Isolation and Identification of Antiarthritic Constituents from Glycine tabacina and Network Pharmacology-Based Prediction of Their Protective Mechanisms against Rheumatoid Arthritis.

Glycine tabacina (Labill.) Benth is an edible medicinal herb for rheumatoid arthritis (RA) treatment in folk medicine. Current phytochemical research on this dried herb led to the isolation of eight new coumestans, named glytabastan A-H (1-8), and twenty-three known compounds 9-31. Their structures were elucidated using spectroscopic methods. The antiarthritic activities of all isolates were evaluated, and the results showed that coumestans 1-6 and 8-10 could inhibit arthritic inflammation in vitro, while coumestans 1, 2, 9, and 10 significantly blocked the osteoclastogenesis induced by receptor activator of nuclear factor (NF) κB ligand (RANKL). Moreover, network pharmacological analysis revealed that the anti-RA effect of G. tabacina involved multitargets, multipathways such as PI3K/Akt and MAPK signaling pathways, and various biological processes such as inflammatory response and cytokine-mediated signaling pathways. These results suggested that this species and its novel coumestans could serve as potential antiarthritic agents for functional food or medicinal use.

Efficacy of orally administered porcine epidemic diarrhea vaccine-loaded hydroxypropyl methylcellulose phthalate microspheres and RANKL-secreting L. lactis.

Here, we examined the efficacy of are combinant subunit antigen-based oral vaccine for preventing porcine epidemic diarrhea virus (PEDV). First, we generated a soluble recombinant partial spike S1 protein (aP2) from PEDV in E. coli and then evaluated the utility of aP2 subunit vaccine-loaded hydroxypropyl methylcellulose phthalate microspheres (HPMCP) and RANKL-secreting L. lactis (LLRANKL) as a candidate oral vaccine in pregnant sows. Pregnant sows were vaccinated twice (with a 2 week interval between doses) at 4 weeks before farrowing. Titers of virus-specific IgA antibodies in colostrum, and neutralizing antibodies in serum, of sows vaccinated with HPMCP (aP2) plus LL RANKL increased significantly at 4 weeks post-first vaccination. Furthermore, the survival rate of newborn suckling piglets delivered by sows vaccinated with HPMCP (aP2) plus LL RANKL was similar to that of piglets delivered by sows vaccinated with a commercial killed porcine epidemic diarrhea virus (PED) vaccine. The South Korean government promotes a PED vaccine program (live-killed-killed) to increase the titers of IgA and IgG antibodies in pregnant sows and prevent PEDV. The oral vaccine strategy described herein, which is based on a safe and efficient recombinant subunit antigen, is an alternative PED vaccination strategy that could replace the traditional strategy, which relies on attenuated live oral vaccines or artificial infection with virulent PEDV.

Bruton's tyrosine kinase inhibitor BMS-986142 in experimental models of rheumatoid arthritis enhances efficacy of agents representing clinical standard-of-care.

Bruton's tyrosine kinase (BTK) regulates critical signal transduction pathways involved in the pathobiology of rheumatoid arthritis (RA) and other autoimmune disorders. BMS-986142 is a potent and highly selective reversible small molecule inhibitor of BTK currently being investigated in clinical trials for the treatment of both RA and primary Sjögren's syndrome. In the present report, we detail the in vitro and in vivo pharmacology of BMS-986142 and show this agent provides potent and selective inhibition of BTK (IC50 = 0.5 nM), blocks antigen receptor-dependent signaling and functional endpoints (cytokine production, co-stimulatory molecule expression, and proliferation) in human B cells (IC50 ≤ 5 nM), inhibits Fcγ receptor-dependent cytokine production from peripheral blood mononuclear cells, and blocks RANK-L-induced osteoclastogenesis. Through the benefits of impacting these important drivers of autoimmunity, BMS-986142 demonstrated robust efficacy in murine models of rheumatoid arthritis (RA), including collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA). In both models, robust efficacy was observed without continuous, complete inhibition of BTK. When a suboptimal dose of BMS-986142 was combined with other agents representing the current standard of care for RA (e.g., methotrexate, the TNFα antagonist etanercept, or the murine form of CTLA4-Ig) in the CIA model, improved efficacy compared to either agent alone was observed. The results suggest BMS-986142 represents a potential therapeutic for clinical investigation in RA, as monotherapy or co-administered with agents with complementary mechanisms of action.

