二氮嗪后处理对大鼠心肌缺血/再灌注损伤的保护作用及其与线粒体渗透性转换孔相关性的研究
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摘要
目的:大鼠在体心肌缺血-再灌注损伤(IRI)模型上,观察线粒体ATP敏感性钾通道(mitoKATP)开放剂二氮嗪(Dz)后处理对大鼠心肌的保护作用;使用线粒体渗透性转换孔(mPTP)开放剂苍术苷(ATR)及阻断剂环孢霉素A(CsA)进行干预,探讨二氮嗪后处理与mPTP相关性。
方法:冠脉结扎法建立SD大鼠在体心肌IRI模型,按照随机原则分为6组(n=12):①假手术组(Sham组);②缺血再灌注损伤对照组(IRI组);③缺血后处理组(Post组);④Dz后处理组(Dz组);⑤Dz后处理+苍术苷组(Dz+ATR组);⑥Dz后处理+环孢霉素A组(Dz+CsA组)。每组分别于再灌注10分钟和120分钟各处死6只大鼠。再灌注10分钟,取缺血区心肌应用紫外分光光度法在540nm波长检测5 min内光密度值(OD540)下降幅度(△OD/min)表示mPTP开放程度;再灌注120分钟,采静脉血测肌酸激酶(CK)及肌酸激酶同工酶(CKMB),取心脏采用TTC染色法测定心肌梗死范围,TUNEL法检测心肌细胞凋亡,电镜观察心肌超微结构损伤。
结果:1.与Sham组比较,IRI、Post及Dz组血清心肌酶、心肌梗死面积、线粒体损伤评分(Flameng评分)、凋亡指数(AI)明显改变(P<0.05)。2.与IRI组比较,Post组及Dz组心肌酶学水平、心肌梗死面积、Flameng评分及AI降低(P<0.05);Dz组与Post组间心肌酶学水平、心肌梗死面积、Flameng评分及AI无差异(P>0.05)。3.与IRI、Dz+ATR组比较,Dz+CsA和Dz组心肌酶学水平、Flameng评分及AI降低(P<0.05);Dz+CsA组较之Dz组的心肌酶学水平、Flameng评分及AI降低程度更为显著(P<0.05);Dz+ATR较之IRI组心肌酶学水平、Flameng评分及AI低(P<0.05)。4.Dz+CsA组与IRI、Dz、Dz+ATR组比较△OD/ min降低(P<0.05);IRI与Dz+ATR组间比较△OD/ min无差异(P>0.05)。
结论:1.大鼠在体模型缺血后处理及二氮嗪后处理均可降低血清心肌酶水平、减少心梗面积、改善心肌细胞超微结构、减轻细胞凋亡、减轻心肌IRI。2.干预后,大鼠在体IRI模型二氮嗪后处理减轻心肌mPTP开放程度的作用被苍术苷完全逆转,减轻心肌IRI的作用部分被抵消;环孢霉素A增强二氮嗪后处理减轻心肌IRI及mPTP开放程度的作用。提示:①mPTP参与了大鼠在体模型二氮嗪后处理的心肌保护作用;②mitoKATP开放剂二氮嗪后处理减轻心肌IRI的机制与mPTP相关,同时还可能存在与mPTP无关的保护机制;③若mitoKATP开放剂二氮嗪后处理联用mPTP特异性阻断剂可进一步减轻心肌IRI。
Objective: Myocardial ischemia reperfusion injury model rats in vivo were prepared. The present study was aimed to explore the cardioprotective effects of diazoxide(mitoKATP opener) postconditioning on myocardial ischemia reperfusion injury. Furthermore,this study was to investigate the relationship between diazoxide postconditioning and mPTP by means of the intervention of cyclosporin A(mPTP opener) and atractyloside(mPTP inhibitor).
