Study On The Inhibitary Effect And Mechanism Of Lignin-like Compounds In Acorns Tatarinowii Schott For Osteoclast Formation

Acorus tatarinowii Schott is a kind of Chinese herbal medicine with excellent medicinal effect,which can treat many diseases.It possesses multiple bioactivities,such as neuro-protection,anti-inflammation and anti-oxidant characteristics.Acorus tatarinowii Schott has been used to treat rheumatoid arthritis,which is an inflammation-mediated bone disease.Actually,most of inflammation or oxidation related diseases are accompanied with bone-lysis disorder due to excessive inflammation or oxidative stress enhances osteoclast formation.Osteoclasts are multinucleated giant cells responsible for bone resorption.Currently,increasing studies are focusing on osteoclast as potential therapeutic target to bone lysis disease,such as osteoporosis,rheumatoid arthritis and Paget’s disease.However,few studies reported the effects of Acorus tatarinowii Schott on osteoclast-mediated bone lysis diseases.In our previous study,a lignin-like compound Tatarinan O（TO）,which was derived from the root of Acorus tatarinowii Schott,was found to inhibit osteoclastogenesis.There are still several other lignin-like compounds extracted from the root of Acorus tatarinowii Schott,which share the same basic structureα-asarone with TO.The effect of these compounds with common asarone structure remains unclear.The aim of this study is to mine out the active lignin-like compounds in Acorus tatarinowii Schott that can effectively inhibit the formation of osteoclasts induced by RANKL and to clarify the molecular mechanism.In this study,two compounds,Tatarinan N（TN）and Tatarinan T（TT）,were identified by TRAP staining assay to have anti-osteoclastogenesis ability.Several experiments were arranged to further investigate the effects and mechanisms of TN and TT on reducing RANKL-induced osteoclastogenesis in vitro and LPS-induced bone loss in vivo.Morphologically,TRAP staining assay,bone resorption assay and F-actin staining assay showed that both TN and TT concentration-dependently inhibited osteoclast differentiation,F-actin ring formation,and bone resorption area at the early stage of osteoclastogenesis.It indicated that both TN and TT can decrease bone resorption area via mediation of osteoclast differentiation and F-actin ring formation.To investigate the molecular mechanisms underlying the suppressive effect of TN and TT on RANKL-mediated osteoclast formation and bone resorption,several osteoclastic genes（OSCAR,DC-STAMP,αvβ3,Apt6v0d2,TRAP,Cts K,MMP-9）were detected using real-time PCR（RT-PCR）.The results showed that the expressions of the osteoclastic genes induced by RANKL were both down-regulated by TN and TT treatment.It suggested that both TN and TT suppressed osteoclastogenesis,F-actin ring formation,and bone resorption via modulating osteoclast-related genes.NFATc1 is the core transcription factor of osteoclastogenesis and regulates the expression of osteoclast-related genes.We found that both TN and TT suppressed NFATc1 at mRNA and protein levels,respectively.These results indicated that the inhibitory effects of TN and TT on osteoclastic genes expression and osteoclast formation owe to the suppression of NFATc1.Furthermore,c-Fos,an upstreaming transcriptor of NFATc1,has also been determined.The results indicated that both TN and TT can inhibit mRNA and protein expression of c-Fos.Subsequently,the effects of TN and TT on some signaling pathways such as NF-κB,MAPKs and Ca²⁺signaling pathways,which evoked by RANKL to modulate NFATc1,were observed using western blot assay,immunofluorescence and fluo-4AM Ca²⁺fluorescent probe.The results showed that TN reduced activation of NF-κB,ERK,p38 pathways and Syk-Btk-PLCγ2 cascades in RANKL-stimulated osteoclast precursors.Moreover,TN inhibited intracellular[Ca²⁺]and attenuated the expression of calcineurin catalytic subunit PP2B-Aα.However,TT did not alter above signaling pathways.In vivo experiments were performed to explore whether TN and TT could improve LPS-induced bone loss in mice.Micro-CT assay showed that TN and TT administration both improved bone micro-structure parameters including trabecular number（Tb.N）,trabecular separation（Tb.Sp）,bone mineral density（BMD）,and bone volume per tissue volume（BV/TV）.H&E and TRAP staining of bone sections showed that TN and TT treatment both obviously reduced LPS-induced bone fracture destruction and excessive osteoclastogenesis.In conclusion,in vitro study suggested that TN and TT suppressed RANKL-induced osteoclast differentiation,bone resorption and F-actin ring formation via reducing the expression of master transcription factor NFATc1,its upstreaming transcriptor c-Fos and its target genes.The results demonstrated that TN might suppress NFATc1 expression through modulation of NF-κB,MAPKs,and PLCγ2-Ca²⁺-calcineurin signaling pathways.However,TT did not alter above signaling pathways in RANKL-stimulated osteoclast precursors.In vivo study further indicated that both TN and TT improved bone destruction in LPS-induced bone loss mice model.This study indicates that TN and TT have therapeutic potential for treating osteoclast-mediated bone lysis diseases.