Intra-articular lentivirus-mediated gene therapy targeting CRACM1 for the treatment of collagen-induced arthritis.

Abnormal store-operated calcium uptake has been observed in peripheral T lymphocytes of rheumatoid arthritis (RA) patients, and sustained intracellular calcium signalling is known to mediate the functions of many types of immune cells. Thus, it is hypothesized that regulating calcium entry through CRACM1 (the pore-forming subunit of calcium release-activated calcium (CRAC) channels; also known as ORAI1) may be beneficial for the management of RA. Localized CRACM1 knockdown in the joints and draining lymph nodes (DLNs) of mice with collagen-induced arthritis (CIA) was achieved via lentiviral-based delivery of shRNA targeting mouse CRACM1. Consistent with CRACM1 knockdown, calcium influx in synovial cells and the histopathological features of CIA were reduced. These effects were also associated with reduced levels of several notable inflammatory cytokines, such as IL-6, IL-17A, and IFN-γ, in the joints. Additionally, CRACM1-shRNA reduced the number of bone marrow-derived osteoclasts in vitro as well as osteoclasts in CIA joints, which was associated with reduced RANKL levels in the serum and joints. In summary, inhibiting calcium entry by CRACM1 knockdown suppressed arthritis development and may be therapeutically beneficial for RA patients.

Mice Deficient in CIZ/NMP4 Develop an Attenuated Form of K/BxN-Serum Induced Arthritis.

CIZ/NMP4 (Cas interacting zinc finger protein, Nmp4, Zfp384) is a transcription factor that is known to regulate matrix related-proteins. To explore the possible pathophysiological role of CIZ/NMP4 in arthritis, we examined CIZ/NMP4 expression in articular cartilage in arthritis model. CIZ/NMP4 was expressed in the articular chondrocytes of mice at low levels while its expression was enhanced when arthritis was induced. Arthritis induction increased clinical score in wild type mice. In contrast, CIZ/NMP4 deficiency suppressed such rise in the levels of arthritis score and swelling of soft tissue. CIZ/NMP4 deficiency also reduced invasion of inflammatory cells in joint tissue. Quantitative PCR analyses of mRNA from joints revealed that arthritis-induced increase in expressions of IL-1ß was suppressed by CIZ/NMP4 deficiency. CIZ/NMP4 bound to IL-1ß promoter and activated its transcription. The increase in CIZ/NMP4 in arthritis was also associated with enhancement in bone resorption and cartilage matrix degradation. In fact, RANKL, a signaling molecule prerequisite for osteoclastogenesis and, MMP-3, a clinical marker for arthritis were increased in joints upon arthritis induction. In contrast, CIZ/NMP4 deficiency suppressed the arthritis-induced increase in bone resorption, expression of RANKL and MMP-3 mRNA. Thus, CIZ/NMP4 plays a role in the development of arthritis at least in part through regulation of key molecules related to the arthritis.

Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression.

