Aim In diabetic patients, metformin appears to provide cardiovascular protection that cannot be attribu- ted only to its antihyperglycemic effects. Metformin is also known as the AMP-activated protein kinase （AMPK） ...Aim In diabetic patients, metformin appears to provide cardiovascular protection that cannot be attribu- ted only to its antihyperglycemic effects. Metformin is also known as the AMP-activated protein kinase （AMPK） ac- tivator. Our previous study suggested that metformin inhibits transforming growth factor-β1 （TGF-β1） production in a mouse heart failure model of pressure overload. TGF-β1 is a key factor in cardiac fibrosis and is usually induced by Angiotensin Ⅱ （Ang Ⅱ ） in the pressure overload mouse models. This study investigated the effect of metformin on cardiac fibrosis and TGF-β production induced by AngII and the underlying mechanisms. Methods C57/BL6 wild-type and AMPKα2 knockout mice were used. AngII （3 mg · kg-1 · d-1） was infused subcutaneously into mice for 7 days. Adult mouse cardiac fibroblasts were isolated and treated with AngII （ 1 μmol · L-1） and/or met- formin （1 mmol · L-l）. Results In C57/BL6 mice, metformin inhibits AngII-induced cardiac fibrosis. In cardi-ac fibroblasts, metformin inhibits TGF-β1 expression and production induced by AngII. AMPK inhibitor, com- pound C, reversed the effects of metformin. In vivo, AMPKα2 deficiency further increases AngII-induced TGF-β1 production. In cardiac fibroblasts, metformin inhibited AngII induced hepatocyte nuclear factor4 （HNF4ot protein level increase and HNF4α binding with TGF-β1 promoter using chromatin immunoprecipitation assay. In vivo, AMPKα2 deficiency further increased AngII-induced HNF4α protein level. Using HNF4α adenovirus, overexpress- ing HNF4α led to a 1.5-fold increase in TGF-β1 mRNA expression. HNF4a siRNA blocked AngII induced TGF- β1 production. Luciferase reporter with deleted HNF4a binding sites showed decreased TGFbl transcriptional activ- ity induced by AngII. In AMPK or2-/- heart, the inhibition of metformin on HNF4a protein was attenuated. Con- clusion Metformin inhibits AngII induced cardiac fibrosis and TGF-β1 production through AMPK activation. The underlying mechanism is that AMPK activation inhibits AngII induced HNF4α and then decreases TGF-β1 expres- sion.展开更多
目的:探讨转化生长因子β1(TGF-β1)对大鼠心肌细胞肥大和凋亡的影响。方法:建立TGF-β1诱导的体外大鼠心肌细胞肥大模型。透射电镜观察心肌细胞形态。碘化丙啶(PI)染色标记法检测心肌细胞RNA含量。实时荧光定量PCR(Real time PCR)检测...目的:探讨转化生长因子β1(TGF-β1)对大鼠心肌细胞肥大和凋亡的影响。方法:建立TGF-β1诱导的体外大鼠心肌细胞肥大模型。透射电镜观察心肌细胞形态。碘化丙啶(PI)染色标记法检测心肌细胞RNA含量。实时荧光定量PCR(Real time PCR)检测心肌细胞胚心基因肌球蛋白重链β亚型(β-MHC)的表达。TdT-FragEL染色检测心肌细胞凋亡。Annexin/7AAD双染法、直接免疫荧光标记法经流式细胞仪检测心肌细胞凋亡率及心肌细胞中caspase-3水平。结果:TGF-β1刺激24 h后,心肌细胞β-MHC mRNA水平明显高于对照组(P<0.01);PI染色结果TGF-β1组RNA含量明显增高,并呈剂量依赖性(P<0.01)。TdT-FragEL染色观察,TGF-β1可增加心肌细胞凋亡率(P<0.01)。与对照组相比,TGF-β1可明显上调心肌细胞中caspase-3(P<0.01);TGF-β1诱导的心肌细胞凋亡率增加(P<0.01)。透射电镜观察TGF-β1可诱导心肌细胞肥大和凋亡。结论:TGF-β1可同时诱导心肌细胞肥大和凋亡,诱导凋亡可能与caspase-3有关。展开更多
