目的 评价磷脂酰肌醇-3-激酶(PI3K)及细胞外信号调节激酶1/2(ERK1/2)、线粒体ATP敏感性钾(mito-K_(ATP))通道及线粒体膜通透性转换孔(mPTP)在七氟醚后处理减轻大鼠离体心脏缺血再灌注损伤中的作用.方法 雄性清洁级SD大鼠,周龄7～10周,体重250～300 g,采用Langendorff法建立大鼠离体心脏灌注模型,取模型制备成功的心脏180个,随机分为12组(n=15),对照组(C组):持续灌注90 min;缺血再灌注组(IR组):停止灌注K-H液30 min,再灌注60 min;IR+LY组、IR+PD组、IR+ATR组、IR+5-HD组和IR+DMSO组:于再灌注即刻分别灌注PI3K特异性抑制剂LY294002(LV)15μmol/L、ERK1/2特异性抑制剂PD98059(PD)20 μmol/L、mPTP开放剂苍术甙(ATR)20 μmol/L、mito-K_(ATP)通道抑制剂5-羟癸酸(5-HD)100 μmol/L和溶剂二甲基亚砜(DMSO)0.02%15 min;七氟醚后处理组(S组):于再灌注即刻灌注经3%七氟醚饱和的K-H液15 min,随后更换正常K-H液再灌注45 min;S+LY组、S+PD组、S+ATR组和S+5-HD组:于再灌注即刻灌注经七氟醚饱和的K-H液同时分别灌注LY 15 μmol/L、PD 20 μmol/L、ATR 20 μmol/L、5-HD 100 μmol/L 15 min,随后更换为正常K-H液再灌注45 min.于平衡灌注20 min、停灌前即刻、再灌注15、30和60 min(T_(0～4))时测定冠状动脉流量(CF),记录心功能指标;C组、IR组和s组分别于T_0和T_4时收集冠状动脉流出液测定乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)活性和肌钙蛋白I(cTnI)浓度;于T_4时取左心室,测定心肌梗死面积,C组、IR组、IR+ATR组、IR+5.HD组、IR+DMSO组、S组、S+ATR组和S+5-HD组检测细胞凋亡情况,计算凋亡指数(AI),C组、IR组、IR+LY组、IR+PD组、IR+DMSO组、S组、S+LY组和S+PD组测定心肌烟酰胺腺嘌呤二核苷酸(NAD~+)含量.结果 与C组比较,其余各组再灌注时心功能降低,CF降低,心肌梗死面积增大,IR组、IR+ATR组、IR+5-HD组、IR+DMSO组、S组、S+ATR组和S+5-HD组AI升高,IR组、IR+LY组、IR+PD组、IR+DMSO组、S组、S+LY组和S+PD组NAD~+含量降低,IR组和S组LDH、CK-MB活性和cTnI浓度升高(P<0.05);与IR组比较,S组再灌注时心功能提高,CF升高,心肌梗死面积减小,AI降低,NAD~+含量升高,LDH、CK-MB活性和cTnI浓度降低(P<0.05),其余组上述指标差异无统计学意义(P>0.05).结论 七氟醚后处理可能通过激活PI3K及ERK1/2、促进mito-K_(ATP)通道开放、抑制mPTP开放,从而减轻大鼠离体心脏缺血再灌注损伤.
