葛根素治疗去卵巢小鼠骨量丢失的疗效及对RANKL/RANK/OPG信号通路的影响

Effict of puerarin on bone loss in ovariectomized mice and its effect on RANKL/RANK/OPG signaling pathway

目的探讨葛根素治疗去卵巢小鼠骨量丢失的疗效及对核因子κB受体活化因子(RANKL)/核因子κB受体活化因子配体(RANK)/骨保护素(OPG)信号通路的影响。方法采用随机数字表法将30只雌性12周龄C57BL/6小鼠分为假手术组、模型组和葛根素治疗组,每组10只。模型组和葛根素治疗组小鼠切除双侧卵巢并结扎输卵管,假手术组小鼠去除卵巢周围等量的脂肪组织。术后第3天,葛根素治疗组小鼠给予100 mg/kg剂量的葛根素(溶于0.4 ml 0.9%氯化钠溶液)灌胃干预,隔天1次,持续8周。模型组小鼠给予0.4 ml 0.9%氯化钠溶液灌胃,隔天1次,持续8周。假手术组小鼠不作处理。治疗8周后,摘取小鼠双侧股骨,采用微型计算机断层扫描仪扫描检测骨密度(BMD)和骨微结构变化,ELISA法检测血清β-Ⅰ型胶原交联羧基末端肽(β-CTX)和抗酒石酸酸性磷酸酶5b(Trap5b)水平,抗酒石酸酸性磷酸酶(Trap)染色观察破骨细胞数量,qRT-PCR检测骨组织RANKL和OPG mRNA表达情况。结果与假手术组比较,模型组小鼠BMD、骨体积分数(BV/TV)、骨小梁数目(Tb.N)均减少,骨小梁间距(Tb.Sp)增宽,血清β-CTX、Trap5b水平均升高,破骨细胞数量增加,RANKL mRNA表达水平升高,OPG mRNA表达水平、OPG/RANKL比值均降低,差异均有统计学意义(均P<0.05)。与模型组比较,葛根素治疗组BMD、BV/TV、Tb.N均增加,Tb.Sp变窄,血清β-CTX、Trap5b水平均降低,破骨细胞数量减少,RANKL mRNA表达水平降低,OPG mRNA表达水平、OPG/RANKL比值均升高,差异均有统计学意义(均P<0.05)。结论葛根素能够延缓去卵巢小鼠骨量丢失,其机制可能是通过调控RANKL/RANK/OPG信号通路,抑制破骨细胞介导的骨吸收过程。

Objective To investigate the effect of puerarin on bone loss in ovariectomized mice and its effect on RANKL/RANK/OPG signaling pathway.Methods Thirty 12-week old female C57BL/6 mice were randomly divided into the sham-operation group,model group and treatment group.Mice in the model and treatment groups were received resection of bilateral ovaries,while mice in the sham-operation group were received resection of the para-ovarian adipose tissue only.Since 3 days post-surgery,mice in the treatment group were given 100 mg/kg puerarin(0.4 ml)intragastrically every other day,and the model group was given same volume of normal saline.At 8 weeks after the intervention,bilateral femurs were harvested from the mice.Micro-CT scanning was performed to detect bone mineral density and bone microstructure.ELISA analysis was performed to detect serum level ofβ-CTX and Trap5b.TRAP staining was performed to observenumber of osteoclasts.qRT-PCR analysis was performed to detect the expression of RANKL mRNA and OPG mRNA in bone tissue.Results Compared with the shamoperation group,mice in the model group had decreased BMD,BV/TV and Tb.N,widened Tb.SP,increased serumβ-CTX and Trap5b,increased osteoclast number,increased RANKL mRNA,decreased OPG mRNA and OPG/RANKL ratio(all P<0.05).Compared with the model group,mice in the treatment group had increased BMD,BV/TV and Tb.N,narrowed Tb.SP,decreased serum theβ-CTX and Trap5b,decreased osteoclast number,decreased RANKL mRNA,increased OPG mRNA and OPG/RANKL ratio(all P<0.05).Conclusion Puerarin can alleviate bone loss in ovoectomized mice,which may be associated with inhibiting osteoclast mediated bone resorption through RANKL/RANK/OPG signaling pathway.