Effect of safflower seeds supplementation on stimulation of the proliferation, differentiation and mineralization of osteoblastic MC3T3-E1 cells

Anti-bone resorption properties of the Korean herbal formulation, Gami-Honghwain (HJ), which comprises Carthamus tinctorius L. seed and hominis placenta, were investigated. We demonstrate that the production of PGE2 is inhibited by 20-100 microg/ml HJ in nontransformed osteoblastic cells (MC3T3-E1 cells), indicating that HJ inhibits PGE2 production. The effect of HJ on the proliferation and osteoblastic differentiation in MC3T3-E1 was also studied. HJ dose-dependently increased DNA synthesis (significant at 20-100 microg/ml), and increased alkaline phosphatase (ALP) and prolyl hydroxylase activities of MC3T3-E1 cells (20-100 microg/ml), while anti-estrogen tamoxifen eliminated the stimulation of proliferation and ALP activity of MC3T3-E1 which was induced by HJ. These results indicate that HJ directly stimulates cell proliferation and differentiation of osteoblasts. Also, when we assessed the effects of HJ on osteoblastic differentiation in MC3T3-E1, HJ enhanced ALP activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the HJ was observed at relatively low doses (significant at 20-100 microg/ml and maximal at 100 microg/ml). Northern blot analysis showed that the HJ (60 microg/ml) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. HJ (100 microg/ml) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that HJ has anabolic effect on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases.     

Stimulative effects of Drynariae Rhizoma extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cells

Pharmacological factors are needed to prevent bone loss that occurs with increasing age. The chemical compounds that act on bone metabolism in herbal medicines, however, are poorly understood. Effects of traditional Korean medicine, Drynariae Rhizoma [Drynaria fortunei (kunze) J. Sm] extract (DR), on the osteoblastic proliferation and differentiation were investigated. The effect of DR, a natural phyto herb, on the proliferation and osteoblastic differentiation in non-transformed osteoblastic cells (MC3T3-E1) was studied. DR dose-dependently increased DNA synthesis (significant at 50-150 microg/ml). DR increased alkaline phosphatase (ALP) activity and prolyl hydroxylase activity of MC3T3-E1 cells (50-150 microg/ml). Antiestrogen tamoxifen eleminated the stimulation of proliferation and ALP activity of MC3T3-E1, which were induced by DR. DR at concentrations ranged from 30-100 microg/ml inhibited prostaglandin E2 production in MC3T3-E1. These results indicate that DR directly stimulates cell proliferation and differentiation of osteoblasts. These results also suggest and DR is effective for bone anti-resorptive action in bone cells.     

Stimulative effects of Ulmus davidiana Planch (Ulmaceae) on osteoblastic MC3T3-E1 cells

Ulmus davidiana Planch (Ulmaceae) has long been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone among other functions. To treat rheumatoid arthritis (RA), a herbal medicine, Ulmus davidiana Planch (Ulmaceae) extract (UD) is being used in traditional oriental medicine. The effect of UD on the proliferation and osteoblastic differentiation in non-transformed osteoblastic cells (MC3T3-E1) was studied. UD dose-dependently increased DNA synthesis (significant at 5-20 microg/ml). UD increased alkaline phosphatase (ALP) activity and prolyl hydroxylase activity of MC3T3-E1 cells (5-20 microg/ml). Antiestrogen tamoxifen eliminated the stimulation of proliferation and ALP activity of MC3T3-E1, which was induced by UD. UD at concentrations ranged from 30 to 100 microg/ml inhibited prostaglandin E2 production in MC3T3-E1. These results indicate that UD directly stimulates cell proliferation and differentiation of osteoblasts. These results also suggest and UD is effective for bone anti-resorptive action in bone cells.     

Effects of soybean ethanol extract on the cell survival and oxidative stress in osteoblastic cells

Recent evidence suggests that high concentrations of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) are thought to increase the apoptosis in osteoblasts and bone resorption and may have important roles in the regulation of osteoblast and osteoclast metabolism, especially in rheumatoid arthritis. The present study was performed to investigate the effect of soybean ethanol extract on the scavenging properties using DPPH and the TNF-alpha and NO production of osteoblastic MC3T3-E1 cells. The soy extract and its fractions according to polarity displayed a strong free radical scavenger activity at 0.01 approximately 0.1g/L, except for aquous fraction which had no significant effect on the function of MC3T3-E1 cells (p < 0.05). TNF-alpha secretion by MC3T3-E1 cells was reduced significantly when stimulated with soy extract (0.05 g/L). Nitrite accumulation in culture medium and apoptosis of MC3T3-E1 cells were induced by the addition of 10(-10) M TNF-alpha, and inhibited by the simultaneous addition of soy extract (0.05g/L).     

