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.     

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.     

The Effect of the Major Components of Fructus Cnidii on Osteoblasts In Vitro

In traditional Chinese medicine, the cause of weak bones or bone loss is generally regarded as a result of kidney deficiency. Fructus Cnidii (FC), which is also known as She-Chuang-Zi, is a traditional herb that has been claimed to have kidney warming effects that invigorate Yang. In this study, we tried to determine the bone production-inducing effect of FC on osteoblastic cells in vitro using osthole, the main component of FC. Osteoblasts were isolated from neonatal Sprague-Dawley rat calvaria using the tissue piece culture method and treated with various concentrations of osthole ranging from 2.5 to 640 μg/mL, together with a blank control. Cell proliferation, alkaline phosphatase (ALP) activity, and bone nodules were measured. The cells were examined by hematoxylin-eosin staining, the Gomori Calcium-Cobalt method and immunofluorescent staining. The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (or MTT) assay, ALP assay, and bone nodule results indicated significantly enhanced osteoblastic proliferation and differentiation at concentrations of osthole ranging from 40 to 320 μg/mL. Concentrations lower than 40 μg/ mL seemed less effective, and cytotoxicity to osteoblasts was observed at concentrations higher than 320 μg/mL. These results indicate that osthole is effective at inducing osteoblastic bone formation through the up-regulation of ALP activity. FC is a Chinese herb used to treat lumbar pain in clinical practice. Further studies concerning the effects and mechanism of osthole on osteoporotic patients and animals should be performed, as these studies may lead to the development of a drug treatment for osteoporosis in the future.     

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.     

丹酚酸B体外干预骨髓间充质干细胞分化过程中cTnT的表达

[目的]观察丹酚酸B(SalB)体外干预骨髓间充质干细胞(MSCs)后细胞形态及心肌特异性蛋白肌钙蛋白T(cTnT)的表达。[方法]培养、纯化及鉴定MSCs,用SalB、5-氮胞苷组(5-aza)、SalB联合5-aza(SalB 5-aza)分别定向诱导分化,观察此过程中MSCs的形态学变化并采用免疫细胞化学法鉴定诱导后MSCs心肌特异性肌钙蛋白T(cTnT)的表达。[结果]诱导后的MSCs体积增大,增殖减慢,并出现肌管样结构;免疫细胞化学结果显示诱导后MSCs表达心肌特异性蛋白cTnT。[结论]SalB在体外定向诱导MSCs分化为心肌细胞的过程中发挥了一定的作用。     

红芪多糖对骨髓间充质干细胞和成骨细胞的影响

目的 研究3种新型红芪多糖(HPS-1、HPS-2、HPS-3)对大鼠骨髓间充质干细胞rBMSCs和颅骨成骨细胞ROBs成骨性分化的影响.方法 采用MTT法检测rBMSCs细胞和ROBs细胞增殖;采用碱性磷酸酶(alkaline phosphatase,ALP)试剂盒检测rBMSCs细胞和ROBs细胞的ALP活性;采用...     

淫羊藿苷促进体外培养大鼠骨髓间 充质干细胞的成骨性分化

目的:研究淫羊藿苷对大鼠体外培养骨髓间充质干细胞(rat bone marrow stromal cells,rBMSCs)成骨性分化的影响.方法:贴壁筛选法体外培养rBMSCs,采用1×10-5mol·L-1的淫羊藿苷进行药物干预,比较淫羊藿苷组和不加药的对照组之间碱性磷酸酶活性、碱性磷酸酶阳性克隆数(CFU-FALP)及钙化结节数量,提取总RNA,RT Real-time PCR检测bFGF,IGF-1,Osterix(OSX),Runx-2 mRNA的表达情况.结果:1×10-5mol·L-1淫羊藿苷可提高碱性磷酸酶(ALP)活性,增加钙化结节和CFU-FALP数量,促进与成骨性分化相关因子bFGF,IGF-1,OSX,Runx-2 mRNA的表达.结论:淫羊藿苷可显著促进rBMSCs的成骨性分化,提示淫羊藿苷具有开发为抗骨质疏松或促进骨折愈合新药的潜力.     

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.     

骨碎补总黄酮对经诱导的兔骨髓间充质干细胞增殖和钙沉积的影响

目的:观察骨碎补总黄酮对兔骨髓间充质干细胞(MSCs)增殖和骨向分化的影响。方法:用密度梯度离心法分离兔MSCs,并对其形态学特征进行观察。用诱导剂对骨髓MSCs向成骨细胞进行诱导分化,并进行形态学观察和免疫细胞化学检测。结果:用密度梯度离心法成功分离获得了高纯度的骨髓MSCs。经骨碎补总黄酮诱导后,ALP和矿化结节染色阳性。结论:骨碎补总黄酮能够诱导兔MSCs向成骨细胞分化。     

心肌微环境下丹酚酸B诱导MSCs 向心肌样细胞分化的研究

[目的]复制心肌缺血-再灌注损伤模型,体外模拟心肌缺氧复氧微环境,观察丹酚酸B(SalB)诱导骨髓间充质干细胞(MSCs)向心肌样细胞分化的作用。[方法]通过向MSCs培养体系中添加心肌细胞裂解液的方法,体外模拟心肌微环境。自成年大鼠骨髓中分离MSCs,设对照组(A组)、心肌细胞裂解液(TJ)组(B组)、SalB组(C组)、TJ+SalB组(D组)。培养4周,观察细胞形态,并采用免疫荧光技术检测cTnT的表达。[结果]除A组MSCs无明显的肌样细胞形成,各诱导组均有肌样细胞分化,D组与C组相比,cTnT表达率明显增高,差异有统计学意义。[结论]心肌细胞裂解液可以诱导MSCs向心肌样细胞分化;心肌微环境的建立有利于SalB诱导MSCs向心肌样细胞分化。     

