Limits...
Low-Level Laser Therapy (LLLT) in Dystrophin-Deficient Muscle Cells: Effects on Regeneration Capacity, Inflammation Response and Oxidative Stress.

Macedo AB, Moraes LH, Mizobuti DS, Fogaça AR, Moraes Fdos S, Hermes Tde A, Pertille A, Minatel E - PLoS ONE (2015)

Bottom Line: The mdx LA 48 group showed significant increase in Myosin Heavy Chain levels compared to the untreated mdx and mdx LA 24 groups.The mdx LA 24 and mdx LA 48 groups showed significant increase in [Ca2+]i.Together, these results suggest that the laser treatment improved regenerative capacity and decreased inflammatory response and oxidative stress in dystrophic muscle cells, indicating that LLLT could be a helpful alternative therapy to be associated with other treatment for dystrophinopathies.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.

ABSTRACT
The present study evaluated low-level laser therapy (LLLT) effects on some physiological pathways that may lead to muscle damage or regeneration capacity in dystrophin-deficient muscle cells of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD). Primary cultures of mdx skeletal muscle cells were irradiated only one time with laser and analyzed after 24 and 48 hours. The LLLT parameter used was 830 nm wavelengths at 5 J/cm² fluence. The following groups were set up: Ctrl (untreated C57BL/10 primary muscle cells), mdx (untreated mdx primary muscle cells), mdx LA 24 (mdx primary muscle cells - LLLT irradiated and analyzed after 24 h), and mdx LA 48 (mdx primary muscle cells - LLLT irradiated and analyzed after 48 h). The mdx LA 24 and mdx LA 48 groups showed significant increase in cell proliferation, higher diameter in muscle cells and decreased MyoD levels compared to the mdx group. The mdx LA 48 group showed significant increase in Myosin Heavy Chain levels compared to the untreated mdx and mdx LA 24 groups. The mdx LA 24 and mdx LA 48 groups showed significant increase in [Ca2+]i. The mdx group showed significant increase in H2O2 production and 4-HNE levels compared to the Ctrl group and LLLT treatment reduced this increase. GSH levels and GPx, GR and SOD activities increased in the mdx group. Laser treatment reduced the GSH levels and GR and SOD activities in dystrophic muscle cells. The mdx group showed significant increase in the TNF-α and NF-κB levels, which in turn was reduced by the LLLT treatment. Together, these results suggest that the laser treatment improved regenerative capacity and decreased inflammatory response and oxidative stress in dystrophic muscle cells, indicating that LLLT could be a helpful alternative therapy to be associated with other treatment for dystrophinopathies.

No MeSH data available.


Related in: MedlinePlus

Analysis of oxidative stress in control and dystrophic muscle cells.In (A), immunoblot analysis shows several bands of 4-HNE-protein adducts, ranging from 17 to 170 kDa. Graphs show protein level in the muscle cells from Ctrl, mdx untreated, mdx LA 24 and mdx LA 48. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. In (B), quantification of H2O2 production in the muscle cells from normal (Ctrl) and dystrophic primary muscle cells untreated (mdx untreated) and LLLT treatment analyzed after 24 hours (mdx LA 24) and 48 hours (mdx LA 48). In (C) analysis of glutathione levels in the muscle cells from normal (Ctrl) and dystrophic culture cells untreated (mdx untreated) and LLLT treatment analyzed after 24 hours (mdx LA 24) and 48 hours (mdx LA 48). All the experiments were performed in triplicate and data expressed as mean ± SD. The relative value of the band intensity was quantified and normalized by the corresponding Ctrl. a P< 0.05 versus Ctrl; b P< 0.05 versus mdx untreated; c P< 0.05 versus mdx LA 24.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4470633&req=5

pone.0128567.g004: Analysis of oxidative stress in control and dystrophic muscle cells.In (A), immunoblot analysis shows several bands of 4-HNE-protein adducts, ranging from 17 to 170 kDa. Graphs show protein level in the muscle cells from Ctrl, mdx untreated, mdx LA 24 and mdx LA 48. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. In (B), quantification of H2O2 production in the muscle cells from normal (Ctrl) and dystrophic primary muscle cells untreated (mdx untreated) and LLLT treatment analyzed after 24 hours (mdx LA 24) and 48 hours (mdx LA 48). In (C) analysis of glutathione levels in the muscle cells from normal (Ctrl) and dystrophic culture cells untreated (mdx untreated) and LLLT treatment analyzed after 24 hours (mdx LA 24) and 48 hours (mdx LA 48). All the experiments were performed in triplicate and data expressed as mean ± SD. The relative value of the band intensity was quantified and normalized by the corresponding Ctrl. a P< 0.05 versus Ctrl; b P< 0.05 versus mdx untreated; c P< 0.05 versus mdx LA 24.

