Madecassic acid suppresses osteoclast differentiation and bone resorption by inhibiting RANKL-induced NF-κB,JNK and NFAT signaling pathways

Background:Overproduction and activation of osteoclasts result in various bone diseases,such as osteoporosis,Paget's disease,and rheumatoid arthritis.Thus,inhibiting osteoclast formation and overactivation may effectively prevent osteoclast-related bone diseases,especially osteoporosis.Madecassic acid,one of the most important active ingredients in Centella asiatica,has various biological effects,but its role in osteoclastogenesis remains unknown.Methods:RAW 264.7 cells were stimulated with receptor activator of nuclear factor(NF)-κΒligand(RANKL,25 ng/mL)to differentiate into multinucleated osteoclasts.Subsequently,osteoclasts were treated with or without varying concentrations of madecassic acid(1,2.5,5,and 10μmol/L).Results:Madecassic acid significantly inhibited RANKL-induced osteoclastogenesis in a concentration-dependent manner.In addition,it reduced the percentage of bone resorptive area compared with the control,confirming that madecassic acid can inhibit osteoclast function.Furthermore,luciferase reporter gene studies indicate that madecassic acid could decrease the transcriptional activity of NF of activated T cells(NFAT)and NF-κB in a dose-dependent manner.Quantitative real-time polymerase chain reaction results show that madecassic acid attenuated the expression of osteoclast-associated genes,including V-ATPase-d2,cathepsin K,tartrate-resistant acid phosphatase(TRAP),NFAT cytoplasmic 1(NFATc1).Western blot analysis shows that madecassic acid inhibited RANKL-mediated degradation of IκBαand NFATc1 expression,as well as phosphorylation of c-Jun N-terminal kinase(JNK)in RAW 264.7 cells.Conclusion:Madecassic acid inhibited osteoclast formation and function in vitro by suppressing NF-κB,JNK,and NFAT signaling pathways,indicating its potential as a novel drug for the treatment of osteoclast-related bone diseases,especially osteoporosis.