23-Hydroxyursolic acid from Viburnum lutescens inhibits osteoclast differentiation in vitro and lipopolysaccharide-induced bone loss in vivo by suppressing c-Fos and NF-κB signalling.

Bone homeostasis is maintained by a combination of osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Excessive osteoclast activity is linked to several bone-related disorders, including osteoporosis and rheumatoid arthritis. Pharmacological therapy might have a number of adverse effects. Therefore, the development of natural anti-osteoclastogenic drugs with greater efficacy and fewer adverse effects is desirable. In this study, the anti-osteoclastogenic effects of 23-hydroxyursolic acid (HUA), a triterpene isolated from Viburnum lutescens, were investigated in vitro and in vivo. HUA significantly inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)-induced mature osteoclast differentiation by reducing the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and F-actin ring formation. It also inhibited the expression of osteoclast-specific marker genes such OSCAR, MMP-9, TRAP, DC-STAMP, and CtsK, as well as transcription factors, c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) in response to RANKL. Mice orally administered with HUA (25 and 50 mg/kg) exhibited significant protection against bone loss and osteoclast formation induced by lipopolysaccharide (LPS). HUA suppressed RANKL-induced nuclear factor kappa B (NF-κB) activation and phosphorylation of JNK and ERK mitogen-activated protein kinases (MAPKs). These results suggest that HUA attenuates osteoclast formation in vitro and in vivo by suppressing the RANKL-mediated AP1, NF-κB, and NFATc1 pathways. Therefore, HUA may be a lead compound for the prevention or treatment of osteolytic bone disorders.

Mussaendoside O, a N-triterpene cycloartane saponin, attenuates RANKL-induced osteoclastogenesis and inhibits lipopolysaccharide-induced bone loss.

BACKGROUND: Elevated activity of osteoclasts (OCs) is linked to osteolytic bone diseases, such as osteoporosis and rheumatoid arthritis. Developing natural anti-osteoclastogenic compounds with greater efficacy and fewer adverse effects is crucial for preventing or treating osteolytic bone diseases. N-triterpene cycloartane saponins (NTCSs) are rarely found in nature, and their inhibitory effects on OC differentiation in vitro and in vivo have not yet been explored. PURPOSE: This study was aimed to investigate the effect of mussaendoside O, an NTCS isolated from Mussaenda pubescens, on RANKL-induced OC differentiation and its underlying mechanism in vitro, and lipopolysaccharide (LPS)-induced bone resorption in a mouse model. METHODS: The content of mussaendoside O in methanol extract of M. pubescens was determined by HPLC. The inhibitory effects of mussaendoside O on RANKL-induced OC formation were assessed using TRAP staining, western blotting, immunofluorescence staining, and real-time qPCR. Meanwhile, the effects of mussaendoside O on LPS-induced inflammatory responses were assessed using a Griess reagent and qPCR. The effects of mussaendoside O on LPS-induced bone resorption in a mouse model were evaluated using micro-CT and immunohistochemical staining. RESULTS: Mussaendoside O inhibited RANKL-induced TRAP-positive multinucleated OC formation in a concentration-dependent manner without affecting cell viability. However, mussaendoside O did not inhibit LPS-induced mRNA expression of COX-2, iNOS, and TNF-α. Mice orally administrated with mussaendoside O exhibited significant protection from LPS-induced bone resorption and OC formation. At the molecular level, mussaendoside O suppressed RANKL-activated phosphorylation of p38 MAPK and JNK, as well as c-Fos expression. In addition, mussaendoside O suppressed RANKL-induced NFATc1 activation and the expression of its target genes, including OSCAR, DC-STAMP, CtsK, and TRAP. CONCLUSION: Mussaendoside O attenuates OC differentiation in vitro and LPS-induced bone resorption in a mouse model by inhibiting the RANKL-activated c-Fos/NFATc1 signaling pathways. Therefore, mussaendoside O may be a valuable lead compound for preventing or treating of osteolytic bone diseases.

Anti-osteoclastogenic Effects of Indole Alkaloids Isolated from Barley (Hordeum vulgare Var. Hexastichon) Grass.

