祛湿健骨方对MC3T3-E1细胞成骨分化能力的促进作用研究

Study on promotion effect of Qushi Jiangu Formulaon osteogenic differentiation of MC3T3-E1 cells

目的探讨祛湿健骨方对MC3T3-E1细胞的作用机理。方法将祛湿健骨方配成不同浓度溶液(0.005、0.025、0.05、0.1、0.2、0.4 mg/mL),用不同浓度祛湿健骨方对细胞进行干预,干预48 h后,采用CCK-8法检测细胞增殖率,筛选最佳药物浓度。将MC3T3-E1细胞制成细胞悬液,随机分为对照组、地塞米松组、低剂量组、中剂量组、高剂量组,接种至六孔板。待细胞贴壁达到80%后进行造模(对照组不添加药物;地塞米松组使用地塞米松干预;低剂量组、中剂量组、高剂量组分别用相应浓度的祛湿健骨方+地塞米松进行干预)48 h后更换为成骨诱导培养基进行培养。诱导15 d后,测定各组细胞的碱性磷酸酶(Alkaline phosphatase,ALP)含量和Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、骨细胞特异性转录因子(Osterix,OSX)蛋白的表达量。结果经CCK-8实验结果对比,药物筛选前3位存活率浓度为0.1 mg/mL(低浓度)、0.2 mg/mL(中浓度)、0.4 mg/mL(高浓度)。与对照组相比,地塞米松组、低剂量组、中剂量组、高剂量组细胞ALP活性均降低差异有统计学意义(P<0.05);与地塞米松组相比,低剂量组、中剂量组差异无统计学意义(P>0.05),高剂量组细胞ALP活性升高,差异有统计学意义(P<0.05)。与对照组比较,中剂量组、低剂量组、地塞米松组细胞的RUNX2、OSX蛋白表达均降低,差异有统计学意义(P<0.05),高剂量组细胞OSX和RUNX2蛋白表达差异无统计学意义(P>0.05);与地塞米松组比较,高剂量组OSX和RUNX2蛋白表达差异均有统计学意义(P<0.05)。结论祛湿健骨方能够促进MC3T3-E1细胞的成骨分化能力,其可能是通过调节RUNX2、OSX等骨形成相关蛋白,以此纠正骨形成与骨吸收的平衡,达到治疗骨质疏松症的作用。

Objective To explore the mechanism of action on MC3T3-E1 cells.Methods The solution were mixed with different concentrations(0.005 mg/mL,0.025 mg/mL,0.05 mg/mL,0.1 mg/mL,0.2 mg/mL,0.4 mg/mL),and the cells were intervened with different concentrations.After 48 hours of intervention,the cell proliferation rate was detected by Cell Counting Kit-8(CCK-8)method,and the best drug concen-tration was screened.MC3T3-E1 cells were made into cell suspension and randomly divided into blank control,dexamethasone,low dose drug(D),medium dose drug(Z),high dose drug(G)and seeded to a six-well plate.The mold was made after the cell adhesion reached 80%(no drug was added in the blank group;DEX intervention with DEX;D,Z and G intervention with corresponding concentration of bone prescription+DEX)for 48 hours,it replaced with osteogenic induction medium for culture.After 15 days of induction,the alkaline phosphatase(ALP)content and the expression levels of RUNX 2 and OSX protein were measured in each group.Results Compared with the results of CCK-8 experiment,the first three survival concentrations were the drug was selected as 0.1 mg/mL(low concentration),0.2 mg/mL(middle concentration)and 0.4 mg/mL(high concentration).Compared with control group,the ALP ac-tivity in the dexamethasone group(P<0.05),the low-dose group,the middle-dose group and the high-dose group was decreased significantly ALP,there was no significant difference between low-dose and mid-dle-dose groups(P>0.05),but the ALP activity in high-dose groups were increased significantly(P<0.05).Compared with the control group,the expression of RUNX2 and OSX protein in the middle-dose group,low-dose group and dexamethasone group was decreased significantly ALP,there was no signifi-cant difference in the expression of OSX and RUNX2 protein between the high-dose group and the control group(P<0.05),but there was significant difference in the expression of OSX and RUNX2 protein be-tween the high-dose group(P<0.05).Conclusion Through the study,it can be found that the bone pre-scription can promote the osteogenic differentiation ability of MC3T3-E1 cells.Meanwhile,it can be in-ferred that it may regulate bone formation related proteins such as RUNX 2,OSX to correct the balance between bone formation and bone resorption and achieve the treatment of osteoporosis.