Peniditerpenoids A and B: Oxidized Indole Diterpenoids with Osteoclast Differentiation Inhibitory Activity from a Mangrove-Sediment-Derived Penicillium sp.

An unprecedented di-seco-indole diterpenoid, peniditerpenoid A (1), and a rare N-oxide-containing indole diterpenoid derivative, peniditerpenoid B (2), together with three known ones (3-5), were obtained from the mangrove-sediment-derived fungus Penicillium sp. SCSIO 41411. Their structures were determined by the analysis of spectroscopic data, quantum chemical calculations, and X-ray diffraction analyses. Peniditerpenoid A (1) inhibited lipopolysaccharide-induced NF-κB with an IC50 value of 11 µM and further effectively prevented RANKL-induced osteoclast differentiation in bone marrow macrophages. In vitro studies demonstrated that 1 exerted significant inhibition of NF-κB activation in the classical pathway by preventing TAK1 activation, IκBα phosphorylation, and p65 translocation. Furthermore, 1 effectively reduced the level of NFATc1 activation, resulting in the attenuation of osteoclast differentiation. Our findings suggest that 1 holds promise as an inhibitor with significant potential for the treatment of diseases related to osteoporosis.

Erianin serves as an NFATc1 inhibitor to prevent breast cancer-induced osteoclastogenesis and bone destruction.

INTRODUCTION: Breast cancer-related bone metastasis can lead to skeletal-related events (SREs), which decrease patient quality of life. Inhibition of osteoclastogenesis is a key treatment for SREs; however, the availability of clinical drugs remains limited, and all existing ones disrupt physiological bone formation, while exhibiting no effect on patient survival time. OBJECTIVES: This study aimed to identify a novel osteoclast inhibitor for the treatment of breast cancer-induced SREs. METHODS: The MDA-MB-231 breast cancer cell-induced bone loss model was used to investigate the therapeutic effects of erianin in vivo. Then, we evaluated the inhibitory effects of erianin on osteoclastogenesis and signalling in bone marrow-derived macrophages (BMMs) induced by conditioned medium from MDA-MB-231 breast cancer cells (231 CM) and receptor activator of nuclear factor-κB ligand (RANKL) in vitro. Next, a Cellular Thermal Shift Assay and siRNA-mediate knockdown were performed, to investigate the target of erianin during osteoclast formation. The effects of erianin on human osteoclastogenesis were evaluated using CD14+ monocytes obtained from patients with breast cancer. RESULTS: Erianin effectively improved breast cancer cells-induced bone destruction at doses of 2 and 20 mg/kg/day in vivo, while suppressing osteoclastogenesis and the upregulation of SRC-NFATc1, INTEGRIN ß3-MMP9 signals induced by 231 CM and RANKL in vitro. Furthermore, erianin interacted with NFATc1 but not SRC, and Nfatc1 knockdown eliminated the inhibitory effects of erianin on osteoclastogenesis. Notably, lower expression of NFATc1 positively correlated with longer survival in patients with cancer and a high risk of bone metastasis. We further revealed that 62.5-250 nM erianin suppresses NFATc1 and excessive osteoclastogenesis in CD14+ monocytes from patients with breast cancer. CONCLUSION: Erianin acts as an NFATc1 inhibitor that attenuates breast cancer-induced osteoclastogenesis and bone destruction.

Identifying Marine-Derived Tanzawaic Acid Derivatives as Novel Inhibitors against Osteoclastogenesis and Osteoporosis via Downregulation of NF-κB and NFATc1 Activation.

To discover novel osteoclast-targeting antiosteoporosis leads from natural products, we identified 40 tanzawaic acid derivatives, including 22 new ones (1-8, 14-19, 27-32, 37, and 38), from the South China Sea mangrove-derived fungus Penicillium steckii SCSIO 41025. Penicisteck acid F (2), one of the new derivatives showing the most potent NF-κB inhibitory activity, remarkably inhibited osteoclast generation in vitro. Mechanistically, 2 reduced RANKL-induced IκBα degradation, NF-κB p65 nuclear translocation, the activation and nuclear translocation of NFATc1, and the relevant mRNA expression. NF-κB p65 could be a potential molecular target for 2, which has been further determined by the cellular thermal shift assay, surface plasmon resonance, and the gene knock-down assay. Moreover, 2 could also alleviate osteoporosis in ovariectomized mice by reducing the quantities of osteoclasts. Our finding offered a novel potential inhibitor of osteoclastogenesis and osteoporosis for further development of potent antiosteoporosis agents.

