左、右归丸调控AMPK/mTOR信号通路促进绝经后骨质疏松症大鼠骨形成的研究

Study on mechanism of bone formation of PMOP rats by Zuogui Pills and Yougui Pills based on AMPK/mTOR signaling pathway

目的:研究左、右归丸对绝经后骨质疏松症(PMOP)大鼠骨髓腺苷酸活化蛋白激酶A(MPK)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路的影响,并基于该信号通路探讨左、右归丸对PMOP大鼠骨形成的作用。方法:将60只2月龄未生育雌性SD大鼠随机分为空白组、假手术组、模型组、左归丸组、右归丸组、补佳乐组,采用去卵巢方法建立PMOP大鼠,给药12周后,取材。采用micro-CT检测大鼠腰椎L3-L5骨密度(BMD)、骨体积分数(BV/TV);Real-time PCR法检测大鼠Runx2、COLⅠ、AMPK、mTOR mRNA表达;Western Blot法检测大鼠Runx2、COLⅠ、骨钙素(BGP)、AMPK、mTOR、p-AMPK、p-mTOR蛋白表达。结果:与空白组比较,模型组的腰椎BMD、BV/TV显著降低(P<0.01),COLⅠ、Runx2 mRNA和蛋白及BGP蛋白表达显著下调(P<0.01),AMPK mRNA和p-AMPK蛋白表达上调,mTOR mRNA和p-mTOR蛋白表达显著下调(P<0.01);与模型组比较,各给药组的腰椎BMD、BV/TV显著升高(P<0.01,P<0.05),COLⅠ、Runx2 mRNA和蛋白及BGP蛋白表达显著上调(P<0.01),AMPK mRNA和p-AMPK蛋白表达显著下调(P<0.01),mTOR mRNA和p-mTOR蛋白表达显著上调(P<0.01)。结论:雌性大鼠去卵巢12周后,AMPK表达异常激活,mTOR表达异常抑制;左、右归丸能明显改善异常,并促进PMOP大鼠的骨形成,这可能是左、右归丸防治PMOP的机制之一。

Objective:To analyze the effects of Zuogui Pills and Yougui Pills on AMPK/mTOR signaling pathway in PMOP rat bone marrow.Based on this signal pathway,to explore the effects of the bone formation of PMOP rats.Methods:Sixty SD barren female rats were randomly divided into blank group,sham operation group,model group,Zuogui Pills group,Yougui Pills group and Bujiale group.The other four groups except the blank group and the sham operation group were treated with oophorectomy.After 12 weeks of treatment,the bone mineral density,bone volume fraction and bone morphology of the lumbar vertebrae were measured by micro-CT;The Runx2,COLⅠ,AMPK and mTOR mRNA in bone marrow were detected by fluorescence quantitative-PCR;The Runx2,COLⅠ,BGP,AMPK,mTOR,p-AMPK and p-mTOR protein expression were detected by Western Blot.Results:Compared with the blank group,the bone mineral density,bone volume fraction in the model group were significantly decreased(P<0.01),Runx2,COLⅠmRNA and protein and BGP protein expression were significantly decreased(P<0.01),AMPK mRNA and p-AMPK protein expression were increased(P<0.01,P<0.05),mTOR mRNA and p-mTOR protein expression were decreased(P<0.01).Compared with the model group,bone mineral density,bone volume fraction was significantly increased in each treatment group(P<0.01,P<0.05),Runx2,COLⅠmRNA and protein and BGP protein were significantly increased in each treatment group(P<0.01,P<0.05),AMPK mRNA and p-AMPK protein expression were decreased(P<0.01),mTOR mRNA and p-mTOR protein expression were increased in each treatment group(P<0.01).Conclusion:After 12 weeks of ovaries removal,AMPK expression was abnormally activated and mTOR expression was abnormally inhibited in rats.Zuogui Pills and Yougui Pills can affect the bone formation in PMOP rats based on AMPK/mTOR signaling pathway,which may be one of the mechanisms regulating bone formation by Zuogui Pills and Yougui Pills.