CAJ | 학술논문

目的评价七氟醚预处理联合后处理对新生大鼠心肌细胞缺氧复氧损伤的影响.方法健康新生SD大鼠,1～3d龄,处死后取心室肌组织,分离心肌细胞,将心肌细胞于DMEM培养液中培养,细胞密度3×105个/ml,接种于24孔培养板(1 ml/孔)、35 mm培养皿(5 ml/皿)或50 ml培养瓶(8ml/瓶),采用随机数字表法,将其随机分为9组(n=24):正常对照组(C组)常规培养160 min；缺氧复氧组(A/R组)、七氟醚预处理组(S1组)、七氟醚预处理+SB203580组(S1+SB组)、七氟醚后处理组(S2组)、七氟醚后处理+ SB203580组(S2+SB组)、七氟醚预处理+七氟醚后处理组(S3组)、七氟醚预处理+七氟醚后处理+ SB203580组(S3+SB组)和SB203580组(SB组)均进行缺氧120 min,复氧20 min.S1组、S1+ SB组、S3组和S3+SB组缺氧前用2.5％七氟醚孵育20 min,S1+SB组和S3+SB组七氟醚预处理同时加入5 μmol/L p38丝裂原激活蛋白激酶(p38MAPK)特异性抑制剂SB203580；S2组、S2+SB组、S3组和S3+ SB组复氧开始时采用2.5％七氟醚孵育20 min,S2+SB组和S3+SB组于七氟醚后处理同时加入5 μmol/L SB203580；SB组缺氧前20 min和复氧开始时均用5μmol/L SB203580孵育20 min.复氧结束时测定细胞培养液乳酸脱氢酶活性、细胞存活率和细胞凋亡率,分别于七氟醚预处理结束和后处理结束时测定磷酸化p38MAPK (p-p38MAPK)表达水平.结果与C组比较,其余各组乳酸脱氢酶活性和细胞凋亡率升高,细胞存活率降低(P＜0.05)；与A/R组比较,S1组、S2组和S3组LDH活性和细胞凋亡率降低,细胞存活率升高(P＜0.05)；S1组、S2组和S3组间上述指标差异无统计学意义(P＞0.05)；SB203580可取消七氟醚预处理、后处理及二者联合应用时心肌保护效应(P＜0.05).七氟醚预处理和后处理均可上调p-p38MAPK表达,且二者联合应用时上调p-p38MAPK表达的效应并未增强,SB203580可取消其上调p-p38MAPK表达的效应(P＜0.05).结论七氟醚预处理联合后处理减轻新生大鼠心肌细胞缺氧复氧损伤效应与单独一种措施的效应相似,其机制与二者均通过p38MAPK信号转导通路发挥心肌保护作用有关. 目的評價七氟醚預處理聯閤後處理對新生大鼠心肌細胞缺氧複氧損傷的影響.方法健康新生SD大鼠,1～3d齡,處死後取心室肌組織,分離心肌細胞,將心肌細胞于DMEM培養液中培養,細胞密度3×105箇/ml,接種于24孔培養闆(1 ml/孔)、35 mm培養皿(5 ml/皿)或50 ml培養瓶(8ml/瓶),採用隨機數字錶法,將其隨機分為9組(n=24):正常對照組(C組)常規培養160 min；缺氧複氧組(A/R組)、七氟醚預處理組(S1組)、七氟醚預處理+SB203580組(S1+SB組)、七氟醚後處理組(S2組)、七氟醚後處理+ SB203580組(S2+SB組)、七氟醚預處理+七氟醚後處理組(S3組)、七氟醚預處理+七氟醚後處理+ SB203580組(S3+SB組)和SB203580組(SB組)均進行缺氧120 min,複氧20 min.S1組、S1+ SB組、S3組和S3+SB組缺氧前用2.5％七氟醚孵育20 min,S1+SB組和S3+SB組七氟醚預處理同時加入5 μmol/L p38絲裂原激活蛋白激酶(p38MAPK)特異性抑製劑SB203580；S2組、S2+SB組、S3組和S3+ SB組複氧開始時採用2.5％七氟醚孵育20 min,S2+SB組和S3+SB組于七氟醚後處理同時加入5 μmol/L SB203580；SB組缺氧前20 min和複氧開始時均用5μmol/L SB203580孵育20 min.複氧結束時測定細胞培養液乳痠脫氫酶活性、細胞存活率和細胞凋亡率,分彆于七氟醚預處理結束和後處理結束時測定燐痠化p38MAPK (p-p38MAPK)錶達水平.結果與C組比較,其餘各組乳痠脫氫酶活性和細胞凋亡率升高,細胞存活率降低(P＜0.05)；與A/R組比較,S1組、S2組和S3組LDH活性和細胞凋亡率降低,細胞存活率升高(P＜0.05)；S1組、S2組和S3組間上述指標差異無統計學意義(P＞0.05)；SB203580可取消七氟醚預處理、後處理及二者聯閤應用時心肌保護效應(P＜0.05).七氟醚預處理和後處理均可上調p-p38MAPK錶達,且二者聯閤應用時上調p-p38MAPK錶達的效應併未增彊,SB203580可取消其上調p-p38MAPK錶達的效應(P＜0.05).結論七氟醚預處理聯閤後處理減輕新生大鼠心肌細胞缺氧複氧損傷效應與單獨一種措施的效應相似,其機製與二者均通過p38MAPK信號轉導通路髮揮心肌保護作用有關.
