松果菊苷对骨质疏松症模型小鼠破骨细胞活性的影响

Effects of echinacoside on osteoclast activity in a mouse model of osteoporosis

目的研究松果菊苷(ECH)对骨质疏松症(OP)模型小鼠破骨细胞活性的影响。方法选取10周龄雌性C57/BL6小鼠28只,随机分为假手术(Sham)组、OP组和ECH低、高剂量组,每组7只。OP组和ECH两组小鼠通过去除双侧卵巢建立OP模型,之后ECH两组分别采用10、20 mg/kg ECH进行腹腔注射干预,2 d/次,连续干预8周后,处死各组小鼠获取完整的股骨。股骨远端切片后进行阿利新蓝(ABH)染色、抗酒石酸酸性磷酸酶(TRAP)染色以及免疫组化检测组织蛋白酶K(CTSK)、核因子κB磷酸化P65(NF-κB p-P65)蛋白表达情况。结果股骨远端ABH染色:与Sham组比较,OP组股骨远端骨小梁变薄、稀疏,髓腔内脂肪细胞堆积;与OP组比较,ECH组股骨远端骨小梁数量增加,骨小梁变粗,髓腔内脂肪细胞减少,尤其是高剂量组ECH作用更加明显。股骨远端TRAP染色:与Sham组比较,OP组股骨远端破骨细胞分化显著增加[(6.79±0.66)N.Oc/T.A比(2.15±0.28)N.Oc/T.A,P<0.01];与OP组比较,ECH低、高剂量组股骨远端破骨细胞分化减少,尤其是高剂量组ECH抑制作用更加明显[(4.35±1.03)N.Oc/T.A、(3.75±0.60)N.Oc/T.A比(6.79±0.66)N.Oc/T.A,P<0.01]。股骨远端免疫组化(CTSK、NF-κB p-P65)染色:与Sham组比较,OP组股骨远端CTSK、NF-κB p-P65表达显著上调[CTSK:(0.031±0.005)%比(0.010±0.003)%;NF-κB p-P65:(0.013±0.002)%比(0.003±0.003)%;P<0.01];与OP组比较,ECH低、高剂量组股骨远端CTSK、NF-κB p-P65表达显著下调,高剂量组ECH抑制作用更加明显[CTSK:(0.018±0.002)%、(0.015±0.003)%比(0.031±0.005)%;NF-κB p-P65:(0.009±0.001)%、(0.007±0.002)%比(0.013±0.002)%;P<0.01]。结论ECH可能通过抑制NF-κB信号通路,减弱破骨细胞活性,改善OP模型小鼠骨量丢失。

Objective To investigate the effect of echinacoside(ECH)on osteoclast activity in a mouse model of osteoporosis(OP).Methods Twenty-eight female C57/BL6 mice of ten-week-old were randomly divided into Sham,model,and ECH-high or low groups(n=7 each group).The OP model was established by removing bilateral ovaries,after which the ECH groups of mice were injected intraperitoneally with 10(low)or 20(high)mg/kg ECH once every two days for 8 weeks.At the end of the experiments,mice were sacrificed to obtain the femurs.The distal femur was sectioned and stained for Alcian blue/Hematoxylin(ABH),tartrate-resistant acid phosphatase(TRAP),and immunohistochemistry of Cathepsin K(CTSK)and nuclear factor kappa-B(NF-κB)p-P65.Results The ABH staining of the distal femur showed that the trabecular bone of the distal femur in the model mice was thinner and sparse and adipocytes were accumulated in the medullary cavity compared with the Sham group.In contrast,the number of bone trabeculae was increased,the bone trabeculae became thicker,and the adipocytes in the medullary cavity were decreased after treatment compared with those of the model mice,especially after high dose of ECH treatment.The TRAP staining of the distal femur revealed that the osteoclast differentiation of the distal femur in the model mice was significantly increased compared with the Sham mice(6.79±0.66 vs.2.15±0.28 N.Oc/T.A;P<0.01);however,osteoclast differentiation was reduced after ECH treatment vs.the model mice,especially in ECH-high group(4.35±1.03 and 3.75±0.60 vs.6.79±0.66 N.Oc/T.A;P<0.01).Immunohistochemical staining of the distal femur showed that expression of CTSK and NF-κB p-P65 was significantly upregulated in model mice vs.control mice(CTSK,0.031±0.005 vs.0.010±0.003%;NF-κB p-P65,0.013±0.002 vs.0.003±0.003%;P<0.01).However,expression of CTSK and NF-κB p-P65 was significantly downregulated after ECH treatment compared with the OP mice,especially in ECH-high group(CTSK,0.018±0.002 and 0.015±0.003 vs.0.031±0.005%;NF-κB p-P65,0.009±0.001 and 0.007±0.002 vs.0.013±0.002%;P<0.01).Conclusion ECH can attenuate bone loss in a mouse model of osteoporosis by inhibition of the osteoclast activity and down-regulation of the NF-κB signaling pathway.