自噬在二氮嗪减轻大鼠离体心脏缺血再灌注损伤中的作用

目的 评价自噬在二氮嗪减轻大鼠离体心脏缺血再灌注损伤中的作用.方法 清洁级健康雄性SD大鼠32只,体重220～250 g,采用随机数字表法,将大鼠随机分为4组(n=8):缺血再灌注组(I/R组)、二氮嗪组(D组)、自噬抑制剂渥曼青霉素+二氮嗪组(WD组)和渥曼青霉素组(W组).WD组与W组腹腔注射渥曼青霉素15μg/kg,I/R组与D组给予等容量生理盐水,30 min后处死大鼠,制备Langendorff灌流模型,I/R组和w组灌注K-H液、D组和WD组灌注含二氮嗪100μmol/L的K-H液10 min,随后停灌20 min,再灌注30 min.于灌注二氮嗪前即刻、停灌前即刻、再灌注30 min时记录心率(HR)、左室舒张末期压(LVEDP)、左心室发展压(LVDP),于再灌注30 min时测定心肌组织SOD活性和MDA含量,检测自噬相关蛋白Beclin-1的表达,透射电镜下观察自噬小体形成情况.结果 与I/R组比较,D组LVDP和HR、心肌SOD活性和心肌Beclin-1表达水平升高,LVEDP和心肌MDA含量降低(P＜0.05),WD组和W组心肌Beclin-1表达水平降低(P＜0.05),其余指标差异无统计学意义(P＞0.05);与D组比较,WD组和W组LVDP和心肌Beclin-1表达水平降低,MDA含量升高,W组LVEDP升高,心肌SOD活性降低(P＜0.05).D组可见大量自噬小体,WD组与I/R组可见少量自噬小体,W组见极少量自噬小体.结论 自噬参与了二氮嗪减轻大鼠心肌缺血再灌注损伤的过程.

Abstract：

Objective To evaluate the role of autophagy in attenuation of myocardial ischemia-reperfusion (I/R) injury by diazoxide in the isolated rat heart.Methods Thirty-two male SD rats were randomly assigned into 4 groups ( n = 8 each) : I/R group, diazoxide group (group D), an inhibitor of autophagy wortmannin + diazoxide group (group WT＞) and wortmannin group (group W) . The animals were anesthetized with intraperitoneal pento-barbital sodium 40 mg/kg. Their hearts were excised and passively perfused in a Langendorff apparatus with an oxygenated (95% O2-5% CO2 ) K-H solution at 37 °C . The isolated hearts were made globally ischemic for 20 min followed by 30 min reperfusion. In I/R and W groups, the isolated hearts were perfused with K-H solution for 10 min before ischemia, while the isolated hearts were perfused with K-H solution containing diazoxide 100 /xmol/L for 10 min before ischemia in D and WD groups. The HR, left ventricular end-diastolic pressure (LVEDP) and left ventricular developed pressure (LVDP) were recorded immediately before perfusion with diazoxide, immediately before the end of perfusion and at 30 min of reperfusion.Myocardial tissues were obtained at 30 min of reperfusion for determination of SOD activity, MDA content and autophagy-related protein Beclin-1 expression (by immunohistochemistry). The formation of autophagosomes was observed by transmission electron microscopy. ResultsCompared with group I/R, LVDP, HR, SOD activity and Beclin-1 expression were significantly increased at 30 min of reperfusion, while LVEDP and MDA content were significantly decreased at 30 min of reperfusion in group D(P＜0.05),and Beclin-1 expression was significantly decreased in WD and W groups(P＜0.05).Compared with group D, LVDP and Beclin-1 expression were significantly decreased, and MDA content was significantly increased in WD and W groups, and LVEDP was significantly increased, while SOD activity decreased in group W (P＜0.05). Microscopic examination showed that a large number of autophagosomes, a small number of autophagosomes and an extremely small number of autophagosomes were observed in D, WD and I/R groups respectively. Conclusion Autophagy is involved in attenuation of myocardial I/R injury by diazoxide in the isolated rat heart.     

