中华心血管病杂志
中華心血管病雜誌
중화심혈관병잡지
Chinese Journal of Cardiology
2015年
8期
718-723
,共6页
肌细胞,心脏%肥大%血管紧张素Ⅱ%白藜芦醇
肌細胞,心髒%肥大%血管緊張素Ⅱ%白藜蘆醇
기세포,심장%비대%혈관긴장소Ⅱ%백려호순
Myocytes,cardiac%Hypertrophy%Angiotensin Ⅱ%Resveratrol
目的 观察白藜芦醇对血管紧张素Ⅱ(AngⅡ)诱导的心肌细胞肥大的作用,并研究其对心肌细胞叉头转录因子O1(FoxO1)/锰超氧化物歧化酶(MnSOD)信号通路的影响.方法 应用细胞培养技术培养新生大鼠心肌细胞,胰酶消化,差数贴壁法体外分离培养大鼠心肌细胞.将心肌细胞分为5组,分别为对照组(不加任何干预因素)、AngⅡ(10-6 mol/L)组(AngⅡ组)、AngⅡ(10-6mol/L)+白藜芦醇(10 μmol/L)组[AngⅡ+白藜芦醇(10 μmol/L)组]、AngⅡ(10-6 μmol/L)+白藜芦醇(25 μmol/L)组[AngⅡ+白藜芦醇(25 μmol/L)组]和AngⅡ(10-6 μmol/L)+白藜芦醇(50μmol/L)组[AngⅡ+白藜芦醇(50 μmol/L)组].3H-亮氨酸掺入法测定心肌细胞蛋白质合成速率(心肌细胞肥大的指标),相差显微镜测量细胞大小(细胞直径),实时定量聚合酶链反应检测A型利钠肽(ANP) mRNA表达水平,蛋白免疫印迹法检测心肌细胞MnSOD、FoxO1和乙酰化FoxO1蛋白表达水平,免疫共沉淀法检测Sirt1与FoxO1的相互结合水平.结果 AngⅡ组心肌细胞蛋白质合成速率明显大于对照组[(1 971±175)计数·min-1·孔-1比(1 216±136)计数·min-1·孔-1,P＜0.05],AngⅡ+白藜芦醇(10 μmol/L)组、AngⅡ+白藜芦醇(25 μmol/L)组和AngⅡ+白藜芦醇(50μmol/L)组则均明显低于AngⅡ组(P均＜0.05),且以AngⅡ+白藜芦醇(25 μmol/L)组最为显著[(1 374±143)计数·min-1·孔-1].AngⅡ组心肌细胞直径大于对照组[(29.3±3.2) μm比(19.4±1.8) μm,P＜0.05],AngⅡ+白藜芦醇(10 μmol/L)组、AngⅡ+白藜芦醇(25 μmol/L)组和AngⅡ+白藜芦醇(50 μmol/L)组则均小于AngⅡ组(P均＜0.05),且以AngⅡ+白藜芦醇(25μmol/L)组最为显著[(20.8±2.1)μm].AngⅡ组心肌细胞ANP mRNA表达水平高于对照组(4.4±0.4比1.0 ±0.1,P＜0.05),AngⅡ+白藜芦醇(10 μmol/L)组、AngⅡ+白藜芦醇(25 μmol/L)组和AngⅡ+白藜芦醇(50 μmol/L)组则均低于AngⅡ组(P均＜0.05),且以AngⅡ+白藜芦醇(25μmol/L)组最为显著(2.2±0.2).AngⅡ组心肌细胞MnSOD蛋白表达水平和活性均明显低于对照组(P均＜0.05),AngⅡ+白藜芦醇(25 μmol/L)组心肌细胞MnSOD蛋白表达水平和活性则均明显高于AngⅡ组(P均＜0.05).AngⅡ组心肌细胞Sirt1与FoxO1的相互结合水平低于对照组(1.00±0.11比1.63±0.16,P＜0.05),而乙酰化FoxO1的表达水平则高于对照组(1.48±0.16比1.00±0.13,P＜0.05),而AngⅡ+白藜芦醇(25 μmol/L)组心肌细胞Sirt1与FoxO1的相互结合水平高于AngⅡ组(1.64±0.16,P＜0.05),乙酰化FoxO1的表达水平则低于AngⅡ组(0.79±0.07,P＜0.05).结论 白藜芦醇可以抑制AngⅡ诱导的心肌细胞肥大,其作用机制可能与激活Sirt1,降低乙酰化FoxO1的表达有关,提示Sirt1可能是治疗心肌细胞肥大的潜在靶点.
