Developing an in vitro screening assay platform for evaluation of antifibrotic drugs using precision-cut liver slices.

BACKGROUND: Precision-cut liver slices present different cell types of liver in a physiological context, and they have been explored as effective in vitro model systems to study liver fibrosis. Inducing fibrosis in the liver slices using toxicants like carbon tetrachloride is of less relevance to human disease conditions. Our aim for this study was to establish physiologically relevant conditions in vitro to induce fibrotic phenotypes in the liver slices. RESULTS: Precision-cut liver slices of 150 µm thickness were obtained from female C57BL/6 J mice. The slices were cultured for 24 hours in media containing a cocktail of 10 nM each of TGF-ß, PDGF, 5 µM each of lysophosphatidic acid and sphingosine 1 phosphate and 0.2 µg/ml of lipopolysaccharide along with 500 µM of palmitate and were analyzed for triglyceride accumulation, stress and inflammation, myofibroblast activation and extracellular matrix (ECM) accumulation. Incubation with the cocktail resulted in increased triglyceride accumulation, a hallmark of steatosis. The levels of Acta2, a hallmark of myofibroblast activation and the levels of inflammatory genes (IL-6, TNF-α and C-reactive protein) were significantly elevated. In addition, this treatment resulted in increased levels of ECM markers - collagen, lumican and fibronectin. CONCLUSIONS: This study reports the experimental conditions required to induce fibrosis associated with steatohepatitis using physiologically relevant inducers. The system presented here captures various aspects of the fibrosis process like steatosis, inflammation, stellate cell activation and ECM accumulation and serves as a platform to study the liver fibrosis in vitro and to screen small molecules for their antifibrotic activity.

Exogenous administration of spermine improves glucose utilization and decreases bodyweight in mice.

Polyamines are highly charged low molecular weight aliphatic polycations and are ubiquitously present in all living cells. In addition to their previously reported role in cell proliferation and cancer, recent studies support their role in energy homeostasis and glucose metabolism. In the present study we have evaluated a polyamine-spermine for its effect on glycemic, lipid and body weight parameters. High fat diet induced obese mice (6 week old male C57B6/J mice fed on high fat diet for 22 weeks) were dosed with spermine intraperitoneally at two different doses (5mg/kg and 10mg/kg body weight) for 4 weeks and its effect on body weight, glycemic and lipid parameters was monitored. We found that at a dose of 10mg/kg bodyweight, spermine treatment resulted in a 24% reduction in the body weight and 18% reduction in the fasting glucose compared to untreated controls. Besides, spermine treated mice exhibited improved glucose utilization associated with improved fat oxidation and loss of white adipose mass. Our study is promising in the direction of exploring the spermine and their analogs for treatment of metabolic syndrome.

Sphingolipid metabolic pathway: an overview of major roles played in human diseases.

Sphingolipids, a family of membrane lipids, are bioactive molecules that participate in diverse functions controlling fundamental cellular processes such as cell division, differentiation, and cell death. Given that most of these cellular processes form the basis for several pathologies, it is not surprising that sphingolipids are key players in several pathological processes. This review discusses the role of the sphingolipid metabolic pathway in diabetes, Alzheimer's disease, and hepatocellular carcinoma, with a special emphasis on the changes in gene expression pattern in these disease conditions. For convenience, the sphingolipid metabolic pathway is divided into hypothetical compartments (modules) with each compartment representing a physiological process and changes in gene expression pattern are mapped to each of these modules. It appears that alterations in the gene expression pattern in these disease conditions are biased to manipulate the system in order to result in a particular disease.