热敏灸对骨质疏松小鼠破骨细胞PI3K/AKT/mTOR通路的影响

Effect of thermal moxibustion on PI3K/AKT/mTOR pathway in osteoclasts of osteoporotic mice

目的 探讨热敏灸对骨质疏松症(osteoporosis, OP)小鼠及破骨细胞PI3K/AKT/mTOR信号通路的干预效果。方法 雌性KM小鼠随机分为假手术组、模型组(双侧卵巢切除术)、雌激素组(0.205 5 mg/kg戊酸雌二醇灌胃)、艾灸组(悬灸肾俞穴,根据小鼠尾温变化分为热敏灸组和非热敏灸组)。干预8周后取材,使用双能X线骨密度仪测量小鼠股骨骨密度(bone mineral density, BMD)。通过RANKL体外诱导RAW264.7细胞,设为空白血清组、模型血清组、雌激素血清组、热敏灸血清组及非热敏灸血清组,分别加入10%对应组别小鼠血清。干预6 d后,采用TRAP染色检测破骨细胞分化情况,实时荧光定量PCR(qPCR)和蛋白免疫印迹法(Western blot)分别检测各组细胞中MMP-9、CTSK与p-PI3K、p-AKT、p-mTOR表达量。结果 与假手术组比较,模型组股骨BMD显著下降(P<0.05),雌激素组、热敏灸组、非热敏灸组股骨BMD较模型组显著升高。组间比较,热敏灸组小鼠的股骨BMD增加程度较雌激素组降低,较非热敏灸组升高(P<0.05)。血清干预6 d后,与空白血清组比较,模型血清组破骨细胞中MMP-9、CTSK、p-PI3K、p-AKT、p-mTOR表达水平上调,破骨细胞数显著增多(P<0.05)。与模型血清组比较,雌激素血清组、热敏灸血清组、非热敏灸血清组上述指标均显著逆转。组间比较,热敏灸血清组破骨细胞数较雌激素血清组增多,较非热敏灸血清组减少(P<0.05)。结论 热敏灸治疗OP小鼠可能是通过调控破骨细胞PI3K/AKT/mTOR信号通路抑制破骨细胞分化实现的,其干预效果优于传统艾灸。

Objective To investigate the effect of thermal moxibustion on PI3K/AKT/mTOR signaling pathway in osteoporosis(OP)mice and osteoclasts.Methods Female KM mice were randomly divided into the sham operation group,model group(bilateral ovariectomy),estrogen group(0.2055 mg/kg estradiol valerate orally),moxibustion group(moxibustion at the Shenshu acupoint,dividing into thermal moxibustion group and non-thermal moxibustion group based on the tail temperature change of the mice).After 8 weeks of intervention,the femoral bone mineral density(BMD)of mice was measured with dual energy X-ray absorptiometry.RAW264.7 cells were induced by RANKL in vitro and divided into blank serum group,model serum group,estrogen serum group,thermal moxibustion serum group,and non-thermal moxibustion serum group,which were additioned with 10%serum of corresponding mice,respectively.After 6 days of intervention,osteoclast differentiation was detected with TRAP staining.MMP-9,CTSK,as well as p-PI3K,p-AKT,and p-mTOR expressions were detected with real-time fluorescent quantitative PCR(qPCR)and Western blotting in each cell group.Results Compared to those in the sham operation group,BMD in the model group decreased significantly(P<0.05),while BMD in the estrogen group,thermal moxibustion group,and non-thermal moxibustion group significantly increased.The increase degree of femoral BMD in the thermal moxibustion group was lower than that in the estrogen group,and higher than that in the non-thermal moxibustion group(P<0.05).After 6 days of serum intervention,compared to those in the blank serum group,the expression levels of CTSK,MMP-9,p-PI3K,p-AKT,and p-mTOR in osteoclasts from the model serum group were up-regulated,and the number of osteoclasts increased significantly(P<0.05).The above indexes in estrogen serum group,thermal moxibustion serum group and non-thermal moxibustion serum group were significantly reversed.The number of osteoclasts in the thermal moxibustion serum group was higher than that in the estrogen serum group,and lower than that in the non-thermal moxibustion serum group(P<0.05).Conclusion Thermal moxibustion inhibits osteoclast differentiation by regulating the PI3K/AKT/mTOR signaling pathway in osteoclasts,