Impact of duration of chronic migraine on long-term effectiveness of monoclonal antibodies targeting the calcitonin gene-related peptide pathway-A real-world study.

OBJECTIVE: We assessed whether the effectiveness of monoclonal antibodies (mAbs) targeting the calcitonin gene-related peptide (CGRP) pathway changes according to the duration of chronic migraine (CM) over 12 months. BACKGROUND: In most patients, CM is a progressive disease starting with episodic migraine. Longer CM duration might be associated with more difficult treatment, probably because the mechanisms underlying chronicization are strengthened. Therefore, early treatment of CM could lead to better outcomes compared with later treatment. METHODS: This cohort study included individuals with CM treated with anti-CGRP mAbs in two tertiary headache centers from April 2019 to May 2023. The primary outcome included a change in monthly migraine days (MMDs) from baseline to the third trimester of treatment, 10-12 months. Secondary outcomes established whether response to anti-CGRP mAbs has a more rapid onset in individuals with shorter CM duration compared with longer duration; they included change in MMDs, monthly headache days (MHDs), and days and number of intakes of acute medication during each trimester compared to baseline. Additional outcomes included persisting MMDs, MHDs, and days and number of intakes of acute medication during each trimester of treatment. Patients were compared across tertiles of the overall CM duration. RESULTS: The study included 335 individuals with CM, with a median (interquartile range [IQR]) age of 48 (39-57) years; 270 (80.6%) were women. Patients in the highest tertile of CM duration (aged 18-60 years) were older than patients in the lower duration tertiles (0-7 years and 8-18 years, respectively), with a median (IQR) age of 56 (48-64) years compared with 42 (31-50) years, and 48 (39-56)years, respectively (p = 0.025); however, this difference was likely due to a correlation between age and disease duration. The change in MMDs from baseline to the last trimester of treatment (10-12 months) was comparable across tertiles of CM duration (median [IQR] -12 [-18 to -5] days, -12 [-17 to -6] days, and -12 [-18 to -4] days; p = 0.946). No difference emerged in secondary outcomes, suggesting a similar time to onset of anti-CGRP mAbs effect across all tertiles of CM duration. CONCLUSIONS: Our data showed that anti-CGRP mAbs are effective and have a rapid onset of action in CM regardless of disease duration.

Unveiling nuclear chromatin distribution using IsoConcentraChromJ: A flourescence imaging plugin for IsoRegional and IsoVolumetric based ratios analysis.

Chromatin exhibits non-random distribution within the nucleus being arranged into discrete domains that are spatially organized throughout the nuclear space. Both the spatial distribution and structural rearrangement of chromatin domains in the nucleus depend on epigenetic modifications of DNA and/or histones and structural elements such as the nuclear envelope. These components collectively contribute to the organization and rearrangement of chromatin domains, thereby influencing genome architecture and functional regulation. This study develops an innovative, user-friendly, ImageJ-based plugin, called IsoConcentraChromJ, aimed quantitatively delineating the spatial distribution of chromatin regions in concentric patterns. The IsoConcentraChromJ can be applied to quantitative chromatin analysis in both two- and three-dimensional spaces. After DNA and histone staining with fluorescent probes, high-resolution images of nuclei have been obtained using advanced fluorescence microscopy approaches, including confocal and stimulated emission depletion (STED) microscopy. IsoConcentraChromJ workflow comprises the following sequential steps: nucleus segmentation, thresholding, masking, normalization, and trisection with specified ratios for either 2D or 3D acquisitions. The effectiveness of the IsoConcentraChromJ has been validated and demonstrated using experimental datasets consisting in nuclei images of pre-adipocytes and mature adipocytes, encompassing both 2D and 3D imaging. The outcomes allow to characterize the nuclear architecture by calculating the ratios between specific concentric nuclear areas/volumes of acetylated chromatin with respect to total acetylated chromatin and/or total DNA. The novel IsoConcentrapChromJ plugin could represent a valuable resource for researchers investigating the rearrangement of chromatin architecture driven by epigenetic mechanisms using nuclear images obtained by different fluorescence microscopy methods.

The Anti-Inflammatory Potential of an Ethanolic Extract from Sarcopoterium spinosum Fruits for Protection and/or Counteraction against Oxidative Stress in Dysfunctional Endothelial Cells.