目的 觀察白藜蘆醇對血管緊張素Ⅱ(AngⅡ)誘導的心肌細胞肥大的作用,併研究其對心肌細胞扠頭轉錄因子O1(FoxO1)/錳超氧化物歧化酶(MnSOD)信號通路的影響.方法 應用細胞培養技術培養新生大鼠心肌細胞,胰酶消化,差數貼壁法體外分離培養大鼠心肌細胞.將心肌細胞分為5組,分彆為對照組(不加任何榦預因素)、AngⅡ(10-6 mol/L)組(AngⅡ組)、AngⅡ(10-6mol/L)+白藜蘆醇(10 μmol/L)組[AngⅡ+白藜蘆醇(10 μmol/L)組]、AngⅡ(10-6 μmol/L)+白藜蘆醇(25 μmol/L)組[AngⅡ+白藜蘆醇(25 μmol/L)組]和AngⅡ(10-6 μmol/L)+白藜蘆醇(50μmol/L)組[AngⅡ+白藜蘆醇(50 μmol/L)組].3H-亮氨痠摻入法測定心肌細胞蛋白質閤成速率(心肌細胞肥大的指標),相差顯微鏡測量細胞大小(細胞直徑),實時定量聚閤酶鏈反應檢測A型利鈉肽(ANP) mRNA錶達水平,蛋白免疫印跡法檢測心肌細胞MnSOD、FoxO1和乙酰化FoxO1蛋白錶達水平,免疫共沉澱法檢測Sirt1與FoxO1的相互結閤水平.結果 AngⅡ組心肌細胞蛋白質閤成速率明顯大于對照組[(1 971±175)計數·min-1·孔-1比(1 216±136)計數·min-1·孔-1,P＜0.05],AngⅡ+白藜蘆醇(10 μmol/L)組、AngⅡ+白藜蘆醇(25 μmol/L)組和AngⅡ+白藜蘆醇(50μmol/L)組則均明顯低于AngⅡ組(P均＜0.05),且以AngⅡ+白藜蘆醇(25 μmol/L)組最為顯著[(1 374±143)計數·min-1·孔-1].AngⅡ組心肌細胞直徑大于對照組[(29.3±3.2) μm比(19.4±1.8) μm,P＜0.05],AngⅡ+白藜蘆醇(10 μmol/L)組、AngⅡ+白藜蘆醇(25 μmol/L)組和AngⅡ+白藜蘆醇(50 μmol/L)組則均小于AngⅡ組(P均＜0.05),且以AngⅡ+白藜蘆醇(25μmol/L)組最為顯著[(20.8±2.1)μm].AngⅡ組心肌細胞ANP mRNA錶達水平高于對照組(4.4±0.4比1.0 ±0.1,P＜0.05),AngⅡ+白藜蘆醇(10 μmol/L)組、AngⅡ+白藜蘆醇(25 μmol/L)組和AngⅡ+白藜蘆醇(50 μmol/L)組則均低于AngⅡ組(P均＜0.05),且以AngⅡ+白藜蘆醇(25μmol/L)組最為顯著(2.2±0.2).AngⅡ組心肌細胞MnSOD蛋白錶達水平和活性均明顯低于對照組(P均＜0.05),AngⅡ+白藜蘆醇(25 μmol/L)組心肌細胞MnSOD蛋白錶達水平和活性則均明顯高于AngⅡ組(P均＜0.05).AngⅡ組心肌細胞Sirt1與FoxO1的相互結閤水平低于對照組(1.00±0.11比1.63±0.16,P＜0.05),而乙酰化FoxO1的錶達水平則高于對照組(1.48±0.16比1.00±0.13,P＜0.05),而AngⅡ+白藜蘆醇(25 μmol/L)組心肌細胞Sirt1與FoxO1的相互結閤水平高于AngⅡ組(1.64±0.16,P＜0.05),乙酰化FoxO1的錶達水平則低于AngⅡ組(0.79±0.07,P＜0.05).結論 白藜蘆醇可以抑製AngⅡ誘導的心肌細胞肥大,其作用機製可能與激活Sirt1,降低乙酰化FoxO1的錶達有關,提示Sirt1可能是治療心肌細胞肥大的潛在靶點.