Discovery of a novel anti-osteoporotic agent from marine fungus-derived structurally diverse sirenins.

Thirteen new sirenin derivatives named eupenicisirenins C-O (1-13), along with a biosynthetically related known one (14), were isolated from the mangrove sediment-derived fungus Penicillium sp. SCSIO 41410. The structures, which possessed a rare cyclopropane moiety, were confirmed by extensive analyses of the spectroscopic data, quantum chemical calculations, and X-ray diffraction. Among them, eupenicisirenin C (1) exhibited the strongest NF-κB inhibitory activities, as well as suppressing effects on cGAS-STING pathway. Moreover, 1 showed the significant inhibitory effect on RANKL-induced osteoclast differentiation in bone marrow macrophages cells, and also displayed the therapeutic potential on prednisolone-induced zebrafish osteoporosis. Transcriptome analysis and the following verification tests suggested that its anti-osteoporotic mechanism is related to the extracellular matrix receptor interaction-related pathways. This study provided a promising marine-derived anti-osteoporotic agent for the treatment of skeletal disease.

Tanzawaic Acid Derivatives from the Marine-Derived Penicillium steckii as Inhibitors of RANKL-Induced Osteoclastogenesis.

Seven new tanzawaic acid derivatives, steckwaic acids E-K (1-7), and one new benzene derivate (8), together with seven known tanzawaic acid analogues (9-16) were isolated from the marine algicolous fungus Penicillium steckii SCSIO 41040. The structures and absolute configurations of these new compounds (1-8) were determined by spectroscopic analyses, X-ray diffraction, and comparison of ECD spectra to calculations. Compounds 2, 10, and 15 inhibited lipopolysaccharide (LPS)-induced nuclear factor kappa-B (NF-κB) with IC50 values of 10.4, 18.6, and 15.2 µM, respectively. Compound 2 could suppress the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation in bone marrow macrophage cells (BMMCs). To the best of our knowledge, this is the first report of osteoclastogenesis inhibitory activity for tanzawaic acid derivatives.

Loss of LXN promotes macrophage M2 polarization and PD-L2 expression contributing cancer immune-escape in mice.

Latexin (LXN) plays an important role in tumorigenesis and inflammatory response and as a tumor suppressor in many tumors. However, whether LXN regulates tumorigenesis through immune regulation remains uncertain. Here, we demonstrate that LXN deficiency increases hematopoietic stem cells, as well as affects the proportion of immune cells in the peripheral system. Animal studies show that mice loss of LXN promotes tumor growth in subcutaneous tumor model and AOM/DSS-induced colorectal cancer model. We found that loss of LXN promotes macrophage M2 polarization and PD-L2 expression in macrophage, thus, inhibits the function of T cells. Adoptive transfer of wild-type macrophage rescues the function of T cells in LXN-deficient mice. LXN deficiency in hematopoietic lineage exacerbates colorectal carcinogenesis, and targeted inhibition of PD-L2 ameliorates cancer growth in LXN-deficient mice. Mechanistically, we demonstrate that LXN inhibits STAT3 transcriptional activity by targeting inhibition of JAK1 in macrophages. LXN deficiency enhances PD-L2 expression rather than PD-L1 in macrophages, which lead to inhibition of T cells in tumor microenvironment. Collectively, we define a critical role of LXN/JAK1/STAT3 signal in macrophage and highlights the potential role of LXN in tumor immune-escape by regulating macrophage polarization, as well as the expression of immune checkpoint PD-L2.

Ruthenium polypyridine complexes with triphenylamine groups as antibacterial agents against Staphylococcus aureus with membrane-disruptive mechanism.

