摘要
目的 探究二甲双胍对2型糖尿病大鼠心房重构的影响及作用机制.方法 本研究为随机对照研究.以H2O2处理的HL-1细胞和2型糖尿病大鼠为研究模型.高脂喂养联合30mg/kg链脲佐菌素腹腔注射构建2型糖尿病模型.采用单纯随机化法将40只Wistar大鼠随机分为对照组、糖尿病组、二甲双胍治疗组、5氨基4甲酰胺咪唑核糖核苷酸(AICAR)治疗组、二甲双胍联合多柔比星(Compound C)治疗组.增补的Claycomb培养基培养HL-1细胞.细胞长至90％以上时分为对照组、H2O2组、二甲双胍+H2O2组、AICAR+H2O2组、二甲双胍+Compound C+H2O2组.动物水平进行超声心动图、无创血压、病理组织结构、超微结构及电生理测量.细胞水平进行线粒体膜电位(MMP)及活性氧(ROS)测定.蛋白质免疫印迹分析检测锰超氧化物歧化酶(Mn-SOD)、细胞间连接蛋白43(Cx43)和线粒体生物合成相关蛋白单磷酸腺苷激活的蛋白激酶(AMPK)、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、过氧化物酶体增殖物激活受体γ辅助活化因子-1α(PGC-1α)和线粒体转录因子A(TFAM)表达.结果 ①动物水平:与对照组相比,糖尿病组左心房内径(4.11 mm对4.83 mm,P＜0.001)、细胞横截面积(94 μm2对123 μm2,P＜0.001)显著增大,心肌间质纤维化增加(2.53％对5.22％,P＜0.001),线粒体形态异常,窦房结恢复时间(187.5 ms对222.5 ms,P＜0.001)、传导时间(12.16 ms对22.87 ms,P＜0.001)延长,心房有效不应期(91.25 ms对74.89 ms,P＜0.001)缩短,心房颤动(房颤)诱发率(16.67％对50.00％,P＜0.001)升高.②细胞水平:与对照组相比,H2O2组MMP(1.00对0.63,P＜0.001)降低,ROS(1.00对1.47,P＜0.001)增加.二甲双胍可改善上述情况.与糖尿病组相比,二甲双胍可以使窦房结恢复时间(222.5 ms 对 205.2 ms,P=0.041)、窦房结传导时间(22.87 ms 对 18.44 ms,P=0.002)缩短,心房有效不应期延长(74.89 ms对85.58 ms,P=0.045),减少房颤诱发率(50.00％对33.33％,P=0.028),增加Cx43表达(0.62对0.78,P=0.003).此外,在动物、细胞水平二甲双胍均可使蛋白Mn-SOD、Cx43、p-AMPK/AMPK、PGC-1α及TFAM表达升高.但二甲双胍的作用均被Compound C抑制.结论 二甲双胍通过激活AMPK信号通路促进线粒体生物合成改善线粒体功能、减少氧化应激进而改善糖尿病心房重构,最终减少房颤的发生.
Abstract
Objective To investigate the effect and mechanism of metformin on the atrial remodeling of type 2 diabetic rats.Methods H2O2-treated HL-1 cells and type 2 diabetic rats were studied in a randomized controlled experiment.Type 2 diabetic model was established by high-fat feeding combined with intraperitoneal injection of 30 mg/kg streptozotocin.Forty Wistar rats were randomly divided into 5 groups:control group,diabetic group,metformin-treated diabetic group,AICA ribonucleotide(AICAR)-treated diabetic group,and metformin combined with Compound C-treated diabetic group.HL-1 cells were cultured with supplemented Claycomb medium.When the cells reached over 90％confluence,they were divided into 5 groups:control group,H2O2 group,metformin+H2O2 group,AICAR+H2O2 group,and metformin+Compound C+H2O2 group.In vivo assessments included echocardiography,non-invasive hemodynamics,histological tissue structure,ultrastructure,and electrophysiological measurements.In vitro assessments included mitochondrial membrane potential(MMP)level and reactive oxygen species(ROS)generation.Western blotting was used to detect the expression of(Mn-superoxide dismutase,Mn-SOD),connexin 43(Cx43),adenosine 5'-monophate activated protein(AMPK),p-AMPK,peroxisome proliferator-activated receptor-γ coactivator 1α(PGC-1α),and mitochondrial transcription factor A(TFAM)at both in vivo and vitro.Results At the animal level,compared with the control group,the diabetic group showed a significant increase in left atrial diameter(4.11 mm vs.4.83 mm,P＜0.001),myocyte cross-sectional area(94 µm2 vs.123 µm2,P＜0.001),and myocardial interstitial fibrosis(2.53％vs.5.22％,P＜0.001),abnormal mitochondrial morphology,prolonged sinus node recovery time(SNRT,187.5 ms vs.222.5 ms,P＜0.001)and conduction time(SNCT,12.16 ms vs.22.87 ms,P＜0.001),shortened atrial effective refractory period(AERP,91.25 ms vs.74.89 ms,P＜0.001),as well as increase in atrial fibrillation(AF)induction(16.67％vs.50.00％,P＜0.001).At the cellular level,there was a significant decrease in MMP level(1.00 vs.0.63,P＜0.001)and increase in ROS production(1.00 vs.1.47,P＜0.001)in the H2O2 group compared with the control group.Metformin treatment improved these conditions.Compared with the diabetic group,metformin shortened SNRT(222.5 ms vs.205.2 ms,P=0.041),SNCT(22.87 ms vs.18.44 ms,P=0.002),prolonged AERP(74.89 ms vs.85.58 ms,P=0.045),reduced AF induction(50.00％vs.33.33％,P=0.028),and increased the expression of Cx43(0.62 vs.0.78,P=0.003).Moreover,metformin elevated the expression of proteins Mn-SOD,Cx43,p-AMPK,AMPK,PGC-1α,and TFAM at both animal and cellular levels.However,all the above effects of metformin were inhibited by Compound C.Conclusion Metformin improves mitochondrial function by activating the AMPK signaling pathway to promote mitochondrial biosynthesis,reduces oxidative stress and thereby improves diabetic atrial remodeling and ultimately reduces the occurrence of AF.
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