Abstract

Bone marrow mesenchymal stem cells (BMSCs) are multipotent cells that play a critical role in bone formation and are vulnerable to oxidative stress-induced dysfunction in osteoporosis (OP). This study investigates the pro-osteogenic potential of ajugoside, a naturally occurring iridoid monoterpene from Ajuga reptans , through a two-phase experimental design involving both in vitro and in vivo models. In the in vitro phase, an oxidative stress model was established in BMSCs using H 2 O 2 , followed by ajugoside treatment or lentiviral overexpression of solute carrier family 5 member 1 (Slc5a1). Cell viability, osteogenic differentiation, oxidative stress, and mitophagy were assessed. In the in vivo phase, an ovariectomy-induced OP mouse model was utilized to examine the therapeutic effects of ajugoside, resveratrol (a positive control), or adeno-associated virus-mediated Slc5a1 overexpression. Ajugoside inhibited Slc5a1 expression and activated AMPK signalling, leading to enhanced mitophagy and reduced oxidative stress. Ajugoside demonstrated a significant capacity to alleviate H 2 O 2 -induced injury in BMSCs, exhibiting a comparable mitigating effect on oxidative stress and mitophagy impairment in OP mice. However, the reactivation of Slc5a1 led to the reversal of these effects. Collectively, these findings demonstrate that ajugoside promotes osteogenic differentiation by suppressing Slc5a1 and activating AMPK-mediated mitophagy, offering a promising therapeutic strategy for OP.