基于MAPK信号通路的丹参注射液体外调控小鼠前成骨细胞的增殖分化作用

In vitro Regulation of Proliferation and Differentiation of Mouse Osteoblasts by Danshen Injection Based on MAPK Signal Pathway

目的:探究丹参注射液对小鼠前成骨细胞的增殖分化的影响及其与有丝分裂原活性蛋白激酶(MAPK)信号通路的关系。方法:将小鼠前成骨细胞分为4组,分别采用丹参稀释液浓度为0(对照组),75,150,300 mg·L-1进行干预。采用细胞计数(CCK-8)试剂盒检测细胞光密度(OD)值;采用0.1%的茜素红溶液对细胞进行染色,并观察各组钙结节生成情况;采用实时荧光定量PCR法检测γ-羟基谷氨酸骨蛋白(BGP)及Runt相关基因2(Run×2) mRNA表达情况;采用蛋白免疫印迹法检测细胞外信号调节激酶2(ERK2)及其磷酸化状态(p-ERK1/2)、p-p38蛋白表达情况。采用抑制剂PD98059抑制ERK1/2通路,采用实时荧光定量PCR检测BGP及Run×2 mRNA表达。结果:各浓度丹参注射液组的OD值均高于对照组,其中300 mg·L-1丹参组各个孵育时间差异均有统计学意义(P<0.01)。各浓度丹参组钙结节数量均显著高于对照组,且呈浓度依赖性(P<0.05)。7 d后各浓度丹参注射液组的BGP及Run×2 mRNA表达显著高于对照组(P<0.01),且呈剂量依赖性(P<0.05)。经PD98059抑制后,300 mg·L-1丹参注射液(SM)+PD组BGP及Run×2 mRNA表达显著低于SM组(P<0.05或P<0.01)。300mg·L-1丹参组p-ERK1/2蛋白表达量和相对蛋白密度显著高于对照组(P<0.01);300 mg·L-1丹参组p-p38蛋白表达与对照组相比差异无统计学意义(P>0.05)。结论:丹参注射液对成骨细胞增殖、分化及矿化具有促进作用,其作用机制可能与激活MAPK通路中的ERK1/2细胞信号相关。

Objective: To investigate the effect of Danshen injection on the proliferation and differentiation of mouse osteoblasts and its relationship with mitogen activated protein kinase( MAPK) signal pathway. Methods: The proosteoblasts of mice were divided into 4 groups,and the concentration of Danshen injection was 0( control group),75,150 and 300 mg·L-1 for intervention,respectively. The cell optical density( OD) value was measured by CCK8;0.1% alizarin red solution was used to stain the cells and observe the formation of calcium nodules;real-time fluorescence quantitative PCR method was used to detect the expressions of γ-hydroxyglutamate bone protein( BGP) and Runt-related gene 2( Run×2) mRNA;Western blotting was used to detect the expressions of extracellular signal-regulated kinase 2( ERK2),its phosphorylation status( p-ERK1/2) and p-p38 protein. Inhibitor PD98059 was used to inhibit ERK1/2 pathway,and real-time fluorescence quantitative PCR was used to detect the expressions of BGP and Run×2 mRNA. Results: The OD value of Danshen injection at each concentration was higher than that in the control group,and the difference in the incubation time of 300 mg·L-1 Danshen injection group was statistically significant( P<0.01). The number of calcium nodules in Danshen injection groups was significantly higher than that in the control group,and it was concentration-dependent( P < 0.05). After 7 days,the expressions of BGP and Run×2 mRNA in Danshen injection groups were significantly higher than those in the control group( P<0.01),and there was a dose-dependent manner( P<0.05). After the inhibition by PD98059,the expressions of BGP and Run×2 mRNA in 300 mg·L-1 Danshen injection( SM) +PD group was significantly lower than that in SM group( P<0.05 or P<0.01). The pERK1/2 protein expression and relative protein density in 300 mg·L-1 Danshen injection group were significantly higher than those in the control group( P<0.01);the p-p38 protein expression in 300 mg·L-1 Danshen injection group was not significantly different from that in the control group( P>0.05). Conclusion: Danshen injection can promote the proliferation,differentiation and mineralization of osteoblasts,and its mechanism may be related to the activation of ERK1/2 signal in MAPK pathway by the preparation.