AIM: Aconiti Lateralis Radix Preparata is a traditional Chinese medicine used to treat chronic arthritis and is highly effective against rheumatoid arthritis. However, the effects of aconine, a derivative of aconitum alkaloids, on osteoclasts, which can absorb bone, remain unknown. Here, we investigated the effects of aconine on osteoclast differentiation and bone resorption in vitro. METHODS: The viability of mouse leukemic monocyte/macrophage cell line RAW264.7 was measured using CCK-8 assays. Osteoclast differentiation was induced by incubation of RAW264.7 cells in the presence of RANKL, and assessed with TRAP staining assay. Bone resorption was examined with bone resorption pits assay. The expression of relevant genes and proteins was analyzed using RT-PCR and Western blots. The activation of NF-κB and nuclear factor of activated T-cells (NFAT) was examined using stable NF-κB and NFATc1 luciferase reporter gene systems, RT-PCR and Western blot analysis. RESULTS: Aconine (0.125, 0.25 µmol/L) did not affect the viability of RAW264.7 cells, but dose-dependently inhibited RANKL-induced osteoclast formation and bone resorptive activity. Furthermore, aconine dose-dependently inhibited the RANKL-induced activation of NF-κB and NFATc1 in RAW264.7 cells, and subsequently reduced the expression of osteoclast-specific genes (c-Src, ß3-Integrin, cathepsin K and MMP-9) and the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which played an important role in cell-cell fusion. CONCLUSION: These findings suggest that aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing the activation of NF-κB and NFATc1 and the expression of the cell-cell fusion molecule DC-STAMP.

Periodontitis and Rheumatoid Arthritis: What Do We Know?

BACKGROUND: Currently, in the field of rheumatology, there is much attention given towards the possible causality between periodontitis and rheumatoid arthritis (RA), specifically regarding the role of Porphyromonas gingivalis (Pg). This bacterium is unique, having a citrullinating enzyme. Antibodies against citrullinated proteins are rather specific for RA. METHODS: Because causality is ultimately tested in longitudinal cohort studies which currently do not exist for periodontitis and RA, this commentary applied Bradford Hill criteria on the existing literature to assess causality as the most likely interpretation of this association. CONCLUSIONS: From an epidemiologic point of view, patients with RA have a higher incidence of periodontal disease than individuals without RA. In addition, there is a dose-response pattern in the association between the severity of periodontitis and RA disease activity. There are indications that periodontitis precedes RA, but there is no evidence yet available to show that Pg plays a direct role in this temporal relationship. The role of the unique characteristic of citrullination by Pg remains unexplained. However, in animal models, citrullination by Pg plays a distinct role in the development and aggravation of experimental arthritis. Although the role of Pg in RA remains speculative, a causative role for periodontitis as a chronic inflammatory disease caused by infectious agents in RA seems biologically plausible.

Jolkinolide B inhibits RANKL-induced osteoclastogenesis by suppressing the activation NF-κB and MAPK signaling pathways.

Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. The unique function and ability of osteoclasts to resorb bone makes them critical in both normal bone homeostasis and pathologic bone diseases such as osteoporosis and rheumatoid arthritis. Thus, new compounds that may inhibit osteoclastogenesis and osteoclast function may be of great value in the treatment of osteoclast-related diseases. In the present study, we examined the effect of jolkinolide B (JB), isolated from the root of Euphorbia fischeriana Steud on receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation. We found that JB inhibited RANKL-induced osteoclast differentiation from bone marrow macrophages (BMMs) without cytotoxicity. Furthermore, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CtsK), and calcitonin receptor (CTR), was significantly inhibited. JB inhibited RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκBα degradation. Moreover, JB inhibited RANKL-induced phosphorylation of mitogen-activated protein kinases (p38, JNK, and ERK). This study thus identifies JB as an inhibitor of osteoclast formation and provides evidence that JB might be an alternative medicine for preventing and treating osteolysis.

Swertiamarin ameliorates inflammation and osteoclastogenesis intermediates in IL-1ß induced rat fibroblast-like synoviocytes.