Due to the emergence and wide spread of methicillin-resistant Staphylococcus aureus, the treatment of this kind of infection becomes more and more difficult. To solve the problem of drug resistance, it is urgent to develop new antibiotics to avoid the most serious situation of no drug available. Three new Ru complexes [Ru (dmob)2PMA] (PF6)2 (Ru-1) [Ru (bpy)2PMA] (PF6)2 (Ru-2) and [Ru (dmb)2PMA] (PF6)2 (Ru-3) (dmob = 4,4'-dimethoxy-2,2'-bipyridine, bpy = 2,2'-bipyridine, dmb = 4,4'-dimethyl-2,2'-bipyridine and PMA = N-(4-(1H-imidazo [4,5-f] [1,10] phenanthrolin-2-yl) -4-methyl-N-(p-tolyl) aniline) were synthesized and characterized by 1H NMR, 13C NMR and HRMS. The detailed molecular structure of Ru-3 was determined by single crystal X-ray diffraction. Their antibacterial activities against Staphylococcus aureus (Staphylococcus aureus) were obvious and Ru-3 showed the best antibacterial effect with the minimum inhibitory concentration value of 4 µg ml-1. Therefore, further study on its biological activity showed that Ru-3 can effectively inhibit the formation of biofilm and destroy cell membrane. In vitro hemolysis test showed that Ru-3 has almost negligible cytotoxicity to mammalian red blood cells. In the toxicity test of wax moth insect model, Ru-3 exhibited low toxicity in vivo. These results, combined with histopathological studies, strongly suggest that Ru-3 was almost non-toxic. In addition, the synergistic effect of Ru-3 with common antibiotics such as ampicillin, chloramphenicol, tetracycline, kanamycin and gentamicin on Staphylococcus aureus was detected by chessboard method. Finally, in vivo results revealed that Ru-3 could obviously promote the wound healing of Staphylococcus aureus infected mice.

Synthesis of ruthenium polypyridine complexes with benzyloxyl groups and their antibacterial activities against Staphylococcus aureus.

Four new ruthenium polypyridyl complexes, [Ru(bpy)2(BPIP)](PF6)2 (Ru(II)-1), [Ru(dtb)2(BPIP)](PF6)2 (Ru(II)-2), [Ru(dmb)2(BPIP)](PF6)2 (Ru(II)-3) and [Ru(dmob)2(BPIP)](PF6)2 (Ru(II)-4) (bpy = 2,2'-bipyridine, dtb = 4,4'-di-tert-butyl-2,2'-bipyridine, dmb = 4,4'-dimethyl-2,2'-bipyridine, dmob = 4,4'-dimethoxy-2,2'-bipyridine and BPIP = 2-(3,5-bis(benzyloxyl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline) had been synthesized and characterized. Their antimicrobial activities were investigated against Staphylococcus aureus (S. aureus) and four complexes showed obvious antibacterial effect, especially the minimum inhibition concentration (MIC) value of Ru(II)-3 was only 4 µg/mL. In addition, Ru(II)-3 was able to kill bacteria quickly and inhibit the formation of biofilm. Meanwhile, the cooperative effect between Ru(II)-3 and general antibiotics were tested and the results showed that Ru(II)-3 could enhance the susceptibility of S. aureus to different types of antibiotics. Most importantly, Ru(II)-3 hardly showed cytotoxicity to mammalian erythrocytes both in homelysis experiment and G. mellonella model. After being injected with high doses of the Ru(II)-3in vivo, the G. mellonella worms still exhibited high survival rates. Finally, a mouse skin infection model and G. mellonella infection model was built to determine the antibacterial activity of Ru(II)-3in vivo. The antibacterial mechanism of Ru(II)-3 was probably related to the membrane-disruption. Taken together, ruthenium polypyridine complexes with benzyloxyl groups had the potential to develop an attractive and untraditional antibacterial agent with new mode of action.

Osteoclastogenesis inhibitory phenolic derivatives produced by the Beibu Gulf coral-associated fungus Acremonium sclerotigenum GXIMD 02501.

Three new chlorinated orsellinic aldehyde derivatives, orsaldechlorins A - C (1-3) and a naturally new brominated orsellinic acid (7), along with ten known biosynthetically related phenolic (4-6, 8-13) and cyclohexanone (14) derivatives, were identified from the Beibu Gulf coral-derived fungus Acremonium sclerotigenum GXIMD 02501. Their structures were determined by spectroscopic data interpretation and comparison with those reported in the literature. Several of them showed inhibition of lipopolysaccharide (LPS)-induced NF-κB activation in RAW 264.7 macrophages at 20 µM. Moreover, the two new potent inhibitors (1 and 2) suppressed RANKL-induced osteoclast differentiation without cytotoxicity in bone marrow macrophages cells (BMMs). Our findings reveal that the phenolic compounds could be potential candidates for the prevention and treatment of osteolytic bone diseases.

