瑞香素通过PI3K/AKT和BMP/Smad信号通路保护糖皮质激素诱导的成骨细胞增殖和分化抑制研究

The inhibitory effects of daphnetin on proliferation and differentiation of osteoblasts induced by dexamethasone and its molecular mechanism

目的探讨瑞香素对地塞米松诱导的成骨细胞增殖和分化抑制的作用及分子机制。方法体外培养MC3T3-E1细胞,分为对照组、地塞米松组、地塞米松+瑞香素40μmol/L组、地塞米松+瑞香素60μmol/L组和地塞米松+瑞香素80μmol/L组;采用CCK-8试剂盒测定细胞活力;RT-PCR法测量细胞中碱性磷酸酶(Alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)、Ⅰ型胶原(typeⅠcollagen,COLL-1)和Runx2表达量;茜素红S染色法测量细胞矿化水平;蛋白免疫印迹法测量细胞中PI3K、p-PI3K、AKT、p-AKT、Smad和p-Smad蛋白水平。结果与对照组比较,地塞米松组细胞活力减小,ALP、OCN、COLL1和Runx2表达量降低,细胞矿化水平降低,且p-PI3K/PI3K、p-AKT/AKT、p-Smad/Smad蛋白水平下降,差异有统计学意义(P<0.05)。相对于地塞米松组,地塞米松+瑞香素40μmol/L组、地塞米松+瑞香素60μmol/L组和地塞米松+瑞香素80μmol/L组细胞活力增加,ALP、OCN、COLL1和Runx2表达量增加,细胞矿化水平增加,且p-PI3K/PI3K、p-AKT/AKT、p-Smad/Smad蛋白水平增加,差异均有统计学意义(P<0.05)。结论瑞香素保护了地塞米松诱导的MC3T3-E1细胞增殖和分化抑制,并且与激活PI3K/AKT和BMP/Smad信号通路有关。

Objective To explore the inhibitory effects of daphnetin on proliferation and differentiation of osteoblasts induced by dexamethasone and its molecular mechanism.Methods MC3T3-E1 cells were cultured in vitro and divided into control group,dexamethasone group,dexamethasone+resin 40μmol/L group,dexamethasone+resin 60μmol/L group and dexamethasone+resin 80μmol/L group.CCK-8 kit was used to determine cell viabilit,and RT-PCR method was used to measure the expression of alkaline phosphatase(ALP),osteocalcin(OCN),typeⅠcollagen(COLL-1)and Runx2 in cells.Alizarin Red S staining method was used to measure the level of cell mineralization.Western blotting was used to measure the protein levels of PI3K,p-PI3K,AKT,p-AKT,Smad and p-Smad in cells.Results Compared with those in control group,the cell viability,the expression levels of ALP,OCN,COLL1 and Runx2,and the levels of cell mineralization in dexamethasone group were significantly decreased,and the protein levels of p-PI3K/PI3K,p-AKT/AKT,and p-Smad/Smad proteins were significantly decreased(P<0.05).Compared with those in dexamethasone group,the cell viability,the expression levels of ALP,OCN,COLL1 and Runx2,and the levels of cell mineralization,p-PI3K/PI3K,p-AKT/AKT protein,and p-Smad/Smad were significantly increased in dexamethasone+resin 40μmol/L group,the dexamethasone+resin 60μmol/L group and the dexamethasone+resin 80μmol/L group(P<0.05).Conclusion Daphnetin can protect the cell proliferation and differentiation of MC3T3-E1 cells induced by dexamethasone,which is related to the activation of PI3K/AKT and BMP/Smad signaling pathways.