目的 評價燐脂酰肌醇-3-激酶(PI3K)及細胞外信號調節激酶1/2(ERK1/2)、線粒體ATP敏感性鉀(mito-K_(ATP))通道及線粒體膜通透性轉換孔(mPTP)在七氟醚後處理減輕大鼠離體心髒缺血再灌註損傷中的作用.方法 雄性清潔級SD大鼠,週齡7～10週,體重250～300 g,採用Langendorff法建立大鼠離體心髒灌註模型,取模型製備成功的心髒180箇,隨機分為12組(n=15),對照組(C組):持續灌註90 min;缺血再灌註組(IR組):停止灌註K-H液30 min,再灌註60 min;IR+LY組、IR+PD組、IR+ATR組、IR+5-HD組和IR+DMSO組:于再灌註即刻分彆灌註PI3K特異性抑製劑LY294002(LV)15μmol/L、ERK1/2特異性抑製劑PD98059(PD)20 μmol/L、mPTP開放劑蒼術甙(ATR)20 μmol/L、mito-K_(ATP)通道抑製劑5-羥癸痠(5-HD)100 μmol/L和溶劑二甲基亞砜(DMSO)0.02%15 min;七氟醚後處理組(S組):于再灌註即刻灌註經3%七氟醚飽和的K-H液15 min,隨後更換正常K-H液再灌註45 min;S+LY組、S+PD組、S+ATR組和S+5-HD組:于再灌註即刻灌註經七氟醚飽和的K-H液同時分彆灌註LY 15 μmol/L、PD 20 μmol/L、ATR 20 μmol/L、5-HD 100 μmol/L 15 min,隨後更換為正常K-H液再灌註45 min.于平衡灌註20 min、停灌前即刻、再灌註15、30和60 min(T_(0～4))時測定冠狀動脈流量(CF),記錄心功能指標;C組、IR組和s組分彆于T_0和T_4時收集冠狀動脈流齣液測定乳痠脫氫酶(LDH)、肌痠激酶同工酶(CK-MB)活性和肌鈣蛋白I(cTnI)濃度;于T_4時取左心室,測定心肌梗死麵積,C組、IR組、IR+ATR組、IR+5.HD組、IR+DMSO組、S組、S+ATR組和S+5-HD組檢測細胞凋亡情況,計算凋亡指數(AI),C組、IR組、IR+LY組、IR+PD組、IR+DMSO組、S組、S+LY組和S+PD組測定心肌煙酰胺腺嘌呤二覈苷痠(NAD~+)含量.結果 與C組比較,其餘各組再灌註時心功能降低,CF降低,心肌梗死麵積增大,IR組、IR+ATR組、IR+5-HD組、IR+DMSO組、S組、S+ATR組和S+5-HD組AI升高,IR組、IR+LY組、IR+PD組、IR+DMSO組、S組、S+LY組和S+PD組NAD~+含量降低,IR組和S組LDH、CK-MB活性和cTnI濃度升高(P<0.05);與IR組比較,S組再灌註時心功能提高,CF升高,心肌梗死麵積減小,AI降低,NAD~+含量升高,LDH、CK-MB活性和cTnI濃度降低(P<0.05),其餘組上述指標差異無統計學意義(P>0.05).結論 七氟醚後處理可能通過激活PI3K及ERK1/2、促進mito-K_(ATP)通道開放、抑製mPTP開放,從而減輕大鼠離體心髒缺血再灌註損傷.
목적 평개린지선기순-3-격매(PI3K)급세포외신호조절격매1/2(ERK1/2)、선립체ATP민감성갑(mito-K_(ATP))통도급선립체막통투성전환공(mPTP)재칠불미후처리감경대서리체심장결혈재관주손상중적작용.방법 웅성청길급SD대서,주령7～10주,체중250～300 g,채용Langendorff법건립대서리체심장관주모형,취모형제비성공적심장180개,수궤분위12조(n=15),대조조(C조):지속관주90 min;결혈재관주조(IR조):정지관주K-H액30 min,재관주60 min;IR+LY조、IR+PD조、IR+ATR조、IR+5-HD조화IR+DMSO조:우재관주즉각분별관주PI3K특이성억제제LY294002(LV)15μmol/L、ERK1/2특이성억제제PD98059(PD)20 μmol/L、mPTP개방제창술대(ATR)20 μmol/L、mito-K_(ATP)통도억제제5-간계산(5-HD)100 μmol/L화용제이갑기아풍(DMSO)0.02%15 min;칠불미후처리조(S조):우재관주즉각관주경3%칠불미포화적K-H액15 min,수후경환정상K-H액재관주45 min;S+LY조、S+PD조、S+ATR조화S+5-HD조:우재관주즉각관주경칠불미포화적K-H액동시분별관주LY 15 μmol/L、PD 20 μmol/L、ATR 20 μmol/L、5-HD 100 μmol/L 15 min,수후경환위정상K-H액재관주45 min.우평형관주20 min、정관전즉각、재관주15、30화60 min(T_(0～4))시측정관상동맥류량(CF),기록심공능지표;C조、IR조화s조분별우T_0화T_4시수집관상동맥류출액측정유산탈경매(LDH)、기산격매동공매(CK-MB)활성화기개단백I(cTnI)농도;우T_4시취좌심실,측정심기경사면적,C조、IR조、IR+ATR조、IR+5.HD조、IR+DMSO조、S조、S+ATR조화S+5-HD조검측세포조망정황,계산조망지수(AI),C조、IR조、IR+LY조、IR+PD조、IR+DMSO조、S조、S+LY조화S+PD조측정심기연선알선표령이핵감산(NAD~+)함량.결과 여C조비교,기여각조재관주시심공능강저,CF강저,심기경사면적증대,IR조、IR+ATR조、IR+5-HD조、IR+DMSO조、S조、S+ATR조화S+5-HD조AI승고,IR조、IR+LY조、IR+PD조、IR+DMSO조、S조、S+LY조화S+PD조NAD~+함량강저,IR조화S조LDH、CK-MB활성화cTnI농도승고(P<0.05);여IR조비교,S조재관주시심공능제고,CF승고,심기경사면적감소,AI강저,NAD~+함량승고,LDH、CK-MB활성화cTnI농도강저(P<0.05),기여조상술지표차이무통계학의의(P>0.05).결론 칠불미후처리가능통과격활PI3K급ERK1/2、촉진mito-K_(ATP)통도개방、억제mPTP개방,종이감경대서리체심장결혈재관주손상.
