补肾活血复方含药血清对大鼠成骨细胞磷酸化GSK-3β、p38MAPK及ERK1/2蛋白的影响

目的:观察补肾活血复方含药血清对大鼠成骨细胞磷酸化糖原合成激酶3β(GSK-3β)、p38丝裂原活化蛋白激酶(p38 MAPK)及细胞外信号调节激酶1/2(ERK1/2)蛋白的作用。方法:3月龄SD雌性大鼠灌胃制备空白血清和含药血清,并分离培养1日龄新生鼠颅骨成骨细胞。实验分为空白血清组和含药血清组,分别采用MTT、ELISA、Western-blot等方法检测两组成骨细胞的增殖、ALP和BGP的分泌及磷酸化GSK-3β、p38 MAPK和ERK1/2蛋白的变化。结果:与空白血清组相比,含药血清组可明显促进成骨细胞的增殖、ALP和BGP的分泌,并促进胞内磷酸化p38 MAPK和ERK1/2蛋白的表达,对磷酸化GSK-3β蛋白的表达未见明显影响。结论:补肾活血复方含药血清可促进大鼠成骨细胞的增殖和分化,提高p38 MAPK和ERK1/2蛋白磷酸化水平。     

淫羊藿苷对骨髓间充质干细胞骨向诱导的实验研究

目的通过研究淫羊藿苷(icariin,ICA)对骨髓间充质干细胞(bone mesenchymal stem cells,BM-SCs)增殖和骨向分化能力的影响,并观察其促BMSCs骨向分化过程中对细胞因子转化生长因子β1(TGF-β1)、骨形成蛋白-2(BMP-2)表达的影响,分析可能的作用途径,为阐释其防治骨质疏松(osteoporosis,OP)的作用机理提供实验依据,并为以TGF-β1、BMP-2作为筛选防治OP药物进行实验研究。方法以碱性磷酸酶(ALP)等指标确定干预药物不同浓度梯度中最佳促BMSCs骨向分化剂量;分组实验,通过检测诱导分化过程中ALP和钙化结节等指标的表达,来观察其对BMSCs骨向分化能力的影响;采用ELISA法检测骨向分化过程中TGF-β1、BMP-2的表达。结果(1)ICA最佳促BMSCs骨向分化浓度为1×10-9mol/L。ICA10-9组能促进成骨指标的表达和TGF-β1和BMP-2分泌增加;(2)能促BMSCs骨向分化能力的ICA各诱导组均伴随TGF-β1和BMP-2分泌增加。结论ICA能促进BMSCs骨向分化;在BMSCs骨向分化过程中,上调TGF-β、BMP-2的表达量可能是各干预药物促进BMSCs骨向分化的作用机制之一。     

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.     

戴村骨伤膏对人成骨样细胞(MG-63细胞)增殖及BMP-2表达的影响

目的:为了研究戴村骨伤膏对骨折愈合的影响,我们将其运用于人成骨样细胞(MG-63细胞)体外的细胞试验,观察其对细胞增殖及BMP-2表达的影响。方法:在体外培养的MG-63细胞中,加入不同浓度的戴村骨伤膏药物溶液作用后,使用四甲基偶氮唑蓝比色法(MTT)检测细胞增殖以及RT-PCR反应法检测细胞中BMP-2表达情况。结果:戴村骨伤膏药在早期能较明显的促进成骨细胞MG-63的增殖分化及BMP-2的表达,并且效果随着药物浓度的升高而增强,但是随着时间延长药物对MG-63细胞增殖反而起抑制作用。结论:说明戴村骨伤膏对MG-63细胞增殖具有促进和抑制双重效应,其效应强度随作用时间不同而不同。     

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.     