Thiodiketopiperazines and Alkane Derivatives Produced by the Mangrove Sediment-Derived Fungus Penicillium ludwigii SCSIO 41408.

A new trithiodiketopiperazine derivative, adametizine C (1), and five new alkane derivatives (7-11), were isolated from the mangrove sediment-derived fungus Penicillium ludwigii SCSIO 41408, together with five known dithiodiketopiperazine derivatives (2-6). Their structures were elucidated on the basis of spectroscopic analysis, and the absolute configuration of 1 was determined by X-ray crystallographic analysis. In a variety of bioactivity screening, 1-5 exhibited some selective antifungal or antibacterial activities. Compounds 1-3 showed cytotoxicity against prostate cancer cell line 22Rv1 with half maximal inhibitory concentration (IC50) values of 13.0-13.9 µM; moreover, 3 showed obvious activity against another prostate cancer PC-3 cells with an IC50 value of 5.1 µM. Further experiments revealed that 3 could significantly reduce PC-3 cells colony formation and induce apoptosis in a dose-dependent manner. Several compounds also exhibited obvious inhibitory activities of lipopolysaccharide-induced nuclear factor-κB with IC50 values range from 8.2 to 21.5 µM, and 1, 5, and 9 were further evaluated for their effects on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. Adametizine C (1), with the strongest inhibitory activity against RANKL-induced osteoclast differentiation in bone marrow macrophage cells with 10 µM, was suggested to be the promising lead compound for the treatment of osteoclast-related diseases.

Anti-Osteoclastogenic and Antibacterial Effects of Chlorinated Polyketides from the Beibu Gulf Coral-Derived Fungus Aspergillus unguis GXIMD 02505.

One new depsidone derivative, aspergillusidone H (3), along with seven known biosynthetically related chlorinated polyketides, were obtained from the Beibu Gulf coral-derived fungus Aspergillus unguis GXIMD 02505. Their structures were determined by comprehensive physicochemical and spectroscopic data interpretation. Notably, the X-ray crystal structure of 2 and the previously unknown absolute configuration of 8, assigned by ECD calculations, are described here for the first time. Compounds 1-5, 7 and 8 exhibited inhibition of lipopolysaccharide (LPS)-induced NF-κB in RAW 264.7 macrophages at 20 µM. In addition, the two potent inhibitors (2 and 7) dose-dependently suppressed RANKL-induced osteoclast differentiation without any evidence of cytotoxicity in bone marrow macrophages cells (BMMs). This is the first report of osteoclastogenesis inhibitory activity for the metabolites of these kinds. Besides, compounds 1, 2, 4, and 6-8 showed inhibitory activity against marine biofilm-forming bacteria, methicillin-resistant Staphylococcus aureus, Microbulbifer variabilis, Marinobacterium jannaschii, and Vibrio pelagius, with their MIC values ranging from 2 to 64 µg/mL. These findings provide a basis for further development of chlorinated polyketides as potential inhibitors of osteoclast differentiation and/or for use as anti-fouling agents.

Dendrobine attenuates osteoclast differentiation through modulating ROS/NFATc1/ MMP9 pathway and prevents inflammatory bone destruction.

BACKGROUND: Osteolytic diseases share symptoms such as bone loss, fracture and pain, which are caused by over-activated osteoclasts. Targeting osteoclast differentiation has emerged as a therapeutic strategy clinically. Dendrobine is an alkaloid isolated from Chinese herb Dendrobium nobile, with knowing effects of analgesia and anti-inflammation. The roles of dendrobine on osteoclasts and osteolysis remain unclear. PURPOSE: Herein, the possible roles of dendrobine in osteoclastogenesis, inflammatory osteolysis and the underlying mechanism were explored. METHODS: Bone marrow-derived macrophages (BMMs) and RAW264.7 cells were employed to evaluate the roles of dendrobine on osteoclastogenesis, bone absorption and the underlying mechanism in vitro. LPS injection was used to cause inflammatory osteolysis in vivo. RESULTS: Dendrobine repressed osteoclastogenesis, bone resorption induced by receptor activator of nuclear factor kappa B ligand (RANKL) in vitro. Mechanistically, dendrobine inhibited RANKL-upregulated intracellular (ROS), p-p38, c-Fos expression and nuclear factor of activated T cells (NFATc1) nuclear translocation. Osteoclastic genes were reduced, and among them matrix metalloproteinase 9 (MMP9) mRNA was dramatically blocked by dendrobine. Moreover, it substantially suppressed MMP9 protein expression during osteoclastogenesis in vitro. Accordingly, oral 20 mg/kg/day dendrobine was capable of preventing LPS-induced osteolysis with decreased osteoclasts in vivo. CONCLUSION: Taken together, dendrobine suppresses osteoclastogenesis through restraining ROS, p38-c-Fos and NFATc1-MMP9 in vitro, thus attenuates inflammatory osteolysis in vivo. This finding supports the discover of dendrobine as a novel osteoclast inhibitor for impeding bone erosion in the future.

