In vitro evaluation of the potential therapeutic role of Dendropanax morbifera extract in ameliorating osteoporosis and resultant bone impairment using MC3T3-E1 cells.

Osteoporosis is a widespread musculoskeletal deformity that affects thousands of older people every year, leading to bone abnormalities and ultimately increasing the risk of bone fractures in both genders. It is considered a lethal disease causing death in thousands of people at the late stage of life. Dendropanax morbifera Leveille is a subtropical broad-leaved prevalent species in Korea. Extracts of the leaves, stems, roots, and seeds of D. morbifera have been used in traditional medicine for the treatment of numerous diseases such as diabetes, atherogenesis, skin disorders, and headaches. However, the anti-osteoporosis effects of D. morbifera have not been examined. The primary objectives of this study were to elucidate the anti-osteoporosis effect of D. morbifera extract through an in vitro study using pre-osteoblastic MC3T3-E1 cells. We found that D. morbifera strongly increased the expression of bone metabolic markers such as alkaline phosphatase (ALP) activity, type I collagen (Col-I) level, and mineralization. Additionally, D. morbifera extract also upregulated the mRNA expression levels of osteogenic genes including ALP, osteocalcin (OCN), osterix (Osx), and runt-related transcription factor 2 (Runx2) in MC3T3-E1 cells via upregulation of bone morphogenetic protein 2 (BMP-2)/p38 MAPK/JNK and Smad1/5/8 signaling pathways. Moreover, addition of D. morbifera significantly suppressed the inhibitory effect of SB203580 (p38 inhibitor). In conclusion, the current study demonstrated that D. morbifera extract significantly increased osteoblast differentiation and mineralization in MC3T3-E1 cells by regulating BMP-2/p38/JNK and Smad1/5/8. Our study might be helpful in the discovery and development of new anti-osteoporosis therapeutic agents.

Therapeutic potential of compound K as an IKK inhibitor with implications for osteoarthritis prevention: an in silico and in vitro study.

Ginsenosides have been used traditionally as an oriental medicine. However, the anti-osteoarthritic effect of ginsenoside compound K (hereafter referred to as CK) has not been reported. Therefore, in this study, the protective effects of CK were evaluated in silico and in vitro using H2O2-stimulated MC3T3-E1 cells by measuring the levels of proinflammatory cytokines responsible for articular cartilage degradation. In silico results demonstrated that, among the selected ginsenosides, CK is a non-toxic drug-like molecule with strong binding affinity for selected cytokine-activated kinase such as IkBα kinase (IKK). The molecular binding energy of CK with the active sites of IKK suggests anti-osteoarthritic functions. Cultured H2O2-stimulated MC3T3-E1 cells that were exposed to CK showed dramatically increased expression of osteoblast differentiation markers such as alkaline phosphatase (ALP) activity, type I collagen (Col-I) content, and mineralization. During aging, H2O2 also leads to the production of reactive oxygen species (ROS) and nitric oxide (NO), which play important roles in the development of osteoarthritis (OA). Therefore, the effect of CK on ROS and NO generation was also examined. Our results showed that CK dose-dependently inhibited H2O2-induced ROS and NO production in MC3T3-E1 cells. Moreover, qRT-PCR data showed that CK increased expression of osteogenic markers such as ALP and Col-I but decreased expression of inflammatory-related genes including IKK and interleukin 1ß (IL-1ß) in a dose-dependent manner in H2O2-stimulated MC3T3-E1 cells. The findings of this study suggest the use of CK as a novel protective and therapeutic agent in AO.

Suppression of MAPKs/NF-κB Activation Induces Intestinal Anti-Inflammatory Action of Ginsenoside Rf in HT-29 and RAW264.7 Cells.

This study investigated the intestinal anti-inflammatory action of ginsenoside Rf in inflammatory bowel disease (IBD). IBD is a chronic inflammatory disease that affects the intestinal tract. It is associated with elevated levels of various inflammatory mediators, including interleukin (IL)-1ß, IL-6, tumor necrosis factor-α (TNF-α), nitric oxide (NO), and reactive oxygen species (ROS). Ginsenosides, the main active constituents of ginseng, have been reported to exert potent therapeutic effects against diverse diseases. However, ginsenoside Rf treatment for inflammation has not yet been examined. In this study, we evaluated the inhibitory effect of ginsenoside Rf on the inflammatory mediators downstream of p38/NF-kB activation on TNF-α-stimulated intestinal epithelial cells (HT-29) and mouse macrophage cells (RAW264.7). Our results showed that ginsenoside Rf significantly reduced the production of IL-1ß, IL-6, TNF-α, NO, and ROS, which are most highly activated in IBD. In addition, ginsenoside Rf significantly suppressed TNF-α/LPS-induced NF-κB transcriptional activity. These results suggest that ginsenoside Rf contains a compound that has potent intestinal anti-inflammatory effects that could be used to treat diseases such as IBD.