As part of our continuous program to identify new potential candidates for controlling osteolytic bone diseases from natural products, the alkaloid fraction of barley (Hordeum vulgare var. hexastichon) grass (HVA) significantly inhibited RANKL-induced osteoclast formation and protected mice from LPS-induced bone loss. A phytochemical investigation of HVA afforded nine indole alkaloids, including one new compound [hordeumin A (1)] and eight known analogues (2-9). Of them, four (1, 2, 4, and 5) were anti-osteoclastogenic compounds. Of these four, compound 5 significantly suppressed RANKL-induced osteoclast formation, actin ring formation, and bone resorption in a concentration-dependent manner. It also suppressed the RANKL-induced NF-κB and MAPK signaling pathways and the activation of c-Fos and NFATc1. Compound 5 also reduced the expression levels of osteoclast-specific marker genes, including TRAP, CtsK, DC-STAMP, OSCAR, and MMP9. Our findings suggest that HVA and its alkaloid constituents could be valuable candidates for the prevention and treatment of osteolytic bone diseases.

Albanol B from Mulberries Exerts Anti-Cancer Effect through Mitochondria ROS Production in Lung Cancer Cells and Suppresses In Vivo Tumor Growth.

Albanol B (ABN-B), an arylbenzofuran derivative isolated from mulberries, has been shown to have anti-Alzheimer's disease, anti-bacterial and antioxidant activities. The aim of this study was to investigate the anti-cancer effect of this compound against lung cancer cells. The results show that ABN-B inhibited the proliferation of four human lung cancer cell lines (A549, BZR, H1975, and H226) and induced apoptosis, based on the cleavage of caspase-7 and PARP (poly (ADP-ribose) polymerase), as well as the downregulation of Bcl-2. ABN-B also induced cell cycle arrest at G2/M by down-regulating the expression of CKD1 (cyclin-dependent kinase 1) and cyclin B1, but up-regulating p21 (cyclin-dependent kinase inhibitor 1) expression. Notably, ABN-B increased the production of mitochondrial reactive oxygen species (ROS); however, treatment with mito-TEMPO (a specific mitochondrial antioxidant) blocked ABN-B-induced cell cycle arrest at G2/M and apoptosis, as well as the up-regulation of p21 and down-regulation of CDK1 and cyclin B1 induced by ABN-B. At the molecular level, ABN-B-induced mitochondrial ROS production increased the phosphorylation levels of AKT (protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2), while the inhibition of these kinases blocked the ABN-B-induced up-regulation of p21 and down-regulation of CDK1 and cyclin B1. Moreover, ABN-B significantly suppressed tumor growth in Ex-3LL (Lewis lung carcinoma) tumor-bearing mice. Taken together, these results suggest that ABN-B can exert an anti-cancer effect by inducing apoptosis and cell cycle arrest at G2/M through mitochondrial ROS production in lung cancer cells.

3-Hydroxyolean-12-en-27-oic Acids Inhibit RANKL-Induced Osteoclastogenesis in Vitro and Inflammation-Induced Bone Loss in Vivo.

Olean-12-en-27-oic acids possess a variety of pharmacological effects. However, their effects and underlying mechanisms on osteoclastogenesis remain unclear. This study aimed to investigate the anti-osteoclastogenic effects of five olean-12-en-27-oic acid derivatives including 3α,23-isopropylidenedioxyolean-12-en-27-oic acid (AR-1), 3-oxoolean-12-en-27-oic acid (AR-2), 3α-hydroxyolean-12-en-27-oic acid (AR-3), 23-hydroxy-3-oxoolean-12-en-27-oic acid (AR-4), and aceriphyllic acid A (AR-5). Among the five olean-12-en-27-oic acid derivatives, 3-hydroxyolean-12-en-27-oic acid derivatives, AR-3 and AR-5, significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced mature osteoclast formation by reducing the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, F-actin ring formation, and mineral resorption activity. AR-3 and AR-5 decreased RANKL-induced expression levels of osteoclast-specific marker genes such as c-Src, TRAP, and cathepsin K (CtsK) as well as c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Mice treated with either AR-3 or AR-5 showed significant protection of the mice from lipopolysaccharide (LPS)-induced bone destruction and osteoclast formation. In particular, AR-5 suppressed RANKL-induced phosphorylation of JNK and ERK mitogen-activated protein kinases (MAPKs). The results suggest that AR-3 and AR-5 attenuate osteoclast formation in vitro and in vivo by suppressing RANKL-mediated MAPKs and NFATc1 signaling pathways and could potentially be lead compounds for the prevention or treatment of osteolytic bone diseases.