Plants and plant extracts are a relevant source of bioactive compounds widely employed as functional foods. In the Mediterranean area, the shrub Sarcopoterium spinosum is traditionally used as an herbal medicine for weight loss and a diabetes treatment. Inflammation is a protective mechanism involved in the development of many pathological conditions, including cardiovascular diseases. The present study aimed to investigate in vitro the antioxidant and cytoprotective properties of an ethanolic extract from S. spinosum fruits (SEE) in a cellular model of endothelium dysfunction. Corilagin and quercetin are two polyphenols abundant in SEE and were tested for comparison. The exposure of HECV cells for 24 h to 30 µM hydrogen peroxide (H2O2) lead to an oxidative stress condition. When HECV cells were treated with 10 µg/mL of SEE or single compounds after or before the oxidative insult, the results showed their ability to (i) decrease the reactive oxygen species (ROS) production quantified using fluorometric analysis and the lipid peroxidation measured with a spectrophotometric assay; (ii) rescue both the glutathione reduced to oxidized (GSH/GSSG) ratio and nitric oxide impair and the protein denaturation; and (iii) accelerate the wound repair measured using a T-scratch assay. Taken together, our findings indicate that the ethanolic extract from S. spinosum fruits could be a potential candidate for nutraceutical application.

Polyphenol-enriched extracts of Sarcopoterium spinosum fruits for counteracting lipid accumulation and oxidative stress in an in vitro model of hepatic steatosis.

Sarcopoterium spinosum (L.) Spach is a Rosaceae shrub employed in the folk medicine in the Eastern Mediterranean basin. The previous few studies have focused on the S. spinosum roots, while the fruits have been poorly investigated. The present study aims to assess the biological properties of S. spinosum fruits collected in Lebanon and subjected to ethanolic, water or boiling water extraction. The extracts were compared for the phenol and flavonoid contents, and for the in vitro radical scavenging ability. The ethanolic extract (SEE) was selected and characterized by high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS) showing a phenolome rich in tannins (ellagitannins), flavonoids (quercetin derivatives), and triterpenes. The biological activity of SEE was tested on a cellular model of moderate steatosis consisting of lipid-loaded hepatic cells treated with increasing concentrations of SEE (1-25 µg/mL), or with corilagin or quercetin as comparison. In steatotic hepatocytes the SEE was able (i) to ameliorate the hepatosteatosis; (ii) to counteract the excess ROS and lipid peroxidation; (iii) to restore the impaired catalase activity. The results indicate that the ethanolic extract from S. spinosum fruits is endowed with relevant antisteatotic and antioxidant activities and might find application as nutraceutical product.

Nuclear and chromatin rearrangement associate to epigenome and gene expression changes in a model of in vitro adipogenesis and hypertrophy.

Hypertrophy of adipocytes represents the main cause of obesity. We investigated in vitro the changes associated with adipocyte differentiation and hypertrophy focusing on the nuclear morphometry and chromatin epigenetic remodelling. The 3 T3-L1 pre-adipocytes were firstly differentiated into mature adipocytes, then cultured with long-chain fatty acids to induce hypertrophy. Confocal and super-resolution stimulation emission depletion (STED) microscopy combined with ELISA assays allowed us to explore nuclear architecture, chromatin distribution and epigenetic modifications. In each condition, we quantified the triglyceride accumulation, the mRNA expression of adipogenesis and dysfunction markers, the release of five pro-inflammatory cytokines. Confocal microscopy revealed larger volume and less elongated shape of the nuclei in both mature and hypertrophic cells respect to pre-adipocytes, and a trend toward reduced chromatin compaction. Compared to mature adipocytes, the hypertrophic phenotype showed larger triglyceride content, increased PPARγ expression reduced IL-1a release, and up-regulation of a pool of genes markers for adipose tissue dysfunction. Moreover, a remodelling of both epigenome and chromatin organization was observed in hypertrophic adipocytes, with an increase in the average fluorescence of H3K9 acetylated domains in parallel with the increase in KAT2A expression, and a global hypomethylation of DNA. These findings making light on the nuclear changes during adipocyte differentiation and hypertrophy might help the strategies for treating obesity and metabolic complications.