Objective To evaluate the role of phosphatidylinusitol-3-kinase (PI3K) and extracellular signal-regulated kinase 1/2 (ERK 1/2), mitochondrial ATP-sensitive potassium (mito-K_(ATP)) channel and mitochondrial permeability transition pore (mPTP) in cardioprotection induced by sevoflurane postconditioning against ischemia-reperfusion (IR) injury to isolated rat hearts. Methods Healthy male SD rats 7-10 weeks old weighing 250-300 g were anesthetized with intraperitoneal 3 % pentobarbital 40 mg/kg. Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95% O_2-5% CO_2 at 36.5-37.5℃. One hundred and nighty isolated rat hearts with I/R injury were randomly assigned to one of 12 groups (n=15) after 20 min of equilibration: control group (group C), IR group, IR + LY group, IR + PD group, IR + ATR group, IR+5-HD group, IR + DMSO group, sevoflurane postconditioning group (group S), S + LY group, S + PD group, S + ATR group and S + 5-HD group. Group C received continuous perfusion for another 90 min. Group IR was made ischemic for 30 min followed by 60 min of reperfusion. Group IR + LY, IR + PD, IR + ATR, IR+5-HD and IR+DMSO received perfusion with PI3K inhibitor LY294002(LY)15 μmol/L,ERK 1/2 inhibitor PD98059 (PD) 20μmol/L,mPTP opener atractyloside (ATR)20μmol/L,mito-KATP channel blocker 5-hybroxydecanoate (5-HD) 100μmol/L and 0.02% the vehicle dimethyl sulfoxide (DMSO)respectively for 15 min immediately before reperfusion. Group S received perfusion with K-H solution saturated with 3% sevoflurane for 15 min and then with plain K-H solution for 45 min. Group S + LY, S + PD, S + ATR and S + 5-HD received perfusion with K-H solution saturated with 3% sevoflurane and with LY 15 μmol/L,PD 20 μmol/L,ATR 20μmol/L and 5-HD 100 μmol/L respectively at the same time for 15 min immediately before reperfusion,and then with plain K-H solution for 45 min. Coronary flow (CF) was measured and left ventriculur developed pressure (LVDP), dp/dt_(max), left ventricular end-diastolic pressure (LVEDP) and HR were recorded at 20 min of equilibration, immediately before suspension of perfusion and at 15, 30 and 60 min of reperfusian (T_(0-4)). Coronary effluent was collected at T_0 and T_4 in group C, IR and S for determination of lactate dehydrogenase(LDH) and creatine kinase-MB (CK-MB) activities and cardiac troponin I (cTnI) concentration. Left ventricle was taken at T_4 for determination of infarct size (IS). The apoptosis was detected using TUNEL and apoptosis index (AI) was calculated in group C, IR, IR + ATR, IR + 5-HD, IR + DMSO, S, S + ATR and S + 5-HD. NAD~+ content in myocardium was determined in group C, IR, IR + LY, IR + PD, IR + DMSO, S, S + LY and S + PD.Results Compared with group C, LVDP,±dp/dt_(max), HR and CF were significantly decreased and LVEDP was significantly increased during reporfuion and IS was significantly increased in the other groups, AI was significantly increased in group IR, IR + ATR, IR + 5-HD, IR + DMSO, S, S + ATR and S + 5-HD, NAD~+ content was significantly decreased in group IR, IR + LY, IR + PD, IR + DMSO, S, S + LY and S + PD, and activities of LDH and CK-MB and cTnI concentration were significantly increased in group IR and S (P < 0.05). Compared with group IR,LVDP,±dp/dt_(max),HR and CF were significantly increased and LVEDP was significantly decreased during reperfusion, IS and AI were significantly decreased, NAD~+ content was significantly increased, and activities of LDH and CK-MB and cTnI concentration were significantly decreased in group S (P < 0.05), but there was no significant difference in the indices mentioned above between the left ten groups and group IR (P >0.05). Conclusion Sevoflurane postconditioning may attenuate IR injury to isolated rat hearts through activating PI3K and ERK 1/2, promoting mito-K_(ATP) channel opening and inhibiting mPTP opening.
