Differentiation of muscle cells treated with laser irradiated M2a macrophages supernatants

Abstract

The occurrence of a muscle injury triggers a complex repair process. Macrophages have a crucial role in the initial and later steps of muscle repair in which are characterized by a pro-inflammatory (M1) and anti-inflammatory (M2) profile, respectively. Concomitantly, myogenic precursor cells (MPC) need to be activated to differentiate in to muscle cells that will relace the injured tissue. In this context, it is well known that mediators produced by M1 macrophages are related to the proliferation of MPC cells as well as M2 macrophages products with their differentiation. On the other hand, the role of Low Level Laser Therapy (LLLT) in the muscle repair process has been extensively studied, but its effects on macrophages products that may influence MPC differentiation are still unclear. The objective of this study is to evaluate the effects of the supernatant of macrophage cultures induced for M2a phenotype and irradiated with red laser (660 nm) and infrared (780 nm) using the same dosimetric parameters (70 mW, 17,5 J/ cm2, 1J) on the MPC differentiation. J774 macrophages will be cultured in DMEM with fetal bovine serum (10%) and L-glutamine (2 Mm). These cells will be treated with IL-4 (0.1 ¼g / mL) for 24h to activate the M2a profile. After this period, the cells will be irradiated with LLLT (660 nm and 780 nm) and cultured for another 24 hours. Non-irradiated and non-activated macrophages will be used as controls. The supernatant of each group will be added to the myogenic precursor cell line C2C12 and incubated for 72 hours. Cells will be collected with TRIzol and stored at -70 ºC to perform RNA extraction, complementary DNA synthesis and Real Time PCR to evaluate gene expression of myogenin and MyoD. Three independent experiments will be performed. All results will be submitted to statistical analysis. (AU)