左归丸干预卵巢摘除骨质疏松模型小鼠骨形态发生蛋白2信号通路的变化

Effect of Zuogui Wan on bone morphogenetic protein 2 signaling pathway in ovariectomized osteoporosis mice

背景:绝经后骨质疏松症在中老年女性中属于高发疾病,严重影响其生活质量,发现并探索出一种有效的防治手段是非常必要的。中医可通过“肾主骨”理论选用补肾药治疗绝经后骨质疏松症,但其分子机制尚不清楚。目的:观察左归丸对骨形态发生蛋白2/Runx-2/Osterix信号通路的影响。方法:采用卵巢摘除的方法建立绝经后骨质疏松症小鼠模型,然后将造模成功的84只小鼠分为模型组、假手术组、雌二醇组、左归丸高、中、低(0.936,0.468,0.234 g/L)剂量组、阻断剂组,每组12只。左归丸高、中、低剂量组给予相应质量浓度的左归丸水煎液;假手术组、模型组给予等体积的生理盐水;雌二醇组给予雌二醇水溶液;阻断剂组给予ICI182780+左归丸高剂量水煎液,持续灌胃8周。采用免疫组化和Western Blot法检测左归丸干预后卵巢摘除骨质疏松症模型小鼠胫骨和股骨中骨形态发生蛋白信号通路的关键蛋白骨形态发生蛋白2、Runx-2和Osterix的表达;通过ICI182780阻断雌激素受体后,检测骨形态发生蛋白通路中蛋白表达的变化。结果与结论:①免疫组化结果显示,与模型组小鼠相比,左归丸高、中剂量均可以明显提高模型组小鼠骨形态发生蛋白2的表达水平(P<0.01,P<0.05);左归丸高、中、低剂量组Runx-2的表达水平相比差异均有显著性意义(P<0.01);②免疫印迹实验结果显示,与模型组小鼠相比,左归丸高、中剂量均可以明显提高模型组小鼠骨形态发生蛋白2的表达水平(P<0.05);左归丸高、中、低剂量组Runx-2的表达水平均显著高于模型组(P<0.01,P<0.05,P<0.05);左归丸高、中剂量组Osterix的表达水平均显著高于模型组(P<0.01,P<0.05);③ICI182780干预后,与左归丸高剂量组相比,阻断剂组小鼠胫骨骨形态发生蛋白2和Runx-2蛋白的表达水平显著降低(P<0.05,P<0.01);阻断剂组小鼠股骨中骨形态发生蛋白2、Runx-2和Osterix的表达水平均显著降低(P<0.01);④提示左归丸可以通过雌激素受体激活骨形态发生蛋白2/Runx-2/Osterix信号通路进而参与骨代谢过程,实现骨保护的作用。

BACKGROUND:Postmenopausal osteoporosis is a high-incidence disease in middle-aged and elderly women,which seriously affects the quality of life.It is very necessary to discover and develop an effective prevention and treatment method.Traditional Chinese medicine can treat postmenopausal osteoporosis with tonifying kidney medicines based on the theory of“kidney governs bone,”but the molecular mechanism is still unclear.OBJECTIVE:To observe the effect of Zuogui Wan on bone morphogenic protein 2(BMP-2)/Runx-2/Osterix signaling pathway.METHODS:The postmenopausal osteoporosis model was established in mice by ovarian removal.Then the successfully modeled mice were divided into model group,sham operation group,estradiol group,high-,medium-and low-dose Zuogui Wan(0.936,0.468,0.234 g/L)groups,and blocker group,with 12 mice per group.In the high-,medium-and low-dose Zuogui Wan groups,the corresponding concentrations of Zuogui Wan decoction were given.Sham operation group and model group were given the equal volume of normal saline.Estradiol group was given estradiol aqueous solution.The blocker group was given ICI182780 plus high-dose Zuogui Wan decoction.Intervention in each group was given via gavage for 8 continuous weeks.Immunohistochemistry and western blot were used to detect the expression of key proteins BMP-2,Runx-2 and Osterix in the tibia and femur of ovariectomized osteoporosis mice after intervention by Zuogui Wan.Changes of protein expressions in the BMP pathway were detected after blocking estrogen receptor using ICI182780.RESULTS AND CONCLUSION:Immunohistochemical findings indicated that compared with the model group,the high and medium doses of Zuogui Wan could significantly increase the expression of BMP-2 in ovariectomized osteoporosis mice(P<0.01,P<0.05);the expression of Runx-2 in the medium-and low-dose Zuogui Wan groups was significantly different(P<0.01).Western blot results showed that compared with the model group,the high and middle doses of Zuogui Wan significantly increased the expression of BMP-2 in ovariectomized osteoporosis mice(P<0.05);the expression of Runx-2 was significantly higher in the high-,medium-and low-dose Zuogui Wan groups(P<0.01,P<0.05,P<0.05);and the expression of Osterix was significantly higher in the high-and medium-dose Zuogui Wan groups(P<0.01,P<0.05).After ICI182780 intervention,compared with the high-dose group,the expression of BMP-2 and Runx-2 in the mouse tibia was significantly reduced in the blocker group(P<0.05,P<0.01)and the expression of BMP-2,Runx-2 and Osterix in the mouse femoral bones was significantly reduced in the blocker group(P<0.01).To conclude,Zuogui Wan can participate in the regulation of bone metabolism by increasing the expression of important proteins in the BMP/Runx-2/Osterix signaling pathway in ovariectomized osteoporosis mice,thereby achieving the protection of bones.