含不同浓度乳化异氟醚的停跳液对大鼠离体心脏缺血再灌注损伤的影响

Objective To investigate the effects of cardioplegic solution containing different concentrations of emulsified isoflurane on myocardial ischemia-reperfusion injury in isolated rat hearts. Methods Fifty-six male SD rats, weighing 180-250 g, were anesthetized with intraperitoneal 20% urethane 1 g/kg and heparin 1 000 U/kg. Their hearts were excised and perfused in a Langendorff apparatus. Fifty-six isolated hearts were randomly divided into 7 groups ( n = 8 each) : St. Thomas cardioplegic solution group (group C) and St. Thomas cardioplegic solution containing 6 different concentrations of emulsified isoflurane groups (group E1-6 ). After 20 min equilibration, cardiac arrest was induced with St. Thomas cardioplegic solution 20 ml and St. Thomas cardioplegic solution containing 0.28, 0.56, 1.12, 1.68, 2.24 and 2.80 mmol/L emulsified isoflurane 20 ml at 4℃for 45 min followed by 60 min reperfusion in group C and E1-6 respectively. HR, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and + dp/dtmax were recorded at the end of 20 min equilibration, 20, 40 and 60 min of reperfusion. Coronary effluent 1.5 ml was collected for determination of LDH and SOD activity and the concentration of cTnI. At the end of 60 min reperfusion, the area of myocardial infarction was calculated. Results Compared with group C, HR, LVDP, + dp/dtmax and SOD activity were significantly higher, LVEDP, LDH activity and cTnI concentration lower, and the area of myocardial infarction lower in group E4, and HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E6 and E6 ( P < 0.05) , but there was no significant difference in the above indices between group E1-3 and group C ( P > 0.05) . HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E1-3-5-6 than in group E4 (P < 0.05 ). Conclusion St. Thomas cardioplegic solution containing 1.68 mmol/L emulsified isoflurane can attenuate myocardial ischemia-reperfusion injury in isolatede rat hearts.     

含不同浓度乳化异氟醚的停跳液对大鼠离体心脏缺血再灌注损伤的影响

Objective To investigate the effects of cardioplegic solution containing different concentrations of emulsified isoflurane on myocardial ischemia-reperfusion injury in isolated rat hearts. Methods Fifty-six male SD rats, weighing 180-250 g, were anesthetized with intraperitoneal 20% urethane 1 g/kg and heparin 1 000 U/kg. Their hearts were excised and perfused in a Langendorff apparatus. Fifty-six isolated hearts were randomly divided into 7 groups ( n = 8 each) : St. Thomas cardioplegic solution group (group C) and St. Thomas cardioplegic solution containing 6 different concentrations of emulsified isoflurane groups (group E1-6 ). After 20 min equilibration, cardiac arrest was induced with St. Thomas cardioplegic solution 20 ml and St. Thomas cardioplegic solution containing 0.28, 0.56, 1.12, 1.68, 2.24 and 2.80 mmol/L emulsified isoflurane 20 ml at 4℃for 45 min followed by 60 min reperfusion in group C and E1-6 respectively. HR, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and + dp/dtmax were recorded at the end of 20 min equilibration, 20, 40 and 60 min of reperfusion. Coronary effluent 1.5 ml was collected for determination of LDH and SOD activity and the concentration of cTnI. At the end of 60 min reperfusion, the area of myocardial infarction was calculated. Results Compared with group C, HR, LVDP, + dp/dtmax and SOD activity were significantly higher, LVEDP, LDH activity and cTnI concentration lower, and the area of myocardial infarction lower in group E4, and HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E6 and E6 ( P < 0.05) , but there was no significant difference in the above indices between group E1-3 and group C ( P > 0.05) . HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E1-3-5-6 than in group E4 (P < 0.05 ). Conclusion St. Thomas cardioplegic solution containing 1.68 mmol/L emulsified isoflurane can attenuate myocardial ischemia-reperfusion injury in isolatede rat hearts.     

含不同浓度乳化异氟醚的停跳液对大鼠离体心脏缺血再灌注损伤的影响

Objective To investigate the effects of cardioplegic solution containing different concentrations of emulsified isoflurane on myocardial ischemia-reperfusion injury in isolated rat hearts. Methods Fifty-six male SD rats, weighing 180-250 g, were anesthetized with intraperitoneal 20% urethane 1 g/kg and heparin 1 000 U/kg. Their hearts were excised and perfused in a Langendorff apparatus. Fifty-six isolated hearts were randomly divided into 7 groups ( n = 8 each) : St. Thomas cardioplegic solution group (group C) and St. Thomas cardioplegic solution containing 6 different concentrations of emulsified isoflurane groups (group E1-6 ). After 20 min equilibration, cardiac arrest was induced with St. Thomas cardioplegic solution 20 ml and St. Thomas cardioplegic solution containing 0.28, 0.56, 1.12, 1.68, 2.24 and 2.80 mmol/L emulsified isoflurane 20 ml at 4℃for 45 min followed by 60 min reperfusion in group C and E1-6 respectively. HR, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and + dp/dtmax were recorded at the end of 20 min equilibration, 20, 40 and 60 min of reperfusion. Coronary effluent 1.5 ml was collected for determination of LDH and SOD activity and the concentration of cTnI. At the end of 60 min reperfusion, the area of myocardial infarction was calculated. Results Compared with group C, HR, LVDP, + dp/dtmax and SOD activity were significantly higher, LVEDP, LDH activity and cTnI concentration lower, and the area of myocardial infarction lower in group E4, and HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E6 and E6 ( P < 0.05) , but there was no significant difference in the above indices between group E1-3 and group C ( P > 0.05) . HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E1-3-5-6 than in group E4 (P < 0.05 ). Conclusion St. Thomas cardioplegic solution containing 1.68 mmol/L emulsified isoflurane can attenuate myocardial ischemia-reperfusion injury in isolatede rat hearts.     