Ginsenoside Rg5:Rk1 attenuates TNF-α/IFN-γ-induced production of thymus- and activation-regulated chemokine (TARC/CCL17) and LPS-induced NO production via downregulation of NF-κB/p38 MAPK/STAT1 signaling in human keratinocytes and macrophages.

Atopic dermatitis (AD) is a chronic skin disease that affects millions of people worldwide. Keratinocytes and macrophages are two cells types that play a pivotal role in the development of AD. These cells produced different chemokines and cytokines, especially thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), as well as nitric oxide (NO) through inducible nitric oxide synthase (iNOS) and COX2 in response to stimulation by TNF-α/IFN-γ and lipopolysaccharide (LPS) respectively. These mediators are thought to be crucial regulators of the pathogenesis of AD. Although several natural compounds to treat AD have been studied, the effect of Rg5:Rk1 from Panax ginseng (P. ginseng) on AD has not yet been investigated. In this study, we evaluated the inhibitory effect of Rg5:Rk1 on TNF-α/IFN-γ stimulated keratinocytes (HaCaT cells) and LPS-stimulated macrophages (RAW 264.7 cells). Enzyme-linked immunosorbent assay (ELISA) data showed that pretreatment of HaCaT cells with Rg5:Rk1 significantly reduced the TNF-α/IFN-γ-induced increase in TARC/CCL17 expression in a dose-dependent manner. In addition, Rg5:Rk1 decreased LPS-mediated nitric oxide (NO) and reactive oxygen species (ROS) production in RAW 264.7 cells. A considerable reduction in messenger RNA (mRNA) expression of the aforementioned AD mediators was also observed. Pretreatment with Rg5:Rk1 attenuated the TNF-α/IFN-γ-induced phosphorylation of p38 MAPK, STAT1, and NF-κB/IKKß in HaCaT cells. Together, these findings suggest that ginsenoside Rg5:Rk1 may have a potential anti-AD effect by suppressing NF-κB/p38 MAPK/STAT1 signaling.

Paenibacillus kyungheensis sp. nov., isolated from flowers of magnolia.

A Gram-staining-positive, catalase-positive, oxidase-negative, facultatively anaerobic, rod-shaped bacterium designated strain DCY88T, was isolated from flowers of magnolia. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that the strain formed a distinct lineage within the genus Paenibacillus that was closely related to Paenibacillus hordei RH-N24T (97.8 %). The other most closely related species were Paenibacillus illinoisensis NRRL NRS-1356T (94.3 %), Paenibacillus hunanensis DSM 22170T (94.2 %), Paenibacillus peoriae DSM 8320T (93.9 %), Paenibacillus kribbensis Am49T (93.8 %) and the type species of the genus, Paenibacillus polymyxa ATCC 842T (93.3 %). Cells of the strain were endospore-forming and motile by peritrichous flagella. Strain DCY88T formed pink-pigmented colonies on trypticase soy agar and R2A agar medium. Growth of strain DCY88T occurs at temperatures 5-37 °C, at pH 4-9 and 0.5-5.5 % NaCl (w/v). The menaquinone was MK-7.The cell wall peptidoglycan of strain DCY88T contained meso-diaminopimelic acid. The major fatty acids were anteiso-C15 : 0 (61.0 %) and C16 : 0 (11.0 %). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified polar lipid. The strain DCY88T contained spermidine as the major polyamine. The DNA G+C content was 51.6 mol%. The DNA-DNA hybridization relatedness between strain DCY88T and P. hordei RH-N24T was 48 ± 2 %. The phenotypic, phylogenetic and chemotaxonomic results indicate that the strain DCY88T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus kyungheensis sp. nov. is proposed. The type strain is DCY88T ( = JCM 19886T = KCTC 33429T).

Inhibition of Osteoclast Differentiation by Ginsenoside Rg3 in RAW264.7 Cells via RANKL, JNK and p38 MAPK Pathways Through a Modulation of Cathepsin K: An In Silico and In Vitro Study.

