In vitro assessment of nutraceutical compounds and novel nutraceutical formulations in a liver-steatosis-based model.

BACKGROUND: Steatosis is a chronic liver disease that depends on the accumulation of intracellular fatty acids. Currently, no drug treatment has been licensed for steatosis; thus, only nutritional guidelines are indicated to reduce its progression. The aim of this study is to combine different nutraceutical compounds in order to evaluate their synergistic effects on a steatosis in vitro model compared to their separate use. In particular, three different formulations based on silymarin, curcumin, vitamin E, docosahexaenoic acid (DHA), choline, and phosphatidylcholine were assayed. METHODS: Human hepatocellular carcinoma cells (HepG2 cell line) were treated with a mixture of fatty acids in order to induce an in vitro model of steatosic cells, and then the amount of intracellular fat was evaluated by Oil Red O staining. The peroxisome proliferator-activated receptors α and γ (PPARα and γ) expression, closely correlated to lipid metabolism, was evaluated. The efficiency of these receptors was evaluated through the study of LPL mRNA expression, a marker involved in the lipolysis mechanism. Superoxide dismutase (SOD-2) and malondialdehydes (MDA) in lipid peroxidation were assayed as specific biomarkers of oxidative stress. In addition, experiments were performed using human liver cells stressed to obtain a steatosis model. In particular, the content of the intracellular fat was assayed using Oil Red O staining, the activation of PPARα and γ was evaluated through western blotting analyses, and the LPL mRNA expression level was analyzed through qRT-PCR. RESULTS: All formulations proved effective on lipid content reduction of about 35%. The oxidative stress damage was reduced by all the substances separately and even more efficiently by the same in formulation (i.e. Formulation 1 and Formulation 3, which reduced the SOD-2 expression and induced the PPARs activation). Lipid peroxidation, was reduced about 2 fold by foormulation2 and up to 5 fold by the others compared to the cells pretreated with H2O2.Formulation 1, was more effective on PPARγ expression (2.5 fold increase) respect to the other compounds on FA treated hepathocytes. Beside, LPL was activated also by Formulation 3 and resulted in a 5 to 9 fold-increase respect to FA treated control. CONCLUSIONS: Our results proved that the formulations tested could be considered suitable support to face steatosis disease beside the mandatory dietetic regimen.

Myclobutanil worsens nonalcoholic fatty liver disease: An in vitro study of toxicity and apoptosis on HepG2 cells.

Myclobutanil is a conazole class fungicide widely used as an agrichemical. It is approved for use on fruit, vegetables and seed commodities in the EU and elsewhere to control fungi such as Ascomycetes, Fungi Imperfecti and, Basidiomycetes. Its widespread use has raised the issue of possible health risks for agrarian communities and the general population, which can be exposed to residues present in food and drinking water. The toxicities identified include adverse effects on liver and kidney and on the development of male reproductive organs. Since the liver is the first-line organ in the defense against xenobiotics, toxic effects on hepatic metabolism cause degeneration, necrosis, and tissue hypertrophy. Therefore, we investigated myclobutanil's effects on the human liver cell line HepG2. We found that myclobutanil increases the amount of fatty acids in these hepatic cells, as evaluated with Oil Red O staining, and progressively reduces cell viability from 1ppm to 500ppm. Analysis of biomarkers such as Bcl-xL/Bak and Mcl-1/Bak confirmed activation of cell death pathways at low doses. Therefore, myclobutanil may play an important role in the pathogenesis and progression of chronic hepatocellular diseases in humans.

Hyaluronan viscosupplementation: state of the art and insight into the novel cooperative hybrid complexes based on high and low molecular weight HA of potential interest in osteoarthritis treatment.

Osteoarthritis (OA) represents a group of chronic, painful, disabling conditions affecting synovial joints. It is characterized by degeneration of articular cartilage, alterations of peri-articular and subchondral bone, low-grade synovial inflammation (synovitis). Despite OA is commonly described as a non-inflammatory disease, it is known that its progression and the subsequent increment of symptoms correlate to the production of inflammatory factors that induce the secretion of enzymes responsible for cartilage degradation. In clinical practice, to alleviate pain and stiffness, not only during acute phases but also as maintenance therapy, intra-articular injections of corticosteroids or similar drugs are used, besides it is well diffused the viscosupplementation procedure based on hyaluronan gel. There are many different products containing high molecular weight linear HA or cross-linked derivatives, however the novelty in the field consist in the hybrid cooperative complexes derived from high and low molecular weight HA through a patented processing. This technique permit to double the amount of HA delivered to the injured site without increasing the injected volume, beside in vitro assay on human chondrocytes suggested hybrid complexes as effective in the modulation of several inflammatory cytokines in joints.

Biotechnological Chondroitin a Novel Glycosamminoglycan With Remarkable Biological Function on Human Primary Chondrocytes.

Cartilage tissue engineering, with in vitro expansion of autologus chondrocytes, is a promising technique for tissue regeneration and is a new potential strategy to prevent and/or treat cartilage damage (e.g., osteoarthritis). The aim of this study was (i) to investigate and compare the effects of new biotechnological chondroitin (BC) and a commercial extractive chondroitin sulfate (CS) on human chondrocytes in vitro culture; (ii) to evaluate the anti-inflammatory effects of the innovative BC compared to extractive CS. A chondrogenic cell population was isolated from human nasoseptal cartilage and in vitro cultures were studied through time-lapse video microscopy (TLVM), immunohistochemical staining and cytometry. In order to investigate the effect of BC and CS on phenotype maintainance, chondrogenic gene expression of aggrecan (AGN), of the transcriptor factor SOX9, of the types I and II collagen (COL1A1 and COL1A2), were quantified through transcriptional and protein evaluation at increasing cultivation time and passages. In addition to resemble the osteoarthritis-like in vitro model, chondrocytes were treated with IL-1ß and the anti-inflammatory activity of BC and CS was assessed using cytokines quantification by multiplex array. BC significantly enhances cell proliferation also preserving chondrocyte phenotype increasing type II collagen expression up to 10 days of treatment and reduces inflammatory response in IL-1ß treated chondrocytes respect to CS treated cells. Our results, taken together, suggest that this new BC is of foremost importance in translational medicine because it can be applied in novel scaffolds and pharmaceutical preparations aiming at cartilage pathology treatments such as the osteoarthritis. J. Cell. Biochem. 117: 2158-2169, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.