Unsaturated Fatty Acids Complex Regulates Inflammatory Cytokine Production through the Hyaluronic Acid Pathway.

In this study, we aimed to develop natural and/or functional materials with antioxidant and anti-inflammatory effects. We obtained extracts from natural plants through an oil and hot-water extraction process and prepared an extract composite of an effective unsaturated fatty acid complex (EUFOC). Furthermore, the antioxidant effect of the extract complex was evaluated, and the anti-inflammatory effect was explored by assessing its inhibitory effect on nitric oxide production through its HA-promoting effect. We conducted a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay to evaluate the cell viability of the EUFOC, and the results showed that EUFOC was not cytotoxic at the test concentrations. In addition, it showed no endogenous cytotoxicity in HaCaT (human keratinocyte) cells. The EUFOC showed excellent 1,1-diphenyl-2-picrylhydrazyl- and superoxide-scavenging abilities. Moreover, it exerted an inhibitory effect on NO production at concentrations that did not inhibit cell viability. The secretion of all the cytokines was increased by lipopolysaccharide (LPS) treatment; however, this was inhibited by the EUFOC in a concentration-dependent manner. In addition, hyaluronic acid content was markedly increased by the EUFOC in a dose-dependent manner. These results suggest that the EUFOC has excellent anti-inflammatory and antioxidant properties, and hence, it can be used as a functional material in various fields.

Ginseng-derived exosome-like nanovesicles extracted by sucrose gradient ultracentrifugation to inhibit osteoclast differentiation.

Plant-derived extracellular nanovesicles contain RNA and proteins with unique and diverse pharmacological mechanisms. The extracellular nanovesicles encapsulating plant extracts resemble exosomes as they have a round, lipid bilayer morphology. Ginseng is anti-inflammatory, anti-cancer, immunostimulant, and osteogenic/anti-osteoporotic. Here, we confirmed that ginseng-derived extracellular nanovesicles (GDNs) inhibit osteoclast differentiation and elucidated the associated molecular mechanisms. We isolated GDNs by centrifugation with a sucrose gradient. We measured their dynamic light scattering and zeta potentials and examined their morphology by transmission electron microscopy. We used bone marrow-derived macrophages (BMMs) to determine the potential cytotoxicity of GDNs and establish their ability to inhibit osteoclast differentiation. The GDNs treatment maintained high BMM viability and proliferation whilst impeding osteoclastogenesis. Tartrate-resistant acid phosphatase and F-actin staining revealed that GDNs at concentrations >1 µg mL-1 strongly hindered osteoclast differentiation. Moreover, they substantially suppressed the RANKL-induced IκBα, c-JUN n-terminal kinase, and extracellular signal-regulated kinase signaling pathways and the genes regulating osteoclast maturation. The GDNs contained elevated proportions of Rb1 and Rg1 ginsenosides and were more effective than either of them alone or in combination at inhibiting osteoclast differentiation. In vivo bone analysis via microcomputerized tomography, bone volume/total volume ratios, and bone mineral density and bone cavity measurements demonstrated the inhibitory effect of GDNs against osteoclast differentiation in lipopolysaccharide-induced bone resorption mouse models. The results of this work suggest that GDNs are anti-osteoporotic by inhibiting osteoclast differentiation and are, therefore, promising for use in the clinical prevention and treatment of bone loss diseases.

Effects of Scoparone on differentiation, adhesion, migration, autophagy and mineralization through the osteogenic signalling pathways.

Scoparone (SCOP), an active and efficient coumarin compound derived from Artemisia capillaris Thunb, has been used as a traditional Chinese herbal medicine. Herein, we investigated the effects of SCOP on the osteogenic processes using MC3T3-E1 pre-osteoblasts in in vitro cell systems. SCOP (C11 H10 O4 , > 99.17%) was purified and identified from A. capillaries. SCOP (0.1 to 100 µM concentrations) did not have cytotoxic effects in pre-osteoblasts; however, it promoted alkaline phosphatase (ALP) staining and activity, and mineralized nodule formation under early and late osteogenic induction. SCOP elevated osteogenic signals through the bone morphogenetic protein 2 (BMP2)-Smad1/5/8 pathway, leading to the increased expression of runt-related transcription factor 2 (RUNX2) with its target protein, matrix metallopeptidase 13 (MMP13). SCOP also induced the non-canonical BMP2-MAPKs pathway, but not the Wnt3a-ß-catenin pathway. Moreover, SCOP promoted autophagy, migration and adhesion under the osteogenic induction. Overall, the findings of this study demonstrated that SCOP has osteogenic effects associated with cell differentiation, adhesion, migration, autophagy and mineralization.

