Objective To investigate the expression, function and related molecular mechanisms of Asporin (ASPN) in the repair process of injuried skeletal muscle. Methods An acute muscle injury mice model was prepared by intramuscular injection of Cardiotoxin (CTX). For interfering in ASPN levels in injured muscle, the recombinant ASPN protein (rmASPN) or neutralizing antibody (anti-ASPN) was treated respectively. Muscle samples were collected on 0 d, 4 d, 7 d, and 10 d post myoinjury, and the expression of ASPN, muscle transcription factors (MyoD, Myogenin), and TGF-β/Smad2/3 pathway molecules were detected by immunofluorescence, Western blot, or RT-qPCR separately. In differentiated C2C12 myotubes cultured in vitro, Western blot was used to detect p-Smad2/3 protein. Results Under physiological condition, muscle fibers stably expressed ASPN protein. During the muscle inflammatory phase (D4) and early stage of muscle regeneration (D7), significant upregulation of ASPN protein was observed in muscle tissue, and in Dystrophin+ centra-nucleus regenerating myofibers. The level of ASPN gradually decreased with the completion of myorepair. rmASPN treatment induced the increase of number and area of regenerated myofibers, and upregulated MyoD and Myogenin levels, but decreased the level of p-Smad2/3 in myofibers. As expected, anti-ASPN treatment resulted in the opposite outcome. As well, inflammatory stimuli induced ASPN upregulation, but inhibited p-Smad2/3 expression in C2C12 myotubes. Conclusions After myoinjury, regenerated myofibers synthesize and secrete ASPN. In inflamed muscle, ASPN may intervene and inhibit the activation of TGF-β-p-Smad2/3 signaling in myofibers, thereby promoting the functional differentiation of regenerated myofibers.
Key words
Asporin /
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Myoinjury /
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Myofiber differentiation /
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TGF-β/Smad2/3 pathway
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