Protective effects of extract of Cleistocalyx operculatus flower buds and its isolated major constituent against LPS-induced endotoxic shock by activating the Nrf2/HO-1 pathway.

The flower buds of Cleistocalyx operculatus are used as an important ingredient in herbal tea and herbal products in several tropical countries. However, their protective effects and underlying mechanisms on lipopolysaccharide (LPS)-induced endotoxic shock remain unclear. The aim of this study was to investigate the anti-inflammatory effects of ethanol extract of C. operculatus flower buds (ECO) and its major constituent 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) in macrophages and in an experimental LPS-induced sepsis mouse model. ECO inhibited the LPS-induced production and expression of pro-inflammatory mediators in macrophages. In an endotoxic shock mouse model, the oral administration of ECO rescued LPS-induced mortality, and attenuated LPS-induced increases in the serum levels of pro-inflammatory mediators, and damage of the lung and liver tissues. ECO increased the nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2), as well as the expression of Nrf2 target genes, including heme oxygenase-1 (HO-1), in macrophages. Similar to the effects of ECO, DMC also inhibited the LPS-induced inflammatory response in macrophages and endotoxic shock in mice, and activated the Nrf2/HO-1 pathway. In conclusion, our findings suggested that ECO and its major constituent, DMC, attenuated LPS-induced endotoxic shock by activating the Nrf2/HO-1 pathway.

Triterpenoids from Ziziphus jujuba induce apoptotic cell death in human cancer cells through mitochondrial reactive oxygen species production.

Ziziphus jujuba var. inermis Rehder is an edible fruit-producing species of the Rhamnaceae family. In the present study, we isolated eight triterpenoids (1-8) from the fruits of Z. jujuba var. inermis and evaluated their apoptotic cell-death-inducing activities in human cancer cell lines (A549, PC-3, and MDA-MB-231). The structures of compounds 1-8 were determined by spectroscopic methods. Among these, four isomers of coumaroyl alphitolic acid showed potent cytotoxic activities on these cancer cells: 3-O-cis-p-coumaroyl alphitolic acid (3), 3-O-trans-p-coumaroyl alphitolic acid (4), 2-O-trans-p-coumaroyl alphitolic acid (5), and 2-O-cis-p-coumaroyl alphitolic acid (6). Moreover, compounds 3-6 induced apoptotic cell death in a concentration-dependent manner. We further investigated the apoptosis-inducing effects of compound 4 in PC-3 cells which triggered the cleavage of procaspase-3, procaspase-7, procaspase-8, bid, and PARP. Compound 4 increased both the mitochondrial reactive oxygen species (ROS) production and the phosphorylation of p38 MAPK (mitogen-activated protein kinase), but decreased the mitochondrial membrane potential. Pretreatment with Mito-TEMPO (a specific mitochondrial-targeted antioxidant) or a specific p38 inhibitor (SB203580) attenuated apoptotic cell death triggered by compound 4 which suggests that compound 4 may induce apoptotic cell death in these cancer cells by increasing the mitochondrial ROS production as well as the subsequent p38 MAPK activation. The study findings provide a rational base to use Ziziphus extracts for cancer treatments in traditional oriental medicine.

A prenylated flavonoid, 10-oxomornigrol F, exhibits anti-inflammatory effects by activating the Nrf2/heme oxygenase-1 pathway in macrophage cells.

Prenylated flavonoids are a unique class of naturally occurring flavonoids that have various pharmacological activities. In the present study, we investigated the anti-inflammatory effect in murine macrophages of a prenylated flavonoid, 10-oxomornigrol F (OMF), which was isolated from the twigs of Morus alba (Moraceae). OMF inhibited the lipopolysaccharide (LPS)-induced production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 in RAW264.7 cells, as well as in mouse bone marrow-derived macrophages (BMMs). OMF also rescued LPS-induced septic mortality in ICR mice. LPS-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α and IL-6 was also significantly suppressed by OMF treatment in RAW264.7 cells. Treatment of RAW264.7 cells with OMF induced heme oxygenase (HO)-1 mRNA and protein expression and increased the nuclear translocation of the nuclear factor-E2-related factor 2 (Nrf2) as well as the expression of Nrf2 target genes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1). Treatment of RAW264.7 cells with OMF increased the intracellular level of reactive oxygen species (ROS) and the phosphorylation levels of p38 mitogen-activated protein kinase (MAPK); co-treatment with the antioxidant N-acetyl-cysteine (NAC) blocked this OMF-induced p38 MAPK phosphorylation. Moreover, NAC, or SB203580 (a p38 MAPK inhibitor), blocked the OMF-induced nuclear translocation of Nrf2 and HO-1 expression, suggesting that OMF induces HO-1 expression by activating Nrf2 through the p38 MAPK pathway. Consistent with the notion that the Nrf2/HO-1 pathway has anti-inflammatory properties, inhibiting HO-1 significantly abrogated the anti-inflammatory effects of OMF in LPS-stimulated RAW264.7 cells. Taken together, these findings suggest that OMF exerts its anti-inflammatory effect by activating the Nrf2/HO-1 pathway, and may be a potential Nrf2 activator to prevent or treat inflammatory diseases.