塞隆骨提取物对体外大鼠成骨样细胞ROS17/2.8的影响

目的研究塞隆骨提取物对体外培养大鼠成骨样细胞ROS17/2.8代谢功能的调节作用,从而在细胞水平上阐述塞隆骨防治骨质疏松的作用机制及药效成分的筛选。方法采用超临界萃取法制备塞隆骨的脂、醇、水提液及水煎提取液,用MTT法测定塞隆骨脂、醇、水提液及水煎液对成骨样细胞的增殖作用;测定碱性磷酸酶(ALP)活性,观察上述提取液对细胞分化的影响。结果10mg/mL塞隆骨脂提液和水提液对大鼠成骨样细胞ROS17/2.8有显著的增殖作用(P<0.01);ALP检测结果显示,10mg/mL脂提液能极显著增强成骨样细胞内ALP活性(P<0.01),1.0、0.1mg/mL能提高ALP活性(P<0.05);10mg/mL水提液能使ALP活性升高(P<0.05)。结论塞隆骨脂和水提取液中分别存在有较高活性的促成骨样细胞增殖、分化的物质,可作为虎骨的替代中药。     

骨康方对大鼠MSCs体外向成骨细胞分化和ALP的影响

目的:观察骨康方对大鼠骨髓基质细胞(marrow stromal cells,MSCs)体外增殖并向成骨细胞分化和其对碱性磷酸酶(ALP)活性的作用。方法:用流式细胞仪鉴定第3代MSCs表面抗原,采用不同浓度的含骨康方药血清联合诱导剂对大鼠MSCs定向诱导分化,观察MSCs诱导前后形态学变化,MTT法观察各组对MSCs的增殖作用并绘制细胞生长曲线,检测ALP活性。结果:CD34表达为阴性,CD44表达为阳性。原代MSCs及诱导后形态正常。细胞生长曲线示各组MSCs数量均随时间延长增加,联合使用高、中浓度骨康方药加诱导剂可显著提高MSCs增殖,并向成骨细胞增殖。联合使用高、中、低不同浓度骨康方药加诱导剂均可显著提高MSCs分化为成骨细胞的ALP活性。结论:骨康方能促进大鼠体外MSCs增殖,并向成骨细胞分化。且能提高成骨细胞的ALP活性,从而增强其成骨能力。     

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.     

Dual Effects of Liquiritigenin on the Proliferation of Bone Cells: Promotion of Osteoblast Differentiation and Inhibition of Osteoclast Differentiation

Bone is constantly controlled by a balance between osteoblastic bone formation and osteoclastic bone resorption. Liquiritigenin is a plant‐derived flavonoid and has various pharmacological effects, such as antioxidative, antitumor, and antiinflammatory effects. Here, we show that liquiritigenin has dual effects on the proliferation of bone cells, regarding the promotion of osteoblast differentiation and the inhibition of osteoclast differentiation. Liquiritigenin‐treated murine osteoblastic MC3T3‐E1 cells showed an increased alkaline phosphatase activity and enhanced phosphorylation of Smad1/5 compared with untreated cells. Moreover, liquiritigenin inhibited osteoclast differentiation, its bone‐resorption activity through slightly decreased the phosphorylation of extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase, and inhibitor of nuclear factor kappa Bα; however, the phosphorylation of Akt and p38 slightly increased in bone marrow‐derived osteoclasts. The expression levels of the osteoclast marker proteins nuclear factor of activated T‐cell cytoplasmic‐1, Src, and cathepsin K diminished. These results suggest that liquiritigenin may be useful as a therapeutic and/or preventive agent for osteoporosis or inflammatory bone diseases. Copyright © 2015 John Wiley & Sons, Ltd.     

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。     

丹酚酸B在大鼠骨髓间充质干细胞分化过程对心肌早期基因Nkx2.5 GATA-4 mRNA表达的影响

目的:探讨丹参单体丹酚酸B(Salvianolic acid B,SalB)在体外诱导大鼠骨髓间充质干细胞(bone marrowmes-enchymal stem cells,MSCs)分化为心肌样细胞的情况及Nkx2.5、GATA-4 mRNA的表达,从而确定大鼠MSCs体外诱导为心肌样细胞的条件、规律及转化细胞的特点。方法:选用成年近交系Wistar大鼠,利用percoll分离液进行密度梯度离心法和直接贴壁法进行分离、提纯MSCs,并进行培养扩增。免疫组化方法对第9代MSCs表面抗原进行鉴定。分别应用10μmol/L5-氮胞苷(5-azacytidine,5-aza)及250μg/L SalB联合5-aza(5-aza+SalB)对第9代的MSCs进行联合诱导24h,于诱导后第4周,用实时荧光定量RT-PCR法检测GATA-4和Nkx2.5基因表达。结果:①第9代MSCs免疫组化染色CD44呈阳性表达,CD34呈阴性表达。②诱导4周后,细胞体积变小,可见由几个细胞连接形成的多核肌管样结构。5-aza组、5-aza+SalB组均出现明确的心肌早期分化基因与5-aza组相比,5-aza+SalB组的NKx2.5、GA-TA-4 mRNA表达明显增加。结论:SalB具有促进5-aza诱导的MSCs向心肌样细胞分化的作用,并且在Nkx2.5基因表达上丹酚酸B的优势更加明显。