Low intensity laser irradiation (LILI) was found to promote the regeneration of skeletal muscle
Lasers have been widely used in biological and medical fields for many years [
In the sight of traditional histopathology, mature skeletal muscle contains postmitotic muscle fibers where they themselves have very limited remodeling capability [
Previously, it was shown that, relative to nonirradiated control, LILI of the injured site markedly accelerated tissue repair and regeneration of skeletal muscle [
Primary myoblasts were derived from hindlimb muscles of neonatal Wistar rats and cultured in Ham’s F-10 nutrient mixture supplemented with 20% fetal bovine serum (FBS) and incubated in a CO2-enriched (5%) humid atmosphere at 37°C. During the first several passages of the primary cultures, myoblasts were enriched by preplating method. Immunocytochemical identification of myoblasts was performed by using a monoclonal antibody to desmin, the muscle-specific intermediate filament protein, and screened under the laser scanning confocal microscope (LSCM).
In this experiment, a He-Ne laser with a wavelength of 632.8 nm was used. Prior to laser irradiation, myoblasts were initially seeded on the cell-culture plates at a density of 4 × 104 cells/mL. At the desired time points, irradiation was performed in the dark at room temperature. Laser irradiation was delivered to the culture plate from above via combination of optical lens. The distance between cells sample and lens was adjusted to make the laser beam diameter the same as the width of culture plates. The power density of irradiation on the cells was measured to be 6 mW/cm2 and the irradiation time was set to 3 min based on our preexperiment. The nonirradiated control cells were subjected to the same experimental conditions as the irradiated cells, except for the irradiation.
To simulate the initial physiological state of myoblasts
Myoblasts were harvested 24 h after laser irradiation or serum refeeding. Following fixation with 75% ethanol, the cells were digested with DNase-free RNase in phosphate-buffered saline (PBS) containing 5
Myoblasts were fixed with 4% paraformaldehyde and then incubated with mouse mAb to proliferate cell nuclear antigen (PCNA) at 4°C for 20 hours. After thorough rinsing with PBS, cells were incubated with biotinylated goat anti-mouse IgG and HRP-conjugated streptavidin successively. The peroxidase reaction was performed using AEC as chromogen and examined under a microscope. Three independent experiments in each condition were performed and a total of at least 100 cells were counted for each specimen.
After irradiation/refeeding, cells were harvested quickly at desired time points, and the total protein was extracted with lysis buffer. Extracts were quantified with a protein assay kit, fractionated by 6% SDS/PAGE, and then transferred to a poly membrane. After blocking, membranes were incubated with mouse mAb to cyclin A and cyclin D, respectively, and then treated with goat anti-mouse IgG conjugated with horseradish peroxidase. Immune complexes on the membrane were visualized by using diaminobenzidine system. The relative gray values of corresponding bands and
All of the experimental data were from experiments that were repeated at least three times, unless otherwise indicated. Statistical analysis was performed by
The enriched primary myoblasts displayed remarkable proliferation potential. Pure myoblast cultures have been expanded beyond 30 population doublings. There were no detectable changes of growth rate or cell morphology even after extensive proliferation
PCNA, proliferating cell nuclear antigen, is the necessary component for DNA duplication of cell chromatosome. Its synthesis and expression were related to cell proliferative cycle. Quantification of PCNA expression is one kind of simple and feasible method for evaluating cell proliferative activity. In this study, the expression of PCNA was detected by immunohistochemical method (IHC) and assessed using semiquantitative analysis. Ours results showed that, 24 hours after laser irradiation, PCNA expression in the laser group was significantly higher than in the control group and the same in the 10% FBS group but less than that of 20% FBS group (Figure
Immunohistochemical detection of PCNA expression in myoblasts after preconditioning with serum starvation
Proliferation index (PI) is defined as the total number of cells in all the phases of the cell cycle (G0, G1, S, G2, and M phase) divided by the number of cells that went into division (S, G2, and M phase); that is, PI = (S + G2/M)/(G0/G1 + S + G2/M). The proliferation index reflects the percentage of proliferative cells in the S phase and G2/M phase of the cell cycle and can be used with the PCNA expression assay to give a more complete understanding of growth characteristics of myoblasts. As shown in Figures
Effects of LLLI on the proliferation index of myoblasts 24 h after irradiation/refeeding. Typical flow cytometric DNA histograms of the myoblasts from control (a), laser irradiation (b), 10% FBS (c), and 20% FBS (d) groups, respectively.
Cartogram of flow cytometric analysis about the proliferation index of myoblasts from each group as described above. Data are means ± SEM.
The division cycle of eukaryotic cells is regulated by a family of cell-cycle regulatory proteins (also known as cyclin) and the cyclin-dependent kinases (CDK) complex, of which cyclin D and cyclin A are the key molecules that can trigger cell-cycle entry, thus driving the cells from quiescence to the mitotic cycle. The periodic change of their expression is synchronized to cell-cycle progression. As presented in Figure
Western blot analysis of cyclin D and cyclin A protein expression after irradiation/refeeding (a). The expression levels of cyclin D and cyclin A were subsequently measured by densitometric analysis (b). The relative protein expression values were normalized to that of
It is well known that skeletal muscle repair is a highly synchronized process involving the activation of various cellular responses [
So far, however, many laboratory results remain controversial regarding the laser-induced photobiomodulatory effects, and differences of the irradiation parameters and growth conditions used in various studies complicate the issue of making meaningful comparisons [
However, in this paper, an interesting aspect of the nonirradiated control myoblasts was that these cells, which were maintained throughout in serum-free cultures conditions, also display a little of cell proliferation as shown in Figure
Researches have shown that the expression of PCNA, an early cell-cycle protein, which is upregulated in the late G1 phase, could be markedly affected by low intensity laser irradiation [
As is well known, cyclin D and cyclin A are the initiating factors for G0/G1 conversion and their inducible expression contributed to cell-cycle progression of the LILI-activated myoblasts. Previous studies [
Further, in a preliminary experiment we also found that LILI with different energy densities had dissimilar influences on myoblasts growth
In our experiment, the ability of irradiated myoblasts to survive and proliferate in the absence of serum sustenance is remarkable as compared with the nonirradiated cells. Laser irradiation appears to be analogous to the components of serum responsible for myoblasts survival and growth whether myoblasts being dormant
Taken together, our studies suggest that myoblasts in culture can be promoted from quiescence into cell proliferation cycle in response to laser irradiation at a certain fluence. These results also indicate that LILI affects early cell-cycle regulatory genes and enhances proliferative potential of myoblasts, thereby increasing cell proliferation and contributing to muscle regeneration
The authors declare that there is no conflict of interests regarding the publication of this paper.
Cui-Ping Zhang, Shao-Dan Li, Yan Chen, and Yan-Ming Jiang contributed equally to this work.
This work was supported by the National Natural Science Foundation of China (81171798, 81121004, and 81230041), Beijing Municipal Natural Science Foundation (7142124), and the National Basic Science and Development Program (973 Program, 2012CB518105).