alcoholic fatty liver disease.

Affecting one fourth of the global population, non-alcoholic fatty liver disease (NAFLD) is the commonest chronic liver disorder. It encompasses the simple liver fat accumulation to more progressive steatosis, inflammation, and fibrosis characterized as non-alcoholic steatohepatitis (NASH) and in some cases cirrhosis and hepatocellular carcinoma. NAFLD regularly coexists with metabolic disorders, such as obesity and mostly type 2 diabetes mellitus (T2DM). A relatively new class of antidiabetic drugs, the sodium glucose co-transporter 2 (SGLT2) inhibitors exert their action by increasing the urinary glucose and calorie excretion leading to ameliorated plasma glucose levels and lower bodyweight. Recently, several animal studies and human clinical trial have emphasized the possible beneficial impact of SGLT2 inhibitors on NAFLD and its progression to NASH. In this present review, we summarize the current literature regarding the efficacy of the aforementioned category of drugs on anthropometric, laboratory, and histological features of patients with NAFLD. Conclusively, as SGLT2 inhibitors seem to be an appealing therapeutic opportunity for NAFLD management, we identify the open issues and questions to be addressed in order to clarify the impact in choosing antidiabetic medication to treat NAFLD patients associated with T2DM.

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

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

The Effects of Muscle Cell Aging on Myogenesis.

The process of myogenesis gradually deteriorates as the skeletal muscle ages, contributing to muscle mass loss. The aim of this study is to investigate the effect of senescence/aging on skeletal myogenesis, in vitro. A model of multiple cell divisions of C2C12 myoblasts was used to replicate cell senescence. Control and aged myoblasts were investigated during myogenesis, i.e., at days 0, 2, and 6of differentiation. SA-ß-gal activity and comet assay were used as markers of aging and DNA damage. Flow cytometry was performed to characterize potential differences in cell cycle between control and aged cells. Alterations in the mRNA and/or protein expression of myogenic regulatory factors (MRFs), IGF-1 isoforms, apoptotic, atrophy, inflammatory, metabolic and aging-related factors were evaluated. Compared with the control cells, aged myoblasts exhibited G0/G1 cell cycle arrest, DNA damage, increased SA-ß-gal activity, and increased expression of aging-related factors p16 and p21 during differentiation. Moreover, aged myoblasts showed a reduction in the expression of MRFs and metabolic/anabolic factors, along with an increased expression of apoptotic, atrophy and inflammatory factors. A diminished differentiation capacity characterized the aged myoblasts which, in combination with the induction of apoptotic and atrophy factors, indicated a disrupted myogenic lineage in the senescent muscle cells.

Hepatic Senescence Accompanies the Development of NAFLD in Non-Aged Mice Independently of Obesity.

Senescence is considered to be a cardinal player in several chronic inflammatory and metabolic pathologies. The two dominant mechanisms of senescence include replicative senescence, predominantly depending on age-induced telomere shortening, and stress-induced senescence, triggered by external or intracellular harmful stimuli. Recent data indicate that hepatocyte senescence is involved in the development of nonalcoholic fatty liver disease (NAFLD). However, previous studies have mainly focused on age-related senescence during NAFLD, in the presence or absence of obesity, while information about whether the phenomenon is characterized by replicative or stress-induced senescence, especially in non-aged organisms, is scarce. Herein, we subjected young mice to two different diet-induced NAFLD models which differed in the presence of obesity. In both models, liver fat accumulation and increased hepatic mRNA expression of steatosis-related genes were accompanied by hepatic senescence, indicated by the increased expression of senescence-associated genes and the presence of a robust hybrid histo-/immunochemical senescence-specific staining in the liver. Surprisingly, telomere length and global DNA methylation did not differ between the steatotic and the control livers, while malondialdehyde, a marker of oxidative stress, was upregulated in the mouse NAFLD livers. These findings suggest that senescence accompanies NAFLD emergence, even in non-aged organisms, and highlight the role of stress-induced senescence during steatosis development independently of obesity.

Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in High Fat Diet Fed ApoE(-/-) Mice by Activating Autophagy and Reducing ER Stress and Apoptosis.

AIMS/HYPOTHESIS: SGLT-2 inhibitors (SGLT-2i) have been studied as potential treatments against NAFLD, showing varying beneficial effects. The molecular mechanisms mediating these effects have not been fully clarified. Herein, we investigated the impact of empagliflozin on NAFLD, focusing particularly on ER stress, autophagy and apoptosis. METHODS: Five-week old ApoE(-/-) mice were switched from normal to a high-fat diet (HFD). After five weeks, mice were randomly allocated into a control group (HFD + vehicle) and Empa group (HFD + empagliflozin 10 mg/kg/day) for five weeks. At the end of treatment, histomorphometric analysis was performed in liver, mRNA levels of Fasn, Screbp-1, Scd-1, Ppar-γ, Pck-1, Mcp-1, Tnf-α, Il-6, F4/80, Atf4, Elf2α, Chop, Grp78, Grp94, Χbp1, Ire1α, Atf6, mTor, Lc3b, Beclin-1, P62, Bcl-2 and Bax were measured by qRT-PCR, and protein levels of p-EIF2α, EIF2a, CHOP, LC3II, P62, BECLIN-1 and cleaved CASPASE-8 were assessed by immunoblotting. RESULTS: Empagliflozin-treated mice exhibited reduced fasting glucose, total cholesterol and triglyceride serum levels, as well as decreased NAFLD activity score, decreased expression of lipogenic enzymes (Fasn, Screbp-1c and Pck-1) and inflammatory molecules (Mcp-1 and F4/80), compared to the Control group. Empagliflozin significantly decreased the expression of ER stress molecules Grp78, Ire1α, Xbp1, Elf2α, Atf4, Atf6, Chop, P62(Sqstm1) and Grp94; whilst activating autophagy via increased AMPK phosphorylation, decreased mTOR and increased LC3B expression. Finally, empagliflozin increased the Bcl2/Bax ratio and inhibited CASPASE-8 cleavage, reducing liver cell apoptosis. Immunoblotting analysis confirmed the qPCR results. CONCLUSION: These novel findings indicate that empagliflozin treatment for five weeks attenuates NAFLD progression in ApoE(-/-) mice by promoting autophagy, reducing ER stress and inhibiting hepatic apoptosis.

Cellular senescence and hepatitis B-related hepatocellular carcinoma: An intriguing link.

Chronic hepatitis B is mainly responsible for the morbidity and mortality from hepatitis B virus (HBV)-related complications, including hepatocellular carcinoma (HCC) and decompensated cirrhosis. Hepatocellular carcinoma remains the main challenge in the management of not only undiagnosed and/or untreated but also diagnosed and treated patients with chronic HBV infection, as its incidence decreases but is not eliminated even after many years of effective anti-HBV therapy. The exact mechanisms used by HBV to cause malignant transformation remain uncertain, although much of the available data are in favour of a pathogenetic role of HBx protein. Senescence is a cellular state, in which cells lose their ability to proliferate. This biological mechanism may function in a dual mode, namely being both cancer-protective as a result of reduced cellular proliferation, but also cancer-enhancing as a result of modulation of the tissular microenvironment by immune cells during persistent accumulation of senescent cells. Protein X of HBV protein exhibits many similarities in terms of the implemented mechanisms of action and pathways related to the biological process of cellular senescence. Concurrently, insufficient clearance of both senescent and precancerous hepatocytes combined with inadequate immune surveillance as a result of immunosenescence caused by chronic HBV infection may lead to hepatocarcinogenesis. Thus, the effect of HBV seems to be critical as a connecting link between cellular senescence and development of HCC. An ongoing research is underway towards identifying and validating markers of hepatocyte senescence, which could improve the landscape for evaluation of chronic liver disease, thereby providing valuable information in terms of HBV-related carcinogenesis.