含不同浓度乳化异氟醚的停跳液对大鼠离体心脏缺血再灌注损伤的影响

Objective To investigate the effects of cardioplegic solution containing different concentrations of emulsified isoflurane on myocardial ischemia-reperfusion injury in isolated rat hearts. Methods Fifty-six male SD rats, weighing 180-250 g, were anesthetized with intraperitoneal 20% urethane 1 g/kg and heparin 1 000 U/kg. Their hearts were excised and perfused in a Langendorff apparatus. Fifty-six isolated hearts were randomly divided into 7 groups ( n = 8 each) : St. Thomas cardioplegic solution group (group C) and St. Thomas cardioplegic solution containing 6 different concentrations of emulsified isoflurane groups (group E1-6 ). After 20 min equilibration, cardiac arrest was induced with St. Thomas cardioplegic solution 20 ml and St. Thomas cardioplegic solution containing 0.28, 0.56, 1.12, 1.68, 2.24 and 2.80 mmol/L emulsified isoflurane 20 ml at 4℃for 45 min followed by 60 min reperfusion in group C and E1-6 respectively. HR, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and + dp/dtmax were recorded at the end of 20 min equilibration, 20, 40 and 60 min of reperfusion. Coronary effluent 1.5 ml was collected for determination of LDH and SOD activity and the concentration of cTnI. At the end of 60 min reperfusion, the area of myocardial infarction was calculated. Results Compared with group C, HR, LVDP, + dp/dtmax and SOD activity were significantly higher, LVEDP, LDH activity and cTnI concentration lower, and the area of myocardial infarction lower in group E4, and HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E6 and E6 ( P < 0.05) , but there was no significant difference in the above indices between group E1-3 and group C ( P > 0.05) . HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E1-3-5-6 than in group E4 (P < 0.05 ). Conclusion St. Thomas cardioplegic solution containing 1.68 mmol/L emulsified isoflurane can attenuate myocardial ischemia-reperfusion injury in isolatede rat hearts.     

含不同浓度乳化异氟醚的停跳液对大鼠离体心脏缺血再灌注损伤的影响

Objective To investigate the effects of cardioplegic solution containing different concentrations of emulsified isoflurane on myocardial ischemia-reperfusion injury in isolated rat hearts. Methods Fifty-six male SD rats, weighing 180-250 g, were anesthetized with intraperitoneal 20% urethane 1 g/kg and heparin 1 000 U/kg. Their hearts were excised and perfused in a Langendorff apparatus. Fifty-six isolated hearts were randomly divided into 7 groups ( n = 8 each) : St. Thomas cardioplegic solution group (group C) and St. Thomas cardioplegic solution containing 6 different concentrations of emulsified isoflurane groups (group E1-6 ). After 20 min equilibration, cardiac arrest was induced with St. Thomas cardioplegic solution 20 ml and St. Thomas cardioplegic solution containing 0.28, 0.56, 1.12, 1.68, 2.24 and 2.80 mmol/L emulsified isoflurane 20 ml at 4℃for 45 min followed by 60 min reperfusion in group C and E1-6 respectively. HR, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and + dp/dtmax were recorded at the end of 20 min equilibration, 20, 40 and 60 min of reperfusion. Coronary effluent 1.5 ml was collected for determination of LDH and SOD activity and the concentration of cTnI. At the end of 60 min reperfusion, the area of myocardial infarction was calculated. Results Compared with group C, HR, LVDP, + dp/dtmax and SOD activity were significantly higher, LVEDP, LDH activity and cTnI concentration lower, and the area of myocardial infarction lower in group E4, and HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E6 and E6 ( P < 0.05) , but there was no significant difference in the above indices between group E1-3 and group C ( P > 0.05) . HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E1-3-5-6 than in group E4 (P < 0.05 ). Conclusion St. Thomas cardioplegic solution containing 1.68 mmol/L emulsified isoflurane can attenuate myocardial ischemia-reperfusion injury in isolatede rat hearts.     

