中华心血管病杂志
中華心血管病雜誌
중화심혈관병잡지
Chinese Journal of Cardiology
2012年
6期
516-521
,共6页
涂荣会%陈立%钟国强%曾志羽%黎庆捷%何艳%何燕%李金轶
塗榮會%陳立%鐘國彊%曾誌羽%黎慶捷%何豔%何燕%李金軼
도영회%진립%종국강%증지우%려경첩%하염%하연%리금질
细胞凋亡%活性氧%缺氧后处理
細胞凋亡%活性氧%缺氧後處理
세포조망%활성양%결양후처리
Apoptosis%Reactive oxygen species%Postconditioning
目的 观察缺氧后处理(PC)对缺氧/复氧心肌细胞凋亡的影响及氧自由基清除剂[超氧化物歧化酶(SOD)及过氧化氢酶(CAT)]后处理对PC抗凋亡效应的影响,探讨PC中线粒体氧化应激平衡对细胞膜和线粒体Bcl-2和Bax蛋白调控心肌细胞凋亡的作用.方法 大鼠乳鼠心肌细胞缺氧3h后,分别进行:(1)复氧6h(缺氧/复氧组,H/R组),(2)复氧5min、缺氧5min,反复3次,再复氧6 h(PC组),(3)应用SOD 100 U/ml后立即进行(2)的操作(SOD +PC组),(4)应用CAT 120U/ml后立即进行(2)的操作(CAT+ PC组),(5)应用SOD联合CAT后立即进行(2)的操作(SOD+CAT+ PC组),对照组心肌细胞在复氧条件下培养9h.应用荧光探针测定线粒体活性氧含量,流式细胞仪检测心肌细胞凋亡率,Western blot检测细胞膜和线粒体Bcl-2和Bax蛋白表达水平.结果 H/R组线粒体活性氧含量(61.53±4.73)显著高于对照组(13.25±1.07)和PC组(32.72±2.86),分别是他们的4.6和1.9倍(P均＜0.01),SOD+ PC组(23.05±1.13)、CAT+ PC组(23.82±1.88)和SOD+ CAT +PC组(16.58±0.74)则明显低于PC组(P均＜0.01),SOD+ PC组与CAT+ PC组比较差异无统计学意义(P＞0.05).各处理组心肌细胞凋亡率均显著高于对照组(P均＜0.01),SOD+CAT+ PC组[(44.60±3.12)％]和H/R组[(45.55±3.75)％]显著高于PC组[(26.42±2.96)％]、SOD+PC组[(26.01±4.24)％]和CAT+ PC组[(26.98±3.66)％],P均＜0.01,PC组、SOD+ PC组和CAT +PC组比较差异无统计学意义(P＞0.05).PC组、SOD+ PC组和CAT+ PC组细胞膜和线粒体Bcl-2蛋白表达水平显著高于对照组(P均＜0.01),Bax蛋白表达水平则显著低于对照组(P均＜0.01).H/R组和SOD+ CAT +PC组Bcl-2蛋白表达水平显著低于对照组(P均＜0.01),Bax蛋白表达水平显著高于对照组(P均＜0.01).结论 PC抑制线粒体活性氧爆发,减轻缺氧复氧心肌细胞凋亡,其抗凋亡机制可能与线粒体和细胞膜Bcl-2蛋白表达上调和Bax蛋白表达下调有关.单独应用SOD或CAT不影响PC抗凋亡作用,而二者联合则减弱PC抗凋亡作用.线粒体活性氧在PC的心脏保护作用发挥着重要作用.
目的 觀察缺氧後處理(PC)對缺氧/複氧心肌細胞凋亡的影響及氧自由基清除劑[超氧化物歧化酶(SOD)及過氧化氫酶(CAT)]後處理對PC抗凋亡效應的影響,探討PC中線粒體氧化應激平衡對細胞膜和線粒體Bcl-2和Bax蛋白調控心肌細胞凋亡的作用.方法 大鼠乳鼠心肌細胞缺氧3h後,分彆進行:(1)複氧6h(缺氧/複氧組,H/R組),(2)複氧5min、缺氧5min,反複3次,再複氧6 h(PC組),(3)應用SOD 100 U/ml後立即進行(2)的操作(SOD +PC組),(4)應用CAT 120U/ml後立即進行(2)的操作(CAT+ PC組),(5)應用SOD聯閤CAT後立即進行(2)的操作(SOD+CAT+ PC組),對照組心肌細胞在複氧條件下培養9h.應用熒光探針測定線粒體活性氧含量,流式細胞儀檢測心肌細胞凋亡率,Western blot檢測細胞膜和線粒體Bcl-2和Bax蛋白錶達水平.結果 H/R組線粒體活性氧含量(61.53±4.73)顯著高于對照組(13.25±1.07)和PC組(32.72±2.86),分彆是他們的4.6和1.9倍(P均＜0.01),SOD+ PC組(23.05±1.13)、CAT+ PC組(23.82±1.88)和SOD+ CAT +PC組(16.58±0.74)則明顯低于PC組(P均＜0.01),SOD+ PC組與CAT+ PC組比較差異無統計學意義(P＞0.05).各處理組心肌細胞凋亡率均顯著高于對照組(P均＜0.01),SOD+CAT+ PC組[(44.60±3.12)％]和H/R組[(45.55±3.75)％]顯著高于PC組[(26.42±2.96)％]、SOD+PC組[(26.01±4.24)％]和CAT+ PC組[(26.98±3.66)％],P均＜0.01,PC組、SOD+ PC組和CAT +PC組比較差異無統計學意義(P＞0.05).PC組、SOD+ PC組和CAT+ PC組細胞膜和線粒體Bcl-2蛋白錶達水平顯著高于對照組(P均＜0.01),Bax蛋白錶達水平則顯著低于對照組(P均＜0.01).H/R組和SOD+ CAT +PC組Bcl-2蛋白錶達水平顯著低于對照組(P均＜0.01),Bax蛋白錶達水平顯著高于對照組(P均＜0.01).結論 PC抑製線粒體活性氧爆髮,減輕缺氧複氧心肌細胞凋亡,其抗凋亡機製可能與線粒體和細胞膜Bcl-2蛋白錶達上調和Bax蛋白錶達下調有關.單獨應用SOD或CAT不影響PC抗凋亡作用,而二者聯閤則減弱PC抗凋亡作用.線粒體活性氧在PC的心髒保護作用髮揮著重要作用.
