二甲双胍对华法令诱导大鼠动脉钙化的影响及其机制初探  

Effect of Metformin on the Aortic Calcification Induced by Warfarin in Rats and Preliminary Investigation of Its Mechanism

作　　者：马文瑞[1] 李寰[2] 陶惠人[1] 沙鑫[1] 沈鹏 吴太林 李锋[1] 罗卓荆[1] 

机构地区：[1]第四军医大学西京医院全军骨科研究所,陕西西安710032 [2]第四军医大学西京医院心脏内科,陕西西安710032 [3]第四军医大学口腔医院口腔修复科军事口腔医学国家重点实验室,陕西西安710032

出　　处：《现代生物医学进展》2016年第12期2231-2235,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(8170256);国家重点基础发展规划项目"973项目"(2014CB542206);长江学者和创新团队发展计划(IRT1053)

摘　　要：目的:研究二甲双胍(metformin,MET)对华法令(Warfarin,WFN)诱导大鼠动脉钙化的影响及其机制。方法:将28只SD大鼠随机分为正常对照组、8周(W)钙化组、8W钙化+8W MET 100 mg/kg治疗组、8W钙化+8W MET 200 mg/kg治疗组。采用Von Kossa染色法检测胸主动脉组织中钙结节;邻甲酚肽络合酮比色法测定颈总动脉组织中钙沉积含量;免疫组化染色检测血管壁中成骨基因Runx2及血管平滑肌标志物α-SMA的表达;Western Blot检测血管壁中Runx2及自噬标志物LC3II的表达。结果:WFN干预8 W后,大鼠动脉中钙沉积含量显著增加(P<0.01),MET治疗组主动脉钙含量与钙化组相比均显著降低(P<0.01)。Von Kossa染色可见钙化组(WFN组)动脉壁中层黑色连续钙盐沉积条带,而进行MET治疗后,黑色条带明显减少,对照组未见黑色条带。免疫组化显示钙化组血管壁Runx2表达阳性,棕褐色染色较深,而α-SMA表达则显著下降,基本未见棕褐色染色沉积;MET治疗后能够逆转上述趋势。Western Blot显示钙化组血管壁Runx2表达明显上升,MET治疗后Runx2表达被抑制。此外,钙化过程中伴随着自噬标志物LC3II表达轻度上升;随着MET浓度升高,血管壁中自噬水平呈剂量依赖性显著升高。结论:二甲双胍能够有效抑制大鼠动脉钙化,减轻血管平滑肌细胞由收缩表型向成骨样表型转换,其机制可能与诱导自噬有关。Objective： To investigate the effect of metformin on the aortic calcification induced by warfarin in rats and its possible mechanisms. Methods： 28 SD rats were divided into the normal group（n=7, normal diet）, 8-week calcification group（n=7, 3.0 and 1.5mg WK1/g for 8 weeks）, 8 weeks calcification ＋ 8 weeks metformin 100 mg/kg group（n=7, 3.0 and 1.5 mg WK1/g and metformin100 mg/kg for 8 weeks） and 8 weeks calcification ＋ 8 weeks metformin 200 mg/kg group（n=7, 3.0 and 1.5 mg WK1/g and metformin200 mg/kg for 8 weeks）. Von Kossa staining was applied to show the calcium nodes. Calcium deposition measurement was used to determine the calcium content. Immunohistochemistry was applied to visualize the expression of Runx2 and α-SMA. The protein expression was detected by Western Blot. Results： 8-week warfarin diet led to an almost linear increase in arterial calcium deposition as assessed by Von Kossa staining. Metformin intake at both low and high dose can prevent the progress of calcium accumulation.Compared to the calcification group, the calcium content of metformin treatment groups were significantly deceased（P0.01） and the count of calcium nodes were also reduced（P0.01）. Calcification marker Runx2 was significantly increased in calcification group while the α-SMA expression was significantly reduced（P0.01）. After metformin treatment, the expression of Runx2 was inhibited and the expression of α-SMA was restored. Moreover, during the metformin treatment, the expression of LC3 was continuously increasing.Conclusions： Metformin could effectively inhibit the aorta calcification induced by warfarin, decrease the phenotypic transition of vascular smooth muscle cell, and its mechanism might be associated with the induction of autophagy in the wall of aorta.

关 键 词：二甲双胍 华法令 动脉钙化 细胞自噬 

分 类 号：R-33[医药卫生] R543