含不同浓度乳化异氟醚的停跳液对大鼠离体心脏缺血再灌注损伤的影响

Objective To investigate the effects of cardioplegic solution containing different concentrations of emulsified isoflurane on myocardial ischemia-reperfusion injury in isolated rat hearts. Methods Fifty-six male SD rats, weighing 180-250 g, were anesthetized with intraperitoneal 20% urethane 1 g/kg and heparin 1 000 U/kg. Their hearts were excised and perfused in a Langendorff apparatus. Fifty-six isolated hearts were randomly divided into 7 groups ( n = 8 each) : St. Thomas cardioplegic solution group (group C) and St. Thomas cardioplegic solution containing 6 different concentrations of emulsified isoflurane groups (group E1-6 ). After 20 min equilibration, cardiac arrest was induced with St. Thomas cardioplegic solution 20 ml and St. Thomas cardioplegic solution containing 0.28, 0.56, 1.12, 1.68, 2.24 and 2.80 mmol/L emulsified isoflurane 20 ml at 4℃for 45 min followed by 60 min reperfusion in group C and E1-6 respectively. HR, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and + dp/dtmax were recorded at the end of 20 min equilibration, 20, 40 and 60 min of reperfusion. Coronary effluent 1.5 ml was collected for determination of LDH and SOD activity and the concentration of cTnI. At the end of 60 min reperfusion, the area of myocardial infarction was calculated. Results Compared with group C, HR, LVDP, + dp/dtmax and SOD activity were significantly higher, LVEDP, LDH activity and cTnI concentration lower, and the area of myocardial infarction lower in group E4, and HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E6 and E6 ( P < 0.05) , but there was no significant difference in the above indices between group E1-3 and group C ( P > 0.05) . HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E1-3-5-6 than in group E4 (P < 0.05 ). Conclusion St. Thomas cardioplegic solution containing 1.68 mmol/L emulsified isoflurane can attenuate myocardial ischemia-reperfusion injury in isolatede rat hearts.     

含不同浓度乳化异氟醚的停跳液对大鼠离体心脏缺血再灌注损伤的影响

Objective To investigate the effects of cardioplegic solution containing different concentrations of emulsified isoflurane on myocardial ischemia-reperfusion injury in isolated rat hearts. Methods Fifty-six male SD rats, weighing 180-250 g, were anesthetized with intraperitoneal 20% urethane 1 g/kg and heparin 1 000 U/kg. Their hearts were excised and perfused in a Langendorff apparatus. Fifty-six isolated hearts were randomly divided into 7 groups ( n = 8 each) : St. Thomas cardioplegic solution group (group C) and St. Thomas cardioplegic solution containing 6 different concentrations of emulsified isoflurane groups (group E1-6 ). After 20 min equilibration, cardiac arrest was induced with St. Thomas cardioplegic solution 20 ml and St. Thomas cardioplegic solution containing 0.28, 0.56, 1.12, 1.68, 2.24 and 2.80 mmol/L emulsified isoflurane 20 ml at 4℃for 45 min followed by 60 min reperfusion in group C and E1-6 respectively. HR, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and + dp/dtmax were recorded at the end of 20 min equilibration, 20, 40 and 60 min of reperfusion. Coronary effluent 1.5 ml was collected for determination of LDH and SOD activity and the concentration of cTnI. At the end of 60 min reperfusion, the area of myocardial infarction was calculated. Results Compared with group C, HR, LVDP, + dp/dtmax and SOD activity were significantly higher, LVEDP, LDH activity and cTnI concentration lower, and the area of myocardial infarction lower in group E4, and HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E6 and E6 ( P < 0.05) , but there was no significant difference in the above indices between group E1-3 and group C ( P > 0.05) . HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E1-3-5-6 than in group E4 (P < 0.05 ). Conclusion St. Thomas cardioplegic solution containing 1.68 mmol/L emulsified isoflurane can attenuate myocardial ischemia-reperfusion injury in isolatede rat hearts.     

含不同浓度乳化异氟醚的停跳液对大鼠离体心脏缺血再灌注损伤的影响

Objective To investigate the effects of cardioplegic solution containing different concentrations of emulsified isoflurane on myocardial ischemia-reperfusion injury in isolated rat hearts. Methods Fifty-six male SD rats, weighing 180-250 g, were anesthetized with intraperitoneal 20% urethane 1 g/kg and heparin 1 000 U/kg. Their hearts were excised and perfused in a Langendorff apparatus. Fifty-six isolated hearts were randomly divided into 7 groups ( n = 8 each) : St. Thomas cardioplegic solution group (group C) and St. Thomas cardioplegic solution containing 6 different concentrations of emulsified isoflurane groups (group E1-6 ). After 20 min equilibration, cardiac arrest was induced with St. Thomas cardioplegic solution 20 ml and St. Thomas cardioplegic solution containing 0.28, 0.56, 1.12, 1.68, 2.24 and 2.80 mmol/L emulsified isoflurane 20 ml at 4℃for 45 min followed by 60 min reperfusion in group C and E1-6 respectively. HR, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and + dp/dtmax were recorded at the end of 20 min equilibration, 20, 40 and 60 min of reperfusion. Coronary effluent 1.5 ml was collected for determination of LDH and SOD activity and the concentration of cTnI. At the end of 60 min reperfusion, the area of myocardial infarction was calculated. Results Compared with group C, HR, LVDP, + dp/dtmax and SOD activity were significantly higher, LVEDP, LDH activity and cTnI concentration lower, and the area of myocardial infarction lower in group E4, and HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E6 and E6 ( P < 0.05) , but there was no significant difference in the above indices between group E1-3 and group C ( P > 0.05) . HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E1-3-5-6 than in group E4 (P < 0.05 ). Conclusion St. Thomas cardioplegic solution containing 1.68 mmol/L emulsified isoflurane can attenuate myocardial ischemia-reperfusion injury in isolatede rat hearts.     