A cassaine diterpene alkaloid, 3ß-acetyl-nor-erythrophlamide, suppresses VEGF-induced angiogenesis and tumor growth via inhibiting eNOS activation.

Angiogenesis is one of the hallmarks of cancer, playing an essential role in tumor growth, invasion, and metastasis. 3ß-Acetyl-nor-erythrophlamide (3-ANE), a cassaine diterpene alkaloid compound from Erythrophleum fordii, exerts various pharmacological effects, including antitumor activity. However, the effects of 3-ANE on tumor angiogenesis and its potential molecular mechanism are still unknown. Here, we demonstrated that 3-ANE inhibited the vascular endothelial growth factor (VEGF)-mediated proliferation, migration, invasion, and capillary-like tube formation of human umbilical vascular endothelial cells (HUVECs), without inducing apoptosis. We also found that 3-ANE blocked angiogenesis in vivo, and suppressed tumor angiogenesis and human lung adenocarcinoma growth in the xenograft tumor model. Furthermore, we showed that 3-ANE blocked VEGF-mediated endothelial nitric oxide synthase (eNOS) phosphorylation, vascular permeability and NO production in HUVECs, via disrupting the VEGF-induced association of eNOS and heat-shock protein 90 (HSP90). Our studies therefore provide the first evidence that 3-ANE inhibits tumor angiogenesis by inhibiting the VEGF-mediated eNOS activation and NO production, and 3-ANE could be a potential candidate in angiogenesis-related disease therapy.

Sappanone A inhibits RANKL-induced osteoclastogenesis in BMMs and prevents inflammation-mediated bone loss.

Receptor activator of nuclear factor-kB ligand (RANKL) is a key factor in the differentiation and activation of osteoclasts. Suppressing osteoclastogenesis is considered an effective therapeutic approach for bone-destructive diseases, such as osteoporosis and rheumatoid arthritis. Sappanone A (SPNA), a homoisoflavanone compound isolated from the heartwood of Caesalpinia sappan, has been reported to exert anti-inflammatory effects; however, the effects of SPNA on osteoclastogenesis have not been investigated. In the present study, we describe for the first time that SPNA inhibits RANKL-induced osteoclastogenesis in mouse bone marrow macrophages (BMMs) and suppresses inflammation-induced bone loss in a mouse model. SPNA inhibited the formation of osteoclasts from BMMs, osteoclast actin-ring formation, and bone resorption in a concentration-dependent manner. At the molecular level, SPNA significantly inhibited RANKL-induced activation of the AKT/glycogen synthase kinase-3ß (GSK-3ß) signaling pathway without affecting its activation of the mitogen-activated protein kinases (MAPKs) JNK, p38, and ERK. In addition, SPNA suppressed the induction of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), which is a crucial transcription factor in osteoclast differentiation. As a result, SPNA decreased osteoclastogenesis-related marker gene expression, including CtsK, TRAP, dendritic cell-specific transmembrane protein (DC-STAMP), MMP-9 and osteoclast-associated receptor (OSCAR). In a mouse inflammatory bone loss model, SPNA significantly inhibited lipopolysaccharide (LPS)-induced bone loss by suppressing the number of osteoclasts. Taken together, these findings suggest that SPNA inhibits osteoclastogenesis and bone resorption by inhibiting the AKT/GSK-3ß signaling pathway and may be a potential candidate compound for the prevention and/or treatment of inflammatory bone loss.