Synthesis and biological evaluation of ruthenium polypyridine complexes with 18ß-glycyrrhetinic acid as antibacterial agents against Staphylococcus aureus.

Four new ruthenium(II) polypyridine complexes bearing 18ß-glycyrrhetinic acid derivatives, [Ru(bpy)2L](PF6)2 (Ru1), [Ru(dmb)2L](PF6)2 (Ru2), [Ru(dtb)2L](PF6)2 (Ru3) and [Ru(phen)2L](PF6)2 (Ru4) (bpy = 2,2-bipyridine, dmb = 4,4'-dimethyl-2,2'-bipyridine, dtb = 4,4'-di-tert-butyl-2,2'-bipyridine, phen = 1,10-phenanthroline and L is the GA modified new ligand) were designed and synthesized. Their antimicrobial activities against Staphylococcus aureus (S. aureus) were evaluated and all complexes showed an obvious inhibitory effect, especially, the minimum inhibitory concentration (MIC) value of Ru2 was 3.9 µg mL-1. Moreover, Ru2 was found to significantly inhibit the formation of biofilms. The membrane-compromising action mode was suggested to be their potential antibactericidal mechanism. In hemolysis experiments, Ru2 hardly showed cytotoxicity to mammalian erythrocytes. Furthermore, the synergism between Ru2 and common antibiotics, such as ampicillin, chloramphenicol, tetracyclines and ofloxacin, against S. aureus was also detected using the checkerboard method. Finally, a mouse skin infection model was established to evaluate the antibacterial activity of Ru2in vivo, and the results showed that Ru2 could effectively promote wound healing in mice infected with S. aureus. Moreover, the results of histopathological research were consistent with the results of the hemolysis test, indicating that the Ru2 complex was almost non-toxic. Thus, it was demonstrated that the polypyridine ruthenium complexes modified with glycyrrhetinic acid (GA) are a promising strategy for developing interesting antibacterial agents.

Bioactive Polyketide and Diketopiperazine Derivatives from the Mangrove-Sediment-Derived Fungus Aspergillus sp. SCSIO41407.

Ten polyketide derivatives (1-10), including a new natural product named (E)-2,4-dihydroxy-3-methyl-6-(2-oxopent-3-en-1-yl) benzaldehyde (1), and five known diketopiperazines (11-15), were isolated from the mangrove-sediment-derived fungus Aspergillus sp. SCSIO41407. The structures of 1-15 were determined via NMR and MS spectroscopic analysis. In a variety of bioactivity screening, 3 showed weak cytotoxicity against the A549 cell line, and 2 exhibited weak antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Compounds 3, 5, and 6 showed inhibition against acetylcholinesterase (AChE) with IC50 values of 23.9, 39.9, and 18.6 µM. Compounds 11, 12, and 14 exhibited obvious inhibitory activities of lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) with IC50 values of 19.2, 20.9, and 8.7 µM, and they also suppressed RANKL-induced osteoclast differentiation in bone marrow macrophages cells (BMMCs), with the concentration of 5 µM. In silico molecular docking with AChE and NF-κB p65 protein were also performed to understand the inhibitory activities, and 1, 11-14 showed obvious protein/ligand-binding effects to the NF-κB p65 protein.

A Nitrobenzoyl Sesquiterpenoid Insulicolide A Prevents Osteoclast Formation via Suppressing c-Fos-NFATc1 Signaling Pathway.