Falcarindiol Stimulates Apoptotic and Autophagic Cell Death to Attenuate Cell Proliferation, Cell Division, and Metastasis through the PI3K/AKT/mTOR/p70S6K Pathway in Human Oral Squamous Cell Carcinomas.

Human oral squamous cell carcinomas (OSCCs) have high cancer mortality and a 5-year survival rate lower than that of most other carcinomas. New therapeutic strategies are required for the treatment and prevention against OSCCs. An approach to cancer therapy using plant-derived natural compounds has been actively in progress as a trend. Falcarindiol (FALC), or its isolated form Ostericum koreanum Kitagawa (O. koreanum), is present in many food and dietary plants, especially in carrots, and this compound has a variety of beneficial effects. However, biological activity of FALC has not been reported in OSCCs yet. This study aimed to demonstrate the antitumor effects of FALC against OSCCs, YD-10B cells. In this study, FALC was selected as a result of screening for compounds isolated from various natural products in YD-10B cells. FALC suppressed cell growth, and FALC-induced apoptotic cell death was mainly accompanied by the dephosphorylation of PI3K, AKT, mTOR, and p70S6K. The apoptotic cell death was also associated with autophagy as evidenced by the expression of Beclin-1, the conversion of LC3-II, and the formation of autophagosome. FALC-induced autophagy was accompanied by MAPKs including ERK1/2 and p38. Furthermore, FALC caused the antimetastatic effects by inhibiting the migration and invasion of YD-10B cells. Taken together, the findings suggest the potential value of FALC as a novel candidate for therapeutic strategy against OSCCs.

11-O-Galloyl Bergenin from Corylopsis coreanas Leaves Induces Autophagy and Apoptosis in Human Osteosarcoma.

Osteosarcoma is the most common malignant bone-forming tumor, wherein most patients with high grade osteosarcomas are treated with chemotherapy. Despite this, survival for metastatic or relapsed osteosarcoma patients has remained at an overall 5-year survival rate of 20%. In particular, the extracts of Corylopsis coreana (Korean winter hazel), a cultivated woody plant in South Korea, have shown beneficial anti-inflammatory, anti-oxidative, anti-osteoclastic, and antihyperuricemic properties. Therefore, this study aimed to demonstrate the antitumor activities and underlying mechanism of 11-O-Galloyl bergenin (OGAL) isolated from Corylopsis coreanas leaves in human osteosarcoma cells. Herein, we found that OGAL inhibited MG63 cell proliferation and induced cellular apoptosis as evidenced by cleaved-PARP, cleaved-caspase 3, TUNEL-positive cells, and Annexin V-positive cells. Specifically, OGAL-induced apoptosis was accompanied by p53 and p21 upregulation, BAX expression, and decreased Bcl-2 and cdk2. Moreover, OGAL induced autophagy via AKT inactivation, LC3II upregulation, and MG63 cell autophagosome formation. OGAL-induced autophagy was also accompanied by increased p38 phosphorylation, whereas JNK and ERK1/2 activities were found to be unaffected upon examining the MAPK signaling pathway. Furthermore, wound healing and Boyden chamber assays showed that OGAL suppressed MG63 cell migration and invasion. Given these findings, this study provided evidence that OGAL has antitumor effects by apoptosis and autophagy enhancement through increased p53, AKT, and p38 signaling, suggesting that OGAL may be a potential therapeutic strategy for osteosarcoma treatment.

