Photobiomodulation mitigates Bothrops jararacussu venom-induced damage in myoblast cells by enhancing myogenic factors and reducing cytokine production

Abstract

Background: Photobiomodulation has exhibited promise in mitigating the local effects induced by Bothrops snakebite envenoming; however, the mechanisms underlying this protection are not yet fully understood. Herein, the effectiveness of photobiomodulation effects on regenerative response of C2C12 myoblast cells following exposure to Bothrops jararacussu venom (BjsuV), as well as the mechanisms involved was investigated.

Methodology/principal findings: C2C12 myoblast cells were exposed to BjsuV (12.5 μg/mL) and irradiated once for 10 seconds with laser light of 660 nm (14.08 mW; 0.04 cm2; 352 mW/cm2) or 780 nm (17.6 mW; 0.04 cm2; 440 mW/ cm2) to provide energy densities of 3.52 and 4.4 J/cm2, and total energies of 0.1408 and 0.176 J, respectively. Cell migration was assessed through a wound-healing assay. The expression of MAPK p38-α, NF-Кβ, Myf5, Pax-7, MyoD, and myogenin proteins were assessed by western blotting analysis. In addition, interleukin IL1-β, IL-6, TNF-alfa and IL-10 levels were measured in the supernatant by ELISA. The PBM applied to C2C12 cells exposed to BjsuV promoted cell migration, increase the expression of myogenic factors (Pax7, MyF5, MyoD and myogenin), reduced the levels of proinflammatory cytokines, IL1-β, IL-6, TNF-alfa, and increased the levels of anti-inflammatory cytokine IL-10. In addition, PBM downregulates the expression of NF-kB, and had no effect on p38 MAKP.

Conclusion/significance: These data demonstrated that protection of the muscle cell by PBM seems to be related to the increase of myogenic factors as well as the modulation of inflammatory mediators. PBM therapy may offer a new therapeutic strategy to address the local effects of snakebite envenoming by promoting muscle regeneration and reducing the inflammatory process.

Fig 1. Effect of photobiomodulation on the migration of C2C12 myoblasts cells.

Cells were seeded in plates and then incubated with 12.5 μg/mL of BjsuV in the presence or absence of PBM irradiation for 1 h. Then cells were washed, the medium replaced, scratched with micropipette and then incubated for 24 h. Then phase contrast microscope was used to take the photos (50x). The representative pictures are shown in A (a-h). Cells migration is represented as percentage of the cell that migrated to the scratched area. The scratched area was quantified by ImageJ software and data are shown in B. The picture represents one independent experiment (n = 3). #P < 0.05; compared to control; *P < 0.05; compared with the BjsuV group. BjsuV, Bothrops jararacussu venom; PBM, photobiomodulation. Figure diagram built using https://commons.wikimedia.org as source of the imagens or icons.

Fig 2. Effect of photobiomidulation on the expression of myogenic regulatory factors.

Representative immunoblot analyses and corresponding bar graphs depicting the protein levels of Myf5 (A and B), Pax7 (A and C), MyoD (A and D), and Myogenin (A and E) in C2C12 myoblast cells. The cells were incubated with 12.5 μg/mL of BjsuV, and the impact of photobiomodulaton treatment was assessed after 6 hours. β-actin served as the loading control. All experiments were performed in triplicate, and the relative intensity of the bands was quantified. Each value represents the mean ± SEM of three independent experiments, #p< 0.05 vs the control group; *p< 0.05 vs the venom-exposed group. BjsuV, Bothrops jararacussu venom; PBM, photobiomodulation. Figure diagram built using https://commons.wikimedia.org as source of the imagens or icons.

Fig 3. Effect of photobiomodulation on cytokine levels in C2C12 cells exposed to B. jararacussu venom.

The supernatant of C2C12 cells was analyzed for interleukin-1β (IL-1β) (A, B, C), interleukin-6 (IL-6) (D, E, F), tumor necrosis factor-α (TNF-α) (G, H, I), and interleukin-10 (IL-10) (J, K, L) at 3, 6, and 24 hours. All the experiments were performed in triplicate and the data were expressed as mean ± SEM. #p < 0.05 vs. control group *p < 0.05 vs the venom-exposed group (ANOVA). BjsuV, Bothrops jararacussu venom; PBM, photobiomodulation. Figure diagram built using https://commons.wikimedia.org as source of the imagens or icons.

Fig 4. Effect of photobiomidulation on the expression of NF-kB and p38 MAPK.

Representative immunoblot analyses and corresponding bar graphs depicting the protein levels of NF-kB (A and B), and p38MAPK (A and C) in C2C12 myoblast cells. The cells were incubated with 12.5 μg/mL of BjsuV, and the impact of photobiomodulation treatment was assessed after 6 hours. β-actin served as the loading control. All experiments were performed in triplicate, and the relative intensity of the bands was quantified. #p< 0.05 vs the control group; *p< 0.05 vs the venom-exposed group. BjsuV, Bothrops jararacussu venom; PBM, photobiomodulation. Figure diagram built using https://commons.wikimedia.org as source of the imagens or icons.