竹节参对MC3T3-E1成骨样细胞增殖与分化的影响

目的：研究竹节参不同提取物对体外培养成骨样细胞（MC3T3-E1）增殖、分化作用的影响。方法：采用MC3T3-E1细胞为体外药物筛选的细胞模型,用MTT法测定药物对成骨细胞的增殖作用,碱性磷酸酶（ALP）试剂盒测定ALP的活性,考察竹节参不同提取物对MC3T3-E1细胞增殖、分化作用的影响。结果：10^-1mg／mL的竹节参水提物和10^-4mg／mL的95％乙醇提取物能显著促进MC3T3-E1细胞的增殖和分化（P〈0．05）。结论：一定浓度的竹节参水提物和95％乙醇提取物能显著促进MC3T3-E1细胞的增殖和分化,该药物具有开发抗骨质疏松药物的潜力。     

仙茅苷促MC3T3-E1成骨样细胞增殖、分化及钙化作用的研究

目的:考察仙茅苷对MC3T3-E1成骨样细胞的增殖、分化及钙化功能的影响。方法:用不同浓度仙茅苷加入MC3T3-E1细胞培养体系中,MTT法检测细胞增殖水平;用茜素红染色法考察骨小结形成能力;以对硝基苯二钠基质动力学法检测碱性磷酸酶的活性。结果:仙茅苷(10-4～10-8mol.L-1)对细胞增殖有促进作用,高浓度(10-4～10-6 mol.L-1时作用明显,其中48h为最佳作用时间;仙茅苷以10-7、10-9 mol.L-1浓度在96h时可促进MC3T3-E1细胞碱性磷酸酶活性;仙茅苷浓度为10-9 mol.L-1时对于骨小结形成最为有效。结论:仙茅苷对MC3T3-E1成骨样细胞的增殖、分化及骨小结形成均有促进作用。     

JNK通路对左归丸含药血清调控MC3T3 成骨细胞Runx2 mRNA表达的影响

目的:探讨c-Jun氨端激酶(JNK)信号通路在左归丸含药血清调控成骨前体细胞(MC3T3-E1)增殖和成骨特异转录因子核心结合因子(Runx2) mRNA表达中的作用.方法:以MC3T3-E1为研究对象,制备左归丸含药血清,选用JNK特异抑制剂SP 600125,实验分为空白对照组、SP 600125组、左归丸组、左归丸加SP 600125组、倍美力组、倍美力加SP 600125组.孵育48 h后,采用噻唑蓝(MTT)法检测SP600125对左归丸含药血清干预MC3T3-E1成骨前体细胞增殖作用的影响,采用Western blot法分析JNK蛋白磷酸化水平,采用Real Time RT-PCR法检测成骨细胞特异转录因子Runx2 mRNA表达情况.结果:与空白对照组比较,左归丸含药血清组显著促进细胞增殖,明显上调p-JNK蛋白和Runx2 mRNA表达(P＜0.01);SP600125显著抑制左归丸含药血清诱导的增殖和p-JNK蛋白表达(P＜0.01),对Runx2 mRNA表达的影响不显著.结论:JNK信号通路的激活可能参与了左归丸含药血清诱导的MC3T3-E1成骨前体细胞增殖,但左归丸含药血清诱导的Runx2mRNA高表达对JNK信号通路依赖不显著.     

Rubus coreanus Miq. extract promotes osteoblast differentiation and inhibits bone-resorbing mediators in MC3T3-E1 cells

To prevent bone loss that occurs with increasing age, certain nutritional and pharmacological factors are needed. In the present study, the ethanol extract from the fruit of Rubus coreanus Miq. (RCE) was investigated for its effect on the function of osteoblastic MC3T3-E1 cells. RCE (10approximately50 microg/ml) caused a significant elevation in cell viability, alkaline phosphatase (ALP) activity, collagen content, and osteocalcin secretion in the cells. The effect of RCE (50 microg/ml) in increasing cell viability, ALP activity, and collagen content was prevented by the presence of 10(-6) M cycloheximide and 10(-6) M tamoxifen, suggesting that RCE's effect results from a newly synthesized protein component and might be partly involved in estrogen action. We then examined the effect of RCE on the H(2)O(2)-induced apoptosis and production of local factors in osteoblasts. Treatment with RCE (10approximately50 microg/ml) decreased the 0.2 mM H(2)O(2)-induced apoptosis and production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and nitric oxide (NO) in osteoblasts. Our data indicate that the enhancement of osteoblast function by Rubus coreanus Miq. may result in the prevention of osteoporosis and inflammatory bone diseases.     