Anti-tumor effects of jaceosidin on apoptosis, autophagy, and necroptosis in human glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most aggressive and common malignant neoplasm. Nevertheless, a 5-year survival rate of patients with GBM has remained below 5%. Artemisia princeps PAMPANINI, used as a food and traditional medicine, have shown beneficial properties including anti-inflammatory, anti-oxidative, and anti-cancer activities. Thus, this study aimed to investigate biological mechanism of a bioactive compound, jaceosidin (JAC), isolated from A. princeps in human GBM T98G cells. Herein, as a result of analysis in terms of cancer survival and death, we found that JAC significantly reduced cell survival against T98G cells. In addition, JAC increased apoptotic cell death via changes on morphological and molecular phenotypes in T98G cells as evidenced by cellular shapes and DNA fragmentation. The apoptotic cell death was confirmed by the cleavage of caspase-3 and PARP, the downregulation of survivin and Bcl-2. Moreover, JAC decreased the expression of cyclinD1 and Cdks and increased the phosphorylation of EKR, JNK, and p38 MAPKs. Specifically, JAC suppressed the PI3K/AKT signaling and its downstream molecules including p70S6, GSK3ß, and ß-catenin. In addition, as a result of analysis in terms of metastasis using wound healing and Boyden chamber assays, JAC showed anti-migrative and anti-invasive activities. Finally, we analyzed in terms of autophagy and necroptosis that are modes of programmed cell survival and death different from apoptosis in T98G cells. We found that JAC inhibited autophgic regulatory proteins including Beclin-1, Atgs, and LC3A/B, thereby reducing autophagic-mediated cell survival, whereas JAC did not affect phosphorylation of key proteins in necroptosis, especially MLKL. Given these findings, our results provided novel evidences on the biological mechanisms of JAC in T98G cells, suggesting that JAC may be a therapeutic agent for patients with GBM.

Calycosin-7-O-ß-Glucoside Isolated from Astragalus membranaceus Promotes Osteogenesis and Mineralization in Human Mesenchymal Stem Cells.

Stem cells have received attention in various diseases, such as inflammatory, cancer, and bone diseases. Mesenchymal stem cells (MSCs) are multipotent stem cells that are critical for forming and repairing bone tissues. Herein, we isolated calycosin-7-O-ß-glucoside (Caly) from the roots of Astragalus membranaceus, which is one of the most famous medicinal herbs, and investigated the osteogenic activities of Caly in MSCs. Caly did not affect cytotoxicity against MSCs, whereas Caly enhanced cell migration during the osteogenesis of MSCs. Caly increased the expression and enzymatic activities of ALP and the formation of mineralized nodules during the osteogenesis of MSCs. The osteogenesis and bone-forming activities of Caly are mediated by bone morphogenetic protein 2 (BMP2), phospho-Smad1/5/8, Wnt3a, phospho-GSK3ß, and phospho-AKT, inducing the expression of runt-related transcription factor 2 (RUNX2). In addition, Caly-mediated osteogenesis and RUNX2 expression were attenuated by noggin and wortmannin. Moreover, the effects were validated in pre-osteoblasts committed to the osteoblast lineages from MSCs. Overall, our results provide novel evidence that Caly stimulates osteoblast lineage commitment of MSCs by triggering RUNX2 expression, suggesting Caly as a potential anabolic drug to prevent bone diseases.

Anti-cancer effects of Hederoside C, a pentacyclic triterpene saponin, through the intrinsic apoptosis and STAT3 signaling pathways in osteosarcoma.

Natural compounds have emerged as an approach in cancer therapy. Pulsatilla koreana Nakai is used as a traditional medicinal plant that found throughout China and Korea. However, anti-cancer effects of Hederoside C (HedC) isolated from P. koreana has not been investigated in osteosarcoma. The present study aimed to demonstrate anti-cancer functions of HedC against human osteosarcoma cells. Herein, we found that HedC suppressed the proliferation of MG63 cells and U2OS cells in the dose- and time-dependent manner, and caused intrinsic apoptosis pathways as evidenced by morphological changes, TUNEL-positive cells, cleaved-PARP, and cleaved-caspase 9 and 3. HedC increased p53, Bax, and p21, whereas HedC reduced Bcl-2. HedC-mediated apoptosis was accompanied by decreases in the mitogen-activated protein kinases (MAPKs) and STAT3 phosphorylation. Wound healing and Boyden chamber assays also showed the anti-metastatic effects of HedC by suppressing migration and invasion. In addition, the anti-cancer effects of HedC were observed in in vivo xenograft mice model, and HedC treatment induced the decreased PCNA and p-STAT3 as well as the increased p53 and cleaved caspase-3. Taken together, our results provide evidence that HedC might be an attractive therapeutic strategy against osteosarcoma.