文摘Aim In diabetic patients, metformin appears to provide cardiovascular protection that cannot be attribu- ted only to its antihyperglycemic effects. Metformin is also known as the AMP-activated protein kinase （AMPK） ac- tivator. Our previous study suggested that metformin inhibits transforming growth factor-β1 （TGF-β1） production in a mouse heart failure model of pressure overload. TGF-β1 is a key factor in cardiac fibrosis and is usually induced by Angiotensin Ⅱ （Ang Ⅱ ） in the pressure overload mouse models. This study investigated the effect of metformin on cardiac fibrosis and TGF-β production induced by AngII and the underlying mechanisms. Methods C57/BL6 wild-type and AMPKα2 knockout mice were used. AngII （3 mg · kg-1 · d-1） was infused subcutaneously into mice for 7 days. Adult mouse cardiac fibroblasts were isolated and treated with AngII （ 1 μmol · L-1） and/or met- formin （1 mmol · L-l）. Results In C57/BL6 mice, metformin inhibits AngII-induced cardiac fibrosis. In cardi-ac fibroblasts, metformin inhibits TGF-β1 expression and production induced by AngII. AMPK inhibitor, com- pound C, reversed the effects of metformin. In vivo, AMPKα2 deficiency further increases AngII-induced TGF-β1 production. In cardiac fibroblasts, metformin inhibited AngII induced hepatocyte nuclear factor4 （HNF4ot protein level increase and HNF4α binding with TGF-β1 promoter using chromatin immunoprecipitation assay. In vivo, AMPKα2 deficiency further increased AngII-induced HNF4α protein level. Using HNF4α adenovirus, overexpress- ing HNF4α led to a 1.5-fold increase in TGF-β1 mRNA expression. HNF4a siRNA blocked AngII induced TGF- β1 production. Luciferase reporter with deleted HNF4a binding sites showed decreased TGFbl transcriptional activ- ity induced by AngII. In AMPK or2-/- heart, the inhibition of metformin on HNF4a protein was attenuated. Con- clusion Metformin inhibits AngII induced cardiac fibrosis and TGF-β1 production through AMPK activation. The underlying mechanism is that AMPK activation inhibits AngII induced HNF4α and then decreases TGF-β1 expres- sion.
文摘目的:探讨转化生长因子β1(TGF-β1)对大鼠心肌细胞肥大和凋亡的影响。方法:建立TGF-β1诱导的体外大鼠心肌细胞肥大模型。透射电镜观察心肌细胞形态。碘化丙啶(PI)染色标记法检测心肌细胞RNA含量。实时荧光定量PCR(Real time PCR)检测心肌细胞胚心基因肌球蛋白重链β亚型(β-MHC)的表达。TdT-FragEL染色检测心肌细胞凋亡。Annexin/7AAD双染法、直接免疫荧光标记法经流式细胞仪检测心肌细胞凋亡率及心肌细胞中caspase-3水平。结果:TGF-β1刺激24 h后,心肌细胞β-MHC mRNA水平明显高于对照组(P<0.01);PI染色结果TGF-β1组RNA含量明显增高,并呈剂量依赖性(P<0.01)。TdT-FragEL染色观察,TGF-β1可增加心肌细胞凋亡率(P<0.01)。与对照组相比,TGF-β1可明显上调心肌细胞中caspase-3(P<0.01);TGF-β1诱导的心肌细胞凋亡率增加(P<0.01)。透射电镜观察TGF-β1可诱导心肌细胞肥大和凋亡。结论:TGF-β1可同时诱导心肌细胞肥大和凋亡,诱导凋亡可能与caspase-3有关。
