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/metabolismoRESUMO
Six diterpenoid alkaloids, namely, pachycentine (1), deacetylswinanine A (2), siwanine A (3), tatsiensine (4), deacetyltatsiensine (5), and 6-deoxydeltamine (6), were isolated from a China-specific Delphinium plant (family Ranunculaceae), Delphinium pachycentrum Hemsl. Their structures were established via detailed spectroscopic analyses, including IR, HR-ESI-MS, 1D and 2D NMR techniques. Pachycentine (1) is a previously undescribed hetisine-type C20-diterpenoid alkaloid, and compounds 5 and 6 were synthetic intermediates newly identified as natural products. In addition, compounds 2-4 were isolated from this species for the first time. The chemotaxonomic significance of all the isolates was summarized. Moreover, the new compound was evaluated for its potential anti-inflammatory effect using LPS-stimulated RAW 264.7 macrophages.
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 fármacosRESUMO
OBJECTIVE: To screen the activated fraction from Celastrus aculeatus and study its effect on T lymphocyte apoptosis. METHODS: Different polarity fractions were isolated from Celastrus aculeatus extract. Flow Cytometry method was used to detect apoptosis ratio of the active fractions. RESULTS: Different fractions extract from Celastrus aculeatus and GSF-A could significantly inhibit T lymphocyte proliferation and induce T lymphocyte apoptosis. CONCLUSION: The activated fractions isolated from Celastrus aculeatus have anti-inflammatory effect. Its mechanism may be related to the apoptosis effect on T lymphocyte.