紫草素通过调节RANKL/RANK/TRAF6及其介导的NF-κB/MAPKs信号通路与氧化应激来改善激素性骨质疏松

Shikonin Ameliorates Glucocorticoid Induced Osteoporosis by Regulating RANKL/RANK/TRAF6 and Its Mediated NF-κB/MAPKs Signaling Pathway and Oxidative Stress

目的评估紫草素对激素诱导的大鼠骨质疏松的影响及其潜在机制。方法不同浓度紫草素处理RAW264.7细胞系,CCK8实验评估紫草素对细胞活力的影响;将细胞分为5个组:对照组、地塞米松(骨质疏松诱导剂)组、地塞米松+重组人甲状旁腺(抗骨质疏松剂)组、地塞米松+紫草素0.3μmol·L^(-1)组,及地塞米松+紫草素1.2μmol·L^(-1)组,通过TRAP染色及骨吸收面积测定评估紫草素对破骨细胞功能影响。构建地塞米松诱导大鼠骨质疏松大鼠模型,并分为5个组(n=8):地塞米松组、地塞米松+重组人甲状旁腺素组、地塞米松+紫草素0.3μmol·L^(-1)组与地塞米松+紫草素1.2μmol·L^(-1)组,设置正常对照组,分别给予:0.9%NaCl6 mL·kg^(-1),每3天PTH 20μg·kg^(-1),每天紫草素0.5 mg·kg^(-1)(0.3μmol·L^(-1)),每天紫草素2 mg·kg^(-1)(1.2μmol·L^(-1)),0.9%NaCl 6 mL·kg^(-1)共3周;ELISA检测血清I型胶原蛋白(CTX)、血清组织蛋白酶K(Cathepsin K)及氧化应激相关指标变化;HE染色检测大鼠骨结构变化,双能X线扫描检测大鼠骨密度(BMD)变化;Western blot及免疫荧光检测紫草素体外对NF-κB/MAPKs信号通路的影响,Western blot及qRT-PCR检测RANKL相关蛋白及mRNA表达,Western blot检测紫草素对RANKL/TRAF6的阻断作用。结果1.2μmol·L^(-1)紫草素对细胞存活率影响最小;紫草素降低了TRAP阳性细胞数量(P<0.05);紫草素降低了骨吸收面积(P<0.05);紫草素降低了CTX及Cathepsin K表达(P<0.05),且1.2μmol·L^(-1)紫草素组降低更显著(P<0.05);紫草素提高了大鼠股骨BMD(P<0.05),且1.2μmol·L^(-1)紫草素组上升更显著(P<0.01);紫草素可以逆转地塞米松引起的骨小梁减少与变薄;紫草素提高了大鼠体内SOD及GSH表达水平(P<0.05),并降低了MDA表达水平(P<0.05);紫草素体外抑制NFκB通路相关蛋白磷酸化;紫草素抑制RANK、RANKL、TRAF6、c-fos及NFATc1蛋白和mRNA表达;且紫草素阻断RANKL诱导剂促进TRAF6表达。结论紫草素可以通过抑制RANKL/RANK/TRAF6及其介导的NF-κB/MAPKs信号通路来改善大鼠激素诱导的骨质疏松。

Objective To evaluate the effect and potential mechanism of Shikonin on hormone-induced osteoporosis in rats.Methods RAW264.7 cell lines were treated with indicated concentrations of Shikonin,and the effects of Shikonin on cell viability were evaluated by CCK8 assay.Then the cells were divided into five groups:control group,dexamethasone(osteoporosis agent)group,dexamethasone+recombinant human parathyroid(anti-osteoporosis agent)group,dexamethasone+Shikonin 0.3μmol·L^(-1)group,and dexamethasone+Shikonin 1.2μmol·L^(-1)group.The effect of Shikonin on osteoclast function was evaluated by TRAP staining and bone resorption area measurement.The rat model of osteoporosis induced by dexamethasone was established,and divided into five groups(n=8):dexamethasone group,dexamethasone+PTH group,dexamethasone+Shikonin 0.3μmol·L^(-1)group and dexamethasone+Shikonin 1.2μmol·L^(-1)group,while control group was set up,then each group given separately 0.9%NaCl 6 mL·kg^(-1),PTH 20μg·kg^(-1)3 days,Shikonin 0.5 mg·kg^(-1)day(0.3μmol·L^(-1)),Shikonin 2 mg·kg^(-1)day(1.2μmol·L^(-1)),and 0.9%NaCl 6 mL·kg^(-1)for 3 weeks.ELISA was used to detect Serum type I collagen(CTX),Cathepsin K(Cathepsin K)and oxidative stress related indicators.HE staining was used to detect the changes of bone structure,and dual-energy X-ray scanning was used to detect the changes of bone density(BMD).Western blot and immunofluorescence were used to detect the effect of Shikonin on NF-κB/MAPKs signaling pathway,Western blot and qRT-PCR were used to detect the expression of RANKL-related proteins and mRNA,and Western blot was used to detect the blocking effect of Shikonin on RANKL/TRAF6.Results 1.2μmol·L^(-1)Shikonin had the least damage to cells.Shikonin decreased the number of TRAP positive cells(P<0.05),decreased bone absorption area(P<0.05).and decreased the expression of CTX and Cathepsin K(P<0.05),and in 1.2μmol·L^(-1)group decreased more significantly(P<0.05).Shikonin increased rat femur BMD(P<0.05),and increased more significantly in 1.2μmol·L^(-1)group(P<0.01).Shikonin reversed the reduction and thinning of bone trabeculae induced by dexamethasone.The expression of SOD and GSH in rats increased(P<0.05),while the expression of MDA decreased(P<0.05).Shikonin inhibited the phosphorylation of NF-κB pathway related proteins in vitro.Shikonin inhibited the expression of RANK,RANKL,TRAF6,c-fos and NFATc1 protein and mRNA.Moreover,Shikonin blocked the expression of TRAF6.Conclusion Shikonin can ameliorate glucocorticoid induced osteoporosis by inhibiting RANKL/RANK/TRAF6 and its mediated NF-κB/MAPKs signaling pathway and oxidative stress.