Mume Fructus reduces interleukin-1 beta-induced cartilage degradation via MAPK downregulation in rat articular chondrocytes.

Osteoarthritis is the most prevalent type of degenerative arthritis. It is characterized by persistent pain, joint dysfunction, and physical disability. Pain relief and inflammation control are prioritised during osteoarthritis treatment Mume Fructus (Omae), a fumigated product of the Prunus mume fruit, is used as a traditional medicine in several Asian countries. However, its therapeutic mechanism of action and effects on osteoarthritis and articular chondrocytes remain unknown. In this study, we analyzed the anti-osteoarthritis and articular regenerative effects of Mume Fructus extract on rat chondrocytes. Mume Fructus treatment reduced the interleukin-1ß-induced expression of matrix metalloproteinase 3, matrix metalloproteinase 13, and a disintegrin and metalloproteinase with thrombospondin type 1 motifs 5. Additionally, it enhanced collagen type II alpha 1 chain and aggrecan accumulation in rat chondrocytes. Furthermore, Mume Fructus treatment regulated the inflammatory cytokine levels, mitogen-activated protein kinase phosphorylation, and nuclear factor-kappa B activation. Overall, our results demonstrated that Mume Fructus inhibits osteoarthritis progression by inhibiting the nuclear factor-kappa B and mitogen-activated protein kinase pathways to reduce the levels of inflammatory cytokines and prevent cartilage degeneration. Therefore, Mume Fructus may be a potential therapeutic option for osteoarthritis.

Development of Anti-OSCAR Antibodies for the Treatment of Osteoarthritis.

Osteoarthritis (OA) is the most common joint disease that causes local inflammation and pain, significantly reducing the quality of life and normal social activities of patients. Currently, there are no disease-modifying OA drugs (DMOADs) available, and treatment relies on pain relief agents or arthroplasty. To address this significant unmet medical need, we aimed to develop monoclonal antibodies that can block the osteoclast-associated receptor (OSCAR). Our recent study has revealed the importance of OSCAR in OA pathogenesis as a novel catabolic regulator that induces chondrocyte apoptosis and accelerates articular cartilage destruction. It was also shown that blocking OSCAR with a soluble OSCAR decoy receptor ameliorated OA in animal models. In this study, OSCAR-neutralizing monoclonal antibodies were isolated and optimized by phage display. These antibodies bind to and directly neutralize OSCAR, unlike the decoy receptor, which binds to the ubiquitously expressed collagen and may result in reduced efficacy or deleterious off-target effects. The DMOAD potential of the anti-OSCAR antibodies was assessed with in vitro cell-based assays and an in vivo OA model. The results demonstrated that the anti-OSCAR antibodies significantly reduced cartilage destruction and other OA signs, such as subchondral bone plate sclerosis and loss of hyaline cartilage. Hence, blocking OSCAR with a monoclonal antibody could be a promising treatment strategy for OA.

Polygonatum sibiricum improves menopause symptoms by regulating hormone receptor balance in an ovariectomized mouse model.

Female menopause is a hormone deficiency phenomenon that causes hot flashes, vaginal dryness, depression, nervous tension, insomnia, obesity, and bone loss. There are various hormone replacement therapy (HRT)-based menopausal treatments, but they are accompanied by side effects such as endometrial cancer and hyperplasia. To confirm the menopausal improvement effect of Polygonatum sibiricum (PS), we prepared an ovariectomized animal model, administered 17ß-estradiol (E2) and PS, and analyzed various menopausal symptoms. PS restored vaginal epithelium thickness, by increasing the expression of estrogen receptors ERα (ESR1) and ERß (ESR2), and increased serotonin concentration by reducing serotonin receptor 1 A (5-HT1A) and glucocorticoid receptor (Gr) expression. In addition, PS suppressed obesity by increasing HDL-C and decreasing LDL-C levels and improved the osteoporosis induced by ovariectomy. In particular, by controlling Hand2, Fgf2, and Faf9 expression through PR, the antiproliferative signal was suppressed in uterine epithelium, thereby reducing the risk of side effects of the administration of E2 alone. These results demonstrate that PS alleviates menopausal symptoms without causing endometrial hyperplasia.