Adipocyte-hepatocyte crosstalk in cellular models of obesity: Role of soluble factors.

Hepatic steatosis is often a consequence of obesity. Adipose tissue is an important endocrine regulator of metabolic homeostasis in the body. In obesity, adipocytes become hypertrophic and develop an inflammatory phenotype, altering the panel of secreted adipokines. Moreover, excess fatty acids are, in part, released by adipocytes and delivered to the liver. These multiple pathways of adipose-liver crosstalk contribute to the development and progression of liver disease: TNFα induces hepatocyte dysfunction, excess of circulating fatty acids promotes hepatic steatosis and inflammation, whilst adipokines mediate and exacerbate liver injury. In this study, we investigated in vitro the effects and mechanisms of the crosstalk between adipocytes and hepatocytes, as a function of the different adipocyte status (mature vs hypertrophic) being mediated by soluble factors. We employed the conditioned medium method to test how mature and hypertrophic adipocytes distinctively affect the liver, leading to metabolic dysfunction. The media collected from adipocytes were characterized by high triglyceride content and led to lipid accumulation and fat-dependent dysfunction in hepatocytes. The present findings seem to suggest that, in addition to triglycerides, other soluble mediators, cytokines, are released by mature and hypertrophic adipocytes and influence the metabolic status of liver cells. Understanding the precise factors involved in the pathogenesis and pathophysiology of NAFLD in obesity will provide important insights into the mechanisms responsible for the metabolic complications of obesity, paving the way for new possible approaches.

The Potential of Lamiaceae Herbs for Mitigation of Overweight, Obesity, and Fatty Liver: Studies and Perspectives.

Numerous plants, plant extracts, and plant-derived compounds are being explored for their beneficial effects against overweight and liver diseases. Obesity is associated with the increased prevalence of non-alcoholic fatty liver disease (NAFLD), becoming the most common liver disease in Western countries. Obesity and NAFLD are closely associated with many other metabolic alternations such as insulin resistance, diabetes mellitus, and cardiovascular diseases. Many herbs of the Lamiaceae family are widely employed as food and spices in the Mediterranean area, but also in folk medicine, and their use for the management of metabolic disorders is well documented. Hereby, we summarized the scientific results of the medicinal and nutraceutical potential of plants from the Lamiaceae family for prevention and mitigation of overweight and fatty liver. The evidence indicates that Lamiaceae plants may be a cost-effective source of nutraceuticals and/or phytochemicals to be used in the management of metabolic-related conditions such as obesity and NAFLD. PubMed, Google Scholar, Scopus, and SciFinder were accessed to collect data on traditional medicinal plants, compounds derived from plants, their reported anti-obesity mechanisms, and therapeutic targets.

Relationship between Liver Stiffness and Steatosis in Obesity Conditions: In Vivo and In Vitro Studies.

Obesity is a major risk factor for metabolic dysfunction such as non-alcoholic fatty liver disease (NAFLD). The NAFLD spectrum ranges from simple steatosis, to steatohepatitis, fibrosis, and cirrhosis. The aim of this study is to characterize the grade of steatosis being associated with overnutrition and obesity, both at the level of single hepatocyte and whole liver, and to correlate it with the hepatocyte/liver stiffness and dysfunction. For the in vivo study, 60 subjects were enrolled and grouped based on the stage of liver steatosis/fibrosis according to biochemical analyses, liver ultrasonography (USG) and acoustic radiation force impulse shear wave elastography (ARFI-SWE). For single hepatocyte analyses we employed in vitro models of moderate and severe steatosis on which to assess the single cell biomechanics by Single Cell Force Spectroscopy (SCFS) and Quantitative Phase Microscopy (QPM). Results show that in vivo liver stiffness depends mainly on the extent of fat accumulation and not on fibrosis. These results parallel the in vitro observations showing that hepatocyte stiffness and dysfunction increase with increasing fat accumulation and lipid droplet enlargement. Our findings indicate that the extent of steatosis markedly affects the biomechanical properties of both liver and single hepatocytes thus proving insights about the role of modulation of liver/hepatocyte elasticity as a physical mechanism transducing the obesity-dependent excess of plasmatic lipids towards liver steatosis and dysfunction.