Methodes: healthy SD rats models of myocardial ischemia reperfusion injury was established according to coronary artery ligation. Rats were randomly divided into 6 groups(n=12): sham operation group(Sham group), ischemia reperfusion injury group(IRI group), ischemic postconditioning group(Post group), diazoxide postconditioning group(Dz group), diazoxide postconditioning + atractyloside group(Dz+ATR group), diazoxide postconditioning + cyclosporin A group(Dz+ CsA group).Six rats in each group were randomly killed at reperfusion 10 minutes or 120 minutes. At the moment of reperfusion 10 minutes, the degree of mPT of ischemic myocardium was detected by ultraviolet spectrophotometry. At the moment of reperfusion 120 minutes, venous blood was sent to measure the concent of CK and CK-MB, myocardial infarction area was measured by TTC staining method, apoptotic cardiomyocytes were detected by in situ end labeling technique (TUNEL).and The severity of MIRI was measured by concent of CK,CK-MB in blood serum, apoptotic cardiomyocytes were detected by in situ end labeling technique(TUNEL), myocardial ultramicrostructures were detected by electro-microscopic visualization.
Results: 1.Compared with sham-operation group, IRI、Post and Dz group induced obviously increasing in myocardial creatase level,, myocardial infarction area, Flameng average score and apoptosis index(AI). 2. Compared with IRI group, Post group and Dz group could significantly decreased myocardial creatase level, myocardial infarct area, Flameng average score and apoptosis index, but with no statistical differences between them. 3. Compared with IRI and Dz+ATR group, Dz+CsA group and Dz group could significantly decreased myocardial creatase level, Flameng average score and apoptosis index; compared with IRI group, Dz+ATR group could significantly decreased myocardial creatase level, Flameng average score and apoptosis index. 4. Compared with IRI and Dz+ATR group, Dz+CsA group and Dz group could significantly decreased△OD/ min; compared with Dz group, Dz+CsA group could significantly decreased△OD/ min; but with no statistical differences between IRI and Dz+ATR group.
Conclusions: Both ischemic postconditioing and diazoxide postconditioning could decrease myocardial creatase level and myocardial infarction area, restrain apoptosis, alleviate myocardial ischemia reperfusion-injury in model rats in vivo;The close of mPTP played a protective role in diazoxide postconditioning;Possibly,the open of mitoKATP channels alleviated myocardial ischemia reperfusion-injury relation to mPTP, and relation to certain mechanism irrelevant to mPTP; these drugs could be a potential therapeutic agent for protecting heart subjected MIRI from synthetic pharmacologic actions including mPTP inhibitor and mitoKATP opener.
方法:冠脉结扎法建立SD大鼠在体心肌IRI模型,按照随机原则分为6组(n=12):①假手术组(Sham组);②缺血再灌注损伤对照组(IRI组);③缺血后处理组(Post组);④Dz后处理组(Dz组);⑤Dz后处理+苍术苷组(Dz+ATR组);⑥Dz后处理+环孢霉素A组(Dz+CsA组)。每组分别于再灌注10分钟和120分钟各处死6只大鼠。再灌注10分钟,取缺血区心肌应用紫外分光光度法在540nm波长检测5 min内光密度值(OD540)下降幅度(△OD/min)表示mPTP开放程度;再灌注120分钟,采静脉血测肌酸激酶(CK)及肌酸激酶同工酶(CKMB),取心脏采用TTC染色法测定心肌梗死范围,TUNEL法检测心肌细胞凋亡,电镜观察心肌超微结构损伤。
结果:1.与Sham组比较,IRI、Post及Dz组血清心肌酶、心肌梗死面积、线粒体损伤评分(Flameng评分)、凋亡指数(AI)明显改变(P<0.05)。2.与IRI组比较,Post组及Dz组心肌酶学水平、心肌梗死面积、Flameng评分及AI降低(P<0.05);Dz组与Post组间心肌酶学水平、心肌梗死面积、Flameng评分及AI无差异(P>0.05)。3.与IRI、Dz+ATR组比较,Dz+CsA和Dz组心肌酶学水平、Flameng评分及AI降低(P<0.05);Dz+CsA组较之Dz组的心肌酶学水平、Flameng评分及AI降低程度更为显著(P<0.05);Dz+ATR较之IRI组心肌酶学水平、Flameng评分及AI低(P<0.05)。4.Dz+CsA组与IRI、Dz、Dz+ATR组比较△OD/ min降低(P<0.05);IRI与Dz+ATR组间比较△OD/ min无差异(P>0.05)。