Up-regulation of VEGF by MC3T3-E1 cells treated with curculigoside

Empirical evidence has shown that curculigoside, the main active compound of the traditionally used Chinese herb, Curculigo orchioides (Amaryllidaceae, rhizome), affects bone formation and fracture healing. However, the mechanistic details of these processes remain unclear. Therefore, the effects of curculigoside on immortalized, pre-osteoblastic mouse MC3T3-E1 cells was investigated. Following treatment with curculigoside, MC3T3-E1 cells exhibited an increased rate of proliferation. Higher levels of vascular endothelial growth factor (VEGF), Fms-like tyrosine kinase-1 (Flt-1) and bone morphogenetic protein-2 (BMP-2) were also detected in cell supernatants and cell lysates by ELISA and western blot analysis, respectively. Furthermore, the stimulatory effect of curculigoside was observed at relatively low doses (i.e. 10-100 μg/mL). In combination, these responses to treatment with curculigoside elucidate mechanistic details underlying the therapeutic effects of Curculigo orchioides on bone, and identifies these molecules as potential targets for the treatment of common metabolic bone diseases.     

金叶子异槲皮苷对MC3T3-E1细胞成骨分化的影响

该研究对从金叶子中分离得到的异槲皮苷的成骨活性进行了系统评价。在1×10-4,1×10-5,1×10-6,1×10-7mol·L-1异槲皮苷浓度作用下检测了MC3T3-E1细胞增殖活力和碱性磷酸酶活性;在异槲皮苷作用的第3天对MC3T3-E1碱性磷酸酶、I型胶原以及转录因子Runx2和Osterix的基因表达水平进行了检测;并通过茜素红染色的方法在第21天对MC3T3-E1进行了胞外基质矿化能力的评价。结果显示,异槲皮苷在1×10-7~1×10-5mol·L-1能促进MC3T3-E1细胞的增殖、分化及矿化能力,上调成骨相关基因的表达,而且该作用呈现出一定的浓度依赖性,在浓度为1×10-6mol·L-1时促进作用最强。在1×10-4mol·L-1时表现出明显的细胞毒性。因此,从金叶子中分离得到的异槲皮苷有一定的成骨活性,这可能是传统中药金叶子治疗骨折的主要药效成分。     

Stimulation of osteoblastic differentiation and mineralization in MC3T3-E1 cells by yeast hydrolysate

In a previous study, it was reported that yeast hydrolysate (YH) was effective in promoting bone growth in Sprague‐Dawley (SD) rats. To further clarify the mechanism of YH, the effects of YH on proliferation, differentiation and gene expression in vitro were investigated using osteoblastic cell lines (MC3T3‐E1). Cell proliferation increased significantly as much as 110% of the basal value when cells were treated with 100 µg/mL of YH. Alkaline phosphatase (ALP) activity increased significantly with a YH concentration of 25–100 µg/mL, and the activity increased 152% that of the control at 100 µg/mL. The calcium content increased as much as 129% at 100 µg/mL YH. The gene expression levels of ALP and collagen type II (COL II) significantly increased approximately 1.3‐fold and 1.7‐fold of control, respectively, at 100 µg/mL. YH increased significantly the mRNA level of bone sialoprotein (BSP) but not in a dose‐dependent manner. The mRNA levels of bone morphogenetic proteins (BMP)‐2, BMP‐4, collagen type I (COL I) and osteonectin (ON) did not increase. In summary, YH increased the proliferation of osteoblasts and directly stimulated ALP and bone matrix proteins (e.g. BSP, COL II), and these increases trigger osteoblastic differentiation (e.g. mineralized nodule formation). Copyright © 2010 John Wiley & Sons, Ltd.     

