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Elucidating the anti-inflammatory mechanism of 5,5-dimethyl-1-pirroline N-oxide (DMPO)

Marcos David Muñoz

National Institute of Health

Obesity is considered as a chronic inflammatory disease. Its affects more than 1, 4 billion adults worldwide. It is known that obesity is the result of an unbalanced diet with an excess in the amount of energy up-take compare to energy expenditure among other genetic and epigenetic factors. The excess of food up-take is kept as triglycerides on the cells of subcutaneous adipose tissue called adipocytes, if the energy up-take does not cease (obese state) the accumulation continues on another compartment called visceral adipose tissue located within organs. In lean adipose tissue (AT), non-inflammatory macrophages (type II) perform the maintenance adipocytes, on the other hand the obese AT is metabolically stressed by free fatty acids, lipopolysaccharide (LPS) and hypoxia activating stress response program in macrophages and giving them an inflammatory (M1-like) phenotype. M1-like AT macrophages (ATM) produces reactive oxygen species and express inflammatory genes that cause type I macrophages infiltration, chronic inflammatory state and tissue dysfunction. A number of reactive oxygen species (ROS) and free radicals are produced inside macrophages primed with LPS. Free radicals are unstable, highly reactive species and their detection in cells was a challenge until the development of spin trapping. Spin trapping makes use of organic compounds to react with the free radicals to produce radical adducts with longer half-lives facilitating his detection and dampening the oxidative stress-related inflammation. 5,5-Dimethy-l-pyrroline N-oxide (DMPO), a nitrone spin trap, passes across cell membranes, effectively traps free radicals, and possesses low toxicity, which make it a good target for alternative treatment studies. Although the AT inflammation is related to oxidative stress increment, the spin-traps have not been considered as a treatment on this scene. In order to understand how DMPO works, studies have been made by our group and shown that DMPO was able to form protein–DMPO nitrone adducts with proteins related to hypoxia response and inflammatory signaling. Furthermore, the anti-inflammatory capabilities of DMPO were tested and shown that at the concentration of 50 mM was able to inhibit inducible nitric oxide synthase expression and other inflammatory genes when added shortly after LPS treatment (≤3 h) in murine macrophages. At this point DMPO shown anti-inflammatory effects on LPS-primed macrophages but these events occurred too early to be considered as a consequence of his ability for spin-trapping. Herein we test the effect of DMPO on the transcriptome and proteome using microarray technology and found that DMPO itself was able to induce anti-inflammatory genes such as PPAR-d and HO-1 and to reduce inflammatory genes such as IRF-7 in macrophages. We aim at understanding the molecular mechanism of phenotypic switch of macrophages by DMPO and how it prevents LPS-induced macrophage polarization towards an inflammatory phenotype. For this purpose, we plan to use in-vitro and in-vivo models, quantitative PCR and bioinformatics tools on the experiments with duration of three months. We hope that this methodology will clarify the mechanism of action and in-vivo effects of DMPO, looking forward to test the spin trap as treatment for systemic inflammation and insulin resistance in obesity.

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