Simple and facile preparation of recombinant human bone morphogenetic protein-2 immobilized titanium implant via initiated chemical vapor deposition technique to promote osteogenesis for bone tissue engineering application.

Over the past few decades, titanium (Ti) implants have been widely used to repair fractured bones. To promote osteogenesis, immobilization of osteoinductive agents, such as recombinant human bone morphogenic protein-2 (rhBMP2), onto the Ti surface is required. In this study, we prepared rhBMP2 immobilized on glycidyl methacrylate (GMA) deposited Ti surface through initiated chemical vapor deposition (iCVD) technique. After preparation, the bio-functionalized Ti surface was characterized by physicochemical analysis. For in vitro analysis, the developed Ti was evaluated by cell proliferation, alkaline phosphatase activity, calcium deposition, and real-time polymerase chain reaction to verify their osteogenic activity against human adipose-derived stem cells (hASCs). The GMA deposited Ti surface was found to effectively immobilize a large dose of rhBMP2 as compared to untreated Ti. Additionally, rhBMP2 immobilized on Ti showed significantly enhanced osteogenic differentiation and increased calcium deposition with nontoxic cell viability. These results clearly confirm that our strategy may provide a simple, solvent-free strategy to prepare an osteoinductive Ti surface for bone tissue engineering applications.

Effects of moderate intensity static magnetic fields on osteoclastic differentiation in mouse bone marrow cells.

Although we recently demonstrated that static magnetic fields (SMFs) of 3, 15, and 50 mT stimulate osteoblastic differentiation, the effects of SMFs on osteoclastogenesis are still poorly understood. This study focused on the suppressive effects of SMFs on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis and bone resorption. Direct SMFs inhibit RANKL-induced multinucleated osteoclast formation, tartrate-resistant acid phosphatase activity, and bone resorption in mouse bone marrow-derived macrophage cells. The conditioned medium from osteoblasts treated with SMFs also resulted in the inhibition of osteoclast differentiation as well as resorption. The RANKL-induced expression of osteoclast-specific transcription factors, such as c-Fos and NFATc1, was remarkably downregulated by SMF at 15 mT. In addition, SMF inhibited RANKL-activated Akt, glycogen synthase kinase 3ß (GSK3ß), extracellular signal-regulated kinase, c-jun N-terminal protein kinase, mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) formation. These findings indicate that SMF-mediated attenuation of RANKL-induced Akt, GSK3ß, MAPK, and NF-κB pathways could contribute to the direct and indirect inhibition of osteoclast formation and bone resorption. Therefore, SMFs could be developed as a therapeutic agent against periprosthetic or peri-implant osteolysis. Additionally, these could be used against osteolytic diseases such as osteoporosis and rheumatoid arthritis. Bioelectromagnetics. 39:394-404, 2018. © 2018 Wiley Periodicals, Inc.

Safflower seed extract inhibits osteoclast differentiation by suppression of the p38 mitogen-activated protein kinase and IκB kinase activity.

Safflower seed has been reported to have a protective effect against bone loss diseases. However, the precise molecular mechanisms underlying the inhibitory effect of safflower seed in osteoclast differentiation remain unclear. In this study, we investigated the inhibitory action of safflower seed extract (SSE) on the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in cultured mouse-derived bone marrow macrophages (BMMs). We found that SSE significantly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in BMMs without cytotoxicity. The gene expressions of nuclear factor of activated T-cells (NFATc1) and TRAP, which are genetic markers of osteoclast differentiation, were substantially decreased by SSE in a dose-dependent manner. Also, SSE diminished RANKL-mediated intracellular reactive oxygen species (ROS) generation on osteoclastogenesis in a dose-dependent manner. The SSE thereafter suppressed RANKL-induced p38 mitogen-activated protein kinase and IκBα kinase signalling activities which were activated by ROS generation for osteoclastogenesis. Additionally, SSE was found to decrease RANKL-induced actin ring formation, which is required for bone resorption activity. Taken together, our results suggest that SSE acts as a RANKL-induced osteoclastogenesis inhibitor by suppression of ROS generation. This induces a remarkable suppression of the p38 and IκBα kinase pathways, thereby suppressing the gene expression of NFATc1 in osteoclast precursors.