Betulinic acid alleviates the inflammatory injury of osteoblasts in osteoporosis by augmenting autophagy via the AMPK-mTOR signaling pathway

Osteoporosis (OP) is a systemic disease characterized by a reduction in the number of trabecular bone structures and damage to the bone microstructure. It is commonly found in people who are aging or have estrogen deficiency. Oxidative stress and chronic inflammation caused by pathological factors such as aging and estrogen deficiency are key pathogenic factors. Betulinic acid (BA), a natural pentacyclic triterpenoid compound, exhibits anti‑inflammatory and antioxidant biological effects. However, its role and potential mechanisms in the inflammatory injury of osteoblasts in OP remain unclear. In the present study, in vivo experiments were conducted using an ovariectomized (OVX) rat model of OP, with bone microstructure analyzed by micro‑CT, protein expression detected by immunohistochemistry, and serum inflammatory factors measured by ELISA. BA was revealed to alleviate bone loss in OVX rats and inhibit the expression of NOD‑like receptor pyrin domain‑containing 3 (NLRP3), Asc and caspase‑1 in the femur of OVX rats, as well as suppress the release of inflammatory factors such as interleukin‑1 β, interleukin‑6, and tumor necrosis factor‑αin the serum of rats. The inflammatory injury osteoblast model of BA intervention was also studied with hydrogen peroxide (H2O2) in vitro, with reactive oxygen species (ROS) levels assessed by fluorescence assay, osteogenic differentiation evaluated by ALP staining and alizarin red staining, and autophagy‑related proteins detected by western blotting. BA pretreatment reduced production of ROS, inhibited expression of NLRP3 and downstream pathway activation, improved alkaline phosphatase activity, mineralization ability, and osteogenic differentiation ability of MC3T3‑E1 cells. Administration of BA increased the autophagy of MC3T3‑E1 cells treated with H2O2, which was confirmed by the increased expression levels of LC3b II and Beclin‑1 and the decreased expression levels of P62. In addition, BA could enhance the phosphorylation of AMPK in MC3T3‑E1 cells treated with H2O2 and reduce the phosphorylation of mTOR, but this effect could be rescued by Compound C (an AMPK blocker). BA can protect osteoblasts from inflammatory injury by reducing the production of ROS and inhibiting the activation of NLRP3 through autophagy mediated by the AMPK/mTOR pathway.