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.     

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.     

Stimulatory effects of extract prepared from the bark of Cinnamomum cassia blume on the function of osteoblastic MC3T3-E1 cells

The ethanol extract from the bark of Cinnamomum cassia Blume (CCE) was tested for estrogenic activity. CCE (4-60 microg/mL) significantly induced the growth of MCF-7 cells, an ER-positive human breast cancer cell line, over that of untreated control cells (p < 0.05). In the ER competitive binding assay, CCE showed higher affinity with ERbeta compared with ERalpha. To investigate the bioactivities of CCE, which act on bone metabolism, the effects of CCE on the function of osteoblastic MC3T3-E1 cells and the production of local factors in osteoblasts were studied. CCE (4-60 microg/mL) dose-dependently increased the survival of MC3T3-E1 cells. In addition, CCE (10 and 50 microg/mL) increased alkaline phosphatase (ALP) activity, collagen synthesis and osteocalcin secretion in MC3T3-E1 cells. Treatment with CCE (10 and 50 microg/mL) prevented apoptosis induced by TNF-alpha (10(-10) m) in osteoblastic cells. In the presence of TNF-alpha, culture with CCE (10-100 microg/mL) for 48 h inhibited the production of IL-6 and nitric oxide in osteoblastic MC3T3-E1 cells. These results suggest that Cinnamomum cassia has a direct stimulatory effect on bone formation in vitro and may contribute to the prevention of osteoporosis and inflammatory bone diseases.     

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。     

Inhibition of Ulmus davidiana Planch (Ulmaceae) on bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts

Ulmus davidiana Planch (Ulmaceae) (UD) has long been known to be antiinflammatory in traditional Korean medicine. This experiment investigated the effects of UD on bone resorption using bone cell culture. Different concentrations of crude extract of UD were added to mouse bone cell culture. The mitochondrial activity of the bone cells after exposure of UD was determined by colorimetric 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). It was demonstrated that UD has potential effects on bone cell culture without cytotoxicity. The most effective concentration of UD in bone cells was 100 microg/mL. Cathepsin K (Cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. When mouse long bone cells including osteoclasts and osteoblasts were treated with UD, UD prevented the osteoclast-mediated intracellular processing of Cat K, suggesting that UD may disrupt the intracellular transport of pro Cat K. Since secreted proenzymes have the potential to reenter the cell via the mannose-6-phosphate (M6P) receptor, to prevent this possibility, UD was tested in the absence or presence of M6P. Inhibition of Cat K processing by UD was observed in a dose-dependent manner. Furthermore, the addition of M6P resulted in enhanced potency of UD. UD dose-dependently inhibited in vitro bone resorption with a potency similar to that observed for inhibition of Cat K processing.     

Suppressive effects of a water extract of Ulmus davidiana Planch (Ulmaceae) on collagen-induced arthritis in mice

Ulmus davidiana Planch (Ulmaceae) has long been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone resorption. Therefore, this study was undertaken to address (1) whether the water extract of the bark of Ulmus davidiana Planch (Ulmaceae) (UD) can modulate the expression of inducible inflammatory cytokines in mice; (2) in order to assess the therapeutic effects of UD in collagen-induced arthritis (CIA) in mice. DBA/1 mice were immunized with bovine type II collagen. After a second collagen immunization, mice were treated with UD orally at 100mg/kg once a day for 3 weeks. Paws were evaluated macroscopically for redness, swelling and deformities. The levels of TNF-alpha and IL-1beta in the ankle were examined. The severity of arthritis within the knee joints was evaluated by histological assessment of cartilage destruction and pannus formation. Administration of UD significantly suppressed the progression of CIA and inhibited the production of TNF-alpha and IL-1beta in the paws. The erosion of cartilage was dramatically reduced in mouse knees after treatment with UD. In the serum of UD-treated mice, the levels of IL-4 and IL-10, anti-inflammatory cytokines, were increased. From the results, it was concluded that administration of UD has therapeutic effects on CIA including protection of cartilage and RA for a potential therapy.     