Osteoclast-associated receptor blockade prevents articular cartilage destruction via chondrocyte apoptosis regulation.

Osteoarthritis (OA), primarily characterized by articular cartilage destruction, is the most common form of age-related degenerative whole-joint disease. No disease-modifying treatments for OA are currently available. Although OA is primarily characterized by cartilage destruction, our understanding of the processes controlling OA progression is poor. Here, we report the association of OA with increased levels of osteoclast-associated receptor (OSCAR), an immunoglobulin-like collagen-recognition receptor. In mice, OSCAR deletion abrogates OA manifestations, such as articular cartilage destruction, subchondral bone sclerosis, and hyaline cartilage loss. These effects are a result of decreased chondrocyte apoptosis, which is caused by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in induced OA. Treatments with human OSCAR-Fc fusion protein attenuates OA pathogenesis caused by experimental OA. Thus, this work highlights the function of OSCAR as a catabolic regulator of OA pathogenesis, indicating that OSCAR blockade is a potential therapy for OA.

Karyotype and B chromosome variation in Lilium amabile Palibin.

OBJECTIVE: Lilium amabile Palibin (2n = 2x = 24) is an endemic lily species in Korea. B chromosomes are supernumerary chromosomes and the presence of B chromosome in L. amabile was known by previous researches. The current research was conducted to characterize the genetical and cytological features of the B chromosome plants in L. amabile. METHODS: Karyotype and B chromosome cytotype analyses were carried out among 135 L. amabile accessions that were collected from six geographical locations in Korea using conventional aceto-carmine staining as well as FISH technique with ribosomal RNA gene probes. RESULTS: The karyotype of L. amabile genome consisted of two large metacentric, four intermediate subtelocentric, and six intermediate to small acrocentric chromosomes in which chromosomes 1, 6 and 7 carried the 45S rRNA gene loci and chromosome 3 carried the 5S rRNA gene. There were 4 types of B chromosomes, two large B chromosomes and two small B chromosomes. The ribosomal RNA gene loci were not present in the B chromosomes. The 135 accessions were classified into 13 cytotypes including diploids and different B chromosome aneuploids. Among the aneuploids, the most frequent cytotype was 24 + 1B, which was followed by 24 + 2B, 24 + 1b, 24 + 1B + 2b, 24 + 1B + 4b, and 24 + 2B + 4b. CONCLUSION: The karyotype of L. amabile was consistent with other species in the genus Lilium without polyploids. The B chromosome cytotypes were highly variable and the occurrences of different cytotypes were random among the six populations, implying that the B and b chromosome occurrence was random in each population.

FlexPro MD, a Mixture of Krill Oil, Astaxanthin, and Hyaluronic Acid, Suppresses Lipopolysaccharide-Induced Inflammatory Cytokine Production Through Inhibition of NF-κB.

FlexPro MD® (FP-MD), a novel multi-ingredient dietary supplement formulation, has been demonstrated to relieve knee joint pain in humans. However, the mechanisms of action responsible for the activity of FP-MD have not been elucidated. In this study, we show the anti-inflammatory effects of FP-MD in RAW264.7 macrophage cells and mice challenged with lipopolysaccharide (LPS). FP-MD significantly inhibited the mRNA levels of pro-inflammatory cytokines, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1ß. In contrast, it elevated the mRNA levels of anti-inflammatory cytokine IL-10 in LPS-stimulated RAW264.7 cells. FP-MD markedly reduced LPS-induced phosphorylation levels of nuclear factor-κB (NF-κB) p65 and inhibitor of κB-α (IκB-α). Importantly, the anti-inflammatory effects of FP-MD were demonstrated in mice with LPS-induced inflammatory arthritis in which FP-MD significantly reduced the expression levels of pro-inflammatory cytokines and inflammatory markers. Thus, this study suggests that FP-MD has anti-inflammatory effects by inhibiting NF-κB that may offer a molecular basis for its pain relief property.

