Styrax Japonica Sieb. et Zucc. has been used as traditional medicine in inflammatory diseases, and isolated compounds have shown pharmacological activities. Pinoresinol glucoside (PIN) belonging to lignins was isolated from the stem bark of S. Japonica. This study aimed to investigate the biological function and mechanisms of PIN on cell migration, osteoblast differentiation, and matrix mineralization. Herein, we investigated the effects of PIN in MC3T3-E1 pre-osteoblasts, which are widely used for studying osteoblast behavior in in vitro cell systems. At concentrations ranging from 0.1 to 100 μM, PIN had no cell toxicity in pre-osteoblasts. Pre-osteoblasts induced osteoblast differentiation, and the treatment of PIN (10 and 30 μM) promoted the cell migration rate in a dose-dependent manner. At concentrations of 10 and 30 μM, PIN elevated early osteoblast differentiation in a dose-dependent manner, as indicated by increases in alkaline phosphatase (ALP) staining and activity. Subsequently, PIN also increased the formation of mineralized nodules in a dose-dependent manner, as indicated by alizarin red S (ARS) staining, demonstrating positive effects of PIN on late osteoblast differentiation. In addition, PIN induced the mRNA level of BMP2, ALP, and osteocalcin (OCN). PIN also upregulated the protein level of BMP2 and increased canonical BMP2 signaling molecules, the phosphorylation of Smad1/5/8, and the protein level of Runt-related transcription factor 2 (RUNX2). Furthermore, PIN activated non-canonical BMP2 signaling molecules, activated MAP kinases, and increased β-catenin signaling. The findings of this study indicate that PIN has biological roles in osteoblast differentiation and matrix mineralization, and suggest that PIN might have anabolic effects in bone diseases such as osteoporosis and periodontitis.
The perennial herb Saposhnikovia divaricata SCHISCHKIN, used as a source of traditional medicines, is known for its anti-bone erosion, anti-inflammatory, and analgesic effects. We examined the osteogenic properties of Sec-O-glucosylhamaudol (SOGH) extracted from S. divaricata roots as a bone-protective compound. We found that SOGH promoted osteoblast differentiation and mineralization and induced the expression of Bsp, Ocn, and Opn without any cytotoxic effects. SOGH also increased nuclear RUNX2 levels in pre-osteoblasts via the BMP2 and Wnt3a signaling pathways, but it had no evident effects on the autophagic and necroptotic signaling pathways. Furthermore, SOGH promoted bone-forming activities by inducing the adhesion and migration of osteoblasts and the expression of RUNX2 target proteins, including VEGF and MMP13. In sum, SOGH has osteogenic properties mediated via the BMP2 and Wnt3a signaling pathways and RUNX2 expression, indicating the bone-protective potential of this compound in diseases such as periodontitis and osteoporosis.
Md. Ataur Rahman, Hanna Kim, Kang Ho Lee, Hyung-Mun Yun, Jung-Hwa Hong, Youngjae Kim, Hyunah Choo, Mikyoung Park, and Hyewhon Rhim. Mol. Cells 2017;40:495-502. https://doi.org/10.14348/molcells.2017.0080
Background: (E)-methyl-cinnamate (EMC), a phytochemical constituent isolated from Alpinia katsumadai Hayata, is a natural flavor compound with anti-inflammatory properties, which is widely used in the food and commodity industry. However, the pharmacological effects of methyl-cinnamate on pre-osteoblasts remain unknown. This study aimed to investigate the pharmacological effects and mechanisms of EMC in pre-osteoblast MC3T3-E1 cells (pre-osteoblasts). Methods: Cell viability and apoptosis were evaluated using the MTT assay and TUNEL staining. Cell migration and osteoblast differentiation were examined using migration assays, as well as alkaline phosphatase activity and staining assays. Western blot analysis was used to examine intracellular signaling pathways and apoptotic proteins. Results: EMC decreased cell viability with morphological changes and increased apoptosis in pre-osteoblasts. EMC also induced the cleavage of Poly (ADP-ribose) polymerase (PARP) and caspase-3 and reduced the expression of anti-apoptotic proteins. In addition, EMC increased TUNEL-positive cells in pre-osteoblasts, decreased the activation of mitogen-activated protein kinases, and suppressed cell migration rate in pre-osteoblasts. Subsequently, EMC inhibited the osteoblast differentiation of pre-osteoblasts, as assessed by alkaline phosphatase staining and activity assays. Conclusion: These findings demonstrate that EMC has a pharmacological and biological role in cell survival, migration, and osteoblast differentiation. It suggests that EMC might be a potential phytomedicine for treating abnormalities of osteoblast function in bone diseases.