Mentions: In order to analyze the laser effects on oxidative stress in dystrophic primary muscle cells, we determined 4-hydroxynonenal (4-HNE)-protein adduct levels; H2O2 production; glutathione (GSH) content and superoxide dismutase (SOD); glutathione peroxidase (GPx); and glutathione reductase (GR) activity. Bands of 4-HNE-protein adducts are shown in Fig 4A. Proteins from 17 to 170 kDa were observed in all groups. The 4-HNE protein adduct levels were significantly higher in mdx primary muscle cells (by 19%) compared to control primary muscle cells (P<0.05; Fig 4A). Reduction of 13% on the 4-HNE protein adduct levels was observed in the mdx LA 48 group (P<0.05; Fig 4A). Mdx primary muscle cells showed a significant increase in H2O2 production (by 20%) compared to control primary muscle cells (Fig 4B). Treatment with laser significantly decreased the H2O2 production (by 17% for mdx LA 24 and 30% for mdx LA 48) in dystrophic primary muscle cells (Fig 4B). GSH levels were significantly higher in mdx primary muscle cells (by six times) compared to control primary muscle cells (P<0.05; Fig 4C). LLLT treatment significantly decreased the GSH levels (by 73% for mdx LA 24 and 60% for mdx LA 48) in dystrophic primary muscle cells (Fig 4C). The effect of the laser treatment on SOD, GPx and GR activities is shown in Table 1. The increase of SOD, GPx and GR activities in mdx group were found to be significant when compared to the Ctrl group (P<0.05; Table 1). LLLT treatment significantly decreased the GR (by 42% for mdx LA 24) and SOD activity (by 47% for mdx LA 24 and 32% for mdx LA 48) in dystrophic primary muscle cells (Table 1).


Low-Level Laser Therapy (LLLT) in Dystrophin-Deficient Muscle Cells: Effects on Regeneration Capacity, Inflammation Response and Oxidative Stress.

Macedo AB, Moraes LH, Mizobuti DS, Fogaça AR, Moraes Fdos S, Hermes Tde A, Pertille A, Minatel E - PLoS ONE (2015)

Analysis of oxidative stress in control and dystrophic muscle cells.In (A), immunoblot analysis shows several bands of 4-HNE-protein adducts, ranging from 17 to 170 kDa. Graphs show protein level in the muscle cells from Ctrl, mdx untreated, mdx LA 24 and mdx LA 48. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. In (B), quantification of H2O2 production in the muscle cells from normal (Ctrl) and dystrophic primary muscle cells untreated (mdx untreated) and LLLT treatment analyzed after 24 hours (mdx LA 24) and 48 hours (mdx LA 48). In (C) analysis of glutathione levels in the muscle cells from normal (Ctrl) and dystrophic culture cells untreated (mdx untreated) and LLLT treatment analyzed after 24 hours (mdx LA 24) and 48 hours (mdx LA 48). All the experiments were performed in triplicate and data expressed as mean ± SD. The relative value of the band intensity was quantified and normalized by the corresponding Ctrl. a P< 0.05 versus Ctrl; b P< 0.05 versus mdx untreated; c P< 0.05 versus mdx LA 24.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4470633&req=5