It is a viable strategy to inhibit osteoclast differentiation for the treatment of osteolytic diseases such as osteoporosis, rheumatoid arthritis and tumor bone metastases. Here we assessed the effects of insulicolide A, a natural nitrobenzoyl sesquiterpenoid derived from marine fungus, on receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis in vitro and its protective effects on LPS-induced osteolysis mice model in vivo. The results demonstrated that insulicolide A inhibited osteoclastogenesis from 1 µM in vitro. Insulicolide A could prevent c-Fos and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) nuclear translocation and attenuate the expression levels of osteoclast-related genes and DC-STAMP during RANKL-stimulated osteoclastogenesis but have no effects on NF-κB and MAPKs. Insulicolide A can also protect the mice from LPS-induced osteolysis. Our research provides the first evidence that insulicolide A may inhibit osteoclastogenesis both in vitro and in vivo, and indicates that it may have potential for the treatment of osteoclast-related diseases.

Kirenol inhibits RANKL-induced osteoclastogenesis and prevents ovariectomized-induced osteoporosis via suppressing the Ca2+-NFATc1 and Cav-1 signaling pathways.

BACKGROUND: Osteoporosis is a threat to aged people who have excessive osteoclast activation and bone resorption, subsequently causing fracture and even disability. Inhibiting osteoclast differentiation and absorptive functions has become an efficient approach to treat osteoporosis, but osteoclast-targeting inhibitors available clinically remain rare. Kirenol (Kir), a bioactive diterpenoid derived from an antirheumatic Chinese herbal medicine Herba Siegesbeckiae, can treat collagen-induced arthritis in vivo and promote osteoblast differentiation in vitro, while the effects of Kir on osteoclasts are still unclear. PURPOSE: We explore the role of Kir on RANKL-induced osteoclastogenesis in vitro and bone loss in vivo. METHODS: The in vitro effects of Kir on osteoclast differentiation, bone resorption and the underlying mechanisms were evaluated with bone marrow-derived macrophages (BMMs). In vivo experiments were performed using an ovariectomy (OVX)-induced osteoporosis model. RESULTS: We found that Kir remarkably inhibited osteoclast generation and bone resorption in vitro. Mechanistically, Kir significantly inhibited F-actinring formation and repressed RANKL-induced NF-κB p65 activation and p-p38, p-ERK and c-Fos expression. Moreover, Kir inhibited both the expression and nuclear translocation of NFATc1. Ca2+ oscillation and caveolin-1 (Cav-1) were also reduced by Kir during osteoclastogenesis in vitro. Consistent with these findings, 2-10 mg/kg Kir attenuated OVX-induced osteoporosis in vivo as evidenced by decreased osteoclast numbers and downregulated Cav-1 and NFATc1 expression. CONCLUSIONS: Kir suppresses osteoclastogenesis and the Cav-1/NFATc1 signaling pathway both in vitro and in vivo and protects against OVX-induced osteoporosis. Our findings reveal Kir as a potential safe oral treatment for osteoporosis.

Oridonin ameliorates inflammation-induced bone loss in mice via suppressing DC-STAMP expression.

Currently, dendritic cell-specific transmembrane protein (DC-STAMP), a multipass transmembrane protein, is considered as the master regulator of cell-cell fusion, which underlies the formation of functional multinucleated osteoclasts. Thus, DC-STAMP has become a promising target for osteoclast-associated osteolytic diseases. In this study, we investigated the effects of oridonin (ORI), a natural tetracyclic diterpenoid compound isolated from the traditional Chinese herb Rabdosia  rubescens, on osteoclastogenesis in vivo and ex vivo. ICR mice were injected with LPS (5 mg/kg, ip, on day 0 and day 4) to induce inflammatory bone destruction. Administration of ORI (2, 10 mg·kg-1·d-1, ig, for 8 days) dose dependently ameliorated inflammatory bone destruction and dramatically decreased DC-STAMP protein expression in BMMs isolated from LPS-treated mice. Treatment of preosteoclast RAW264.7 cells with ORI (0.78-3.125 µM) dose dependently inhibited both mRNA and protein levels of DC-STAMP, and suppressed the following activation of NFATc1 during osteoclastogenesis. Knockdown of DC-STAMP in RAW264.7 cells abolished the inhibitory effects of ORI on RANKL-induced NFATc1 activity and osteoclast formation. In conclusion, we show for the first time that ORI effectively attenuates inflammation-induced bone loss by suppressing DC-STAMP expression, suggesting that ORI is a potential agent against inflammatory bone diseases.

