左归丸与右归丸对骨髓间充质干细胞成骨分化的影响

Comparison of effects of Zuogui Pills and Yougui Pills on osteogenic differentiation of BMSCs

目的:比较左归丸与右归丸促成骨分化的影响及作用机制。方法:无菌培养SD大鼠骨髓间充质干细胞(BMSC),分别制备左归丸和右归丸高、低剂量含药血清,分为对照组(CON组)、左归丸低剂量组(ZD组)、左归丸高剂量组(ZG组)、右归丸低剂量组(YD组)和右归丸高剂量组(YG组)。采用CCK8、碱性磷酸酶(AKP)试剂盒、ALP染色检测各组细胞增殖、成骨活性和分化能力,RT-q PCR检测FNDC5、Runx2、Osterix、BMP2、Tgif2和PPARγm RNA表达,Western blot检测FNDC5、Runx2、BMP2蛋白表达。结果:CCK8显示各组细胞7d内增殖呈上升趋势;含药血清干预3d,ZG组AKP活性明显高于其余各组(P<0.01);干预7d,ZD、ZG、YG组AKP活性明显高于CON组(P<0.01),且ZG组AKP活性明显高于YD、YG组(P<0.05,P<0.01);ALP染色显示干预3、7d各含药血清组ALP染色阳性率均高于CON组,其中ZG组更为明显(P<0.01)。PT-q PCR显示,含药血清干预第3天ZG、YG组BMP2表达高于CON组(P<0.05),第7天ZG组Runx2、Osterix显著高于CON组(P<0.05,P<0.01),各组Tgif2和ZG、YG组PPARγ表达显著低于CON组(P<0.05,P<0.01);Western blot显示,干预3d时ZG组FNDC5蛋白表达水平高于CON、YD和YG组(P<0.05);各组BMP2、Runx2蛋白表达水平明显高于CON组(P<0.05),ZG和YG组Runx2蛋白表达水平较ZD和YD组明显升高(P<0.05);干预7d时,ZG组FNDC5蛋白表达水平明显高于CON、ZD、YD组(P<0.05);各组BMP2、Runx2蛋白表达水平较CON组均明显升高(P<0.05),ZG组BMP2、Runx2蛋白表达水平均高于YD和YG组(P<0.05)。结论:左归丸和右归丸均能一定程度促BMSCs成骨分化,其中左归丸在促进成骨活性及成骨分化能力略强于右归丸。二者促成骨分化的差异可能与其上调FNDC5、BMP2、Runx2表达程度相关。

Objective： To compare the effects and mechanism of Zuogui Pills and Yougui Pills on osteogenic differentiation on rat bone mesenchymal stem cells（BMSCs）. Methods： BMSCs were obtained from SD rats and cultured by aseptic method and the drug-containing serum of high and low dose of Zuogui Pills and Yougui Pills were respectively prepared. MSCs were divided into control group（CON）, Zuogui Pills low dose group（ZD）, Zuogui Pills high dose group（ZG）, Yougui Pills low dose group（YD）, Yougui Pills high dose group（YG）. CCK8, AKP and ALP staining were used to detect the cell proliferation, osteogenic activity and differentiation in each group. RT-q PCR was used to detect the m RNA expression of FNDC5, Runx2, osterix, BMP2, tigf2 and PPARγ; Western blot was used to detect the protein expression of FNDC5, Runx2 and BMP2. Results： CCK8 showed that the proliferation of each group of cells increased in 7 days and revealed an upward trend, and ZD and YG groups increased significantly compared with the other groups, but there was no significant difference. The activity of AKP in ZG group was significantly higher than other four groups on the 3 rd day of intervention（P〈0.01）. The activity of AKP in ZD, ZG and YG groups was significantly higher than CON group after 7 days intervention（P〈0.01） and the activity of AKP in ZG was significantly higher than that in YD and YG group（P〈0.05, P〈0.01）. ALP staining showed the positive rate in each group was higher than CON group on the 3 rd and 7 th day, and ZG group was more obvious. RT-q PCR showed that the expression of BMP2 in ZG and YG groups were significantly higher than CON group on day 3（P〈0.05）. On the 7 th day, Runx2 and Osterix in ZG group were significantly higher than CON group（P〈0.05, P〈0.01）. The expression of Tgif2 in each group and PPARγ in ZG and YG groups were significantly lower than CON group（P〈0.05, P〈0.01）. WB showed that the protein expression of FNDC5 in ZG group was higher than CON, YD and YG groups after 3 days intervention（P〈0.05）. Compared with CON group, the expression levels of BMP2 and Runx2 in the other groups were significantly increased（P〈0.05）. The protein expression of Runx2 in ZG and YG groups was significantly higher than that in the ZD and YD groups（P〈0.05）. After 7 days intervention, the protein expression level of FNDC5 in ZG group was significantly higher than that in CON, ZD and YD groups（P〈0.05）, and ZD group was significantly higher than YD group（P〈0.05）. The protein expressions of BMP2 and Runx2 in each group were significantly higher than CON group（P〈0.05） and protein expressions of BMP2 and Runx2 in ZG group were higher than those in YD and YG groups（P〈0.05）. Conclusion： Both Zuogui Pills and Yougui Pills can promote the osteogenic differentiation of BMSCs. Zuogui Pills is slightly stronger than Yougui Pills in promoting osteogenic activity and differentiation. The differences between the two drugs may be related to the up-regulation of FNDC5, BMP2 and Runx2 expression.