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1.
Acta Pharmacol Sin ; 45(4): 790-802, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38191913

RESUMO

Osteoporosis results from overactivation of osteoclasts. There are currently few drug options for treatment of this disease. Since the successful development of allosteric inhibitors, phosphatases have become attractive therapeutic targets. Protein phosphatase 1, regulatory subunit 15 A (PPP1R15A), is a stress-responsive protein, which promotes the UPR (unfolded protein response) and restores protein homeostasis. In this study we investigated the role of PPP1R15A in osteoporosis and osteoclastogenesis. Ovariectomy (OVX)-induced osteoporosis mouse model was established, osteoporosis was evaluated in the left femurs using micro-CT. RANKL-stimulated osteoclastogenesis was used as in vitro models. We showed that PPP1R15A expression was markedly increased in BMMs derived from OVX mice and during RANKL-induced osteoclastogenesis in vitro. Knockdown of PPP1R15A or application of Sephin1 (a PPP1R15A allosteric inhibitor in a phase II clinical trial) significantly inhibited osteoclastogenesis in vitro. Sephin1 (0.78, 3.125 and 12.5 µM) dose-dependently mitigated the changes in NF-κB, MAPK, and c-FOS and the subsequent nuclear factor of activated T cells 1 (NFATc1) translocation in RANKL-stimulated BMMs. Both Sephin1 and PPP1R15A knockdown increased the phosphorylated form of eukaryotic initiation factor 2α (eIF2α); knockdown of eIF2α reduced the inhibitory effects of Sephin1 on NFATc1-luc transcription and osteoclast formation. Furthermore, Sephin1 or PPP1R15A knockdown suppressed osteoclastogenesis in CD14+ monocytes from osteoporosis patients. In OVX mice, injection of Sephin1 (4, 8 mg/kg, i.p.) every two days for 6 weeks significantly inhibited bone loss, and restored bone destruction and decreased TRAP-positive cells. This study has identified PPP1R15A as a novel target for osteoclast differentiation, and genetic inhibition or allosteric inhibitors of PPP1R15A, such as Sephin1, can be used to treat osteoporosis. This study revealed that PPP1R15A expression was increased in osteoporosis in both human and mice. Inhibition of PPP1R15A by specific knockdown or an allosteric inhibitor Sephin1 mitigated murine osteoclast formation in vitro and attenuated ovariectomy-induced osteoporosis in vivo. PPP1R15A inhibition also suppressed pathogenic osteoclastogenesis in CD14+ monocytes from osteoporosis patients. These results identify PPP1R15A as a novel regulator of osteoclastogenesis and a valuable therapeutic target for osteoporosis.


Assuntos
Guanabenzo , Osteoporose , Animais , Feminino , Humanos , Camundongos , Diferenciação Celular , Guanabenzo/análogos & derivados , Guanabenzo/uso terapêutico , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos , Osteogênese , Osteoporose/tratamento farmacológico , Ovariectomia , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/farmacologia , Ligante RANK/metabolismo
2.
Pharmacol Res ; 185: 106517, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36265554

RESUMO

Regulatory B cells (Bregs) potently suppress immune disorders, including allergic contact hypersensitivity (CHS). IKKß overactivation is prominent in various inflammatory diseases. However, its effect on Bregs has not been defined. This study is to investigate the new regulator and inhibitory mechanism of Bregs. IkkßC46A transgenic mice with a Cys46 mutation, resulting in increased IKKß activation, were employed for analysis. IL-10-competent CD9+ Bregs were expanded in IkkßC46A mice and B cell specific-IkkßC46A mutation mice. IkkßC46A mutant CD9+ Bregs had stronger suppressive effects on CD4+ and CD8+ T cells in vitro and CHS responses in vivo. The inhibitory CD9+ Bregs from IkkßC46A mice were characterized by upregulated Neuropilin 2 (Nrp2) and IL-10 in comparison with that of Ikkßwt mice. Interestingly, increased expression of Nrp2 was observed in CD9+ Bregs compared with that of CD9- B cells in wild-type mice. The suppressive activity of wild-type CD9+ Bregs in vitro was attenuated by inhibition of Nrp2 on Bregs or silencing its ligand Sema3f on CD4+ T cells. Our findings delineate a distinct role of IKKß activation in enhancing Bregs to disturb the immune balance. It identifies Nrp2 as a novel regulatory molecule of Bregs that partly contributes to B cell-mediated immune tolerance.