Sanguisorba officinalis L. (Rosaceae) is a perennial herbaceous plant and its roots have been used as an important traditional medicine for over 2000 years. Ziyuglycoside I (Ziyu), an active compound isolated from the roots of S. officinalis L., has shown biological effects such as anti-oxidant, antiviral, and antiwrinkle activities. This study aimed to elucidate the underlying mechanisms of action of Ziyu on cytotoxicity, migration, and differentiation of pre-osteoblasts. Herein, at concentrations ranging from 1 to 100 [Formula: see text]M, Ziyu was not cytotoxic against pre-osteoblasts. Alkaline phosphatase activity assay and staining, and migration assay showed that Ziyu increased cell migration and promoted early osteoblast differentiation, followed by the enhancement of mineralized nodule formation in a dose-dependent manner, as indicated by Alizarin Red S staining. In addition, Ziyu increased the protein levels of runt-related transcription factor 2 (RUNX2) during osteoblast differentiation, whereas it did not affect the phosphorylation of Smad1/5/8 and GSK3b and expression of [Formula: see text]-catenin. Ziyu also activated ERK1/2 and mitogen-activated protein kinase during osteoblast differentiation, and ERK1/2 inhibitor attenuated Ziyu-mediated RUNX2 expression and nuclear accumulation. Furthermore, Ziyu-mediated early and late osteoblast differentiation was significantly suppressed by the inhibition of ERK1/2, which was accompanied by attenuation in the mRNA levels of osteoblast-related genes including bone sialoprotein, osteopontin, and osteocalcin. Taken together, the findings of this study provide evidence that Ziyu promotes cell migration, osteoblast differentiation, and bone mineralization and suggest a potential role for Ziyu in the treatment of bone diseases.
Continuous identification and validation of novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for proposed targets. Recently, the 5-HT6 receptor (5- HT6R), a member of the class of recently discovered 5-HT receptors, has received considerable attention for its possible implications in depression, cognition, and anxiety. However, the cellular signaling mechanisms of 5-HT6R are poorly understood due to the lack of selective 5-HT6R ligands. In the present study, we examined functional coupling of the human 5-HT6R, 5-HT7AR, or 5-HT7BR with various Gα-proteins (Gα15, Gαqs5, or GαqG66Ds5) to develop a reliable cell-based HTS method for 5-HT receptors. Among variable couplings between 5-HT receptors and G-proteins, we found that functional coupling of human 5-HT6R with GαqG66Ds5 produced the highest levels of Ca2+ signaling in HEK293 cells as measured by the fluorescence-based HTS plate reader, FDSS6000. After validation of this new 5-HT6R HTS system (Z´-factor = 0.56) in 96-well plates and characterization of the pharmacological profile of the 5-HT6R, we screened ∼500 synthetic chemical compounds including butanamide and benzenesulfonamide derivatives. Based on this preliminary screening, we found that the butanamide derivative LSG11104 produced an IC50 value of 6.3 μM. This compound will serve as a lead structure for further chemical modification to develop novel 5-HT6R ligands. Furthermore, we demonstrated that this HTS method can be utilized to identify proteins that modulate 5-HT6R function and present Fyn tyrosine kinase as an example, which is already known as a 5-HT6R interacting protein. Taken together, these results suggest that the 5-HT6R/GαqG66Ds5 FDSS6000 system can be utilized to screen for selective 5-HT6R ligands and to examine any functional relationships between 5-HT6R and its binding proteins. Keywords: 5-hyrdoxytryptamine (5-HT), 5-HT6 receptor, HTS, FDSS6000, chimeric G-protein, fluoresce Ca2+ imaging, Z´-factor, Fyn
