补肾安胎冲剂对复发性流产小鼠胎盘滋养细胞miR-187、VEGF、VEGF-R2表达的影响

Effects of Bushen Antai Granules on Expression of miR-187,VEGF and VEGF-R2 in Placental Trophoblastic Cells of Mice with Recurrent Spontaneous Abortion

目的观察补肾安胎冲剂对复发性流产(RSA)小鼠胎盘滋养细胞miR-187、血管内皮生长因子(VEGF)及其受体VEGF-R2表达的影响,探讨其相关作用机制。方法将雌性CBA/J小鼠与雄性DBA/2小鼠合笼饲养建立RSA小鼠模型,雌性CBA/J小鼠与雄性BALB/c小鼠合笼饲养作为正常妊娠小鼠,分离并培养胎盘滋养细胞,将细胞分为正常对照组(正常妊娠小鼠胎盘滋养细胞+空白血清)、模型组(RSA小鼠胎盘滋养细胞+空白血清)、补肾安胎冲剂组(RSA小鼠胎盘滋养细胞+20%含药血清)、miR-187 inhibitor NC组(RSA小鼠胎盘滋养细胞转染空白miR-187 inhibitor+空白血清)、miR-187 inhibitor组(RSA小鼠胎盘滋养细胞转染miR-187 inhibitor+空白血清)、联合组(RSA小鼠胎盘滋养细胞转染miR-187 inhibitor+20%含药血清)。RT-qPCR检测miR-187、VEGF、VEGF-R2 mRNA表达,Western blot检测VEGF、VEGF-R2蛋白表达,双荧光素酶实验检测miR-187和VEGF靶向关系,采用Spearman法分析miR-187与VEGF、VEGF-R2 mRNA表达量的相关性。结果与正常对照组比较,模型组细胞miR-187表达显著升高,VEGF、VEGF-R2 mRNA和蛋白表达显著降低(P<0.05);与模型组比较,补肾安胎冲剂组细胞miR-187表达显著降低,VEGF、VEGF-R2 mRNA和蛋白表达显著升高(P<0.05);与miR-187 inhibitor NC组比较,miR-187 inhibitor组细胞miR-187表达显著降低,VEGF、VEGF-R2 mRNA和蛋白表达显著升高(P<0.05);与补肾安胎冲剂组及miR-187 inhibitor组比较,联合组细胞miR-187表达显著降低,VEGF、VEGF-R2 mRNA和蛋白表达显著升高(P<0.05)。双荧光素酶实验结果显示,miR-187能够与VEGF靶向结合。Spearman相关性分析显示,miR-187与VEGF、VEGF-R2 mRNA表达呈负相关。结论补肾安胎冲剂可通过下调miR-187活性,靶向上调VEGF、VEGF-R2表达,介导血管新生,促进母胎界面血管重构,从而对RSA发挥治疗作用。

Objective To observe the effects of Bushen Antai Granules on miR-187,VEGF and VEGF-R2 expression in placental trophoblastic cells of mice with recurrent spontaneous abortion(RSA);To explore the related mechanism of action.Methods Female CBA/J mice and male DBA/2 mice were housed together to establish the RSA mice model.Female CBA/J mice and male BALB/c mice were co-caged as normal pregnant mice.The placental trophoblasts were isolated and cultured.Cells were divided into normal control group(placental trophoblast cells of normal pregnant mice+blank serum),model group(RSA mouse placental trophoblast cells+blank serum),Bushen Antai Granules group(RSA mouse placental trophoblast cells+20%medicated serum),miR-187 inhibitor NC group(RSA mouse placental trophoblast cells transfected with blank miR-187 inhibitor+blank serum),miR-187 inhibitor group(RSA mouse placental trophoblast cells transfected with miR-187 inhibitor+blank serum),and combination group(RSA mouse placental trophoblast cells transfected with miR-187 inhibitor+20%medicated serum).The mRNA expressions of miR-187,VEGF and VEGF-R2 were detected by RT-qPCR,the expressions of VEGF and VEGF-R2 protein were detected by Western blot,double luciferase experiment was used to detect the targeting relationship between miR-187 and VEGF,Spearman’s method was used to analyze the correlation between miR-187 and VEGF,VEGF-R2 mRNAexpression.Results Compared with the normal control group,the expression of miR-187 in the model group significantly increased,and the mRNA and protein expressions of VEGF and VEGF-R2 significantly decreased(P<0.05).Compared with the model group,the expression of miR-187 in Bushen Antai Granules group significantly decreased,the mRNA and protein expressions of VEGF and VEGF-R2 significantly increased(P<0.05).Compared with miR-187 inhibitor NC group,the miR-187 expression in miR-187 inhibitor group significantly decreased,the mRNA and protein expressions of VEGF and VEGF-R2 increased significantly(P<0.05).Compared with Bushen Antai Granules group and miR-187 inhibitor group,the expression of miR-187 in combination group decreased,the expressions of VEGF and VEGF-R2 mRNA and protein increased significantly(P<0.05).The results of double luciferase experiment showed that miR-187 could target and bind to VEGF.Spearman correlation analysis showed that miR-187 was negatively correlated with the mRNA expression of VEGF and VEGF-R2.Conclusion Bushen Antai Granules can down-regulate the activity of miR-187,up-regulate the expressions of VEGF and VEGF-R2,mediate angiogenesis and promote the recasting of blood vessels at the maternal-fetal interface,thus playing a therapeutic role in RSA.