Epimedium Brevicornu and Curculigo orchioides inhibit osteoclast autophagy by degrading the level of miRNA-199 to regulate the mTOR signaling pathway.

Abstract

To investigate the effect and mechanism of Epimedium brevicornu Maxim. and Curculigo orchioides Gaertn. (EBCO) on miRNA-199 expression in osteoclasts. EBCO-containing and blank serum was drawn from Wistar rats received EBCO (12 g/kg/d) or the same volume of dH₂O for 7 days intragastrically. RAW264.7 cells were induced to differentiate into osteoclasts. The miRNA-199 and Mtor mRNA level of osteoclasts treated with blank serum and low (5%), medium (10%), high (20%) concentrations of EBCO-containing serum for 48 h were measured. The binding relationship between miRNA-199 and Mtor was verified by dual-luciferase assay. Osteoclasts were divided into control, blank serum, EBCO-containing serum, blank serum, or EBCO-containing serum with 2 µM MHY1485 (mTOR agonist) or 10 nM Dactolisib (mTOR inhibitor) groups, proliferation, ROS level, miRNA-199 mRNA level, the expression of mTOR signaling pathway and autophagy-related proteins expression were observed. Compared with the control group, EBCO-containing serum significantly (P < 0.05) decreased the level of miRNA-199 in osteoclasts, increased Mtor mRNA level. The relative fluorescence level was significantly (P < 0.01) decreased after co-transfection of miRNA-199 mimics and MTOR-3'UTR-WT compared to NC-mimics group. EBCO-containing serum reduced cell viability and ROS level, suppressed light chain 3 (LC3)-II/LC3-I and Beclin-1, enhanced phosphorylated mTOR, mTOR and phosphorylated p70S6 kinase (p-p70S6K) proteins expression. EBCO delays the progression of osteoporosis by reducing miRNA-199 in osteoclasts to regulate mTOR signaling and inhibit autophagy. Further research is needed to explore new avenues of clinical application.