Dihydroartemisinin attenuates lipopolysaccharide-induced osteoclastogenesis and bone loss via the mitochondria-dependent apoptosis pathway

Dihydroartemisinin (DHA) is a widely used antimalarial drug isolated from the plant Artemisia annua. Recent studies suggested that DHA has antitumor effects utilizing its reactive oxygen species (ROS) yielding mechanism. Here, we reported that DHA is inhibitory on lipopolysaccharide (LPS)-induced osteoclast (OC) differentiation, fusion and bone-resorption activity in vitro. Intracellular ROS detection revealed that DHA could remarkably increase ROS accumulation during LPS-induced osteoclastogenesis. Moreover, cell apoptosis was also increased by DHA treatment. We found that DHA-activated caspase-3 increased Bax/Bcl-2 ratio during LPS-induced osteoclastogenesis. Meanwhile, the translocation of apoptotic inducing factor (AIF) and the release of cytochrome c from the mitochondria into the cytosol were observed, indicating that ROS-mediated mitochondrial dysfunction is crucial in DHA-induced apoptosis during LPS-induced osteoclastogenesis. In vivo study showed that DHA treatment decreased OC number, prevents bone loss, rescues bone microarchitecture and restores bone strength in LPS-induced bone-loss mouse model. Together, our findings indicate that DHA is protective against LPS-induced bone loss through apoptosis induction of osteoclasts via ROS accumulation and the mitochondria-dependent apoptosis pathway. Therefore, DHA may be considered as a new therapeutic candidate for treating inflammatory bone loss.Bone is a dynamic organ that undergoes continuous remodeling throughout life. Bone homeostasis is maintained by a balanced bone-resorbing and bone-forming process. In this process, hematopoietic stem cells or monocytes/macrophage progenitor cell-derived osteoclasts (OCs) are mainly responsible for bone resorption.¹ Abnormal OC function is associated with numerous diseases, and most of them are due to excessive osteoclastic activity. These diseases include osteoporosis, rheumatoid arthritis and periodontitis.^{2, 3} Two of the most important regulating factors during OC differentiation are receptor activator of nuclear factor κB ligand (RANKL) and macrophage-colony-stimulating factor (M-CSF).^{4, 5} Binding of RANKL to RANK results in the initiation of the TNF receptor-associated factor 6 signaling, which activates nuclear factor-κB, Akt and MAP kinase (ERk, JNK and p-38), and eventually leads to the proliferation, differentiation and maturation of OCs.^{6, 7}Lipopolysaccharide (LPS) is an important component of the outer membrane of Gram-negative bacteria. In LPS-induced bone loss, many factors are involved including local host response, prostanoids and cytokine production, inflammatory cell recruitment and OC activation.^{8, 9, 10} Experimental evidence have shown that LPS-mediated inflammation is highly dependent on reactive oxygen species (ROS) and the associated downstream MAPK signaling pathways including ERK, JNK and p-38.^{11, 12} ROS has been shown having an important role in the process of OC differentiation, survival, activation and bone resorption.^{13, 14, 15, 16} It has also been proved that ROS production in OC and intracellular hydrogen peroxide accumulation is critical for osteoclastogenesis and skeletal homeostasis.¹⁷ Recently, a study reported that LPS induces OC formation via the ROS-mediated JNK and STAT3 pathway, which could be blocked by peroxiredoxin II.¹⁸Dihydroartemisinin (DHA) is the main active metabolite isolated from the plant Artemisia annua. DHA has been widely used as first-line therapeutics against falciparum malaria.¹⁹ Recent evidence suggested that DHA has antitumor effects because of its unique cytotoxicity mechanism.²⁰ In particular, studies reported that DHA is pro-apoptotic in tumor cell lines regarding breast and prostate cancer.^{21, 22} Although the detailed mechanism of DHA cytotoxicity and pro-apoptotic effects is not fully understood, DHA-mediated ROS production has a central role.^{23, 24} However, the effect of DHA on bone health has not been studied.In the present study, we reported that DHA could attenuate LPS-induced OC differentiation, fusion and bone-resorption activity in vitro. Our data showed that DHA-induced cell apoptosis during LPS-induced osteoclastogenesis via intracellular ROS generation and mitochondria-mediated pathways. DHA administration in LPS-induced mouse models decreased OC number and reversed bone loss in vivo.