pone.0128567.g004: Analysis of oxidative stress in control and dystrophic muscle cells.In (A), immunoblot analysis shows several bands of 4-HNE-protein adducts, ranging from 17 to 170 kDa. Graphs show protein level in the muscle cells from Ctrl, mdx untreated, mdx LA 24 and mdx LA 48. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. In (B), quantification of H2O2 production in the muscle cells from normal (Ctrl) and dystrophic primary muscle cells untreated (mdx untreated) and LLLT treatment analyzed after 24 hours (mdx LA 24) and 48 hours (mdx LA 48). In (C) analysis of glutathione levels in the muscle cells from normal (Ctrl) and dystrophic culture cells untreated (mdx untreated) and LLLT treatment analyzed after 24 hours (mdx LA 24) and 48 hours (mdx LA 48). All the experiments were performed in triplicate and data expressed as mean ± SD. The relative value of the band intensity was quantified and normalized by the corresponding Ctrl. a P< 0.05 versus Ctrl; b P< 0.05 versus mdx untreated; c P< 0.05 versus mdx LA 24.
Mentions: In order to analyze the laser effects on oxidative stress in dystrophic primary muscle cells, we determined 4-hydroxynonenal (4-HNE)-protein adduct levels; H2O2 production; glutathione (GSH) content and superoxide dismutase (SOD); glutathione peroxidase (GPx); and glutathione reductase (GR) activity. Bands of 4-HNE-protein adducts are shown in Fig 4A. Proteins from 17 to 170 kDa were observed in all groups. The 4-HNE protein adduct levels were significantly higher in mdx primary muscle cells (by 19%) compared to control primary muscle cells (P<0.05; Fig 4A). Reduction of 13% on the 4-HNE protein adduct levels was observed in the mdx LA 48 group (P<0.05; Fig 4A). Mdx primary muscle cells showed a significant increase in H2O2 production (by 20%) compared to control primary muscle cells (Fig 4B). Treatment with laser significantly decreased the H2O2 production (by 17% for mdx LA 24 and 30% for mdx LA 48) in dystrophic primary muscle cells (Fig 4B). GSH levels were significantly higher in mdx primary muscle cells (by six times) compared to control primary muscle cells (P<0.05; Fig 4C). LLLT treatment significantly decreased the GSH levels (by 73% for mdx LA 24 and 60% for mdx LA 48) in dystrophic primary muscle cells (Fig 4C). The effect of the laser treatment on SOD, GPx and GR activities is shown in Table 1. The increase of SOD, GPx and GR activities in mdx group were found to be significant when compared to the Ctrl group (P<0.05; Table 1). LLLT treatment significantly decreased the GR (by 42% for mdx LA 24) and SOD activity (by 47% for mdx LA 24 and 32% for mdx LA 48) in dystrophic primary muscle cells (Table 1).

Bottom Line: The mdx LA 48 group showed significant increase in Myosin Heavy Chain levels compared to the untreated mdx and mdx LA 24 groups.The mdx LA 24 and mdx LA 48 groups showed significant increase in [Ca2+]i.Together, these results suggest that the laser treatment improved regenerative capacity and decreased inflammatory response and oxidative stress in dystrophic muscle cells, indicating that LLLT could be a helpful alternative therapy to be associated with other treatment for dystrophinopathies.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.

ABSTRACT
The present study evaluated low-level laser therapy (LLLT) effects on some physiological pathways that may lead to muscle damage or regeneration capacity in dystrophin-deficient muscle cells of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD). Primary cultures of mdx skeletal muscle cells were irradiated only one time with laser and analyzed after 24 and 48 hours. The LLLT parameter used was 830 nm wavelengths at 5 J/cm² fluence. The following groups were set up: Ctrl (untreated C57BL/10 primary muscle cells), mdx (untreated mdx primary muscle cells), mdx LA 24 (mdx primary muscle cells - LLLT irradiated and analyzed after 24 h), and mdx LA 48 (mdx primary muscle cells - LLLT irradiated and analyzed after 48 h). The mdx LA 24 and mdx LA 48 groups showed significant increase in cell proliferation, higher diameter in muscle cells and decreased MyoD levels compared to the mdx group. The mdx LA 48 group showed significant increase in Myosin Heavy Chain levels compared to the untreated mdx and mdx LA 24 groups. The mdx LA 24 and mdx LA 48 groups showed significant increase in [Ca2+]i. The mdx group showed significant increase in H2O2 production and 4-HNE levels compared to the Ctrl group and LLLT treatment reduced this increase. GSH levels and GPx, GR and SOD activities increased in the mdx group. Laser treatment reduced the GSH levels and GR and SOD activities in dystrophic muscle cells. The mdx group showed significant increase in the TNF-α and NF-κB levels, which in turn was reduced by the LLLT treatment. Together, these results suggest that the laser treatment improved regenerative capacity and decreased inflammatory response and oxidative stress in dystrophic muscle cells, indicating that LLLT could be a helpful alternative therapy to be associated with other treatment for dystrophinopathies.

No MeSH data available.


Related in: MedlinePlus