Beneficial Effects of Carvacrol on In Vitro Models of Metabolically-Associated Liver Steatosis and Endothelial Dysfunction: A Role for Fatty Acids in Interfering with Carvacrol Binding to Serum Albumin.

BACKGROUND: Carvacrol, a plant phenolic monoterpene, is largely employed as food additive and phytochemical. OBJECTIVE: We aimed to assess the lipid lowering and protective effects of carvacrol in vitro using cellular models of hepatic steatosis and endothelial dysfunction. We also investigated if and how the binding of carvacrol to albumin, the physiological transporter for small compounds in the blood, might be altered by the presence of high levels of fatty acids (FAs). METHODS: Hepatic FaO cells treated with exogenous FAs mimic hepatosteatosis; endothelial HECV cells exposed to hydrogen peroxide are a model of endothelial dysfunction. In these models, we measured spectrophotometrically lipid accumulation and release, lipoperoxidation, free radical production, and nitric oxide release before and after treatment with carvacrol. The carvacrol binding to albumin in the presence or absence of high levels of FAs was assessed by absorption and emission spectroscopies. RESULTS: Carvacrol counteracted lipid accumulation and oxidative stress in hepatocytes and protected endothelial cells from oxidative stress and dysfunction. Moreover, high levels of FAs reduced the binding of carvacrol to albumin. CONCLUSION: The results suggest the good potential of carvacrol in ameliorating dysfunction of hepatic and endothelial cells in vitro. High levels of circulating FAs might compete with carvacrol for binding to albumin thus influencing its transport and bio-distribution.

Extent and features of liver steatosis in vitro pave the way to endothelial dysfunction without physical cell-to-cell contact.

BACKGROUND AND AIMS: Several chronic multifactorial diseases originate from energy unbalance between food intake and body energy expenditure, including non-alcoholic fatty liver disease (NAFLD), diabetes, and cardiovascular disorders. Vascular endothelium plays a central role in body homeostasis, and NAFLD is often associated with endothelial dysfunction (ED), the first step in atherosclerosis. Both sugars and fatty acids (FAs) are fuel sources for energy production, but their excess leads to liver steatosis which may trigger ED through a network of mechanisms which need to be clarified. Here, we investigated the crosstalk pathways between in vitro cultured steatotic hepatocytes (FaO) and endothelial cells (HECV) being mediated by soluble factors. METHODS AND RESULTS: We employed the conditioned medium approach to test how different extent and features of hepatic steatosis distinctively affect endothelium leading to ED. The steatogenic media collected from steatotic hepatocytes were characterized by high triglyceride content and led to lipid accumulation and fat-dependent dysfunction in HECV cells. We found a parallelism between (i) extent of hepatocyte steatosis and level of lipid accumulation in HECV cells; (ii) type of hepatocyte steatosis (with macro- or microvesicular LDs) and extent of oxidative stress, lipid peroxidation, nitric oxide release and expression of ED markers in HECV cells. CONCLUSIONS: The present findings seem to suggest that, in addition to triglycerides, other soluble mediators should be released by steatotic hepatocytes and may influence lipid accumulation and function of HECV cells. Further studies need to depict the exact profile of soluble factors involved in steatotic hepatocyte-endothelium crosstalk.

Reactive Astrocytosis in a Mouse Model of Chronic Polyamine Catabolism Activation.

BACKGROUND: In the brain, polyamines are mainly synthesized in neurons, but preferentially accumulated in astrocytes, and are proposed to be involved in neurodegenerative/neuroinflammatory disorders and neuron injury. A transgenic mouse overexpressing spermine oxidase (SMOX, which specifically oxidizes spermine) in the neocortex neurons (Dach-SMOX mouse) was proved to be a model of increased susceptibility to excitotoxic injury. METHODS: To investigate possible alterations in synapse functioning in Dach-SMOX mouse, both cerebrocortical nerve terminals (synaptosomes) and astrocytic processes (gliosomes) were analysed by assessing polyamine levels, ezrin and vimentin content, glutamate AMPA receptor activation, calcium influx, and catalase activity. RESULTS: The main findings are as follows: (i) the presence of functional calcium-permeable AMPA receptors in synaptosomes from both control and Dach-SMOX mice, and in gliosomes from Dach-SMOX mice only; (ii) reduced content of spermine in gliosomes from Dach-SMOX mice; and (iii) down-regulation and up-regulation of catalase activity in synaptosomes and gliosomes, respectively, from Dach-SMOX mice. CONCLUSIONS: Chronic activation of SMOX in neurons leads to major changes in the astrocyte processes including reduced spermine levels, increased calcium influx through calcium-permeable AMPA receptors, and stimulation of catalase activity. Astrocytosis and the astrocyte process alterations, depending on chronic activation of polyamine catabolism, result in synapse dysregulation and neuronal suffering.

