RESUMO
Iron overload-associated osteoporosis presents a significant challenge to bone health. This study examines the effects of arecoline (ACL), an alkaloid found in areca nut, on bone metabolism under iron overload conditions induced by ferric ammonium citrate (FAC) treatment. The results indicate that ACL mitigates the FAC-induced inhibition of osteogenesis in zebrafish larvae, as demonstrated by increased skeletal mineralization and upregulation of osteogenic genes. ACL attenuates FAC-mediated suppression of osteoblast differentiation and mineralization in MC3T3-E1 cells. RNA sequencing analysis suggests that the protective effects of ACL are related to the regulation of ferroptosis. We demonstrate that ACL inhibits ferroptosis, including oxidative stress, lipid peroxidation, mitochondrial damage, and cell death under FAC exposure. In this study, we have identified heme oxygenase-1 (HO-1) as a critical mediator of ACL inhibiting ferroptosis and promoting osteogenesis, which was validated by HO-1 knockdown and knockout experiments. The study links ACL to HO-1 activation and ferroptosis regulation in the context of bone metabolism. These findings provide new insights into the mechanisms underlying the modulation of osteogenesis by ACL. Targeting the HO-1/ferroptosis axis is a promising therapeutic approach for treating iron overload-induced bone diseases.
RESUMO
BACKGROUND: Osteoporosis is a systemic skeletal disorder characterized by decreased bone density and the degradation of bone tissue microarchitecture. Ginsenoside Rg1, derived from Panax ginseng, has been a part of traditional Chinese medicine in China for centuries, particularly for treating osteoporosis. However, there remains limited research on the osteogenic potential of Rg1 within the glucocorticoid-induced osteoporosis (GIOP) model and its specific mechanisms. PURPOSE: The primary objective of this study is to investigate the osteogenic potential of Rg1 within the GIOP model and explore the signaling pathways associated with its in vivo and in vitro effects. METHODS: Cell proliferation, differentiation and mineralization were evaluated by the Cell counting kit 8(CCK8) assay, alkaline phosphatase (ALP) test and Alizarin Red S staining, respectively. The qPCR technique was used to determine the relative expression of mRNA and the western blot was used to determine the relative expression of protein. In vivo experiments, spinal vertebrae staining in zebrafish larvae was accomplished by alizarin red S staining. RESULTS: Zebrafish larvae's hatching, survival, malformation, and heart rate were unaffected by 50 µM of Rg1 in vivo, while the MEC3T3-E1 cell line's proliferation was unaffected by 50 µM of Rg1 in vitro. Meanwhile, Rg1 was shown to improve osteogenic differentiation or bone formation as well as the level of mRNA expression of osteogenic markers in vivo and in vitro. Treatment with Rg1 significantly increased the expression of G protein-coupled estrogen receptor (GPER) and pAKT. In addition, the GPER inhibitor G15 could significantly reduce the mRNA and protein expression levels of GPER and phosphorylated AKT, LY294002, a PI3K/AKT pathway inhibitor, markedly suppresses the expression of phosphorylated AKT, yet shows no significant impact on GPER expression. Both G15 and LY294002 can significantly blocked the Rg1-mediated enhancement of osteogenesis capacity in the GIOP model. In contrast, when both the agonists G1 of GPER and LY294002 were added, G1 increased the relative expression of mRNA and protein of GPER, but not the expression of osteogenic capacity and osteogenic markers. CONCLUSIONS: This study investigates the mineralization effects and mechanisms of Ginsenoside Rg1 both in vitro and in vivo. For the first time, we propose that Rg1 might regulate osteogenesis by modulating AKT phosphorylation through mediating GPER expression within the PI3K/AKT pathway in the GIOP model. This discovery introduces novel targets and avenues for osteoporosis treatment.