목적 관찰결양후처리(PC)대결양/복양심기세포조망적영향급양자유기청제제[초양화물기화매(SOD)급과양화경매(CAT)]후처리대PC항조망효응적영향,탐토PC중선립체양화응격평형대세포막화선립체Bcl-2화Bax단백조공심기세포조망적작용.방법 대서유서심기세포결양3h후,분별진행:(1)복양6h(결양/복양조,H/R조),(2)복양5min、결양5min,반복3차,재복양6 h(PC조),(3)응용SOD 100 U/ml후립즉진행(2)적조작(SOD +PC조),(4)응용CAT 120U/ml후립즉진행(2)적조작(CAT+ PC조),(5)응용SOD연합CAT후립즉진행(2)적조작(SOD+CAT+ PC조),대조조심기세포재복양조건하배양9h.응용형광탐침측정선립체활성양함량,류식세포의검측심기세포조망솔,Western blot검측세포막화선립체Bcl-2화Bax단백표체수평.결과 H/R조선립체활성양함량(61.53±4.73)현저고우대조조(13.25±1.07)화PC조(32.72±2.86),분별시타문적4.6화1.9배(P균＜0.01),SOD+ PC조(23.05±1.13)、CAT+ PC조(23.82±1.88)화SOD+ CAT +PC조(16.58±0.74)칙명현저우PC조(P균＜0.01),SOD+ PC조여CAT+ PC조비교차이무통계학의의(P＞0.05).각처리조심기세포조망솔균현저고우대조조(P균＜0.01),SOD+CAT+ PC조[(44.60±3.12)％]화H/R조[(45.55±3.75)％]현저고우PC조[(26.42±2.96)％]、SOD+PC조[(26.01±4.24)％]화CAT+ PC조[(26.98±3.66)％],P균＜0.01,PC조、SOD+ PC조화CAT +PC조비교차이무통계학의의(P＞0.05).PC조、SOD+ PC조화CAT+ PC조세포막화선립체Bcl-2단백표체수평현저고우대조조(P균＜0.01),Bax단백표체수평칙현저저우대조조(P균＜0.01).H/R조화SOD+ CAT +PC조Bcl-2단백표체수평현저저우대조조(P균＜0.01),Bax단백표체수평현저고우대조조(P균＜0.01).결론 PC억제선립체활성양폭발,감경결양복양심기세포조망,기항조망궤제가능여선립체화세포막Bcl-2단백표체상조화Bax단백표체하조유관.단독응용SOD혹CAT불영향PC항조망작용,이이자연합칙감약PC항조망작용.선립체활성양재PC적심장보호작용발휘착중요작용.
Objective To investigate mitochondrial oxidative stress on cardiomyocyte apoptosis and the expression of Bcl-2 and Bax proteins in cardiac sarcolemma and mitochondria after application of hypoxia postconditioning and free radical scavengers.Methods Primary cultured neonatal rat cardiomyocytes were exposed to 3 h hypoxia ( H ) followed by ( 1 ) 6 h of reoxygenation (R) (H/R),(2) 3 intermittent cycles of 5 min H and R before 6 h of R (PC),(3) application of superoxide dismutase (SOD) before PC ( SOD +PC),(4) application of catalase (CAT) before PC ( CAT + PC ),and (5) application of SOD plus CAT before PC (SOD + CAT + PC ).Cardiac sarcnlemma and mitochondria were isolated by differential centrifugation.Mitochondrial reactive oxygen species (ROS) was detected with fluorescent probes ( DCFHDA) and cardiomyocyte apoptosis was detected with flow cytometry.The expressions of Bcl-2 and Bax proteins in cardiac sarcolemma and mitochoncria were measured by Western blot.Results Mitochondrial ROS reduced significantly in PC,SOD + PC,CAT + PC and especially in SOD + CAT + PC groups ( all P ＜0.01 ).The number of apoptotic cardiomyocytes reduced significantly in PC,SOD + PC and CAT + PC ( all P ＜0.01 ) but not in SOD + CAT + PC groups.Bcl-2 levels increased while Bax levels decreased in cardiac sarcolemma and mitochondria in PC,SOD + PC and CAT + PC groups (all P＜0.01 ),Bcl-2 levels decreased and Bax levels increased in H/R and PC + SOD + CAT groups ( all P ＜0.01 ).Conclusions PC attenuated H/R induced ROS and cardiomyocyte apoptosis,which might be mediated by upregulating the expression of Bcl-2 and downregulating the Bax in mitochondria and sarcolemma:SOD or CAT alone did not but SOD plus CAT attenuate the anti-apoptotic effect of hypoxia postconditioning:mitochondrial ROS thus plays an important role in PC's cardioprotection.