结论:1.大鼠在体模型缺血后处理及二氮嗪后处理均可降低血清心肌酶水平、减少心梗面积、改善心肌细胞超微结构、减轻细胞凋亡、减轻心肌IRI。2.干预后,大鼠在体IRI模型二氮嗪后处理减轻心肌mPTP开放程度的作用被苍术苷完全逆转,减轻心肌IRI的作用部分被抵消;环孢霉素A增强二氮嗪后处理减轻心肌IRI及mPTP开放程度的作用。提示:①mPTP参与了大鼠在体模型二氮嗪后处理的心肌保护作用;②mitoKATP开放剂二氮嗪后处理减轻心肌IRI的机制与mPTP相关,同时还可能存在与mPTP无关的保护机制;③若mitoKATP开放剂二氮嗪后处理联用mPTP特异性阻断剂可进一步减轻心肌IRI。
Objective: Myocardial ischemia reperfusion injury model rats in vivo were prepared. The present study was aimed to explore the cardioprotective effects of diazoxide(mitoKATP opener) postconditioning on myocardial ischemia reperfusion injury. Furthermore,this study was to investigate the relationship between diazoxide postconditioning and mPTP by means of the intervention of cyclosporin A(mPTP opener) and atractyloside(mPTP inhibitor).
Methodes: healthy SD rats models of myocardial ischemia reperfusion injury was established according to coronary artery ligation. Rats were randomly divided into 6 groups(n=12): sham operation group(Sham group), ischemia reperfusion injury group(IRI group), ischemic postconditioning group(Post group), diazoxide postconditioning group(Dz group), diazoxide postconditioning + atractyloside group(Dz+ATR group), diazoxide postconditioning + cyclosporin A group(Dz+ CsA group).Six rats in each group were randomly killed at reperfusion 10 minutes or 120 minutes. At the moment of reperfusion 10 minutes, the degree of mPT of ischemic myocardium was detected by ultraviolet spectrophotometry. At the moment of reperfusion 120 minutes, venous blood was sent to measure the concent of CK and CK-MB, myocardial infarction area was measured by TTC staining method, apoptotic cardiomyocytes were detected by in situ end labeling technique (TUNEL).and The severity of MIRI was measured by concent of CK,CK-MB in blood serum, apoptotic cardiomyocytes were detected by in situ end labeling technique(TUNEL), myocardial ultramicrostructures were detected by electro-microscopic visualization.
Results: 1.Compared with sham-operation group, IRI、Post and Dz group induced obviously increasing in myocardial creatase level,, myocardial infarction area, Flameng average score and apoptosis index(AI). 2. Compared with IRI group, Post group and Dz group could significantly decreased myocardial creatase level, myocardial infarct area, Flameng average score and apoptosis index, but with no statistical differences between them. 3. Compared with IRI and Dz+ATR group, Dz+CsA group and Dz group could significantly decreased myocardial creatase level, Flameng average score and apoptosis index; compared with IRI group, Dz+ATR group could significantly decreased myocardial creatase level, Flameng average score and apoptosis index. 4. Compared with IRI and Dz+ATR group, Dz+CsA group and Dz group could significantly decreased△OD/ min; compared with Dz group, Dz+CsA group could significantly decreased△OD/ min; but with no statistical differences between IRI and Dz+ATR group.
Conclusions: Both ischemic postconditioing and diazoxide postconditioning could decrease myocardial creatase level and myocardial infarction area, restrain apoptosis, alleviate myocardial ischemia reperfusion-injury in model rats in vivo;The close of mPTP played a protective role in diazoxide postconditioning;Possibly,the open of mitoKATP channels alleviated myocardial ischemia reperfusion-injury relation to mPTP, and relation to certain mechanism irrelevant to mPTP; these drugs could be a potential therapeutic agent for protecting heart subjected MIRI from synthetic pharmacologic actions including mPTP inhibitor and mitoKATP opener.
引文
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