Sirt6 cooperates with Blimp1 to positively regulate osteoclast differentiation.

Global deletion of the gene encoding a nuclear histone deacetylase sirtuin 6 (Sirt6) in mice leads to osteopenia with a low bone turnover due to impaired bone formation. But whether Sirt6 regulates osteoclast differentiation is less clear. Here we show that Sirt6 functions as a transcriptional regulator to directly repress anti-osteoclastogenic gene expression. Targeted ablation of Sirt6 in hematopoietic cells including osteoclast precursors resulted in increased bone volume caused by a decreased number of osteoclasts. Overexpression of Sirt6 led to an increase in osteoclast formation, and Sirt6-deficient osteoclast precursor cells did not undergo osteoclast differentiation efficiently. Moreover, we showed that Sirt6, induced by RANKL-dependent NFATc1 expression, forms a complex with B lymphocyte-induced maturation protein-1 (Blimp1) to negatively regulate expression of anti-osteoclastogenic gene such as Mafb. These findings identify Sirt6 as a novel regulator of osteoclastogenesis by acting as a transcriptional repressor.

A novel pyrazole derivative protects from ovariectomy-induced osteoporosis through the inhibition of NADPH oxidase.

Osteoclast cells (OCs) are differentiated from bone marrow-derived macrophages (BMMs) by activation of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). Activation of NADPH oxidase (Nox) isozymes is involved in RANKL-dependent OC differentiation, implicating Nox isozymes as therapeutic targets for treatment of osteoporosis. Here, we show that a novel pyrazole derivative, Ewha-18278 has high inhibitory potency on Nox isozymes. Blocking the activity of Nox with Ewha-18278 inhibited the responses of BMMs to RANKL, including reactive oxygen species (ROS) generation, activation of mitogen-activated protein (MAP) kinases and NF-κB, and OC differentiation. To evaluate the anti-osteoporotic function of Ewha-18278, the derivative was applied to estrogen-deficient ovariectomized (OVX) ddY mice. Oral administration of Ewha-18278 (10 mg/kg/daily, 4 weeks) into the mice recovered bone mineral density, trabecular bone volume, trabecular bone length, number and thickness, compared to control OVX ddY mice. Moreover, treatment of OVX ddY mice with Ewha-18278 increased bone strength by increasing cortical bone thickness. We provide that Ewha-18278 displayed Nox inhibition and blocked the RANKL-dependent cell signaling cascade leading to reduced differentiation of OCs. Our results implicate Ewha-18278 as a novel therapeutic agent for the treatment of osteoporosis.

Sirtuin 3 (SIRT3) maintains bone homeostasis by regulating AMPK-PGC-1ß axis in mice.

The mitochondrial sirtuin 3 (SIRT3) is involved in suppressing the onset of multiple pathologies, including cardiovascular disease, fatty liver, age-related hearing loss, and breast cancer. But a physiological role of SIRT3 in bone metabolism is not known. Here we show that SIRT3 is a key regulatory molecule to maintain bone homeostasis. Mice deficient in SIRT3 exhibited severe osteopenia owing to increased numbers of osteoclasts. Osteoclast precursors from Sirt3-/- mice underwent increased osteoclastogenesis in response to receptor activator of nuclear factor-κB ligand (RANKL), an essential cytokine for osteoclast differentiation. SIRT3 expression from RANKL induction depended on the transcription coactivator PGC-1ß (peroxisome proliferator-activated receptor-γ co-activator-1ß) and the nuclear receptor ERRα (estrogen receptor-related receptor α), and that SIRT3 inhibited the differentiation by interfering with the RANKL-induced expression of PGC-1ß. Thus an auto-regulatory feedback mechanism operates to induce its own inhibitor SIRT3 by PGC-1ß. Moreover, Sirt3-/- osteoclast precursors reduced AMP-activated protein kinase (AMPK) phosphorylation through down-regulating the expression of AMPK. Our results suggest that a mitochondrial SIRT3 is an intrinsic inhibitor for RANKL-mediated osteoclastogenesis.

