In vitro transdentinal effect of low-level laser therapy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Low-level laser therapy (LLLT) has been used for the treatment of dentinal hypersensitivity. However, the specific LLL dose and the response mechanisms of these cells to transdentinal irradiation have not yet been demonstrated. Therefore, this study evaluated the transdentinal effects of different LLL doses on stressed odontoblast-like pulp cells MDPC-23 seeded onto the pulpal side of dentin discs obtained from human third molars. The discs were placed in devices simulating in vitro pulp chambers and the whole set was placed in 24-well plates containing plain culture medium (DMEM). After 24 h incubation, the culture medium was replaced by fresh DMEM supplemented with either 5% (simulating a nutritional stress condition) or 10% fetal bovine serum (FBS). The cells were irradiated with doses of 15 and 25 J cm(-2) every 24 h, totaling three applications over three consecutive days. The cells in the control groups were removed from the incubator for the same times as used in their respective experimental groups for irradiation, though without activating the laser source (sham irradiation). After 72 h of the last active or sham irradiation, the cells were evaluated with respect to succinic dehydrogenase (SDH) enzyme production (MTT assay), total protein (TP) expression, alkaline phosphatase (ALP) synthesis, reverse transcriptase polymerase chain reaction (RT-PCR) for collagen type 1 (Col-I) and ALP, and morphology (SEM). For both tests, significantly higher values were obtained for the 25 J cm(-2) dose. Regarding SDH production, supplementation of the culture medium with 5% FBS provided better results. For TP and ALP expression, the 25 J cm(-2) presented higher values, especially for the 5% FBS concentration (Mann-Whitney p < 0.05). Under the tested conditions, near infrared laser irradiation at 25 J cm(-2) caused transdentinal biostimulation of odontoblast-like MDPC-23 cells.235Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [2007/50646-3, 2008/08424-6, 2008/54785-0

Hydrogen peroxide production regulates the mitochondrial function in insulin resistant muscle cells: Effect of catalase overexpression

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The mitochondrial redox state plays a central role in the link between mitochondrial overloading and insulin resistance. However, the mechanism by which the ROS induce insulin resistance in skeletal muscle cells is not completely understood. We examined the association between mitochondrial function and H2O2 production in insulin resistant cells. Our hypothesis is that the low mitochondrial oxygen consumption leads to elevated ROS production by a mechanism associated with reduced PGC1 alpha transcription and low content of phosphorylated CREB. The cells were transfected with either the encoded sequence for catalase overexpression or the specific siRNA for catalase inhibition. After transfection, myotubes were incubated with palmitic acid (500 mu M) and the insulin response, as well as mitochondrial function and fatty acid metabolism, was determined. The low mitochondrial oxygen consumption led to elevated ROS production by a mechanism associated with beta-oxidation of fatty acids. Rotenone was observed to reduce the ratio of ROS production. The elevated H2O2 production markedly decreased the PGC1 alpha transcription, an effect that was accompanied by a reduced phosphorylation of Akt and CREB. The catalase transfection prevented the reduction in the phosphorylated level of Ala and upregulated the levels of phosphaylated CREB. The mitochondrial function was elevated and H2O2 production reduced, thus increasing the insulin sensitivity. The catalase overexpression improved mitochondrial respiration protecting the cells from fatty acid-induced, insulin resistance. This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and beta-oxidation of fatty acids. (C) 2013 Elsevier B.V. All rights reserved.18321015911604Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq