葛根素促进体外培养大鼠颅骨成骨细胞增殖和分化过程中对cAMP/PKA信号通路的影响

目的 研究葛根素对体外培养大鼠颅骨成骨细胞(rats osteoblast,ROBs)增殖和分化及cAMP/PKA信号通路的影响.方法 体外分离培养ROBs,观察不同浓度(1×10-4～1 ×10-8 mol/L)葛根素对ROBs增殖和碱性磷酸酶(alkaline phosphatase,ALP)活性的影响;分析葛根素对骨形态发生蛋白(Bone morphogenetic protein,BMP-2)、Ⅰ型胶原(Col Ⅰ)、成骨特异性转录因子-2(Runx-2)和Oxterix mRNA表达水平的影响;观察1×10-6 mol/L葛根素对BMP-2、Col-Ⅰ、Runx-2、Oxterix、PKA和磷酸化PKA(p-PKA)蛋白表达水平,测定细胞中cAMP的变化.结果 细胞增殖结果显示1×10-7 mol/L ROBs细胞浓度高于对照组,1×10-4 mol/L组ROBs细胞浓度低于对照组(P＜0.01).1×10-5、1×10-6、1×10-7mol/L组ROBs ALP活性均高于对照组(P＜0.05,P＜0.01).1×10-6 mol/L葛根素处理ROBsRunx-2、Oxterix、BMP-2、Col Ⅰ mRNA和蛋白及p-PKA、cAMP表达水平均高于对照组(P＜0.05,P＜0.01).结论 1×10-6 mol/L葛根素为促进ROBs增殖和分化的最适浓度,其可促进体外培养ROBs分化过程中cAMP/PKA信号通路的激活.

Effects of Puerarin in Promoting Proliferation and Differentiation of Rats' Osteoblast by Culture in Vitro on cAMP/PKA signal Pathway

Objective To investigate effects of Puerarin on promoting proliferation and differentiation of rats'osteoblasts (ROBs) by culture in vitro on cAMP/PKA signal pathway.Methods ROBs were isolated and cultured in vitro,and effects of different concentrations Puerarin (1 × 10-4 mol · L-1-× 10-8 mol · L-1) on proliferation and alkaline phosphatase (ALP) activity of ROBs were observed.Effects of Puerarin on bone morphogenetic protein-2 (BPM-2),type Ⅰ collagen (Col Ⅰ),osteogenic specific transfer factor-2 (Runx-2) and Oxterix mRNA expression levels were analyzed.Effects of Puerarin (1 × 10-6 mol/L) on BPM-2,collagen-1,Runx-2,Oxterix,PKA and phosphorylation-PKA (p-PKA) protein expression levels were observed,and cAMP changes was detected.Results The result of cell proliferation showed that cell concentration of ROBs by 1 × 10-7 mol/L Puerarin was significantly increased,but the concentration of ROBs by 1 × 10-4 mol/L was significantly decreased compared with those in control groups (P ＜ 0.01).Values of ROBs ALP activity in 1 × 10-5,1 × 10-6 and 1 × 10-7 mol/L groups were significantly increased than those in control group (P ＜ 0.05,P ＜ 0.01).ROBs Runx-2,Oxterix,BPM-2,collagen-1 mRNA and protein expression levels,and pPKA and cAMP expression levels in 1 × 10-6 mol/L Puerarin group were significantly increased compared with those in control group (P ＜0.05,P ＜0.01).Conclusion The 1 × 10-6 mol/L Puerarin is the best concentration for promoting ROBs proliferation and differentiation,and it may promote activation of cAMP/PKA cell signal pathway during ROBs differentiation by culture in vitro.