목적 관찰백려호순대혈관긴장소Ⅱ(AngⅡ)유도적심기세포비대적작용,병연구기대심기세포차두전록인자O1(FoxO1)/맹초양화물기화매(MnSOD)신호통로적영향.방법 응용세포배양기술배양신생대서심기세포,이매소화,차수첩벽법체외분리배양대서심기세포.장심기세포분위5조,분별위대조조(불가임하간예인소)、AngⅡ(10-6 mol/L)조(AngⅡ조)、AngⅡ(10-6mol/L)+백려호순(10 μmol/L)조[AngⅡ+백려호순(10 μmol/L)조]、AngⅡ(10-6 μmol/L)+백려호순(25 μmol/L)조[AngⅡ+백려호순(25 μmol/L)조]화AngⅡ(10-6 μmol/L)+백려호순(50μmol/L)조[AngⅡ+백려호순(50 μmol/L)조].3H-량안산참입법측정심기세포단백질합성속솔(심기세포비대적지표),상차현미경측량세포대소(세포직경),실시정량취합매련반응검측A형리납태(ANP) mRNA표체수평,단백면역인적법검측심기세포MnSOD、FoxO1화을선화FoxO1단백표체수평,면역공침정법검측Sirt1여FoxO1적상호결합수평.결과 AngⅡ조심기세포단백질합성속솔명현대우대조조[(1 971±175)계수·min-1·공-1비(1 216±136)계수·min-1·공-1,P＜0.05],AngⅡ+백려호순(10 μmol/L)조、AngⅡ+백려호순(25 μmol/L)조화AngⅡ+백려호순(50μmol/L)조칙균명현저우AngⅡ조(P균＜0.05),차이AngⅡ+백려호순(25 μmol/L)조최위현저[(1 374±143)계수·min-1·공-1].AngⅡ조심기세포직경대우대조조[(29.3±3.2) μm비(19.4±1.8) μm,P＜0.05],AngⅡ+백려호순(10 μmol/L)조、AngⅡ+백려호순(25 μmol/L)조화AngⅡ+백려호순(50 μmol/L)조칙균소우AngⅡ조(P균＜0.05),차이AngⅡ+백려호순(25μmol/L)조최위현저[(20.8±2.1)μm].AngⅡ조심기세포ANP mRNA표체수평고우대조조(4.4±0.4비1.0 ±0.1,P＜0.05),AngⅡ+백려호순(10 μmol/L)조、AngⅡ+백려호순(25 μmol/L)조화AngⅡ+백려호순(50 μmol/L)조칙균저우AngⅡ조(P균＜0.05),차이AngⅡ+백려호순(25μmol/L)조최위현저(2.2±0.2).AngⅡ조심기세포MnSOD단백표체수평화활성균명현저우대조조(P균＜0.05),AngⅡ+백려호순(25 μmol/L)조심기세포MnSOD단백표체수평화활성칙균명현고우AngⅡ조(P균＜0.05).AngⅡ조심기세포Sirt1여FoxO1적상호결합수평저우대조조(1.00±0.11비1.63±0.16,P＜0.05),이을선화FoxO1적표체수평칙고우대조조(1.48±0.16비1.00±0.13,P＜0.05),이AngⅡ+백려호순(25 μmol/L)조심기세포Sirt1여FoxO1적상호결합수평고우AngⅡ조(1.64±0.16,P＜0.05),을선화FoxO1적표체수평칙저우AngⅡ조(0.79±0.07,P＜0.05).결론 백려호순가이억제AngⅡ유도적심기세포비대,기작용궤제가능여격활Sirt1,강저을선화FoxO1적표체유관,제시Sirt1가능시치료심기세포비대적잠재파점.
