Masitinib mediates TGF-Beta1 and Nitric Oxide Secretion and Ameliorates MPTP/Microglia-Induced Degeneration of Differentiated SH-SY5Y Cells

Abstract

AbstractIntroductionMicroglia secretome includes not only growth factors and cytokines which support neuronal survival, it includes neurotoxic cytokines/enzymes, as well. MPTP is a neurotoxin which has degenerative effects on SH-SY5Y neuroblastoma cells. Masitinib mesylate is a tyrosine kinase inhibitor which has been shown to have beneficial effects in neurodegenerative diseases.AimWe first aimed to determine the most efficient microglial cell conditioned medium in terms of neurodegenerative effect. Next, we investigated the possible protective/therapeutic effects of masitinib against MPTP/microglia-induced degeneration of differentiated (d)-SH-SY5Y cells, and the role of transforming growth factor (TGF)-β1 and nitric oxide (NO) in these events.Material-MethodsNon-stimulated/LPS-stimulated microglia cells were treated with masitinib or its solvent, DMSO. With or without MPTP-d-SH-SY5Y cell cultures were exposed to the conditioned media (CM) from microglia cell cultures, followed by cell survival analysis. Immunofluorescence staining of microglia and d-SH-SY5Y cells were performed with anti-CD-11b and anti-PGP9.5 antibody, respectively. TGF-β1/NO concentrations in CM of microglia/d-SH-SY5Y cell culture were measured.ResultsThe initial 24 hrs CM of non-stimulated microglia cell culture was found to be the most detrimental microglial medium with lowest survival rates of treated d-SH-SY5Y cells. The toxicity of 48 and 72 hrs’ CM on d-SH-SY5Y cells were both lower than that of 24 hrs’ CM. Masitinib (0.5 µM), significantly prevented MPTP-related cell degeneration of d-SH-SY5Y cells. It also decreased the degenerative effects of both non-induced/LPS-induced microglia CM on with or without MPTP-d-SH-SY5Y cells. Although NO levels in microglia CM showed a negative correlation with survival rates of treated d-SH-SY5Y cells, a positive correlation was seen between TGF-β1 concentrations in microglial CM and rates of treated d-SH-SY5Y cell survival.ConclusionMasitinib ameliorates viability of with/without MPTP-d-SH-SY5Y cells. It does not only reverse the degenerative effects of its solvent, DMSO, but also prevents the degenerative effects of microglial secretions and MPTP. We suggest that masitinib begins to act as a neuroprotective agent via mediating TGF-β1 and NO secretion, as neurons are exposed to over-activated microglia or neurotoxins.