摘要
目的观察二甲双胍对血管紧张素Ⅱ(AngⅡ)诱导的成年大鼠心脏成纤维细胞(CF)增殖的影响,并探讨其可能机制。方法通过胰酶、胶原酶Ⅱ联合消化法获取成年大鼠CF,AngⅡ(100nmol/L)刺激CF建立细胞增殖模型,并给予不同浓度(10、50及200μmol/L)二甲双胍进行干预。采用噻唑蓝法观察CF的增殖情况,EdU掺入法测定CF的DNA合成能力。免疫印迹法检测内皮型一氧化氮合酶(eNOS)、磷酸化内皮型一氧化氮合酶(peNOS)蛋白表达情况,硝酸还原酶法测定CF培养上清一氧化氮(NO)含量。结果AngⅡ刺激48h能够明显促进成年大鼠CF增殖(P<0.05),二甲双胍呈浓度依赖性抑制AngⅡ诱导的CF增殖,二甲双胍呈浓度依赖性增加CF内eNOS磷酸化水平和NO含量(P<0.05),eNOS抑制剂N-硝基-L-精氨酸甲酯(L-NAME)能阻断二甲双胍抑制AngⅡ诱导的CF增殖的作用(P<0.05)。结论二甲双胍能抑制AngⅡ诱导的成年大鼠CF增殖,与二甲双胍激活CF内eNOS/NO通路有关。
Aim To investigate the effect of metformin on angiotensin Ⅱ（AngⅡ）-induced proliferation of adult rat cardiac fibroblasts（CF） and its underlying mechanism.Methods The adult rat CF was isolated by a combination of trypsin and collagenase Ⅱ digestion.The cell proliferation was induced by AngⅡ（100 nmol / L） stimulation,and the CF was treated with metformin in different concentrations（10,50,and 200 μmol / L）.The proliferation of CF was evaluated by MTT assay,and the DNA synthesis was detected by Ed U incorporation.The expression of endothelial nitric oxide synthase（eNOS） and phosphorylated endothelial nitric oxide synthase（p-eNOS） were detected by Western blot.The level of NO in CF culture supernatant fluids was measured by nitrate reductase method.Results Stimulation with AngⅡ for 48 h induced the proliferation of adult rat CF,and this effect was inhibited by pretreatment of CF with metformin in a concentration-dependent manner.Metformin significantly increased the phosphorylation level of eNOS in CF as well as the level of NO in cell culture supernatant fluids in a concentration-dependent manner.In addition,pretreatment of CF with eNOS inhibitor L-NAME markedly attenuated the inhibitory effect of metformin on AngⅡ-induced cell proliferation.Conclusion Metformin inhibits AngⅡ-induced proliferation of adult rat CF,and this effect may be associated with the activation of the eNOS / NO pathway.
出处
《中国动脉硬化杂志》
CAS
北大核心
2015年第7期663-667,共5页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金资助项目(81000055
81070195)
