Quercitrin and Taxifolin stimulate osteoblast differentiation in MC3T3-E1 cells and inhibit osteoclastogenesis in RAW 264.7 cells

Flavonoids are natural antioxidants that positively influence bone metabolism. The present study screened among different flavonoids to identify biomolecules for potential use in bone regeneration. For this purpose, we used MC3T3-E1 and RAW264.7 cells to evaluate their effect on cell viability and cell differentiation. First, different doses of chrysin, diosmetin, galangin, quercitrin and taxifolin were analyzed to determine the optimum concentration to induce osteoblast differentiation. After 48 h of treatment, doses ≥100 μM of diosmetin and galangin and also 500 μM taxifolin revealed a toxic effect on cells. The same effect was observed in cells treated with doses ≥100 μM of chrysin after 14 days of treatment. However, the safe doses of quercitrin (200 and 500 μM) and taxifolin (100 and 200 μM) induced bone sialoprotein and osteocalcin mRNA expression. Also higher osteocalcin secreted levels were determined in 100 μM taxifolin osteoblast treated samples when compared with the control ones. On the other hand, quercitrin and taxifolin decreased Rankl gene expression in osteoblasts, suggesting an inhibition of osteoclast formation. Indeed, osteoclastogenesis suppression by quercitrin and taxifolin treatment was observed in RAW264.7 cells. Based on these findings, the present study demonstrates that quercitrin and taxifolin promote osteoblast differentiation in MC3T3-E1 cells and also inhibit osteoclastogenesis in RAW264.7 cells, showing a positive effect of these flavonoids on bone metabolism.     

盐酸二甲双胍对成骨细胞增殖、BMP-2及Cbfa-1基因表达的影响

目的初步研究二甲双胍（MF）在体外对小鼠颅盖骨成骨细胞增殖、骨形态发生蛋白一2（BMP一2）及核心结合因子（Cbfa一1）mRNA表达的影响,探讨二甲双胍对骨代谢的可能作用机制。方法（1）分离培养原代颅盖骨成骨细胞并对其进行鉴定。（2）以乳鼠成骨细胞为体外实验模型,不同浓度（0、50、100、200、400Ixmol／L）的MF干预体外培养的成骨细胞24h后,M1vr法检测成骨细胞的增殖能力,实时荧光定量PCR法检测成骨细胞BMP-2及Cbfa-1基因表达。结果二甲双胍干预成骨细胞24h后,可促进成骨细胞的增殖,在浓度400μxmol／L的OD值最大为0．298±0．047（P〈0．05）;可促进BMP-2及Cbfa-1mRNA的表达,呈剂量效应关系。结论二甲双胍可促进成骨细胞的增殖和分化,可能通过调节BMP-2及Cbfa-1的表达,从而促进骨的形成。     

染料木素促成骨样细胞增殖作用及其机制研究

目的观察染料木素(genistein)对成骨样细胞MC3T3-E1细胞增殖及骨形成蛋白-2(BMP-2)、β-连环素(β-catenin)表达的影响。方法培养成骨样细胞MC3T3-E1,加入不同浓度的染料木素(1×10-10、1×10-9、1×10-8、1×10-7、1×10-6和1×10-5mol·L-1),以1×10-9mol·L-1雌激素(β-estradiol,E2)作为阳性对照组,采用MTT比色试验法和碱性磷酸酶(ALP)活性检测法,观察染料木素作用后MC3T3-E1的增殖及分化情况,以Westernblot方法检测成骨样细胞中BMP-2和β-catenin蛋白的表达。结果染料木素可促进MC3T3-E1细胞增殖,染料木素作用后细胞的ALP值也明显升高,呈浓度依赖性;染料木素可增加BMP-2和β-catenin表达,且呈浓度依赖性。结论染料木素促进MC3T3-E1细胞增殖与分化,可通过调节BMP-2和Wnt/β-catenin信号通路而影响成骨样细胞的活性。     

Geniposide promotes the proliferation and differentiation of MC3T3-E1 and ATDC5 cells by regulation of microRNA-214

The research plans to make sure how Geniposide (GEN) functions in osteoblast proliferation and differentiation. The MC3T3-E1 and ATDC5 cells were treated with the GEN, XAV-939 and/or transfected with microRNA (miR)-214 mimic or corresponding control. Cell viability was detected with the CCK-8. The CyclinD1, Runx2, Osx, Ocn, Wnt3a and β-catenin were individually quantified via western blot. The cell cycle was tested by cell cycle analysis assay. The ALP activity was tested by ALP assay. qRT-PCR was used to examine the miR-214 expression level. The cell viability and the expressions of the CyclinD1, Runx2, Osx, Ocn Wnt3a and β-catenin, as well as the ALP activity were individually and significantly promoted by the GEN. Besides, miR-214 was down-regulated by the GEN. The XAV-939 or the miR-214 mimic destroyed the promotional effect of GEN on these elements above. In conclusion, GEN induced the proliferation and differentiation of the MC3T3-E1 and ATDC5 cells by targeting the miR-214 through Wnt/β-catenin activation.     

Kaempferol protects MC3T3-E1 cells through antioxidant effect and regulation of mitochondrial function

Kaempferol, a natural flavonoid present in fruits, vegetables, and teas, provides beneficial effects for human health. In this study, we investigated the protective effects of kaempferol on antimycin A (AMA)-induced toxicity in osteoblast-like MC3T3-E1 cells. Exposure of MC3T3-E1 cells to AMA caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, intracellular calcium ([Ca²⁺]_i) elevation, and reactive oxygen species (ROS) production. Pretreatment with kaempferol prior to AMA exposure significantly reduced AMA-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, [Ca²⁺]_i elevation, and ROS production. Kaempferol also induced the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B), and CREB (cAMP-response element-binding protein) inhibited by AMA, which result demonstrates that kaempferol utilizes the PI3K/Akt/CREB pathway to augment metabolic activity inhibited by AMA. All these data indicate that kaempferol may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders.     

Isoliquiritigenin impairs insulin signaling and adipocyte differentiation through the inhibition of protein-tyrosine phosphatase 1B oxidation in 3T3-L1 preadipocytes

Isoliquritigenin (ISL) is an abundant dietary flavonoid with a chalcone structure, which is an important constituent in Glycyrrhizae Radix (GR). ISL exhibits anti-oxidant activity, and this activity has been shown to play a beneficial role in various health conditions. However, it is unclear whether the anti-oxidant activity of ISL affects insulin signaling pathway and lipid accumulation of adipocytes. We sought to investigate the effects and molecular mechanisms of ISL on insulin-stimulated adipogenesis in 3T3-L1 cells. We investigated whether ISL attenuates insulin-induced Reactive Oxygen Species (ROS) generation, and whether ISL inhibits the lipid accumulation and the expression of adipogenic-genes during the differentiation of 3T3-L1 cells. ISL blocked the ROS generation, suppressed the lipid accumulation and the expression of adipocyte-specific proteins, which are increased in response to insulin stimulation during adipocyte differentiation of 3T3-L1 cells. We also investigated whether the anti-oxidant capacity of ISL is involved in regulating the molecular events of insulin-signaling cascade in 3T3-L1 adipocytes. ISL restores PTP1B activity by inhibiting PTP1B oxidation and IR/PI3K/AKT phosphorylation during the early stages of insulin-induced adipogenesis. Our findings show that the anti-oxidant capacity of ISL attenuated insulin IR/PI3K/AKT signaling through inhibition of PTP1B oxidation, and ultimately attenuated insulin-induced adipocyte differentiation of 3T3-L1 cells.