Assuntos
Linfócitos B Reguladores , Doenças do Sistema Imunitário , Animais , Camundongos , Linfócitos T CD8-Positivos/metabolismo , Quinase I-kappa B/metabolismo , Doenças do Sistema Imunitário/metabolismo , Interleucina-10 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuropilina-2/genética , Neuropilina-2/metabolismo
3.
J Leukoc Biol ; 112(4): 835-843, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35188700

RESUMO

Primary bile acids (BAs), products of cholesterol metabolism and clearance, are synthesized in the liver and released into the intestine to facilitate the digestion and absorption of lipids. BAs are further converted by gut commensal bacteria into secondary colonic BAs and the metabolism disorder is closely linked to cholestatic liver diseases via regulating immune response. However, the effect and underlying mechanism of these host-microorganism biliary metabolites on T lymphocyte remain unclear. In the current study, we synthesized a sulfated product of lithocholic acid (LCA), lithocholic acid 3-sulfate (LCA-3-S), and investigated the binding affinity of the BAs metabolites on RORγt, the transcription factor of IL-17A. Our results demonstrated that the sulfate of LCA, LCA-3-S, exhibited better effect than its oxidated metabolite, 3-oxo-LCA, binding to RORγt. The results further demonstrated that LCA-3-S selectively suppressed Th17 cell differentiation without influence on Th1, Th2, and Treg cells. Collectively, we synthesized the sulfated biliary metabolite LCA-3-S and demonstrated that LCA-3-S selectively inhibited Th17 cell differentiation by targeting RORγt, indicating that metabolite disorder of BAs resulting in the decrease of LCA-3-S probably contributes to the pathogenesis of cholestatic liver diseases.


Assuntos
Hepatopatias , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Ácidos e Sais Biliares/farmacologia , Diferenciação Celular , Colesterol , Humanos , Interleucina-17 , Ligantes , Lipídeos , Ácido Litocólico/metabolismo , Ácido Litocólico/farmacologia , Sulfatos/farmacologia , Células Th17/metabolismo , Fatores de Transcrição
4.
Phytomedicine ; 96: 153838, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34801352

RESUMO

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.


Assuntos
Reabsorção Óssea , Osteólise , Alcaloides , Animais , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/prevenção & controle , Diferenciação Celular , Metaloproteinase 9 da Matriz , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B , Fatores de Transcrição NFATC , Osteoclastos , Osteogênese , Osteólise/tratamento farmacológico , Osteólise/prevenção & controle , Ligante RANK , Espécies Reativas de Oxigênio
5.
Angew Chem Int Ed Engl ; 60(17): 9374-9378, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33527661

RESUMO

Cephalodiones A-D (1-4), the first example of C19 -norditerpenoid dimers, were isolated and fully characterized from a Cephalotaxus plant. These new skeletal natural products shared a unique tricyclo[6.4.1.12,7 ]tetradeca-3,5,9,11-tetraene-13,14-dione core that was capped in both ends with rigid multicyclic ring systems either C2 -symmetrically or asymmetrically. Compounds 1-4 were proposed to be biosynthetically produced by the [6+6]-cycloaddition of two identical C19 -norditerpenoid troponoids, which was validated by the semisyntheses of dimers 2-4. Moreover, some compounds showed significant inhibition on Th17 cell differentiation.


