Therapeutic potential of Amentoflavone against myostatin for skeletal muscle atrophy treatment: An in silico, in vitro, and in vivo study

BACKGROUND: Myostatin (MSTN) negatively regulates skeletal muscle (SM) growth, and its over activity suppresses myogenic differentiation, promoting muscle atrophy and aging. Amentoflavone (AMF), a biflavonoid from Ginkgo biloba, possesses anti-atrophy effects. AMF has anti-inflammatory, antioxidant, antitumor, and anti-viral properties. The effect of AMF on SM atrophy and myoblast differentiation has not been explained. PURPOSE: This study was conducted to evaluate the effects of AMF on promoting muscle growth and to elucidate its underlying mechanisms. METHODS: AMF was screened against MSTN using molecular docking and further validated by 100-ns molecular dynamics (MD) simulations. In vitro experiments used C2C12 myoblasts and human muscle satellite cells (MSCs) to assess the AMF effect on the proliferation and differentiation. In vivo, a dexamethasone-induced mouse model of muscle atrophy was used to examine the effect of AMF on muscle mass, fibre morphology, and function. RESULTS: In silico analyses revealed strong AMF-MSTN binding and a stable complex confirmed by MD simulation (100 ns) and cellular thermal shift assay. AMF enhanced myogenic differentiation in C2C12 and human MSCs, increasing MYH and MYOG gene and protein expression while reducing MSTN and phosphorylated SMAD levels. In dexamethasone-induced atrophic C2C12 and human MSCs, AMF restored myogenic markers and suppressed MSTN expression and signaling. In atrophic mice, AMF prevented weight and muscle loss; improved fiber cross-sectional area, strength, and endurance; decreased MSTN, Atrogin-1, MuRF1; and activated MYH and AKT/mTOR pathways. CONCLUSION: AMF interacts with MSTN, inhibiting its downstream signaling, promoting myogenic differentiation, and alleviating muscle atrophy, highlighting its potential as a natural therapeutic for muscle-wasting conditions. Through computational modeling, MD simulations, and experimental validation, AMF demonstrates strong binding to MSTN, with confirmed efficacy in both in vitro and in vivo studies.