含不同浓度乳化异氟醚的停跳液对大鼠离体心脏缺血再灌注损伤的影响

Objective To investigate the effects of cardioplegic solution containing different concentrations of emulsified isoflurane on myocardial ischemia-reperfusion injury in isolated rat hearts. Methods Fifty-six male SD rats, weighing 180-250 g, were anesthetized with intraperitoneal 20% urethane 1 g/kg and heparin 1 000 U/kg. Their hearts were excised and perfused in a Langendorff apparatus. Fifty-six isolated hearts were randomly divided into 7 groups ( n = 8 each) : St. Thomas cardioplegic solution group (group C) and St. Thomas cardioplegic solution containing 6 different concentrations of emulsified isoflurane groups (group E1-6 ). After 20 min equilibration, cardiac arrest was induced with St. Thomas cardioplegic solution 20 ml and St. Thomas cardioplegic solution containing 0.28, 0.56, 1.12, 1.68, 2.24 and 2.80 mmol/L emulsified isoflurane 20 ml at 4℃for 45 min followed by 60 min reperfusion in group C and E1-6 respectively. HR, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and + dp/dtmax were recorded at the end of 20 min equilibration, 20, 40 and 60 min of reperfusion. Coronary effluent 1.5 ml was collected for determination of LDH and SOD activity and the concentration of cTnI. At the end of 60 min reperfusion, the area of myocardial infarction was calculated. Results Compared with group C, HR, LVDP, + dp/dtmax and SOD activity were significantly higher, LVEDP, LDH activity and cTnI concentration lower, and the area of myocardial infarction lower in group E4, and HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E6 and E6 ( P < 0.05) , but there was no significant difference in the above indices between group E1-3 and group C ( P > 0.05) . HR, LVDP, + dp/dtmax and SOD activity were significantly lower, LVEDP, LDH activity and cTnI concentration higher, and the area of myocardial infarction higher in group E1-3-5-6 than in group E4 (P < 0.05 ). Conclusion St. Thomas cardioplegic solution containing 1.68 mmol/L emulsified isoflurane can attenuate myocardial ischemia-reperfusion injury in isolatede rat hearts.     

七氟醚后处理对大鼠离体心脏缺血再灌注时心肌细胞凋亡的影响

目的 探讨七氟醚后处理对大鼠离体心脏缺血再灌注(IR)时心肌细胞凋亡的影响.方法 雄性SD大鼠48只,随机分4组(n=12),制备离体心脏灌注模型,K-H液平衡灌注30 min后,C组灌注K-H液120 min;IR组缺血30 min,K-H液再灌注90 min;缺血后处理组(IP组)缺血30 min后行4个循环复灌20 s/缺血20 s,再灌注K-H液;七氟醚后处理组(SP组)缺血30 min后灌注含七氟醚的K-H液5 min,K-H液冲洗10 min,再灌注K-H液.IP组和SP组再灌注总时间90 min.灌注期间测定心功能,灌注结束时取心脏测定心梗面积、Bcl-2、细胞色素C和Caspase-3蛋白表达水平,计算心肌细胞凋亡指数(AI).结果 与C组比较,其余各组左心室最大上升或下降速率(±dp/dt)、左心室发展压(LVDP)、HR降低,左心室舒张末压(LVEDP)和AI升高,Bel-2、细胞色素C和Caspase-3蛋白表达上调(P<0.05);与IR组比较,IP组和SP组±dp/dt、LVDP升高,LVEDP和AI降低,心梗面积减小,Bcl-2表达上调,细胞色素C和Caspase-3蛋白表达下调(P<0.05).结论 七氟醚后处理可减少大鼠离体心脏IR时心肌细胞凋亡,其机制与其下调细胞色素C和Caspase-3蛋白表达,上调Bcl-2表达有关.     

