CAJ | 학술논문

目的通过在体和离体实验评价七氟醚预处理对小鼠心肌缺血再灌注时环氧化酶-2(COX-2)表达的影响.方法在体实验健康成年小鼠54只,体重约250 g,6～8周龄,采用随机数字表法,将其随机分为3组(n=18):对照组(C组)、心肌缺血再灌注组(I/R组)和七氟醚预处理组(SP组).采用结扎左冠状动脉30 min再灌注的方法制备心肌缺血再灌注模型.SP组小鼠吸入2.0％七氟醚,15min/次,共3次,间隔15 min.七氟醚预处理后90 min制备心肌缺血再灌注模型.再灌注6、9h时,取心肌组织分别采用Western blot法测定COX-2表达,采用分光光度法测定心肌caspase-3的活性.再灌注24h时测定左室舒张末压(LVEDP).细胞实验采用随机数字表法,将H9C2细胞随机分为3组,每组6皿:对照组(C组)、缺氧复氧组(H/R组)和七氟醚预处理组(SP组).H9C2细胞置于95％N2+ 5％CO2培养箱中孵育14 h后,置于95％O2+ 5％CO2培养箱中孵育3h,建立缺氧复氧模型.SP组H9C2细胞培养基中加入2.0 mmol/L七氟醚,每次孵育15 min,共3次,间隔15 min.七氟醚预处理结束后15 min建立缺氧复氧模型.复氧3h时采用Western blot法测定细胞COX-2的表达水平,采用比色法测定上清液和细胞裂解液LDH活性,计算LDH释放量.结果在体实验:与C组比较,I/R组和SP组LVEDP升高,心肌组织caspase-3活性增强,COX-2表达上调(P＜0.01)；与I/R组比较,SP组LVEDP降低,心肌组织caspase-3活性减弱,COX-2表达下调(P＜0.01).细胞实验结果:与C组比较,H/R组和SP组H9C2细胞COX-2表达上调,LDH释放量增加(P＜0.01)；与H/R组比较,SP组H9C2细胞COX-2表达下调,LDH释放量减少(P＜0.01).结论七氟醚预处理可通过直接下调心肌COX-2的表达,抑制细胞凋亡减轻小鼠心肌缺血再灌注损伤.
목적 통과재체화리체실험평개칠불미예처리대소서심기결혈재관주시배양화매-2(COX-2)표체적영향.방법 재체실험건강성년소서54지,체중약250 g,6～8주령,채용수궤수자표법,장기수궤분위3조(n=18):대조조(C조)、심기결혈재관주조(I/R조)화칠불미예처리조(SP조).채용결찰좌관상동맥30 min재관주적방법제비심기결혈재관주모형.SP조소서흡입2.0％칠불미,15min/차,공3차,간격15 min.칠불미예처리후90 min제비심기결혈재관주모형.재관주6、9h시,취심기조직분별채용Western blot법측정COX-2표체,채용분광광도법측정심기caspase-3적활성.재관주24h시측정좌실서장말압(LVEDP).세포실험채용수궤수자표법,장H9C2세포수궤분위3조,매조6명:대조조(C조)、결양복양조(H/R조)화칠불미예처리조(SP조).H9C2세포치우95％N2+ 5％CO2배양상중부육14 h후,치우95％O2+ 5％CO2배양상중부육3h,건립결양복양모형.SP조H9C2세포배양기중가입2.0 mmol/L칠불미,매차부육15 min,공3차,간격15 min.칠불미예처리결속후15 min건립결양복양모형.복양3h시채용Western blot법측정세포COX-2적표체수평,채용비색법측정상청액화세포렬해액LDH활성,계산LDH석방량.결과 재체실험:여C조비교,I/R조화SP조LVEDP승고,심기조직caspase-3활성증강,COX-2표체상조(P＜0.01)；여I/R조비교,SP조LVEDP강저,심기조직caspase-3활성감약,COX-2표체하조(P＜0.01).세포실험결과:여C조비교,H/R조화SP조H9C2세포COX-2표체상조,LDH석방량증가(P＜0.01)；여H/R조비교,SP조H9C2세포COX-2표체하조,LDH석방량감소(P＜0.01).결론 칠불미예처리가통과직접하조심기COX-2적표체,억제세포조망감경소서심기결혈재관주손상.
Objective To evaluate the effect of sevoflurane preconditioning on expression of cyclooxygenase-2 (COX-2) during myocardial ischemia-reperfusion (I/R) in mice in vivo and in vitro.Methods In the in vivo experiment,54 healthy adult mice,aged 6-8 weeks,weighing 250 g,were randomly divided imo 3 groups (n=18 each):control group (group C),I/R group and sevoflurane preconditioning group (group SP).Myocardial I/R was induced by ligation of the left coronary artery for 30 min followed by reperfusion.In SP group,the mice received 3 episodes of 15 min 2.0％ sevoflurane inhalation at 15 min interval 90 min before I/R.The myocardial specimens were obtained at 6 and 9 h of reperfusion for measurement of COX-2 expression (by Western blot) and caspase-3 activity (by spectmphotometry).Left ventricular end-diastolic pressure (LVEDP) was recorded at 24 h of reperfusion.in the cell experiment,H9C2 cells were randomly divided into 3 groups with 6 dishes in each group:control group (group C),hypoxia-reoxygenation (H/R) group,and sevoflurane preconditioning group (SP group).The cells were exposed to 95％ N2 + 5％ CO2 in an incubator for 14 h followed by reoxygenation with 95％ O2 +5％ CO2 for 3 h.Sevoflurane 2.0 mmol/L was added to the culture medium and the cells underwent 3 episodes of 15 min incubation with 2.0％ sevoflurane at 15 min interval in group SP.H/R was induced 15 min after the end of sevoflurane preconditioning.COX-2 expression was measured at 3 h of reoxygenation by Western blot.Lactic dehydrogenase (LDH) activity in the supernatant and cell lysate was measured by colorimetric method.The release of LDH was caculated.Results In the in vivo experiment,compared with group C,LVEDP and caspase-3 activity were significantly increased and COX-2 expression was up-regulated in groups I/R and SP (P ＜0.01).Compared with group I/R,LVEDP and caspase-3 activity were significantly decreased and COX-2 expression was down-regulated in group SP (P ＜ 0.01).In the cell experiment,compared with group C,COX-2 expression was significantly up-regulated and release of LDH was increased in H/R and SP groups (P ＜ 0.01).Compared with group H/R,COX-2 expression was significantly down-regulated and the release of LDH was decreased in group SP (P ＜ 0.01).Conclusion Sevoflurane preconditioning can reduce myocardial I/R injury through downregulating myocardial COX-2 expression and inhibiting cell apoptosis in mice.