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目的:研究紫草素对破骨细胞体外分化的影响,并探讨其对去卵巢(ovariectomized,OVX)诱导的骨质疏松模型小鼠的骨保护作用。方法:体外细胞生物学实验,采用CCK-8法检测不同浓度紫草素对C57BL/6J小鼠骨髓源性单核巨噬细胞的毒性;采用RANKL和M-CSF诱导单核巨噬细胞破骨分化模型,给予不同浓度的紫草素干预后,经TRAP染色对破骨细胞进行形态学观察,并通过Real-Time PCR技术检测破骨细胞特异性基因TRAP、c-Fos和NFATc1的表达。动物体内实验,随机将15只小鼠平均分为假手术组、OVX组、治疗组。造模成功后治疗组给予紫草素干预,假手术组和OVX组以等体积生理盐水处理。连续处理30天后取胫骨,用Micro CT扫描重建观察胫骨近端骨丢失状况。结果:(1)高于250 nmol/L的紫草素显著抑制小鼠单核巨噬细胞生长(P<0.01)。(2)不同浓度的紫草素干预能显著抑制体外破骨细胞形成(P<0.01)。(3)不同浓度的紫草素干预能显著抑制TRAP,c-Fos和NFATc1等参与破骨细胞分化的重要基因表达(P<0.01)。(4)紫草素干预能显著改善去卵巢诱导的骨质疏松模型小鼠的骨丢失(P<0.05)。结论:紫草素能在体外抑制破骨细胞分化并在体内改善去卵巢诱导的小鼠骨质疏松。
OBJECTIVE: To study the effect of shikonin on the differentiation of osteoclasts in vitro and its osteoprotegerie effect on ovariectomized (OVX) -induced osteoporosis model mice. Methods: The cytotoxicity of shikonin in bone marrow-derived mononuclear macrophages of C57BL / 6J mice was detected by CCK-8 assay in vitro. The osteoclasts of mononuclear macrophages were induced by RANKL and M-CSF Differentiation model was induced by different concentrations of shikonin. The morphology of osteoclasts was observed by TRAP staining. The expression of TRAP, c-Fos and NFATc1 in osteoclast were detected by Real-Time PCR. Animals in vivo experiments, 15 mice were randomly divided into sham operation group, OVX group, treatment group. The model group was treated with shikonin after successful modeling. The sham-operated group and OVX group were treated with equal volume of normal saline. The tibia was taken after 30 days of continuous treatment and the bone loss in the proximal tibia was observed by Micro-CT scan reconstruction. Results: (1) Shikonin above 250 nmol / L significantly inhibited the growth of mouse monocyte-macrophage (P <0.01). (2) Different concentrations of shikonin could significantly inhibit the formation of osteoclasts in vitro (P <0.01). (3) Different concentrations of shikonin could significantly inhibit the expression of important genes involved in osteoclast differentiation such as TRAP, c-Fos and NFATc1 (P <0.01). (4) Shikonin intervention can significantly improve bone loss in ovariectomized osteoporosis model mice (P <0.05). CONCLUSIONS: Shikonin inhibits osteoclast differentiation in vitro and improves ovariectomized mouse osteoporosis in vivo.