Effects Of A Marine Fungus-derived Nitrobenzoyl Sesquiterpenoid On RANKL-induced Osteoclastogenesis And Inflammatory Bone Destruction

Background and objectives:Osteoclasts are the sole cells which have bone resorption activity in the body.Excessive activation of osteoclasts results in the bone loss that is found in various osteolytic diseases,including osteoporosis,inflammatory disorders such as sepsis,rheumatoid arthritis(RA)and metastatic cancers.In recent years,targeting osteoclast differentiation has become a feasible therapeutic strategy for preventing bone destruction in osteolytic.RANKL and M-CSF are key factors for osteoclast survival,differentiation and function.Binding of RANKL to its cognate receptor RANK activates intracellular signaling pathways,including the NF-?B pathway,the MAPK and activator protein 1(AP-1)pathway,and the activated T cell nuclear factor 1(NFATc1)pathway.NFATc1 is the master nuclear transcription factor for osteoclast differentiation,and activation of NFATc1 induces the production of osteoclast-associated genes to initiate osteoclast differentiation and maturation.Nitrobenzoyl sesquiterpenoids(NSs)are naturally rare.Only 5 NSs have been reported before our study,and the fifth new nitrophenyl sesquiterpene 6?,9?-dihydroxy-14-p-nitrobenzoylcinnamolide(NS4)was discovered from the seaweed marine fungus Aspergillus ochraceus Jema1F17 by our cooperator in 2014.Therefore,we planned to make further excavation of the secondary metabolites of the Aspergillus ochraceus Jcma1F17,and hoped to obtain more different natural products of NS for deeper understanding of anti-inflammatory and immunological activities.Here,we isolated NSs from the culture of Aspergillus ochraceus Jcma1F17,and evaluated the inhibitory activities of NSs on RANKL-induced NF-?B luciferase activity and osteoclastogenesis in preosteoclastic RAW264.7 cells.The most active compound NS was selected to further study its effects and underlying mechanisms on osteoclast formation.Methods:1.The marine-derived Aspergillus ochraceus Jcma1F17 was fermented for 50L,the EtOAc extrat of the culture was subjected to silica gel,dextran gel,reversed phase C-18 silica gel.Compounds were then purified by HPLC and identified by MS,nuclear magnetic resonance(NMR),optical rotation(OR)and circular dichroism(CD).Then we evaluated the inhibitory activities of NSs on LPS-induced NF-?B luciferase activity in preosteoclastic RAW264.7 cells.2.The inhibition of NSs on RANKL-induced NF-?B luciferase activity and osteoclastogenesis were performed in preosteoclastic RAW264.7 cells,the structure-activity relationship between NSs and anti-osteoclastogenesis activity was determined by molecular docking.Then we performed MTT,CCK-8 and flow cytometry assays on the most active NS to determine the cytotoxicity of NS.The most active NS was further evaluated by RANKL-induced osteoclastogenesis and bone resorption.3.NF-?B and NFATc1 luciferase reporter gene assays were used to evaluate the effects of NS on RANKL-induced NF-?B luciferase activity and NFATc1 luciferase activity;the protein level of p-I?B? p65,Rel-B,MAPKs,DC-STAMP,c-Fos and NFATc1 were detected by Western blot or Confocal.Effects of NS on RANKL-induced osteoclastogenesis-related genes and fusion-related factor DC-STAMP expression were detected by Real-time PCR.4.LPS was used to induce inflammatory bone destruction in mice,micro-CT,HE and TRAP staining were used to evaluate the protective effect of NS on LPS-induced bone destruction.Results:1.The structures of 7 compounds were identified,including 2 new nitrophenyl sesquiterpenoids NS1 and NS3,1 new naturally occurring nitrophenyl sesquiterpene compound NS2,3 known nitrophenyl sesquiterpenoids NS4,NS5,NS6,and a sesquiterpene derivative 7;NS4 was the most active NS to inhibit LPS-induced NF-kB luciferase activity.2.NS4 displayed the strongest inhibition on RANKL-induced NF-kB luciferase activity and osteoclastogenesis among the 7 compounds.Molecular docking indicated that NS4 binds well to NF-?B p65 protein,the hydroxyl group of C-9 and the nitrobenzoyl moiety group of C-14 are the most effective groups to suppress osteoclastogenesis.MTT,CCK-8 and flow cytometry assays showed that NS4 had no obvious toxic effects on RAW264.7 and BMMs cells at 2 ?M.The results of TRAP staining and bone resorption of RAW264.7 and BMMs showed that NS4(0-2 ?M)inhibited osteoclastogenesis and bone resorption in a concentration-dependent manner compared with the RANKL control group.3.NS4 blocks RANKL-induced I?Ba phosphorylation,NF-?B p65 and NF-?B Rel-B nuclear translocation,NFATc1and DC-STAMP expression but not MAPK-c-Fos pathway during ostoclastogenesis in vitro.4.According to the results of Micro-CT,HE and TRAP staining,low dose(1mg/kg)NS4 and high dose(5mg/kg)NS4 can effectively inhibit LPS-induced bone destruction in mice.Conclusions:1.We discovered two new nitrophenyl sesquiterpene compounds NS1 and NS3.2.The new pharmacological function of marine-derived sesquiterpenoids on osteoclasts has been demonstrated.3.NS4 can be used as a potential osteoclast differentiation inhibitor for the treatment of osteolytic diseases.