2-(trimethylammonium) ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate suppresses osteoclast maturation and bone resorption by targeting macrophage-colony stimulating factor signaling.

2-(Trimethylammonium) ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient megakaryopoiesis. Here, we show that (R)-TEMOSPho blocks osteoclast maturation from progenitor cells of hematopoietic origin, as well as blocking the resorptive function of mature osteoclasts. The inhibitory effect of (R)-TEMOSPho on osteoclasts was due to a disruption of the actin cytoskeleton, resulting from impaired downstream signaling of c-Fms, a receptor for macrophage-colony stimulating factor linked to c-Cbl, phosphoinositol-3-kinase (PI3K), Vav3, and Rac1. In addition, (R)-TEMOSPho blocked inflammation-induced bone destruction by reducing the numbers of osteoclasts produced in mice. Thus, (R)-TEMOSPho may represent a promising new class of antiresorptive drugs for the treatment of bone loss associated with increased osteoclast maturation and activity.

Dynein light chain LC8 inhibits osteoclast differentiation and prevents bone loss in mice.

NF-κB is one of the key transcription factors activated by receptor activator of NF-κB ligand (RANKL) during osteoclast differentiation. The 8-kDa dynein L chain (LC8) was previously identified as a novel NF-κB regulator. However, its physiological role as an NF-κB inhibitor remains elusive. In this study, we showed the inhibitory role of LC8 in RANKL-induced osteoclastogenesis and signaling pathways and its protective role in osteolytic animal models. LC8 suppressed RANKL-induced osteoclast differentiation, actin ring formation, and osteoclastic bone resorption. LC8 inhibited RANKL-induced phosphorylation and subsequent degradation of IκBα, the expression of c-Fos, and the consequent activation of NFATc1, which is a pivotal determinant of osteoclastogenesis. LC8 also inhibited RANKL-induced activation of JNK and ERK. LC8-transgenic mice exhibited a mild osteopetrotic phenotype. Moreover, LC8 inhibited inflammation-induced bone erosion and protected against ovariectomy-induced bone loss in mice. Thus, our results suggest that LC8 inhibits osteoclast differentiation by regulating NF-κB and MAPK pathways and provide the molecular basis of a new strategy for treating osteoporosis and other bone diseases.

Inactivation of glycogen synthase kinase-3ß is required for osteoclast differentiation.

Glycogen synthase kinase-3ß (GSK-3ß) is a serine/threonine kinase originally identified as a regulator of glycogen deposition. Although the role of GSK-3ß in osteoblasts is well characterized as a negative regulator of ß-catenin, its effect on osteoclast formation remains largely unidentified. Here, we show that the GSK-3ß inactivation upon receptor activator of NF-κB ligand (RANKL) stimulation is crucial for osteoclast differentiation. Regulation of GSK-3ß activity in bone marrow macrophages by retroviral expression of the constitutively active GSK-3ß (GSK3ß-S9A) mutant inhibits RANKL-induced osteoclastogenesis, whereas expression of the catalytically inactive GSK-3ß (GSK3ß-K85R) or small interfering RNA (siRNA)-mediated GSK-3ß silencing enhances osteoclast formation. Pharmacological inhibition of GSK-3ß further confirmed the negative role of GSK-3ß in osteoclast formation. We also show that overexpression of the GSK3ß-S9A mutant in bone marrow macrophages inhibits RANKL-mediated NFATc1 induction and Ca(2+) oscillations. Remarkably, transgenic mice expressing the GSK3ß-S9A mutant show an osteopetrotic phenotype due to impaired osteoclast differentiation. Further, osteoclast precursor cells from the transgenic mice show defects in expression and nuclear localization of NFATc1. These findings demonstrate a novel role for GSK-3ß in the regulation of bone remodeling through modulation of NFATc1 in RANKL signaling.