Objective To investigate the effect of resveratrol (RSV) on angiotensin Ⅱ (Ang Ⅱ) induced cardiomyocytes hypertrophy and FoxO1/MnSOD signaling pathway.Methods The cardiomyocytes isolated from neonatal Wistar rats were cultured with pancreatin and preplating technique and divided into five groups:control (CON),Ang Ⅱ (1 × 10-6 mol/L,AngⅡ),Ang Ⅱ +RSV (10 μmol/L),Ang Ⅱ + RSV (25 μmol/L),and Ang Ⅱ + RSV (50 μmol/L).3H-Leucine incorporation method were used to determine the cardiomyocyte protein synthesis rate.Cell size was measured by phase contrast microscope.The gene expression of A type natriuretic factor (ANF) was detected by real-time PCR.Western blot was used to measure the expression of MnSOD,FoxO1 and acety-FoxO1.Immunoprecipitation was used to detect the interaction between Sirt1 and FoxO1.Results Cardiomyocyte protein synthesis rate in Ang ⅡⅡ group was significantly higher in Ang Ⅱ group than in the control group ((1 971 ± 175) cpm vs.(1 216 ± 136) cpm,P ＜ 0.05),which could be significantly attenuated by RSV (10 μmol/L,25 μmol/L,50 μmol/L,all P＜0.05),especially in Ang Ⅱ +RSV (25 μmol/L) group((1 374 ±143) cpm).Cardiomyocytes size in Ang Ⅱ group was significantly larger than control group ((29.3 ± 3.2) μm vs.(19.4 ± 1.8) μm,P ＜ 0.05),which could be significantly reduced by cotreatment with RSV (10 μmol/L,25 μmol/L,50 μmol/L,all P ＜ 0.05),especially in Ang Ⅱ + RSV (25 μmol/L) group ((20.8 ± 2.1) μm).Ang Ⅱ also significantly upregulated ANP mRNA expression of cardiomyocytes (4.4 ± 0.4 vs.1.0 ± 0.1 in control group,P ＜ 0.05),which could be significantly inhibited by cotreatment with RSV,especially in Ang Ⅱ + RSV (25 μmol/L) group (2.2 ± 0.2).Ang Ⅱ significantly decreased MnSOD expression of cardiomyocytes compared with control group (P ＜ 0.05),which was reversed by RSV (25 μmol/L).The binding level of Sirt1 and FoxO1 was significantly lower (1.00 ±0.11 vs.1.63 ±0.16,P ＜0.05),and the expression of acetylation of FoxO1 was significantly higher in Ang Ⅱ group than in control group (1.48 ± 0.16 vs.1.00 ± 0.13,P ＜ 0.05),which was significantly reversed by cotreatment with RSV (25 μmol/L).Conclusions Resveratrol treatment can inhibit Ang Ⅱ induced cardiomyocyte hypertrophy.This protective effect is associated with reduced FoxO1 acetylation and activation of Sirt1,suggesting that Sirt1 may serve as a potential therapeutic target of cardiomyocyte hypertrophy.
