Effect of Oenothera odorata Root Extract on Microgravity and Disuse-Induced Muscle Atrophy
Effect of EVP on oxidative stress by microgravity in C2C12 myoblasts. (a) Effect of EVP on SOD1 expression using immunocytochemistry. Representative images (up) and quantitative analysis (down) of fluorescence density of SOD1 in C2C12 myoblasts. C2C12 myoblasts were cultured in 35 mm cover-glass-bottom dish, and the medium was then replaced to serum-free medium with or without NAC (2 mM) or EVP (50 μg/mL). After 24 hr, 3D-clinorotation was subjected for 24 hr with DMEM containing 10% (v/v) FBS. Images were acquired with a LSM710 confocal microscope after immunofluorescence staining with SOD1 and Alexa 488 antibodies (green). C2C12 myoblasts were stained with DAPI to visualize nuclei (blue). Images are shown the mean values (±SD) from three experiments. Scale bar: 20 μm. NAC: N-acetyl cysteine. versus microgravity alone. (b) Effect of EVP on SOD1 expression using immunoblot analysis. C2C12 myoblasts were cultured in 12-well plates until confluent, and the medium was then replaced to serum-free medium with or without the EVP (50 μg/mL) for 24 hr. 3D-clinorotation was subjected for 24 hr with DMEM containing 10% (v/v) FBS. Protein lysate was extracted using the PRO-PREP Protein extraction Kit. The whole lysates were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels. SOD1 and β-actin were analyzed by immunoblot analysis using specific antibodies. Immunoblot was analyzed by densitometry and the inserts display representative blots of four similar independent experiments, respectively.