骨碎补总黄酮对晚期糖基化终末产物作用下成骨细胞分化及p38和ERKl/2蛋白激酶表达的影响

目的 研究骨碎补总黄酮(TFDF)对晚期糖基化终末产物(AGEs)作用下成骨细胞分化活性及对细胞外信号调节蛋白激酶(ERK1/2)和p38丝裂原活化蛋白激酶(p38MAPK)信号蛋白的影响,探讨骨碎补总黄酮防治骨质疏松的作用机理.方法 二次酶消化法分离培养新生SD大鼠颅骨成骨细胞以及活性观察;pNPP、ELISA、茜素红染色法分别检测不同质量浓度晚期糖基化终末产物对成骨细胞碱性磷酸酶活性(ALP)、Ⅰ型胶原(Col I)、骨钙素(BGP)以及矿化能力的影响,并观察骨碎补总黄酮对晚期糖基化终末产物作用下成骨细胞分化的干预;Westem-hlot检测骨碎补总黄酮对晚期糖基化终末产物作用下,成骨细胞p38和ERK1/2蛋白磷酸化的影响.结果 晚期糖基化终末产物(200、400、800 mg/L)可以降低成骨细胞表达ALP、Col I、BGP,降低其矿化能力.骨碎补总黄酮可以剂量依赖性的提高晚期糖基化终末产物(400 mg/L)作用下成骨细胞表达ALP、Col I、BGP和矿化能力.骨碎补总黄酮(50 mg/L)可以提高晚期糖基化终末产物(400 mg/L)作用下p38和ERK1/2的蛋白磷酸化.结论 骨碎补总黄酮可以提高晚期糖基化终末产物作用下成骨细胞分化和矿化能力,其机制可能和提高p38和ERK1/2信号蛋白的磷酸化有关.     

苦参碱通过MAPK信号转导通路促进U937细胞凋亡

目的:探讨苦参碱(mattine,Mat)对人淋巴瘤细胞株(U937)的抗癌作用及其分子机制.方法:用0.1,0.2,0.3,0.4,0.5 g·L~(-1)Mat处理U937细胞后,分别采用倒置显微镜和电子显微镜观察细胞形态学变化;台盼蓝拒染法及MTT实验检测细胞增殖抑制作用;Westem blot方法检测细胞MAPK的磷酸化活性.结果:0.2 g·L~(-1) Mat对人组织淋巴瘤U937细胞的增殖有抑制作用;0.2 g·L~(-1) Mat作用U937细胞48 h后,倒置显微镜下可见细胞数目减少,有些细胞变小、变圆,随着Mat作用浓度的增加,U937细胞逐步出现增多的凋亡细胞;Mat可以抑制U937细胞中ERK的活性,并在一定程度上提高p38和JNK的活性.结论:Mat可以在体外抑制人淋巴瘤细胞U937的增殖作用,诱导其凋亡,并且Mat可能通过抑制U937细胞中ERK的活性,提高p38和JNK的活性发挥其诱导凋亡作用.     

骨碎补总黄酮对高糖作用下成骨细胞分化及ERK_(1/2)和p38蛋白激酶表达的影响

目的:研究骨碎补总黄酮(TFDF)对高浓度葡萄糖作用下成骨细胞(OB)分化活性及对细胞外信号调节蛋白激酶(ERK1/2)和p38丝裂原活化蛋白激酶(p38MAPK)信号蛋白的影响,为TFDF能否用来防治糖尿病性骨质疏松提供细胞分子学依据。方法:二次酶消化法分离培养新生SD大鼠颅骨OB及活性观察;MTT法检测TFDF对OB的细胞毒性,确定实验药物在培养基中的添加剂量;pNPP、ELISA、茜素红染色法分别检测不同浓度葡萄糖对OB碱性磷酸酶活性(ALP)、Ⅰ型胶原(ColⅠ)、骨钙素(BGP)以及矿化能力的影响,并观察TFDF对高糖作用下OB分化活性的干预作用;Western-blot检测TFDF对高糖作用下OB ERK1/2和p38蛋白磷酸化的情况。结果:原代分离培养新生SD大鼠OB,具有典型的OB分化特性;根据TFDF对OB的毒性试验,选择TFDF的浓度25、50、100 mg/L为实验梯度浓度;葡萄糖(25、50 mmol/L)可以降低OB表达ALP、ColⅠ、BGP,降低其矿化能力;TFDF能剂量依赖性的提高高糖(25 mmol/L)作用下OB表达ALP、ColⅠ、BGP和矿化能力;TFDF(50 mg/L)能提高高糖(25 mmol/L)作用下p38和ERK1/2的蛋白磷酸化。结论:高糖可以降低OB分化和矿化能力,TFDF可提高高糖作用下OB的分化和矿化能力,其作用机理可能和提高p38和ERK1/2信号蛋白的磷酸化有关。     

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.     