목적 평개칠불미예처리연합후처리대신생대서심기세포결양복양손상적영향.방법 건강신생SD대서,1～3d령,처사후취심실기조직,분리심기세포,장심기세포우DMEM배양액중배양,세포밀도3×105개/ml,접충우24공배양판(1 ml/공)、35 mm배양명(5 ml/명)혹50 ml배양병(8ml/병),채용수궤수자표법,장기수궤분위9조(n=24):정상대조조(C조)상규배양160 min；결양복양조(A/R조)、칠불미예처리조(S1조)、칠불미예처리+SB203580조(S1+SB조)、칠불미후처리조(S2조)、칠불미후처리+ SB203580조(S2+SB조)、칠불미예처리+칠불미후처리조(S3조)、칠불미예처리+칠불미후처리+ SB203580조(S3+SB조)화SB203580조(SB조)균진행결양120 min,복양20 min.S1조、S1+ SB조、S3조화S3+SB조결양전용2.5％칠불미부육20 min,S1+SB조화S3+SB조칠불미예처리동시가입5 μmol/L p38사렬원격활단백격매(p38MAPK)특이성억제제SB203580；S2조、S2+SB조、S3조화S3+ SB조복양개시시채용2.5％칠불미부육20 min,S2+SB조화S3+SB조우칠불미후처리동시가입5 μmol/L SB203580；SB조결양전20 min화복양개시시균용5μmol/L SB203580부육20 min.복양결속시측정세포배양액유산탈경매활성、세포존활솔화세포조망솔,분별우칠불미예처리결속화후처리결속시측정린산화p38MAPK (p-p38MAPK)표체수평.결과 여C조비교,기여각조유산탈경매활성화세포조망솔승고,세포존활솔강저(P＜0.05)；여A/R조비교,S1조、S2조화S3조LDH활성화세포조망솔강저,세포존활솔승고(P＜0.05)；S1조、S2조화S3조간상술지표차이무통계학의의(P＞0.05)；SB203580가취소칠불미예처리、후처리급이자연합응용시심기보호효응(P＜0.05).칠불미예처리화후처리균가상조p-p38MAPK표체,차이자연합응용시상조p-p38MAPK표체적효응병미증강,SB203580가취소기상조p-p38MAPK표체적효응(P＜0.05).결론 칠불미예처리연합후처리감경신생대서심기세포결양복양손상효응여단독일충조시적효응상사,기궤제여이자균통과p38MAPK신호전도통로발휘심기보호작용유관.
Objective To investigate the effect of sevoflurane preconditioning combined with postconditioning (Spost) on anoxia/reoxygenation (A/R) injury to neonatal rat cardiomyocytes.Methods Primary cultured neonatal rat cardiomyocytes were isolated from SD rats aged 1-3 days and cultured in DMEM liquid culture medium.The cells were seeded in 24-well plates (1 ml/hole),35 mm diameter dishes (5 ml/dish) or in 50 mm culture flasks (8 ml/flask) with a density of 3 × 105/ml and randomly divided into 9 groups (n =24 each):control group (group C),A/R group,Spre group (group S1),Spre + SB203580 group (group S1 + SB),sevoflurane postcon-ditioning (Spost) group (group S2),Spost + SB203580 group(group S2 + SB),Spre + Spost group (group S3),Spre + Spost + SB203580 group (group S3 + SB),and group SB203580 (group SB).The cells were cultured routinely for 160 min in group C and the cells were exposed to 95％ N2-5％ CO2 in an incubator at 37 ℃ for 120 min followed by reoxygenation for 20 min in the other groups.The cells were incubated with 2.5 ％ sevoflurane for 20 min before anoxia in groups S1,S1 + SB,S3 and S3 + SB and in addition SB203580 (specific p38MAPK inhibitor) 5 μmol/L was added simultaneously in groups S1 + SB and S3 + SB.The cells were incubated with 2.5％ sevoflurane for 20 min after beginning of reoxygenation in groups S2,S2-SB,S3 and S3 + SB,and in addition SB203580 5 μmol/L was added simultaneously in groups S2 + SB and S3 + SB.The cells were incubated with SB203580 5 μmol/L for 20 min before anoxia and after beginning of reoxygenation in group SB.The lactate dehydrogenase (LDH) activity,cell survival rate and apoptotic rate were measured at the end of reoxygenation.The levels of phosphor-p38MAPK (p-p38MAPK) was detected at the end of Spre and Spost.Results Compared with group C,the LDH activity and apoptotic rate were significantly increased,while the cell survival rate was significantly decreased in the other groups (P ＜ 0.05).Compared with group A/R,the LDH activity and apoptotic rate were significantly decreased,while the cell survival rate was significantly increased in groups S1,S2 and S3 (P ＜ 0.05).There was no significant difference in the LDH activity,cell survival rate and apoptotic rate between groups S1,S2and S3 (P ＞ 0.05).The myocardial protective effect of Spre or Spost alone or in combination was eliminated by SB203580 (P ＜ 0.05).Spre or Spost alone up-regulated the expression of p-p38MAPK,Spre combined with Spost offered no additional benefit over Spre or Spost alone,and the up-regulative effect was eliminated by SB203580 (P ＜ 0.05).Conclusion Spre combined with Spost produces similar myocardial protective effect with that of either alone and it may because that both Spre and Spost attenuate A/R-induced injury to cardiomyocytes through p38MAPK signaling pathway.