// Hyung-Mun Yun 1 , Kyung-Ran Park 1 , Eun-Cheol Kim 1 and Jin Tae Hong 2 1 Department of Maxillofacial Tissue Regeneration, School of Dentistry and Research Center for Tooth and Periodontal Regeneration (MRC), Kyung Hee University, Dongdaemun-gu, Republic of Korea 2 College of Pharmacy and Medical Research Center, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea Correspondence to: Jin Tae Hong, email: // Keywords : PRDX6, EAE, demyelination, astrocytes, inflammation, multiple sclerosis, Pathology Section Received : July 17, 2015 Accepted : August 12, 2015 Published : August 17, 2015 Abstract Multiple sclerosis (MS) is a complex disease with an unknown etiology and has no effective medications despite extensive research. Antioxidants suppress oxidative damages which are implicated in the pathogenesis of MS. In this study, we showed that the expression of an antioxidant protein peroxiredoxin 6 (PRDX6) is markedly increased in spinal cord of mice with experimental autoimmune encephalomyelitis (EAE) compared to other PRDXs. PRDX6 transgenic (Tg) mice displayed a significant decrease in clinical severity and attenuated demyelination in EAE compared to wide type mice. The increased PRDX6 expression in astrocytes of EAE mice and MS patients reduced MMP9 expression, fibrinogen leakage, chemokines, and free radical stress, leading to reduction in blood-brain-barrier (BBB) disruption, peripheral immune cell infiltration, and neuroinflammation. Together, these findings suggest that PRDX6 expression may represent a therapeutic way to restrict inflammation in the central nervous system and potentiate oligodendrocyte survival, and suggest a new molecule for neuroprotective therapies in MS.
Paeonia suffruticosa is a magnificent and long-lived woody plant that has traditionally been used to treat various diseases including inflammatory, neurological, cancer, and cardiovascular diseases. In the present study, we demonstrated the biological mechanisms of paeonoside (PASI) isolated from the dried roots of P. suffruticosa in pre-osteoblasts. Herein, we found that PASI has no cytotoxic effects on pre-osteoblasts. Migration assay showed that PASI promoted wound healing and transmigration in osteoblast differentiation. PASI increased early osteoblast differentiation and mineralized nodule formation. In addition, PASI enhanced the expression of Wnt3a and bone morphogenetic protein 2 (BMP2) and activated their downstream molecules, Smad1/5/8 and β-catenin, leading to increases in runt-related transcription factor 2 (RUNX2) expression during osteoblast differentiation. Furthermore, PASI-mediated osteoblast differentiation was attenuated by inhibiting the BMP2 and Wnt3a pathways, which was accompanied by reduction in the expression of RUNX2 in the nucleus. Taken together, our findings provide evidence that PASI enhances osteoblast differentiation and mineralized nodules by regulating RUNX2 expression through the BMP2 and Wnt3a pathways, suggesting a potential role for PASI targeting osteoblasts to treat bone diseases including osteoporosis and periodontitis.
Osteosarcoma, which has poor prognosis after metastasis, is the most common type of bone cancer in children and adolescents. Therefore, plant-derived bioactive compounds are being actively developed for cancer therapy. Artemisia apiacea Hance ex Walp. is a traditional medicinal plant native to Eastern Asia, including China, Japan, and Korea. Vitexicarpin (Vitex), derived from A. apiacea, has demonstrated analgesic, anti-inflammatory, antitumour, and immunoregulatory properties; however, there are no published studies on Vitex isolated from the aerial parts of A. apiacea. Thus, this study aimed to evaluate the antitumour activity of Vitex against human osteosarcoma cells. In the present study, Vitex (>99% purity) isolated from A. apiacea induced significant cell death in human osteosarcoma MG63 cells in a dose- and time-dependent manner; cell death was mediated by apoptosis, as evidenced by the appearance of cleaved-PARP, cleaved-caspase 3, anti-apoptotic proteins (Survivin and Bcl-2), pro-apoptotic proteins (Bax), and cell cycle-related proteins (Cyclin D1, Cdk4, and Cdk6). Additionally, a human phosphokinase array proteome profiler revealed that Vitex suppressed AKT-dependent downstream kinases. Further, Vitex reduced the phosphorylation of PRAS40, which is associated with autophagy and metastasis, induced autophagosome formation, and suppressed programmed cell death and necroptosis. Furthermore, Vitex induced antimetastatic activity by suppressing the migration and invasion of MMP13, which is the primary protease that degrades type I collagen for tumour-induced osteolysis in bone tissues and preferential metastasis sites. Taken together, our results suggest that Vitex is an attractive target for treating human osteosarcoma.
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