舒芬太尼后处理和七氟醚后处理对大鼠离体心脏缺血再灌注损伤的影响

目的 评价舒芬太尼后处理和七氟醚后处理对大鼠离体心脏缺血再灌注损伤的影响.方法 雄性SD大鼠,体重230～250 g,成功制备Langendorff离体灌注模型的40个心脏随机分为4组(n=10):缺血再灌注组(Ⅰ组)、七氟醚后处理组(Ⅱ组)、舒芬太尼后处理组(Ⅲ组)和七氟醚联合舒芬太尼后处理组(Ⅳ组).采用K-H液平衡灌注(灌注压10 kPa)30 min,全心缺血40 min再灌注120 min.再灌注即刻时Ⅱ组、Ⅲ组和Ⅳ组进行药物后处理15 min:Ⅱ组K-H液中通入3.0%七氟醚,Ⅲ组K-H液中加入100 nmol/L舒芬太尼,Ⅳ组同时进行七氟醚后处理和舒芬太尼后处理.分别于平衡灌注末(基础状态)、再灌注15 min、30 min、60 min、90 min、120 min时记录左室收缩压(LVSP)、左室舒张末压(LVEDP)、左室发展压(LVDP)、左室内压上升最大速率(+dp/dtmax)、左室内压下降最大速率(-dp/dtmax)、心率(HR)和灌脉流量(CF).再灌注5 min时,收集冠脉流出液,测定肌酸激酶(CK)和乳酸脱氢酶(LDH)的活性.再灌注120 min时取心肌组织,测定心肌梗死体积、Bcl-2和Bax的表达水平,并计算Bcl-2和Bax表达的比值(Bcl-2/Bax).结果 与Ⅰ组比较,Ⅱ组、Ⅲ组和Ⅳ组LVSP、LVDP、+dp/dtmax、-dp/dtmax和CF升高,LVEDP和LDH、CK的活性降低,心肌梗死体积缩小,Bcl-2表达上调,Bax表达下调,Bcl-2/Bax升高(P＜0.05或0.01);Ⅱ组、Ⅲ组和Ⅳ组间上述指标差异无统计学意义(P＞0.05).结论 舒芬太尼后处理可减轻大鼠心肌缺血再灌注损伤,联合七氟醚后处理时心肌保护作用并未增加,其心肌保护的机制与上调Bcl-2表达、下调Bax表达从而抑制细胞凋亡有关.     

心肌细胞缝隙连接蛋白43在线粒体敏感性钾通道介导七氟醚预处理减轻大鼠离体心脏缺血再灌注中的作用

目的 评价心肌细胞缝隙连接蛋白43(Cx43)在线粒体敏感性钾(mito-KATP)通道介导七氟醚预处理减轻大鼠离体心脏缺血再灌注中的作用.方法 健康成年雄性SD大鼠40只,体重200～250 g,采用Langendorff灌注模型进行离体心脏灌注.采用随机数字表法,将心脏随机分为5组(n=8):对照组(C组)、缺血再灌注组(I/R组)、七氟醚预处理组(S组)、七氟醚预处理+5-羟葵酸(5-HD)组(SH组)和5-HD组(H组).采用结扎左冠状动脉前降支(LAD) 30 min,恢复灌注120 min的方法制备心脏缺血再灌注模型.各组平衡灌注10 min;然后C组持续灌注,仅于LAD下穿线而不结扎;I/R组继续灌注30 min后结扎LAD;S组、S+H组和H组结扎LAD前30 min时分别用3％七氟醚预先饱和的K-H液、3％七氟醚预先饱和的K-H液+100 μmol/L 5-HD和K-H液+100 μmol/L 5-HD灌注15 min,然后用K-H液冲洗15 min.分别于给药前(T0)、给药结束即刻(T1)、缺血前即刻(T2)、缺血30 min(T3)和再灌注120 min(T4)时,记录HR、左心室收缩压(LVSP)、左心室舒张压(LVDP)、左心室最大上升速率(+dp/dtmax)和左心室最大下降速率(- dp/dtmax).再灌注结束后,取左心室心肌组织,测定心肌梗死体积,采用免疫组化法测定心肌细胞Cx43表达,采用Western Blot法测定心肌细胞Cx43和磷酸化Cx43(p-Cx43)表达.结果 与C组比较,I/R组、S+H组和H组HR、LVSP、+dp/dtmax和- dp/dtmax降低,LVDP升高,心肌细胞Cx43和p-Cx43表达下调(P＜0.05).与I/R组比较,S组HR、LVSP、+dp/dtmax和- dp/dtmax升高,LVDP和心肌梗死体积降低,心肌细胞Cx43和p-Cx43表达上调(P＜0.05),S+H组和H组各指标差异无统计学意义(P＞0.05).结论 七氟醚预处理可能通过开放mito-KATP通道,促进心肌细胞Cx43磷酸化,减轻大鼠离体心脏缺血再灌注损伤.     