A marine fungus-derived nitrobenzoyl sesquiterpenoid suppresses receptor activator of NF-κB ligand-induced osteoclastogenesis and inflammatory bone destruction.

BACKGROUND AND PURPOSE: Osteoclasts are unique cells to absorb bone. Targeting osteoclast differentiation is a therapeutic strategy for osteolytic diseases. Natural marine products have already become important sources of new drugs. The naturally occurring nitrobenzoyl sesquiterpenoids first identified from marine fungi in 1998 are bioactive compounds with a special structure, but their pharmacological functions are largely unknown. Here, we investigated six marine fungus-derived nitrobenzoyl sesquiterpenoids on osteoclastogenesis and elucidated the mechanisms. EXPERIMENTAL APPROACH: Compounds were first tested by RANKL-induced NF-κB luciferase activity and osteoclastic TRAP assay, followed by molecular docking to characterize the structure-activity relationship. The effects and mechanisms of the most potent nitrobenzoyl sesquiterpenoid on RANKL-induced osteoclastogenesis and bone resorption were further evaluated in vitro. Micro-CT and histology analysis were used to assess the prevention of bone destruction by nitrobenzoyl sesquiterpenoids in vivo. KEY RESULTS: Nitrobenzoyl sesquiterpenoid 4, with a nitrobenzoyl moiety at C-14 and a hydroxyl group at C-9, was the most active compound on NF-κB activity and osteoclastogenesis. Consequently, nitrobenzoyl sesquiterpenoid 4 exhibited suppression of RANKL-induced osteoclastogenesis and bone resorption from 0.5 µM. It blocked RANKL-induced IκBa phosphorylation, NF-κB p65 and RelB nuclear translocation, NFATc1 activation, reduced DC-STAMP but not c-Fos expression during osteoclastogenesis in vitro. Nitrobenzoyl sesquiterpenoid 4 also ameliorated LPS-induced osteolysis in vivo. CONCLUSION AND IMPLICATIONS: These results highlighted nitrobenzoyl sesquiterpenoid 4 as a novel inhibitor of osteoclast differentiation. This marine-derived sesquiterpenoid is a promising lead compound for the treatment of osteolytic diseases.

A mutation of cysteine 46 in IKK-ß promotes mPGES-1 and caveolin-1 expression to exacerbate osteoclast differentiation and osteolysis.

IKK-ß is indispensable for inflammatory osteolysis, the functional residues of IKK-ß are therapeutic drug targets for developing inhibitors to treat multiple diseases now. Thus it remains appealing to find the new residues of IKK-ß to influence osteoclasts for alleviating bone loss diseases such as rheumatoid arthritis (RA). By employing IKK-ß cysteine 46-A transgenic (IKK-ßC46A) mice, we found that mutation of cysteine 46 to alanine in IKK-ß exacerbated inflammatory bone destruction in vivo, and increased osteoclast differentiation and bone resorption ex vivo and in vitro. Consistent with these, IKK-ß kinase activity as well as c-Fos, NFATc1 were up-regulated in bone marrow macrophages (BMMs) from IKK-ßC46A mice during RANKL-induced osteoclastogenesis. Of interesting, we further identified and demonstrated that the expressions of mPGES-1 and caveolin-1 were heightened in BMMs of IKK-ßC46A mice compared to those in WT mice in RANKL-induced osteoclastogenesis. Together, it revealed that mutating cysteine 46 in IKK-ß could increase caveolin-1 and mPGES-1 expression to facilitate osteoclast differentiation and osteolysis. Cysteine 46 can serve as a novel target in IKK-ß for designing inhibitors to treat osteolysis.

Sesquiterpenes and diterpenes from Euphorbia thymifolia.

One new pseudoguaianolide (1), one new megastigmane (6), and one new ent-abietane diterpene (9), together with seven known compounds (2-5, 7, 8, and 10) were isolated from the aerial parts of Euphorbia thymifolia. The structures of the new compounds and their relative configurations were determined by spectroscopic data analysis. The absolute configurations of compounds 1, 6, and 9 were determined by single-crystal X-ray crystallographic analysis, modified Mosher's method and calculated ECD experiment, respectively. All compounds were tested for their inhibitory effects against LPS-induced NO production in BV-2 microglial cells, and pseudoguaianolides (1-5) showed significant activity with IC50 values of 0.41-15.32 µM.