OBJECTIVE AND DESIGN: Rheumatoid arthritis is a chronic inflammatory and autoimmune disease that leads to aggressive joint cartilage and bone destruction. Swertiamarin is a secoiridoid glycoside found in Enicostema axillare (Lam) A. Raynal, a medicinal plant used in the Indian system of traditional medicine. In the present study, the potential of swertiamarin was evaluated in IL-1ß induced fibroblast-like synoviocytes (FLS). METHODS: The FLS were isolated from Freund's Complete Adjuvant induced arthritic (AA) rats and cultured with IL-1ß. The normal FLS and AA-FLS were cultured and used for subsequent experiment in fibroblastic morphology form. The efficacy of swertiamarin (10-50 µg/ml) was evaluated on mRNA and protein expression levels of inflammatory and osteoclastogenesis mediators. The efficacy was also evaluated on p38 MAPKα levels with time course studies (2, 4, 6, 8 and 12 h). RESULTS: IL-1ß induced cell proliferation (149.46 ± 13.73 %) and NO production (162.03 ± 11.03%) in AA-FLS; treatment with swertiamarin controlled proliferation (82.77 ± 4.22%) and NO production (82.06 ± 3.91% at 50 µg/ml) in a dose-dependent manner. It also significantly (P < 0.05) modulated the expression of apoptotic marker (caspase 3), proinflammation mediators (TNFα, IL-6, PGE2, COX-2, iNOS, MMPs) and osteoclastogenic mediator (RANKL) at both the mRNA and protein levels. Treatment with swertiamarin inhibited the levels of p38 MAPKα in a dose-dependent manner and also significantly (P < 0.05) attenuated the release of the same in time dependent mode. CONCLUSION: These findings suggest that treatment with swertiamarin attenuated IL-1ß induced FLS, and it revealed anti-inflammatory potential by attenuating aggressive FLS.

Clinical review: Effect of endocrine therapies on bone in breast cancer patients.

CONTEXT: Two common strategies are used to treat estrogen receptor-positive breast cancer in women: tamoxifen to inhibit estrogen action, and aromatase inhibitors (AIs) to block estrogen biosynthesis. Recent data suggest that AIs are more effective than tamoxifen in the adjuvant and advanced disease settings and are now being more commonly used. Tamoxifen, as a selective estrogen receptor modulator, exerts estrogenic effects to preserve bone, whereas the AIs profoundly lower estrogen levels and cause bone loss. Recent comparative studies of these agents provide extensive data on fracture rates, bone mineral density, and markers of bone formation and resorption. OBJECTIVE: The aim of the study was to review the mechanistic effects of estrogen on bone and clinical data regarding bone density, bone turnover markers, and fracture rates in women with breast cancer taking tamoxifen or AIs. EVIDENCE ACQUISITION AND SYNTHESIS: Data presented reflect a review of the literature and data integration from the perspective of the author's knowledge of the field. RESULTS: Tamoxifen increases bone density and reduces fractures in postmenopausal women with breast cancer, whereas AIs increase rate of fracture, accelerate loss of bone mineral density, and enhance levels of markers of bone formation and resorption. Bisphosphonates and denosumab counteract the effects of the AIs on bone. Guidelines for management of AI-induced bone loss are available from several sources, but a simple algorithm guides decision making most effectively. CONCLUSIONS: Endocrine therapy for postmenopausal women with breast cancer exerts substantial effects on bone, and guidelines are available to assist in the management of bone-related problems.

Effect of denosumab on bone mineral density in women receiving adjuvant aromatase inhibitors for non-metastatic breast cancer: subgroup analyses of a phase 3 study.

Denosumab increased lumbar spine bone mineral density (BMD) versus placebo in a 2-year, randomized, placebo-controlled, phase 3 study of patients with hormone-receptor-positive, non-metastatic breast cancer and low bone mass who were receiving adjuvant aromatase inhibitor therapy. In subgroup analyses at 12 and 24 months, we evaluated factors (duration and type of aromatase inhibitor, tamoxifen use, age, time since menopause, body mass index, T-score) that might influence BMD at the lumbar spine, total hip, femoral neck, and 1/3 radius. Patients were randomized to receive placebo (n = 125) or 60 mg denosumab (n = 127) subcutaneously every 6 months. In all subgroups, 12 or 24 months' treatment with denosumab was associated with larger BMD gains than placebo across multiple skeletal sites. Most increases were statistically significant (P < 0.05). Twice-yearly administration of denosumab, regardless of patient subgroup or skeletal site, resulted in consistent increases in BMD versus placebo at 12 and 24 months.