Efficacy of a Three Drug-Based Therapy for Neuroblastoma in Mice.

High-risk neuroblastoma (HR-NB) still remains the most dangerous tumor in early childhood. For this reason, the identification of new therapeutic approaches is of fundamental importance. Recently, we combined the conventional pharmacological approach to NB, represented by cisplatin, with fendiline hydrochloride, an inhibitor of several transporters involved in multidrug resistance of cancer cells, which demonstrated an enhancement of the ability of cisplatin to induce apoptosis. In this work, we co-administrated acetazolamide, a carbonic anhydrase isoform IX (CAIX) inhibitor which was reported to increase chemotherapy efficacy in various cancer types, to the cisplatin/fendiline approach in SKNBE2 xenografts in NOD-SCID mice with the aim of identifying a novel and more effective treatment. We observed that the combination of the three drugs increases more than twelvefold the differences in the cytotoxic activity of cisplatin alone, leading to a remarkable decrease of the expression of malignancy markers. Our conclusion is that this approach, based on three FDA-approved drugs, may constitute an appropriate improvement of the pharmacological approach to HR-NB.

An Overview of Long Non-Coding (lnc)RNAs in Neuroblastoma.

Neuroblastoma (NB) is a heterogeneous developmental tumor occurring in childhood, which arises from the embryonic sympathoadrenal cells of the neural crest. Although the recent progress that has been done on this tumor, the mechanisms involved in NB are still partially unknown. Despite some genetic aberrations having been identified, the sporadic cases represent the majority. Due to its wide heterogeneity in clinical behavior and etiology, NB represents a challenge in terms of prevention and treatment. Since a definitive therapy is lacking so far, there is an urgent necessity to unveil the molecular mechanisms behind NB onset and progression to develop new therapeutic approaches. Long non-coding RNAs (lncRNAs) are a group of RNAs longer than 200 nucleotides. Whether lncRNAs are destined to become a protein or not, they exert multiple biological functions such as regulating gene expression and functions. In recent decades, different research has highlighted the possible role of lncRNAs in the pathogenesis of many diseases, including cancer. Moreover, lncRNAs may represent potential markers or targets for diagnosis and treatment of diseases. This mini-review aimed to briefly summarize the most recent findings on the involvement of some lncRNAs in NB disease by focusing on their mechanisms of action and possible role in unveiling NB onset and progression.

Antitumor Activity of Ethanolic Extract from Thymbra Spicata L. aerial Parts: Effects on Cell Viability and Proliferation, Apoptosis Induction, STAT3, and NF-kB Signaling.

Thyme-like plants including Thymbra spicata L. are widely used as food and folk medicinal remedies in the Mediterranean area. This study aimed to explore the in vitro antitumor potential of polyphenol-enriched extracts from aerial parts of T. spicata. The ethanolic extract significantly inhibited proliferation of different human tumor cell lines, without significant effects on non-neoplastic cells. A deeper investigation of the molecular mechanism sustaining the in vitro antitumor activity of the extract was carried on the human breast cancer cells MCF-7 in comparison with the normal breast cells MCF-10A. The effects on MCF-7 cells were associated with the following: (i) production of reactive oxygen species (ROS) and release of nitric oxide; (ii) apoptosis induction; and (iii) reduction in STAT3 and NF-kB phosphorylation. The ethanolic extract from T. spicata leaves might represent a novel therapeutic tool in combination with conventional chemotherapy to reduce the adverse side effects and drug resistance.

Adipocyte hypertrophy parallels alterations of mitochondrial status in a cell model for adipose tissue dysfunction in obesity.

