Effects of the combination of Epimedii Folium and Ligustri Lucidi Fructus on apoptosis and autophagy in SOP rats and osteoblasts via PI3K/AKT/mTOR pathway

Background: This study aimed to investigate the effects of the combination of Epimedii Folium (EF) and Ligustri Lucidi Fructus (LLF) on regulating Apoptosis and Autophagy in senile osteoporosis (SOP) rats.

Methods: Firstly, we identified the components in the decoction and drug-containing serum of EL (EF&LLF) by Ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS). Secondly, SOP rats were treated with EF, LLF, EL and caltrate to evaluate the advantages of EL. Finally, H₂O₂-, chloroquine-, and MHY1485-induced osteoblasts were treated with different doses of EL to reveal the molecular mechanism of EL. We detected bone microstructure, oxidative stress levels, ALP activity and the expressions of Bax, Bcl-2, caspase3, P53, Beclin-1, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, mTOR, and LC3 in vivo and in vitro.

Results: 36 compounds in EL decoction and 23 in EL-containing serum were identified, including Flavonoids, iridoid Terpenoids, phenylethanoid glycosides, polyols and triterpenoids. EL could inhibit Apoptosis activity and increase ALP activity. In SOP rats and chloroquine-inhibited osteoblasts, EL could improve bone tissue microstructure and osteoblasts functions by upregulating Bcl-2, Beclin1, and LC3-II/LC3-I, while downregulating p53 in all treatment groups. In H₂O₂-induced osteoblasts, EL could upregulate the protein and mRNA expressions of Bcl-2 while downregulate LC3-II/LC3-I, p53 and Beclin1. Besides, EL was able to down-regulate PI3K/Akt/mTOR pathway which activated in SOP rats and MHY1485-induced osteoblasts.

Conclusions: These findings demonstrate that EL with bone protective effects on SOP rats by regulating Autophagy and Apoptosis via PI3K/Akt/mTOR signaling pathway, which might be an alternative medicine for the treatment of SOP.

Autophagy / apoptosis; Epimedii Folium / Ligustri Lucidi Fructus; Osteoblast; PI3K/Akt/mTOR; Senile osteoporosis; UPLC-Q-TOF-MS.