Assuntos
Alcaloides/farmacologia , Produtos Biológicos/farmacologia , Cephalotaxus/química , Alcaloides/síntese química , Alcaloides/química , Produtos Biológicos/síntese química , Produtos Biológicos/química , Diferenciação Celular/efeitos dos fármacos , Reação de Cicloadição , Humanos , Conformação Molecular , Estereoisomerismo , Células Th17
6.
Phytomedicine ; 80: 153377, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33126167

RESUMO

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.


Assuntos
Caveolina 1/metabolismo , Diterpenos/farmacologia , Fatores de Transcrição NFATC/metabolismo , Osteogênese/efeitos dos fármacos , Osteoporose/prevenção & controle , Administração Oral , Animais , Reabsorção Óssea/prevenção & controle , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diterpenos/administração & dosagem , Feminino , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Osteoclastos/efeitos dos fármacos , Osteoporose/etiologia , Ovariectomia/efeitos adversos , Ligante RANK/metabolismo , Ligante RANK/farmacologia , Transdução de Sinais/efeitos dos fármacos
7.
Acta Pharmacol Sin ; 42(5): 744-754, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-32753731

RESUMO

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.


Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Diterpenos do Tipo Caurano/uso terapêutico , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Osteólise/tratamento farmacológico , Animais , Regulação para Baixo/efeitos dos fármacos , Feminino , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteólise/induzido quimicamente , Osteólise/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos
8.
Br J Pharmacol ; 177(18): 4242-4260, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32608081

RESUMO

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.


Assuntos
Reabsorção Óssea , Osteólise , Receptor Ativador de Fator Nuclear kappa-B , Sesquiterpenos , Reabsorção Óssea/tratamento farmacológico , Diferenciação Celular , Fungos , Humanos , Ligantes , Simulação de Acoplamento Molecular , NF-kappa B , Fatores de Transcrição NFATC , Osteoclastos , Osteogênese , Ligante RANK , Sesquiterpenos/farmacologia
9.
Biochem Pharmacol ; 172: 113762, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31843541

RESUMO

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.


Assuntos
Caveolina 1/metabolismo , Diferenciação Celular/fisiologia , Quinase I-kappa B/genética , Osteoclastos/fisiologia , Osteólise/metabolismo , Prostaglandina-E Sintases/metabolismo , Animais , Células da Medula Óssea , Caveolina 1/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Quinase I-kappa B/metabolismo , Macrófagos , Camundongos , Camundongos Transgênicos , Monócitos , Mutação , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteólise/genética , Prostaglandina-E Sintases/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ligante RANK/administração & dosagem , Ligante RANK/farmacologia , Regulação para Cima
10.
Pharmacol Res ; 148: 104457, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31536782

RESUMO

Dysregulated host immune homeostasis in sepsis is life-threatening even after a successfully treated bacterial infection. Lipopolysaccharide (LPS) is an endotoxin that is a major contributor to the aberrant immune responses and endotoxic shock in gram-negative bacterial sepsis. However, the current knowledge of the role of B cells in endotoxic shock is limited. Here, we report that CD1d expression in B cells and the percentage of CD5+CD1dhi regulatory B (Breg) cells decreased in a mouse model of endotoxic shock. Interestingly, IL-10 but not FasL expression in CD5+CD1dhi Breg cells in response to endotoxin was dramatically reduced in severe septic shock mice, and the regulatory function of CD5+CD1dhi Breg cells in vitro to control the Th1 response was also diminished. Adoptive transfer of CD5+CD1dhi Breg cells from healthy WT mice but not IL-10 deficient mice downregulated the IFN-γ secretion in CD4+ T cells and conferred protection against severe endotoxic shock in vivo. Our findings demonstrate the change and notable therapeutic potential of IL-10-producing Breg cells in endotoxic shock.


Assuntos
Linfócitos B Reguladores/imunologia , Interleucina-10/imunologia , Choque Séptico/imunologia , Animais , Antígenos CD1d/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígenos CD5/imunologia , Feminino , Interferon gama/imunologia , Lipopolissacarídeos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL
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