AIMS: Adipocyte hypertrophy is the main cause of obesity. A deeper understanding of the molecular mechanisms regulating adipocyte dysfunction may help to plan strategies to treat/prevent obesity and its metabolic complications. Here, we investigated in vitro the molecular alterations associated with early adipocyte hypertrophy, focusing on mitochondrial dysfunction. MAIN METHODS: As model of adipocyte hypertrophy, we employed 3T3-L1 preadipocytes firstly differentiated into mature adipocytes, then cultured with long-chain fatty acids. As a function of differentiation and hypertrophy, we assessed triglyceride content, lipid droplet size, radical homeostasis by spectrophotometry and microscopy, as well as the expression of PPARγ, adiponectin and metallothioneins. Mitochondrial status was investigated by electron microscopy, oxygraph 2 k (O2K) high-resolution respirometry, fluorimetry and western blot. KEY FINDINGS: Compared to mature adipocytes, hypertrophic adipocytes showed increased triglyceride accumulation and lipid peroxidation, larger or unique lipid droplet, up-regulated expression of PPARγ, adiponectin and metallothioneins. At mitochondrial level, early-hypertrophic adipocytes exhibited: (i) impaired mitochondrial oxygen consumption with parallel reduction in the mitochondrial complexes; (ii) no changes in citrate synthase and HSP60 expression, and in the inner mitochondrial membrane polarization; (iii) no stimulation of mitochondrial fatty acid oxidation. Our findings indicate that the content, integrity, and catabolic activity of mitochondria were rather unchanged in early hypertrophic adipocytes, while oxygen consumption and oxidant production were altered. SIGNIFICANCE: In the model of early adipocyte hypertrophy exacerbated oxidative stress and impaired mitochondrial respiration were observed, likely depending on reduction in the mitochondrial complexes, without changes in mitochondrial mass and integrity.

MCM2 and Carbonic Anhydrase 9 Are Novel Potential Targets for Neuroblastoma Pharmacological Treatment.

To overcome the lack of effective pharmacological treatments for high-risk neuroblastoma (HR-NB), the development of novel in vitro and in vivo models that better recapitulate the disease is required. Here, we used an in vitro multiclonal cell model encompassing NB cell differentiation stages, to identify potential novel pharmacological targets. This model allowed us to identify, by low-density RT-PCR arrays, two gene sets, one over-expressed during NB cell differentiation, and the other up-regulated in more malignant cells. Challenging two HR-NB gene expression datasets, we found that these two gene sets are related to high and low survival, respectively. Using mouse NB cisplatin-treated xenografts, we identified two genes within the list associated to the malignant stage (MCM2 and carbonic anhydrase 9), whose expression is positively correlated with tumor growth. Thus, we tested their pharmacological targeting as potential therapeutic strategy. We measured mice survival and tumor growth rate after xenografts of human NB treated with cisplatin in the presence of MCM2/carbonic anhydrase 9 inhibitors (ciprofloxacin and acetazolamide). MCM2 or carbonic anhydrase 9 inhibition significantly increased cisplatin activity, supporting their possible testing for NB therapy.

New Perspectives of S-Adenosylmethionine (SAMe) Applications to Attenuate Fatty Acid-Induced Steatosis and Oxidative Stress in Hepatic and Endothelial Cells.

S-adenosylmethionine (SAMe) is an endogenous methyl donor derived from ATP and methionine that has pleiotropic functions. Most SAMe is synthetized and consumed in the liver, where it acts as the main methylating agent and in protection against the free radical toxicity. Previous studies have shown that the administration of SAMe as a supernutrient exerted many beneficial effects in various tissues, mainly in the liver. In the present study, we aimed to clarify the direct effects of SAMe on fatty acid-induced steatosis and oxidative stress in hepatic and endothelial cells. Hepatoma FaO cells and endothelial HECV cells exposed to a mixture of oleate/palmitate are reliable models for hepatic steatosis and endothelium dysfunction, respectively. Our findings indicate that SAMe was able to significantly ameliorate lipid accumulation and oxidative stress in hepatic cells, mainly through promoting mitochondrial fatty acid entry for ß-oxidation and external triglyceride release. SAMe also reverted both lipid accumulation and oxidant production (i.e., ROS and NO) in endothelial cells. In conclusion, these outcomes suggest promising beneficial applications of SAMe as a nutraceutical for metabolic disorders occurring in fatty liver and endothelium dysfunction.

