Evaluation of the effect of LMP-420, an inhibitor of the tumor necrosis factor alpha (TNF-alpha) transcription, in a mouse myoblast cell line and a rat mast cell line

• Grant number: 07/03781-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: December 01, 2007
• End date: November 30, 2008
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Maria Cristina Ramos Costa
• Grantee: Ana Carolina Toschi
• Host Institution: Universidade de Ribeirão Preto (UNAERP). Campus Ribeirão Preto. Ribeirão Preto , SP, Brazil

Abstract

Duchenne Muscular Dystrophy (DMD) is a degenerative disorder caused by mutations in the gene of dystrophin, a subsarcolemmal protein important to connect actin cytoskeleton to the extracellular matrix. DMD is characterized by muscular weakness resulting from skeletal muscle progressive degeneration and culminates in early death by cardiac or respiratory failure. The mdx mouse, the most utilized DMD model, shows susceptibility to injury, increase of serum creatine kinase levels and muscular degeneration and regeneration cycles. The milder phenotype, when compared to DMD, can be intensified by compulsory physical activity. Muscular degeneration, initially caused by sarcolemma fragility due to dystrophin absence, is exacerbated by a chronic inflammatory process, with the participation of immune cells and inflammatory cytokines. Among them, the tumor necrosis factor alpha (TNF-alpha), which is released by mast cells and injured myofibers, has a key role. Recent studies have shown that the blockage of TNF-alpha, using monoclonal antibodies and a soluble receptor, results in diminished muscular degeneration in the mdx mouse. The objective of this project is to evaluate the effect of LMP-420, a recently developed inhibitor of TNF-alpha transcription, in a mouse myoblast cell line and a rat mast cell line. To this end, the following cell lines will be used: i) C2C12 of mouse myoblasts, submitted or not to injury, in the presence or absence of LMP-420; and ii) RBL-2H3 of rat mast cells, which will be used as positive control due to their high expression levels of TNF-alpha, stimulated via FcERI receptor, previously treated or not with LMP-420. Comparative analyses of the expression levels of TNF-alpha will be carried out by semiquantitative RT-PCR and western blot techniques. Immunofluorescence assays will be utilized to verify TNF-alpha cellular location. This study is part of a wider project that aims to evaluate the effect of LMP-420 in the dystrophinopathy progression of the mdx mouse. This project will also contribute to the functional characterization of LMP-420, as it will be possible to estimate the inhibition of the TNF-alpha transcription in mouse myoblasts.