诺卡氏菌属菌株04-5195发酵产物Amicoumacin B对BMP-2基因作用的研究

 目的研究Amicoumacin B对骨形态形成蛋白因子Ⅱ(BMP-2)基因的作用。方法构建以bmp-2启动子为作用靶点、荧光素酶为报告基因、通过测定模型荧光强度而间接反映药物上调活性的检测模型,研究诺卡菌属菌株04-5195发酵产物Amicoumacin B对BMP-2基因的作用。结果Amicoumacin B可上调BMP-2基因的表达。结论Amicoumacin B可由诺卡菌属菌株产生,并具有促进成骨细胞活性的潜能。     

Effects of Tinospora cordifolia (Menispermaceae) on the proliferation, osteogenic differentiation and mineralization of osteoblast model systems in vitro

Ethnopharmacological relevance

Ancient Indian ayurvedic literature prescribes Tinospora cordifolia as a remedy to rheumatoid arthritis, inflammatory and allied diseases of musculo skeletal system.

Aim

To investigate the effects of the alcoholic extract of Tinospora cordifolia (TC) on the proliferation, differentiation and mineralization of bone like matrix on osteoblast model systems in vitro and hence its possible use as a potential anti-osteoporotic agent.

Materials and methods

Two in vitro osteoblast model systems were used in the study viz., human osteoblast-like cells MG-63 and primary osteoblast cells isolated from femur of rats. Cell growth and viability was assessed by standard colorimetric assays like MTT assay. The cell differentiation into osteoblastic lineage was evaluated by the activities of bone marker alkaline phosphatase. The effect of the extract on matrix mineralization was assessed by alizarin red-s staining and Von kossa staining. Cell morphology was studied by phase contrast microscopy and light microscopy (Giemsa/crystal violet staining).

Results

Results indicate that the alcoholic extract of TC at a dosage of 25 μg/ml stimulated the growth of osteoblasts, increased the differentiation of cells into osteoblastic lineage and increased the mineralization of bone like matrix on both the osteoblast model systems used in the study. Cell morphology studies clearly indicated the increase in cell numbers and absence of adverse change in the cell morphology on treatment with the extract.

Conclusion

TC extract has a potential influence on osteogenesis and hence its use could be explored as a potential anti-osteoporotic agent.     

大茴香醛缩氨基均三唑席夫碱对人肝癌细胞分化和凋亡的影响

 目的 观察大茴香醛缩氨基均三唑席夫碱对人肝癌细胞 BEL-7402 的诱导分化和凋亡作用。 方法 用不同浓度的 3( 3- 吡啶 )-4-[ （ 4- 甲氧基 - 苯亚甲基）氨基 ]-5- 甲硫基 -1 , 2 , 4- 三唑（ LH-38 ）与 BEL-7402 细胞体外培养。 四甲基偶氮唑蓝（ MTT ）法检测细胞增殖和 LH-38 对细胞增殖的抑制作用。用 Hoechst 33258 荧光染色细胞形态学和 Annexin V/PI 双标记技术观察细胞凋亡变化。 放射免疫法检测 BEL-7402 细胞甲胎蛋白（ α-FP ）和白蛋白（ Alb ）的分泌量,以了解细胞分化状态。 蛋白质免疫印迹（ Western blotting ）半定量分析 Caspase-3 和 p53 的表达。可见光法测定超氧化物歧化酶（ SOD ）和过氧化氢酶（ CAT ）活力 。 结果 LH-38 在 0.1~10 μmol?L^-1 的浓度范围能抑制细胞增殖,且抑制率随浓度和时间的增加而增高。 10 和 1.0 μmol?L^-1 的 LH-38 使细胞 Alb 、 Caspase-3 和 p53 的蛋白表达明显增加, α-FP 含量下降。 SOD 活性升高 ( P<0.01) ,而 CAT 活性降低,差异有显著性 ( P<0.01, P<0.05) 。药物浓度在 10 μmol?L^-1 作用 48 h , BEL-7402 细胞皱缩,呈现凋亡各期改变,细胞凋亡率显著高于对照组 ( P<0.05) 。 结论 大茴香醛缩氨基均三唑席夫碱对人肝癌 BEL-7402 细胞具有抗增殖、促分化和凋亡作用,作用可能与过氧化氢的氧化损伤作用有关。     