Various studies have demonstrated that overexpression of cathepsin K (Cat-K) causes excessive bone loss, which ultimately leads to a variety of bone diseases including osteoporosis. Therefore, inhibition of Cat-K signifies a potential therapeutic target in osteoporosis treatment. Ginsenoside Rg3 is one of the most promising compound of Panax ginseng Meyer (P. ginseng) with numerous biological activities. Thus, in recent study the inhibitory effect of Rg3 isolated from P. ginseng was investigated in order to impede the osteoclast activity by an in silico approach followed by in vitro study validation using RAW264.7 cells through the investigation of different biological activity prediction such as absorption distribution metabolism and excretion (ADMET) properties against Cat-K protein. The docking results of our study showed that Rg3 is a non-toxic compound and may act as a drug-like molecule. Additionally, the molecular interaction of Rg3 with the active residues of Cat-K markedly describes its inhibitory effects on osteoclastogenesis. Findings of the present study exhibited that Rg3 significantly reduced receptor activator of nuclear factor kappa B ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP) activity, pit formation (actin rings), and TRAP-positive multinucleated cells development in RAW264.7 cells. Furthermore, Rg3 dose-dependently reduced the mRNA expression levels of osteoclast-specific markers such as RANK, TRAP, and Cat-K induced by RANKL through the down regulation of p38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) pathways. In conclusion, in silico docking study and in vitro validation together suggested that Rg3 inhibits osteoclastogenesis and reduces bone resorption through the inhibition of Cat-K. Therefore, Rg3 might be a useful therapeutic agent for the treatment of osteoporosis and proper bone formation. Copyright © 2015 John Wiley & Sons, Ltd.

Effect of Fermented Red Ginseng Extract Enriched in Ginsenoside Rg3 on the Differentiation and Mineralization of Preosteoblastic MC3T3-E1 Cells.

In this study, red ginseng extract (RGE) was converted into high-content minor ginsenosides by fermenting with Bgp1 enzymes at 37°C for 5 days. Compared to the RGE, the minor ginsenoside contents were increased in fermented red ginseng extract (FRGE). Moreover, the amount of minor ginsenosides such as Rh1 (11%) and Rg2 (16%) was slightly augmented, while the level of Rg3 (33%) was significantly increased after bioconversion. Furthermore, we also examined and compared the effect of RGE and FRGE on the differentiation and mineralization of preosteoblastic MC3T3-E1 cells. Similarly, the level of mRNA expression of intracellular alkaline phosphatase (ALP) activity, type-1 collagen (Col-I) was also increased. Based on the comparison, it is clear that the FRGE has improved effects on bone formation and differentiation of preosteoblastic MC3T3-E1 cells.

Ginsenoside Rh1 induces mouse osteoblast growth and differentiation through the bone morphogenetic protein 2/runt-related gene 2 signalling pathway.

OBJECTIVES: This study aimed to investigate the stimulative and pharmacological effects of ginsenoside Rh1 (hereinafter referred to as: Rh1) on differentiation and mineralization of osteoblast and its possible mechanism of action on the expression of bone morphogenetic protein 2 (BMP-2)/Runt-related gene 2 (Runx2) signalling pathways using mouse preosteoblastic MC3T3-E1 cell line as in-vitro model. METHODS: An in-vitro stimulative activity of Rh1 was assessed by analyzing alkaline phosphatase activity (ALP), type-I collagen (Coll-I) synthesis, mineralization and glutathione content. Its antioxidant activity was measured by evaluating the reactive oxygen species (ROS) production in the presence of antimycin A (AMA), one of the mitochondrial dysfunction factors. The level of BMP-2/Runx2 signal-regulated osteoblast-specific proteins such as osteocalcin (OCN), Coll-I and ALP were detected using Western blot analysis. KEY FINDINGS: Rh1 was capable to stimulate cell growth, ALP activity, Coll-I synthesis, mineralization and glutathione content in the MC3T3-E1 cells. BMP-2 and Runx2 expression were also increased by Rh1 concentration dependently. Additionally, Rh1 also showed inhibitory action on the level of ROS production enhanced by AMA in MC3T3-E1 cells. Rh1 could increase the expression level of BMP-2/Runx2 signal-regulated osteogenic markers such as ALP, Coll-I and OCN. CONCLUSIONS: Rh1, a protopanaxatriol type's active ingredients of Panax ginseng Meyer, possesses osteoblast differentiation, osteogenic stimulatory and anti-oxidative activity.

Arthrobacter ginsengisoli sp. nov., isolated from soil of a ginseng field.

