Low-level laser prevents doxorubicin-induced skeletal muscle atrophy by modulating AMPK/SIRT1/PCG-1α-mediated mitochondrial function, apoptosis and up-regulation of pro-inflammatory responses

Figures

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  Fig. 1. Schematic diagram of the animal experimental. Animals were allocated to three groups: Control group (Ctl), Dox-receiving group (Dox), and LLL treatment group (Dox + LLL). In Dox-treat animals, Dox (3 mg/kg) was injected by intraperitoneal injection per week ( total 4 weeks study period, accumulated dosage: 12 mg/ kg). In LLL-exposed animals, the laser was applied 10 min per point, 1 time per day, and 7 days per week. On the first day of the 1st, 2nd, 3rd and 4th week, after 2 h of LLL irradiation, Dox was injected by i.p. injections. Samples were collected on the last day of this 4-weeks experimental

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  Fig. 2. LLL exposure reverse Dox-induced muscle wasting in animals. The effects of LLL on Dox-induced muscle atrophy. The weight change (A), food intake (B), muscle mass (C), and cross-sectional area (D) in Con and Dox animals. Representative hematoxylin–eosin (H&E) and TUNEL stained soleus (E). The arrow heads indicated TUNEL ( brown color). Quantification of the percentage of TUNEL positive nuclei (F). Atrogin-1 (G) and MuRF-1 (H) mRNA expression levels in soleus muscle were investigated by real-time PCR. The data were presented as the mean ± SD of 8 animals. (* indicating p < 0.05 compared with the control group)

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  Fig. 3. Low level laser (LLL) exposure increases the phosphorylation of AMPK. C2C12 cells were exposed to various dosages of LLL for a total of 2 h. The expression levels of phosphorylated AMPK and total AMPK were investigated using Western blot assay (A). Protein expression levels were quantified and presented by a bar chart (B). C2C12 cells were treated with 2 µM doxorubicin (Dox) for 24 h, in the LLL-treated group, cells were exposed to LLL 8 J/m2 before Dox treatment. The expression level of phosphorylated AMPK and total AMPK were investigated using Western blot assay (C). Protein expression levels were quantified and presented by a bar chart (D). The data were presented as the mean ± SD of three biological replicates at three separate times. (* indicating p < 0.05 compared with the control group; #indicating p < 0.05 compared to Dox group)

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  Fig. 4. LLL exposure mitigates Dox-reduced SIRT1 and PGC-1α expression via modulation of AMPK. C2C12 cells were exposed to LLL 8J/m2 for 2 h and incubated in the 37 °C incubator with 5% CO2 for additional 22 h. The expression level of SIRT1, PGC-1α, and β-actin were investigated using Western blot assay (A). Protein expression levels were quantified and presented by a bar chart (B, C). C2C12 cells were treated with 2 µM doxorubicin (Dox) for a total of 24 h, in the LLL-treated group, cells were exposed to LLL 8 J/m2 before Dox treatment. The expression level of SIRT1, PGC-1α, and β-actin were investigated using Western blot assay (D). Protein expression levels were quantified and presented by a bar chart (E, F). In the LLL-treated group, cells were exposed to LLL 8J/m2 for 2 h before Dox treatment. In LLL plus AMPK silencing group, cells were transfected with AMPK siRNA for 36 h before LLL exposure and Dox treatment. The expression level of SIRT1, PGC-1α, and β-actin were investigated using Western blot assay (G). Protein expression levels were quantified and presented by a bar chart (H, I). Western blot analysis of phosphorylated AMPK, SIRT1, and PGC-1α in the left soleus muscle from…

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  Fig. 5. LLL exposure protects against Dox-impaired mitochondria function and biogenesis. C2C12 cells were treated with 2 µM doxorubicin (Dox) for a total of 24 h, in the LLL-treated group, cells were exposed to LLL 8 J/m2 before Dox treatment. In LLL plus AMPK, SIRT1, and PGC-1α silencing groups, cells were transfected with AMPK or SIRT1 or PGC-1α siRNA for 36 h before LLL exposure and Dox treatment. The mitochondrial membrane potential was investigated by JC-1. Results of JC-1 were obtained by a fluorescence microscope (A) and flow cytometry (B). FL-1 (green) and FL-2 (red) from the flow cytometry were quantified and presented by a bar chart (C). The mitochondrial oxygen consumption rate (ORC) was investigated by the Seahorse analyzer (D). The mt DNA copy number, which represents the biogenesis of mitochondrial, was investigated by the real-time PCR (E). The mitochondria superoxide was tested using the MitoSOX (F). Cytosolic ROS was investigated by DCF-DA (G). The data were presented as the mean ± SD of three biological replicates at three separate times. (* indicating p < 0.05 compared with the control group; # indicating p < 0.05 compared to Dox group; & indicating p < 0.05 compared…

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  Fig. 6. LLL exposure reduces Dox-caused proinflammatory responses by regulating AMPK/SIRT1/PGC-1a-mediated mitochondria function. C2C12 cells were treated with 2 µM doxorubicin (Dox) for a total of 24 h, in the LLL-treated group, cells were exposed to LLL 8 J/m2 before Dox treatment. In LLL plus AMPK, SIRT1, and PGC-1α silencing groups, cells were transfected with AMPK or SIRT1 or PGC-1α siRNA for 36 h before LLL exposure and Dox treatment. The expression level of p-p38, p38, nuclear NF-κBp65, and nuclear HADC1 were investigated using Western blot assay (A). Protein expression levels were quantified and presented by a bar chart (B, C). The activity of nuclear NF-κBp65 was investigated by a commercial kit (D). IL-8, Atrogin-1, and MuRF-1 mRNA expression levels were investigated by the real-time PCR. The data were presented as the mean ± SD of three biological replicates at three separate times. (* indicating p < 0.05 compared with the control group; # indicating p < 0.05 compared to Dox group; & indicating p < 0.05 compared to only LLL exposure group)

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  Fig. 7. LLL exposure attenuates Dox-induced apoptosis. C2C12 cells were treated with 2 µM doxorubicin (Dox) for a total of 24 h, in the LLL-treated group, cells were exposed to LLL 8 J/m2 before Dox treatment. In LLL plus AMPK, SIRT1, and PGC-1α silencing groups, cells were transfected with AMPK or SIRT1 or PGC-1α siRNA for 36 h before LLL exposure and Dox treatment. The expression level of Bcl-2, Bax, caspase 3, and β-actin were investigated using Western blot assay (A). Protein expression levels were quantified and presented by a bar chart (B–D). The TUNEL assay was used for detecting apoptotic DNA fragmentation using flow cytometry. The data were presented as the mean ± SD of three biological replicates at three separate times. (* indicating p < 0.05 compared with the control group; # indicating p < 0.05 compared to Dox group; & indicating p < 0.05 compared to only LLL exposure group)

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  Fig. 8. Schematic diagram summarizes the protective effects of LLL irradiation against Dox-induced skeletal muscle atrophy via the activation of AMPK/SIRT1/PGC-1α signaling. The in vivo and in vitro experiments show that LLL exposure promotes AMPK phosphorylation and upregulation of SIRT1 and PGC-1α expression thereby contributing to the preservation of mitochondrial homeostasis, and alleviation of oxidative stress and apoptosis in acute DOX treated skeletal muscles