葛根素对RAW264.7细胞破骨分化的影响

Effects of puerarin on osteoclast differentiation of RAW264.7 cells

背景:葛根素是葛根中含有的一种黄酮类衍生物,其可以预防骨质疏松、促进新骨生成,有望成为治疗骨质破坏相关疾病的潜在药物。目的:观察葛根素对RAW264.7细胞破骨分化能力的影响,探究Notch信号通路在其中的调控作用。方法:将RAW264.7细胞分5组干预培养:对照组加入DMEM高糖培养基;破骨诱导组加入破骨诱导培养基(含巨噬细胞集落刺激因子与核因子κB受体活化因子配体的DMEM高糖培养基);低、中、高浓度葛根素组分别加入含10,25,50μmol/L葛根素的骨诱导培养基。培养7 d后,采用抗酒石酸酸性磷酸酶染色及F-actin染色评估破骨细胞形成情况,RT-PCR检测破骨细胞形成相关基因的表达,Western blot及RT-PCR检测Notch信号通路相关指标表达。结果与结论:①抗酒石酸酸性磷酸酶染色显示,与对照组比较,破骨诱导组破骨细胞形成能力升高(P<0.01);与破骨诱导组比较,低、中、高剂量葛根素组破骨细胞形成能力降低(P<0.05,P<0.01),且呈浓度依赖性;②F-actin染色显示,与对照组比较,破骨诱导组出现边界清晰的F-actin环;与破骨诱导组比较,各浓度葛根素组细胞F-actin环变小,且呈浓度依赖性;③RT-PCR检测显示,与对照组比较,破骨诱导组抗酒石酸酸性磷酸酶、组织蛋白酶K、C-fos mRNA表达升高(P<0.01);与破骨诱导组比较,各浓度葛根素组酒石酸酸性磷酸酶、组织蛋白酶K、C-fos mRNA表达降低(P<0.01),且呈浓度依赖性;④Western blot及RT-PCR检测显示,与对照组比较,破骨诱导组Notch1、Notch2、Hes1、Jagged1、Jagged2的表达升高(P<0.01);与破骨诱导组比较,各浓度葛根素组Notch1、Notch2、Hes1、Jagged1、Jagged2的表达降低(P<0.01),且呈浓度依赖性;⑤结果表明,葛根素通过抑制Notch信号通路来抑制RAW264.7细胞的破骨分化能力。

BACKGROUND:Puerarin is a flavonoid derivative from Pueraria lobata.Studies have found that puerarin can prevent osteoporosis and promote new bone formation,which is expected to become a potential drug for treating diseases related to bone destruction.OBJECTIVE:To observe the effect of puerarin on osteoclast differentiation of RAW264.7 cells and to explore the regulatory effect of Notch signaling pathway.METHODS:RAW264.7 cells were divided into five groups:control group treated with Dulbecco’s modified Eagle medium high sugar medium;osteoclast induction group treated with osteoclast induction medium(Dulbecco’s modified Eagle medium high sugar medium containing macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand);low-,medium-and high-concentration puerarin groups treated with osteoclast induction medium containing 10,25 and 50μmol/L puerarin respectively.After 7 days of culture,tartrate-resistant acid phosphatase staining and F-actin staining were used to observe the role of puerarin in osteoclast formation.RT-PCR was used to detect the expression of genes related to osteoclast formation.Western blot and RT-PCR were used to detect the expression of Notch signaling pathway-related indicators.RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase staining results indicated that osteoclast formation ability was enhanced in the osteoclast induction group compared with the control group(P<0.01),while compared with the osteoclast induction group,low-,middle-,and high-concentration puerarin intervention inhibited osteoclast formation in a concentration-dependent manner(P<0.05,P<0.01).F-actin staining results revealed that clear ring structure could be observed in the osteoclast induction group;compared with the osteoclast induction group,puerarin intervention could inhibit the formation of F-actin ring in a concentration-dependent manner.RT-PCR results showed that compared with the control group,the expression of tartrate-resistant acid phosphatase,cathepsin K and C-fos mRNA was increased in the osteoclast induction group(P<0.01);compared with the osteoclast induction group,puerarin intervention decrease the expression of tartrate-resistant acid phosphatase,cathepsin K and C-fos mRNA in a concentration-dependent manner(P<0.01).Western blot and RT-PCR results showed that the expression of Notch1,Notch2,Hes1,Jagged1 and Jagged2 was increased in the osteoclast induction group compared with the control group(P<0.01);compared with the osteoclast induction group,puerarin intervention reduced the expression of Notch1,Notch2,Hes1,Jagged1,and Jagged2 in a concentration-dependent manner(P<0.01).To conclude,puerarin inhibits the osteoclast differentiation ability of RAW264.7 cells by inhibiting the Notch signaling pathway.