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。     

Asperosaponin VI, a saponin component from Dipsacus asper wall, induces osteoblast differentiation through bone morphogenetic protein-2/p38 and extracellular signal-regulated kinase 1/2 pathway

Osteoporosis is a reduction in skeletal mass because of the loss of osteoblastic activity or an increase in osteoclastic activity. The survival of osteoblast cells plays a crucial role in the development of osteoporosis. Asperosaponin VI (ASA VI) is a kind of saponin in the medicinal herb Dipsacus asper Wall which has long been used as an antiosteoporosis drug. The assay of cell proliferation, alkaline phosphatase (ALP) activity and measurement of mineralized matrix, showed that ASA VI exhibited a significant induction of proliferation, differentiation and mineralization in MC3T3-E1 and primary osteoblastic cells. Induction of differentiation by ASA VI was associated with increased bone morphogenetic protein-2 (BMP-2), indicating that BMP-2 is essential in ASA VI to mediate osteoblast maturation and differentiation. In addition, ASA VI may induce differentiation by increasing the activity of p38 and ERK1/2. In conclusion, ASA VI may induce osteoblast maturation and differentiation, and then increase bone formation via increasing BMP-2 synthesis, and activating p38 and ERK1/2.     

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.     

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。     

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.     

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.     

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).     

补肾活血固齿方对小鼠颅顶前成骨细胞碱性磷酸酶活性的影响

目的观察补肾活血固齿方对小鼠颅顶前成骨细胞(MC3T3-E1)碱性磷酸酶(ALP)活性的影响,探讨其对成骨细胞分化的作用机制。方法将20只SD大鼠随机分为含药血清组和无药血清组,各10只。含药血清组大鼠予补肾活血固齿方灌胃,无药血清组予等容积0.9%氯化钠注射液灌胃,均灌胃7 d后抽取腹主动脉血制备成含药血清和无药血清。胎牛血清组为直接购买的PAA胎牛血清。分别用含10%含药血清、10%无药血清及10%胎牛血清的培养基培养MC3T3-E1细胞24、48、72 h,检测MC3T3-E1细胞中ALP的含量。结果含药血清组培养24、48、72 h后MC3T3-E1细胞ALP含量均高于胎牛血清组及无药血清组同期水平,比较差异均有统计学意义(P0.05),但胎牛血清组与无药血清组同期差异无统计学意义(P0.05);含药血清组MC3T3-E1细胞ALP含量随培养时间的延长而明显增加(P0.05)。结论补肾活血固齿方可增强MC3T3-E1细胞中ALP活性,促进MC3T3-E1细胞向成骨细胞分化,且均有一定时间依赖性。     

The effect of the major components of Salvia Miltiorrhiza Bunge on bone marrow cells

Salvia Miltiorrhiza Bunge (SMB), a traditional Chinese medicinal herb, has been alleged to support bone healing. However, the effects of the isolated major components of SMB on osseous cells and their corresponding effective doses are still unclear. In the present study, the effects of three components of SMB, including tanshinone IIA (Ts), salvianolic acid B (salB) and protocatechuic aldehyde (Pca), on mesenchymal bone marrow cells with the potential for osteoblastic differentiation were investigated. Various concentrations of Ts, salB and Pca were added to a rat bone marrow cell culture. The total metabolic activity and differentiation of bone marrow cells were evaluated by a metabolic assay and alkaline phosphatase (ALP) activity, respectively. The morphology and number of cells was observed by phase contrast microscopy and fluorescent microscopy after propidium iodide staining, respectively. Ts suppressed the growth and differentiation of bone precursor cells. SalB exhibited a biphasic effect: the high concentration of 160 microg/mL significantly depressed the population of bone marrow cells, however, lower concentrations (3-80 microg/mL) enhanced the total metabolic activity and their ALP expression. Pca suppressed the bone marrow cell population in a dose-dependent manner. Therefore, SalB has the potential to ameliorate bone healing by stimulating both the total metabolic activity and ALP activity of osteoblastic cells. Aqueous extracts, which preferably contain salB over Pca and are free of Ts therefore are recommended for bone formation.