Trillin inhibits myoblast differentiation via increasing autophagy

Trillin, an active ingredient in traditional Chinese medicine Trillium tschonoskii, is a potential small molecule compound candidate that affecting myoblast differentiation, which predicting by AI technology in our previous study. Autophagy modulating myoblast differentiation has also been studied. In addition, Trillin was shown to regulate mTOR signaling pathway, a highly conserved kinase important for autophagy regulation. In this research, we aim to clarify the effect and underlying mechanism of Trillin on myoblast differentiation. Using mice C2C12 cell line to establish a myoblast differentiation model in vitro , treated with different concentration and time of Trillin, to explore the effect and latent mechanism of Trillin on myoblast differentiation by qRT-PCR, Western Blot and other molecular biological technique. Results showed that C2C12 differentiation was significantly inhibited by Trillin in a dose-dependent manner. The expression of MyHC, MyOG and MyoD was decreased extremely significant after 10 μM Trillin treatment. Meanwhile, autophagy level was significantly elevated with the supplement of Trillin. And C2C12 differentiation was recovered after ATG7 knockdown. Mechanically, we found that the activity of AKT/mTOR declined during the inhibition of differentiation by Trillin. Our findings suggested that Trillin attenuated C2C12 differentiation via increasing autophagy through AKT/mTOR signaling pathway. Taken together, we introduce a novel physiological function of Trillin in inhibiting skeletal muscle differentiation. Trillin restrains C2C12 differentiation that may through inhibiting AKT/mTOR signaling pathway then decrease autophagy level. The addition of Trillin drives C2C12 to decline AKT / mTORC1 activity during differentiation, on one side reducing the p-S6 and on the other side promoting the activation of autophagy, thereby inhibiting differentiation.