Aquaporin-9 is involved in the lipid-lowering activity of the nutraceutical silybin on hepatocytes through modulation of autophagy and lipid droplets composition.

Hepatic steatosis is the hallmark of non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome and insulin resistance with potential evolution towards non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma. Key roles of autophagy and oxidative stress in hepatic lipid accumulation and NAFLD progression are recognized. Here, we employed a rat hepatoma cell model of NAFLD progression made of FaO cells exposed to oleate/palmitate followed or not by TNFα treatment to investigate the molecular mechanisms through which silybin, a lipid-lowering nutraceutical, may improve hepatic lipid dyshomeostasis. The beneficial effect of silybin was found to involve amelioration of the fatty acids profile of lipid droplets, stimulation of the mitochondrial oxidation and upregulation of a microRNA of pivotal relevance in hepatic fat metabolism, miR-122. Silybin was also found to restore the levels of Aquaporin-9 (AQP9) and glycerol permeability while reducing the activation of the oxidative stress-dependent transcription factor NF-κB, and autophagy turnover. In conclusion, silybin was shown to have molecular effects on signaling pathways that were previously unknown and potentially protect the hepatocyte. These actions intersect TG metabolism, fat-induced autophagy and AQP9-mediated glycerol transport in hepatocytes.

Excess fructose and fatty acids trigger a model of non­alcoholic fatty liver disease progression in vitro: Protective effect of the flavonoid silybin.

Overconsumption of fats and sugars is a major cause of development of non­alcoholic fatty liver disease (NAFLD). The main objectives of the present study were to explore the pathways sustaining the interfering metabolic effects of excess fructose and fatty acids in hepatocytes, and to clarify the mechanisms through which the nutraceutical silybin rescues the functional and metabolic alterations that are associated with the NALFD progression. Cultured hepatocytes were exposed to fructose and fatty acids, alone or in combination, to induce different grades of steatosis in vitro. Cell viability, apoptosis, free radical production, lipid content, lipid peroxidation and activity of lipogenic enzymes were assessed by spectrophotometric assays. Oxygen consumption and mitochondrial respiration parameters were measured using a Seahorse analyzer. Expression of markers for liver steatosis and dysfunction were also evaluated by reverse transcription­quantitative polymerase chain reaction. The data revealed that fructose and fatty acid combination in vitro had a positive interference on lipogenic pathways, leading to more severe steatosis and liver dysfunction, reduced cell viability, increased apoptosis, oxidative stress and mitochondrial respiration. Hepatic cell abnormalities were almost completely alleviated by silybin treatment. These findings suggest that silybin may serve as a novel and cost­effective dietary supplement for treatment and/or prevention of hepatosteatosis associated with NAFLD progression.

Biomechanics of cultured hepatic cells during different steatogenic hits.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease often associated with overnutrition. Number and morphometry of lipid droplets (LDs) define micro vs macrovesicular steatosis, influence the morphology and function of hepatocytes and possibly their stiffness. The link between grade and features of steatosis and biomechanical properties of single hepatocytes requires deeper investigations. In vitro NAFLD models with distinct steatosis conditions were set by exposing FaO hepatoma cells to single or combined fructose (Fru), fatty acids (FA), and tumor necrosis factor (TNF)α. Single Cell Force Spectroscopy and Quantitative Phase Microscopy quantified the single cell stiffness and a series of morphometric parameters; the mRNA expression of genes involved in lipid metabolism was quantified by real-time PCR. In our models, LD size and number increased with Fru and FA as single agents, and more with combined Fru/FA (macrovesicular steatosis), while FA/TNFα combination increased LD number with a reduction in their size (microvesicular steatosis). We found that the changes in LD size and number influenced cell stiffness and morphometry as follows: (i) single cell elasticity increased in macrovesicular steatosis (maximally with combined Fru/FA); (ii) FA-induced steatosis resulted in cells thinner and larger, whereas combined FA/TNFα shrunk the hepatocytes. Taken together the data on hepatocyte biomechanics show that, in addition to extent of lipid accumulation, cell stiffness is mainly influenced by LD size, while cell morphometry directly relates to LD number. Our findings suggest that a novel mechanobiology perspective might provide future contributions in NAFLD research.