新疆马鹿角提取物对MC3T3-E1成骨细胞RANKL/OPGmRNA表达的影响

目的 研究新疆马鹿角提取物对MC3T3-E1成骨细胞核因子-kβ受体活化因子配体(RANKL)/护骨素(OPG)mRNA表达的影响.方法 体外培养MC3T3-E1成骨细胞,分别加入含不同剂量新疆马鹿角提取物的培养液,72 h后分别提取细胞总RNA,用RT-PCR方法检测RANKL/OPG mRNA表达.结果 培养72 h后RANKL/OPG mRNA表达的灰度值(0.312±0.0710),与对照组(2.017±0.1320)比较,新疆马鹿角提取物呈浓度依赖性降低RANKL mRNA的表达,增加OPG mRNA的表达,各剂量组RANKL/OPG mRNA吸光度比值降低.结论 新疆马鹿角提取物可能通过调节成骨细胞RANKL/OPG的基因表达,而抑制破骨细胞介导的骨吸收.     

基于分子对接的方法探讨桑根酮C对MC3T3-E1细胞的作用

目的:观察桑根酮C(SanC)对地塞米松(DEX)作用下小鼠MC3T3-E1成骨细胞增殖与分化的影响,并探讨其作用机制。方法:将SanC与同源建模所得的Runt-相关转录因子2(Runx2)蛋白结构进行分子对接。不同浓度SanC(8,16,32μmol·L^-1)和1μmol·L^-1DEX共同作用MC3T3-E1细胞,而后采用细胞增殖-毒性检测试剂盒(CCK-8)法检测SanC对MC3T3-E1成骨细胞增殖影响。试剂盒测定MC3T3-E1成骨细胞碱性磷酸酶(ALP)活性和茜素红染色检测骨矿化结节的形成。采用实时荧光定量聚合酶链反应(Real-time PCR)检测Runt-相关转录因子2(Runx2),ALP,和锌指结构转录因子(Osterix)mRNA的表达水平。蛋白免疫印迹法(Western blot)检测Runx2蛋白表达。结果:SanC与Runx2对接打分为-9.78。与正常组比较,DEX组显著降低细胞存活率(P<0.01),其中7 d存活率差异达到最大;与DEX组比较,SanC能显著促进MC3T3-E1的细胞增值(P<0.01),其中32μmol·L^-1SanC作用细胞7 d增殖率差异达到最大。与正常组比较,DEX组Runx2,ALP和Osterix mRNA的表达均有一定程度升高(P<0.05);与DEX组比较,不同浓度SanC组依赖性上调Runx2,ALP和Osterix mRNA的表达(P<0.01)。与正常组比较,DEX组Runx2蛋白表达明显下降(P<0.05);与DEX组比较,SanC干预下细胞Runx2蛋白表达显著升高(P<0.01)。结论:桑根酮C能促进MC3T3-E1成骨细胞增殖、分化和矿化,其机制可能与上调Runx2表达有关。     

Extract prepared from the bark of Cinnamomum cassia Blume prevents glutamate-induced neuronal death in cultured cerebellar granule cells

We studied the protective effect of a water extract from the bark of Cinnamomum cassia Blume on glutamate-induced neuronal death by MTT assay and its action on (45)Ca(2+) influx using cultured rat cerebellar granule cells. In a dose-dependent manner, this extract (10(-5)-10(-4) g/mL) significantly protected against glutamate-induced cell death and also inhibited glutamate-induced (45)Ca(2+) influx. These results suggest that the bark of Cinnamomum cassia has a protective effect on glutamate-induced neuronal death through the inhibition of Ca(2+) influx.     

Cuscuta chinensis seeds water extraction protecting murine osteoblastic MC3T3-E1 cells against tertiary butyl hydroperoxide induced injury

Ethnopharmacological relevance

Cuscuta chinensis (C. chinensis) is a well-known traditional Chinese herb that has been used to treat heart disease, diabetes, liver injury, cancer, and aging.

Materials and methods

Murine osteoblastic MC3T3-E1 cells were treated with various concentrations of C. chinensis water extraction at different time intervals. The antioxidant effect of C. chinensis on MC3T3-E1 cells was evaluated using MTT and TUNEL assays. The effect of C. chinensis on cell cycle was analyzed by flow cytometry with propidium iodide. Lipid peroxidation was measured by the HPLC method. The cellular redox status was determined from the reduced glutathione to oxidized glutathione ratio (GSH/GSSG) and the enzymes involved in glutathione metabolism, including glutathione reductase (GR), Glutathione S-transferase (GST), and Glucose-6-phosphate dehydrogenase (G6PD). The changes in relative mitochondrial transmembrane potential (ΔΨm) in the MC3T3-E1 cells were analyzed with rhodamine 123 staining. Western blot analysis was used to evaluate the levels of cytochrome c (cyto c), Bax, Bcl-2, caspase 3, Sirt3, and IDH2 expressions.