淫羊藿苷对人成骨细胞增殖与分化的影响

目的:观察淫羊藿苷对人成骨细胞增殖和分化的影响。方法:将人骨髓基质干细胞定向诱导分化为成骨细胞,取第3,4代细胞,MTT法观察淫羊藿苷对人成骨细胞的增殖作用,碱性磷酸酶(ALP)比活性测定观察淫羊藿苷对人成骨细胞分化的影响,RT-PCR方法检测淫羊藿苷对人成骨细胞 BMP-2 mRNA表达的影响。结果:浓度为20μg·mL^-1的淫羊藿苷能够显著促进人成骨细胞的增殖和分化,且使BMP-2 mRNA的表达升高。结论:淫羊藿苷具有促进人成骨细胞增殖和分化的作用,这一作用可能与其升高人成骨细胞BMP 2 mRNA的表达有关。     

ent-Kaurane diterpenoids from Croton tonkinensis stimulate osteoblast differentiation

Four new ent-kaurane diterpenoids (1-4) were isolated from the leaves of Croton tonkinensis by bioactivity-guided fractionation using an in vitro osteoblast differentiation assay. Their structures were identified as ent-11β-acetoxykaur-16-en-18-ol (1), ent-11α-hydroxy-18-acetoxykaur-16-ene (2), ent-14β-hydroxy-18-acetoxykaur-16-ene (3), and ent-7α-hydroxy-18-acetoxykaur-16-ene (4). Compounds 1-4 significantly increased alkaline phosphatase activity and osteoblastic gene promoter activity. Compounds 1-3 also increased the levels of ALP and collagen type I alpha mRNA in C2C12 cells in a dose-dependent manner. These results suggest that ent-kaurane diterpenoids from C. tonkinensis have a direct stimulatory effect on osteoblast differentiation and may be potential therapeutic molecules against bone diseases such as osteoporosis.     

酢浆草提取物对成骨细胞增殖及分化的影响

目的:探讨酢浆草水提物对体外培养的成骨细胞增殖、分化和矿化的影响。方法:不同质量浓度酢浆草水提物(10,100,200 mg·L-1)作用人成骨肉瘤细胞株SaOS-2后,MTT法测定细胞增殖情况、磷酸对硝基苯酯(PNPP)法检测碱性磷酸酶(ALP)活性,酶联免疫吸附法(ELISA)法检测钙离子(Ca2+)和骨钙素(OTC)的分泌以及用茜素红染色法测定成骨细胞矿化结节数。结果:与空白组比较,酢浆草水提物能明显促进SaOS-2细胞的生长(P<0.01);ALP活性呈时间依赖性增加(P<0.05),以100 mg·L-1剂量组最为显著(P<0.01);增强了细胞分泌Ca2+(P<0.05)和OTC的能力(P<0.01);并且酢浆草中、高剂量组可以明显促进矿化结节形成(P<0.05)。结论:酢浆草水提物在体外可以促进成骨细胞的增殖、分化和矿化,提示酢浆草可能具有促进骨形成的能力。     

葛根素对成骨细胞增殖及骨形态发生蛋白的影响

目的:探讨葛根素对成骨细胞增殖及骨形态发生蛋白的影响。方法:采用血清药理学方法制备葛根素含药血清,分为葛根素组、阳性对照组和阴性对照组。从SD大鼠乳鼠颅骨获取成骨细胞,分别用含药血清培养分离得到的成骨细胞。采用CCK-8检测成骨细胞增殖、酶联免疫吸附试验检测骨形态发生蛋白2(BMP-2)的表达;采用微量酶标法检测碱性磷酸酶的活性。结果药物作用24、48和72 h后,葛根素组D值显著高于阴性对照组(P0.05),且显著低于阳性对照组(P0.05)。随着时间的增加,葛根素组和阳性对照组D值在药物作用48 h达到最高,至72 h后有下降趋势。药物作用3 d和7 d后,葛根素组BMP-2表达水平均显著高于阴性对照组(P0.05),且显著低于阳性对照组(P0.05)。药物作用3d和7 d后,葛根素组BMP-2表达水平均显著高于阴性对照组(P0.05),且显著低于阳性对照组(P0.05)。药物作用3 d和7 d后,葛根素组碱性磷酸酶活性均显著高于阴性对照组(P0.05),且显著低于阳性对照组(P0.05)。结论葛根素可能通过诱导BMP-2表达促进增殖,活化碱性磷酸酶活性促进生物矿化。