性别因素对七氟醚后处理减轻大鼠离体心肌缺血再灌注损伤的影响

目的 探讨性别因素对七氟醚后处理减轻大鼠离体心肌缺血再灌注损伤的影响.方法 SD大鼠60只,雄雌各半,2月龄,雄性大鼠随机分为对照组(MC组)和七氟醚后处理组(MS组),雌性大鼠随机分为对照组(FC组)和七氟醚后处理组(FS组),每组15只.建立大鼠离体心脏灌注模型,采用全心缺血40 min,再灌注2 h的方法制备缺血再灌注模型.对照组心脏再灌注时给予含氧K-H缓冲液,七氟醚后处理组在复灌的前10min灌注经3%七氟醚饱和的含氧K-H缓冲液,余110 min灌注含氧K-H缓冲液.于缺血前、再灌注期间记录HR、左心室舒张末压(LVEDP)和左心室发展压(LVDP),再灌注5 min时测定冠状动脉流出液LDH活性和心肌梗死面积,再灌注10 min时测定心肌总蛋白激酶B(t-Akt)、磷酸化蛋白激酶B(p-Akt)的表达,计算p-Akt与t-Akt比值(p-Akt/t-Akt).结果 与MC组比较,MS组和FC组LVDP升高,LVEDP降低,冠状动脉流出液LDH活性降低,心肌梗死面积减小,心肌p-Akt表达上调,p-Akt/t-Akt升高(P＜0.05);与MS组比较,FS组LVDP降低,LVEDP和冠状动脉流出液LDH活性升高,心肌梗死面积增大(P＜0.05);FC组和FS组LVDP和LVEDP比较差异无统计学意义(P＞0.05).结论 七氟醚后处理减轻大鼠离体心肌缺血再灌注损伤存在性别差异,对雄性大鼠的心肌保护作用强于雌性大鼠,该差异可能与心肌Akt的活化水平有关.     

PI3K/Akt信号通路在七氟醚后处理减轻大鼠局灶性脑缺血再灌注损伤中的作用

目的 评价磷脂酰肌醇3激酶/蛋白质丝氨酸苏氨酸激酶(PI3K/Akt)信号通路在七氟醚后处理减轻大鼠局灶性脑缺血再灌注损伤中的作用.方法 雄性SD大鼠64只,体重300～350 g,随机分为4组(n=16):局灶性脑缺血再灌注组(IR组)、七氟醚后处理组(SP组)、七氟醚后处理+Wortmannin组(SW组)和Wortmannin组(W组).采用电凝法阻断左侧大脑中动脉,夹闭双侧颈总动脉60 min恢复灌注的方法制备大鼠局灶性脑缺血再灌注模型.再灌注即刻SP组和SW组吸入2.5%七氟醚60 min,夹闭双侧颈总动脉30 min时SW组和W组股静脉输注Wortmannin 0.6 mg/kg.于再灌注24、48、72 h时行神经功能评分,最后1次评分后处死大鼠取脑,测定脑梗死体积,采用Western blot法检测左侧脑组织磷酸化Akt(p-Akt)和磷酸化Bad(p-Bad)的表达水平.结果 与IR组相比,SP组和SW组各时点神经功能评分升高,再灌注72 h时脑梗死体积比降低,脑组织p-Akt和p-Bad表达上调(P＜0.05),W组上述指标差异无统计学意义(P＞0.05);与SP组相比,SW组各时点神经功能评分降低,再灌注72 h时脑梗死体积比升高,脑组织p-Akt和p-Bad表达下调(P＜0.05).结论 PI3K/Akt信号通路激活后p-Bad表达上调可能参与了七氟醚后处理减轻大鼠局灶性脑缺血再灌注损伤的过程.     

不同浓度硝酸甘油对大鼠离体心脏缺血再灌注损伤的影响

目的 探讨不同浓度硝酸甘油对大鼠离体心脏缺血再灌注损伤的影响.方法 健康成年雌性SD大鼠,体重220～330 g,周龄7周,建立Langendorff离体心脏灌注模型,取模型制备成功的心脏30个,随机分为3组(n=10):缺血再灌注组(IR组)K-H液灌注15 min,停灌20 min,再灌注60 min;低浓度硝酸甘油组(LN组)采用含有2.5 μg/L硝酸甘油的K-H液灌注15 min,停灌20 min,再灌注60 min;高浓度硝酸甘油组(HN组)采用含有25μg/L硝酸甘油的K-H液灌注15 min,停灌20 min,再灌注60 min.于再灌注10、20、30、40、50、60 min时记录心率(HR)、冠状动脉流量(CF)、左心室舒张末压(LVEDP)、左心室发展压(LVDP)、左心室内压最大上升速率(+dp/dtmax)和左心室内压最大下降速率(-dp/dt_(max)).于平衡灌注末、再灌注5、30和60 min时收集冠状动脉流出液1.5 ml,测定乳酸脱氢酶(LDH)和肌酸激酶(CK)的活性;于再灌注60 min时取心脏,测定心肌梗死面积,计数心肌凋亡细胞.结果 与IR组比较,LN组HR、CF、LVDP,+dp/dt_(max)和-dp/dt_(max)升高,LVEDP、CK活性、LDH活性、心肌梗死面积和细胞凋亡率降低(P<0.05),HN组HR、CF、LVDP、+dp/dt_(max)和-dp/dt_(max)降低,LVEDP、CK活性、LDH活性、心肌梗死面积和细胞凋亡率升高(P<0.05).与LN组比较,HN组HR、CF、LVDP、+dp/dt_(max)和-dp/dt_(max)降低,LVEDP、CK活性、LDH活性、心肌梗死面积和细胞凋亡率升高(P<0.05).结论 低浓度硝酸甘油(2.5μg/L)可减轻大鼠离体心脏缺血再灌注损伤,而高浓度硝酸甘油(25 μg/L)则可加重心脏缺血再灌注损伤.     