Results

The C. chinensis water extraction protects tertiary butyl hydroperoxide (TBHP)-treated MC3T3-E1 cells from death in a dose-dependent manner. C. chinensis treatment significantly inhibited the reactive oxygen species (ROS) generation, malondialdehyde (MDA) production, and increased the activity of superoxide dismutase (SOD), GR, GST, and G6PD. The release of cyto c from mitochondria was reduced by C. chinensis, which increased the expression of antiapoptotic IDH2, Sirt3, and Bcl-2 and decreased the expression of Bax, cyto c, and caspase 3.

Conclusions

C. chinensis modulated the oxidative stress-induced apoptosis in MC3T3-E1 cells, probably due to its antioxidant activity and functioning via mitochondria-dependent pathways.     

In vitro and in vivo inhibition of glucocorticoid-induced osteoporosis by the hexane extract of Poncirus trifoliata

This study was performed to discover a novel herbal therapeutic for effective glucocorticoid-induced osteoporosis (GIO) treatment and further to clarify its molecular mechanism of action. Ethanol or methanol extracts of 68 edible Korean native plants were screened to find effective natural plant sources for the treatment of GIO, and Poncirus trifoliata (L.) (Rutaceae, PT) was selected as a final candidate because of its high inhibitory activity plus its novelty. The hexane extract of PT (PT-H) inhibited apoptotic cell death in dexamethasone-induced osteoblastic cell lines, C3H10T1/2 and MC3T3-E1. In vivo mouse results indicated that PT-H not only had an inhibitory effect on the bone loss caused by glucocorticoid, but also promoted bone formation. The molecular mechanisms behind the effect of PT-H on GIO were further clarified by screening of differentially expressed genes (DEGs) between dexamethasone (Dex)-induced osteoblastic cells with or without PT-H treatment. Finally, it was found that the expression level of AnxA6 in Dex-induced osteoblastic cells and prednisolone (PD)-treated GIO-model mice was significantly decreased by PT-H treatment. These findings suggest that PT-H has a strong in vitro and in vivo inhibitory effect on GIO, and decreased expression of AnxA6 may play a key role in this inhibition.     

Antimicrobial activities of cinnamon oil and cinnamaldehyde from the Chinese medicinal herb Cinnamomum cassia Blume

Both Cinnamomum verum J.S. Presl. and Cinnamomum cassia Blume are collectively called Cortex Cinnamonmi for their medicinal cinnamon bark. Cinnamomum verum is more popular elsewhere in the world, whereas C. cassia is a well known traditional Chinese medicine. An analysis of hydro-distilled Chinese cinnamon oil and pure cinnamaldehyde by gas chromatography/mass spectrometry revealed that cinnamaldehyde is the major component comprising 85% in the essential oil and the purity of cinnamaldehyde in use is high (> 98%). Both oil and pure cinnamaldehyde of C. cassia were equally effective in inhibiting the growth of various isolates of bacteria including Gram-positive (1 isolate, Staphylococcus aureus), and Gram-negative (7 isolates, E. coli, Enterobacter aerogenes, Proteus vulgaris, Pseudomonas aeruginosa, Vibrio cholerae, Vibrio parahaemolyticus and Samonella typhymurium), and fungi including yeasts (four species of Candida, C. albicans, C. tropicalis, C. glabrata, and C. krusei), filamentous molds (4 isolates, three Aspergillus spp. and one Fusarium sp.) and dermatophytes (three isolates, Microsporum gypseum, Trichophyton rubrum and T. mentagraphytes). Their minimum inhibition concentrations (MIC) as determined by agar dilution method varied only slightly. The MICs of both oil and cinnamaldehyde for bacteria ranged from 75 microg/ml to 600 microg/ml, for yeasts from 100 microg/ml to 450 microg/ml, for filamentous fungi from 75 microg/ml to 150 microg/ml, and for dermatophytes from 18.8 microg/ml to 37.5 microg/ml. The antimicrobial effectiveness of C. cassia oil and its major constituent is comparable and almost equivalent, which suggests that the broad-spectrum antibiotic activities of C. cassia oil are due to cinnamaldehyde. The relationship between structure and function of the main components of cinnamon oil is also discussed.