Syntenin promotes VEGF-induced VEGFR2 endocytosis and angiogenesis by increasing ephrin-B2 function in endothelial cells.

Syntenin, a tandem PDZ-domain-containing scaffold protein, is involved in the regulation of diverse biological functions, including protein trafficking, exosome biogenesis, and cancer metastasis. Here, we present the first study to explore the significance of syntenin in endothelial cells. Syntenin knockdown in human umbilical vein endothelial cells (HUVECs) impaired vascular endothelial growth factor (VEGF)-mediated proliferation, migration, invasion, vascular permeability, and nitric oxide (NO) production. Syntenin knockdown also suppressed expression of the VEGFR2 target genes VEGF, MMP2, and Nurr77 as well as VEGF-induced angiogenesis in vitro and in vivo. And it decreased cell-surface levels of ephrin-B2. Biochemical analyses revealed that syntenin exists in complex with VEGFR2 and ephrin-B2. Syntenin knockdown abolished the association between VEGFR2 and ephrin-B2, suggesting syntenin functions as a scaffold protein facilitating their association in HUVECs. Consistent with these observations, knocking down syntenin or ephrin-B2 abolished VEGF-induced endocytosis and VEGFR2 phosphorylation and activation of its downstream signaling molecules. Treatment with MG132, a proteasome inhibitor, rescued the downregulation of ephrin-B2 and VEGFR2 signaling induced by syntenin knockdown. These findings demonstrate that syntenin promotes VEGF signaling and, through its PDZ-dependent interaction with ephrin-B2, enhances VEGF-mediated VEGFR2 endocytosis and subsequent downstream signaling and angiogenesis in endothelial cells.

Osseous Injury Associated With Ligamentous Tear of the Knee.

One of the most common knee injuries is ligament tear, which may initially manifest as an osseous injury in radiographs. Radiologists should therefore be able to recognize ligament tears of the knee as osseous abnormalities in images. This review focuses on the imaging features of knee ligament injuries and their related osseous injuries: anterior cruciate ligament (ACL) tear with Segond fracture; associated marrow contusion; ACL avulsion fracture; posterior cruciate ligament (PCL) tear with osseous avulsion of the ligament including arcuate sign; reverse Segond fracture; PCL avulsion fracture; medial collateral ligament tear with Pellegrini-Stieda disease; lateral collateral ligament tear with avulsion fracture of the fibular head; and patellar ligament injuries with Osgood-Schlatter and Sinding-Larsen-Johansson.

Sappanone A exhibits anti-inflammatory effects via modulation of Nrf2 and NF-κB.

Homoisoflavonoids constitute a small class of natural products. In the present study, we investigated the anti-inflammatory effect of sappanone A (SPNA), a homoisoflavanone that is isolated from the heartwood of Caesalpinia sappan (Leguminosae), in murine macrophages. SPNA inhibited the production of nitric oxide (NO), prostaglandin E2 (PGE2) and interleukin-6 (IL-6) as well as the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and IL-6 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Moreover, SPNA protected C57BL/6 mice from LPS-induced mortality. Treatment of RAW264.7 cells with SPNA induced heme oxygenase (HO)-1 protein and mRNA expression and increased nuclear translocation of the nuclear factor-E2-related factor 2 (Nrf2) as well as the expression of Nrf2 target genes such as NAD(P)H: quinone oxidoreductase 1 (NQO1). Knockdown of Nrf2 by siRNA blocked SPNA-mediated HO-1 induction. SB203580, p38 mitogen-activated protein kinase (MAPK) inhibitor, blocked SPNA-induced HO-1 expression and nuclear translocation of Nrf2, suggesting that SPNA induces HO-1 expression by activating Nrf2 through the p38 MAPK pathway. Consistent with the notion that the Nrf2/HO-1 pathway has anti-inflammatory properties, inhibiting HO-1 significantly abrogated the anti-inflammatory effects of SPNA in LPS-stimulated RAW264.7 cells. Moreover, SPNA suppressed LPS-induced nuclear factor κB (NF-κB) activation via inhibiting Ser 536 phosphorylation and transcriptional activity of RelA/p65 subunit of NF-κB. Taken together, these findings suggest that SPNA exerts its anti-inflammatory effect by modulating the Nrf2 and NF-κB pathways, and may be a valuable compound to prevent or treat inflammatory diseases.