Piperlongumine (PL) is a natural product found in long pepper (Piper longum ). The pharmacological effects of PL are well known, and it has been used for pain, hepatoprotection, and asthma in Oriental medicine. No studies have examined the effects of PL on bone tissue or bone-related diseases, including osteoporosis. The current study investigated for the first time the inhibitory effects of PL on osteoclast differentiation, bone resorption, and osteoclastogenesis-related factors in RAW264.7 macrophages stimulated by the receptor activator for nuclear factor- κB ligand (RANKL). Cytotoxicity was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and osteoclast differentiation and bone resorption were confirmed by tartrate-resistant acid phosphatase (TRAP) staining and pit formation analysis. Osteoclast differentiation factors were confirmed by western blotting. PL exhibited toxicity in RAW264.7 macrophages, inhibiting osteoclast formation and bone resorption, in addition to inhibiting the expression of osteoclastogenesis-related factors, such as tumor necrosis factor receptor-associated factor 6 (TRAF6), c-Fos, and NFATc1, in RANKL-stimulated RAW264.7 macrophages. These findings suggest that PL is suitable for the treatment of osteoporosis, and it serves as a potential therapeutic agent for various bone diseases.

Introduction
Materials and Methods
    1. Cell culture
    2. MTT assay
    3. Tartrate-resistant acid phosphatase staining
    4. Pit formation
    5. Western blot analysis
    6. Statistical analysis
Results
    1. Piperlongumine reduced the cell viability of RAW264.7 cells
    2. Piperlongumine suppressed receptor activator for nuclear factor-κB ligand-induced osteoclast differentiation and resorption in RAW264.7 cells
    3. Piperlongumine inhibited osteoclastogenesis markers of receptor activator for nuclear factor-κB ligand-induced osteoclastogenesis
Discussion
References

• Sun-Mi Jin(Department of Oral and Maxillofacial Surgery, Pusan National University Dental Hospital)
• Hae-Mi Kang(Department of Oral Anatomy, School of Dentistry, Pusan National University, BK21 PLUS Project, School of Dentistry, Pusan National University)
• Dan-Bi Park(Department of Oral Anatomy, School of Dentistry, Pusan National University, BK21 PLUS Project, School of Dentistry, Pusan National University)
• Su-Bin Yu(Department of Oral Anatomy, School of Dentistry, Pusan National University)
• In-Ryoung Kim(Department of Oral Anatomy, School of Dentistry, Pusan National University, BK21 PLUS Project, School of Dentistry, Pusan National University) Correspondence to
• Bong-Soo Park(Department of Oral Anatomy, School of Dentistry, Pusan National University, BK21 PLUS Project, School of Dentistry, Pusan National University) Correspondence to