二甲双胍对糖基化终产物诱导血管平滑肌细胞钙化的抑制作用

摘要

目的:在细胞水平观察二甲双胍对晚期糖基化终产物(AGEs)诱导的血管平滑肌细胞钙化的抑制作用.方法:应用大鼠血管平滑肌细胞株(A7R5)进行体外实验,分别设对照组、牛血清白蛋白(BSA)组、AGE-BSA组、二甲双胍与AGE-BSA共干预组.将200 mg·L-1 AGE-BSA与不同浓度(10-4、5×10-5、10-5 mol·L-1)的二甲双胍在含10 mmol·L-1 β-甘油磷酸的钙化培养液中共培养血管平滑肌细胞,观察不同浓度二甲双胍对血管平滑肌细胞钙化的影响.采用全自动生化分析仪测定细胞层钙含量,磷酸苯二钠法检测碱性磷酸酶活性,RT-PCR 检测骨形成蛋白-2(BMP-2) mRNA 表达,Western blotting检测BMP-2蛋白表达.结果:与对照组相比较,AGE-BSA组细胞层钙含量增加,碱性磷酸酶活性升高,细胞BMP-2 mRNA 表达升高,BMP-2 蛋白表达增加( P<0.01);不同浓度二甲双胍与AGE-BSA共干预可降低AGEs诱导的上述指标升高.结论: AGE-BSA可促进血管平滑肌细胞钙化,二甲双胍可剂量依赖性地抑制AGE-BSA诱导的钙化促进作用.%Objective : To investigate the inhibition effects of metformin on calcification in vascular smooth muscle cells(VSMCs) induced by advanced glycation end products( AGEs) on cell level. Methods : VSMCs( A7R5 ) were used in vitro experiments. The cells were divided into control group, BSA group, AGE-BSA group, common interfere with metformin and AGE-BSA group. VSMCs( A7R5 ) were incubated with 200 mg·L-1 AGE-BSA and different concentrations of metformin ( 10 -4 ， 5 × 10 -5 , 10 -5 mol·L -1 ) in DMEM with 10 mmol·L - β- glycerophosphate.The effects of various concentrations of metformin on VSMCs calcification were observed. Calcium deposition was detected by automatic biochemical analyzer. The activity of ALP was detected by the method of disodium phenyl phosphate. The mRNA and protein expression of BMP-2 were detected by RT-PCR and Western blotting, respectively. Results : Compared with control group , the calcium deposition and ALP activity of AGE-BSA group were increased obviously, and the expression of cell BMP-2 mRNA and protein were all elevated. The expression of these indicators induced by AGE- BSA was declined in different concentration of metformin group. Conclusion: AGE-BSA could promote calcification of VSMCs,and metformin can inhibit VSCMs calcification induced by AGE- BSA in dose- dependently.