A Gram-staining-positive, catalase-positive, oxidase-negative, non-motile, non-flagellate and rod-shaped bacterium, was designated as DCY81(T), and isolated from soil of a ginseng field in Pocheon province, Republic of Korea. The 16S rRNA gene sequence analysis revealed that strain DCY81(T) belonged to the genus Arthrobacter. Major fatty acid was anteiso-C15:0, while major polar lipids were diphosphatidyglycerol, phatidyglycerol, phosphatidylinositol, monogalactosyldiacylglycerol (GL1), and dimannosyldiacylglycerol (GL2). The dominant quinone was MK-9(H2). The peptidoglycan type was A3α with an L-Lys-L-Ala-L-Thr-L-Ala interpeptide bridge. The DNA-DNA hybridization relatedness between strain DCY81(T) and Arthrobacter siccitolerans LMG 27359(T) (98.2 %), Arthrobacter sulfonivorans JCM 13520(T) (97.81 %), Arthrobacter scleromae DSM 17756(T) (97.59 %), Arthrobacter oxydans KCTC 3383(T) (97.3 %) was 39.1 ± 0.2, 62.2 ± 1.6, 36.8 ± 1.1 and 48.3 ± 1.6 %, respectively which show that the genotypic separation of strain DCY81(T) from the closest reference strain of the genus Arthrobacter. The DNA G+C content was 65.2 mol%. The genotypic analysis, physiological, and chemotaxonomic results indicate that strain DCY81(T) represents a novel species of the genus Arthrobacter. Therefore, Arthrobacter ginsengisoli sp. nov., is proposed as the type strain (=KCTC 29225(T) = JCM 19357(T)).

Stimulative effect of ginsenosides Rg5:Rk1 on murine osteoblastic MC3T3-E1 cells.

Panax ginseng C.A. Meyer (P. ginseng), hereafter referred to as P. ginseng, is known to exert a wide range of pharmacological effects both in vitro and in vivo; however, few studies have investigated the effects of ginseng on bone metabolism. We therefore investigated the potential antiosteoporotic properties of ginseng on the growth and differentiation of murine MC3T3-E1 cells. Rg5:Rk1 is a mixture of protopanaxadiol-type ginsenosides, isolated from fresh P. ginseng root, via a repetitive steaming and drying process. In this study, we examined the stimulatory effects of Rg5:Rk1 on the differentiation and mineralization of MC3T3-E1 cells. Undifferentiated cells were treated with a range of concentrations of Rg5:Rk1 (1-50 µg/mL), and cell viability was measured with the 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Treatment with Rg5:Rk1 significantly increased cell viability in a dose-dependent manner. To investigate the possible mechanisms by which Rg5:Rk1 affects the early differentiation phase of MC3T3-E1 cells, the cells were treated with Rg5:Rk1 for 14-24 days before assessing the levels of multiple osteoblastic markers. The markers examined included alkaline phosphatase (ALP) activity type I collagen content (Coll-I), calcium deposition (by Alizarin Red S staining), extracellular mRNA expression of bone morphogenetic protein-2 (BMP-2), and the level of Runt-related transcription factor 2 (Runx2). Rg5:Rk1 treatment also increased the activities of proteins associated with osteoblast growth and differentiation in a dose-dependent manner. Overall, we found that the Rg5:Rk1 mixture of ginsenosides improved the osteoblastic function of MC3T3-E1 cells by increasing their proliferative capacity. This improvement is due to the action of Rg5:Rk1 on BMP-2, which is mediated by Runx2-dependent pathways.

Ginseng saponins and the treatment of osteoporosis: mini literature review.

The ginseng plant (Panax ginseng Meyer) has a large number of active ingredients including steroidal saponins with a dammarane skeleton as well as protopanaxadiol and protopanaxatriol, commonly known as ginsenosides, which have antioxidant, anticancer, antidiabetic, anti-adipocyte, and sexual enhancing effects. Though several discoveries have demonstrated that ginseng saponins (ginsenosides) as the most important therapeutic agent for the treatment of osteoporosis, yet the molecular mechanism of its active metabolites is unknown. In this review, we summarize the evidence supporting the therapeutic properties of ginsenosides both in vivo and in vitro, with an emphasis on the different molecular agents comprising receptor activator of nuclear factor kappa-B ligand, receptor activator of nuclear factor kappa-B, and matrix metallopeptidase-9, as well as the bone morphogenetic protein-2 and Smad signaling pathways.