线粒体心磷脂在二氮嗪预处理减轻大鼠离体心脏缺血再灌注损伤中的作用

目的 评价线粒体心磷脂在二氮嗪预处理减轻大鼠离体心脏缺血再灌注损伤中的作用.方法 清洁级SD大鼠72只,体重200～280 g,雌雄各半,随机分为对照组(C组)、缺血再灌注组(I/R组)、二氮嗪预处理组(DZ组)和5-羟葵酸拮抗二氮嗪组(HD组),每组18只.采用Langendorff灌流装置建立大鼠离体心脏缺血再灌注模型,C组平衡灌注20 min,持续灌注100 min;I/R组平衡灌注20 min,持续灌注30 min,缺血40 min,再灌注30 min;DZ组平衡灌注20 min后,依次灌注K-H液15 min、50 μmol/L二氮嗪10 min和K-H液5 min,其余缺血再灌注同I/R组;HD组二氮嗪预处理前给予含5-羟葵酸100 μmol/L K-H液10 min,其余处理同DZ组.各组分别于平衡灌注末(T1)、缺血前即刻(T2)、再灌注末(T3)时随机取6只大鼠,监测心率(HR)、左心室发展压(LVDP)和左心室舒张末压(LVEDP),采用高效液相色谱仪测定心肌线粒体心磷脂含量.结果 与T1,2时比较,各组T3时HR、LVDP降低,LVEDP升高,心肌线粒体心磷脂含量降低(P<0.05);与C组比较,其余3组T3时HR、LVDP降低,LVEDP升高,心肌线粒体心磷脂含量降低(P<0.05);与I/R组比较,DZ组T3时HR、LVDP升高,LVEDP降低,心肌线粒体心磷脂含量升高(P<0.05);与DZ组比较,HD组T3时HR、LVDP降低,LVEDP升高,心肌线粒体心磷脂含量降低(P<0.05).结论 二氮嗪预处理可减轻大鼠离体心脏缺血再灌注损伤,与维持心肌线粒体心磷脂含量有关.     

线粒体通透性转换孔在七氟醚延迟预处理减轻大鼠心肌缺血再灌注损伤中的作用

目的 探讨线粒体通透性转换孔(mPTP)在七氟醚延迟预处理减轻大鼠心肌缺血再灌注损伤中的作用.方法 雄性SD大鼠80只,体重250～300 g,随机分为5组(n=16):假手术组(S组)、缺血再灌注组(IR组)、七氟醚延迟预处理组(SP组)、mPTP开放剂苍术苷+七氟醚延迟预处理组(A+SP组)和苍术苷组(A组).IR组、SP组、A+SP组和A组采用结扎左冠状动脉前降支30 min后进行再灌注的方法制备心肌缺血再灌注模型.SP组和A+SP组吸入2.5%七氟醚l h,其余组吸入纯氧1 h,停止吸入后24 h进行心肌缺血.A+SP组和A组在缺血前15 min经尾静脉注射苍术苷5 mg/kg.再灌注120 min时采集颈动脉血样,测定血清肌钙蛋白I(cTnI)浓度.然后处死大鼠,测定心肌梗死体积,检测心肌组织Bcl-2及Bax表达水平,电镜下观察心肌超微结构.结果 与S组比较,其他各组血清cTnI浓度升高,心肌梗死体积扩大,Bcl-2表达下调,Bax表达上调(P＜0.05).与IR组比较,SP组血清cTnI浓度降低,心肌梗死体积缩小,Bcl-2表达上调,Bax表达下调(P＜0.05),心肌病理学损伤减轻.苍术苷可取消七氟醚延迟预处理减轻大鼠心肌缺血再灌注损伤的效应(P＜0.05).结论 抑制mPTP开放后可导致Bcl-2表达上调,Bax表达下调,参与了七氟醚延迟预处理减轻大鼠心肌缺血再灌注损伤.     

芬太尼联合七氟烷后处理对离体大鼠缺血/再灌注心功能的影响

目的 研究芬太尼联合七氟烷后处理对离体大鼠缺血/再灌注(ischemia/reperfusion,I/R)心脏心功能的影响.方法 建立离体大鼠心脏缺血40 min,再灌注120 min模型.根据再灌注开始10 min的不同处理,使用随机数字表法将实验动物随机分为4组(n=10):I/R对照组(Con),七氟烷后处理组(...