七氟醚后处理减轻离体大鼠心肌缺血再灌注损伤的作用机制研究

发布时间：2018-06-17 12:13

本文选题：七氟醚 + 后处理　；参考：《苏州大学》2015年博士论文

【摘要】：第一部分七氟醚后处理减轻离体大鼠心肌缺血再灌注损伤目的通过对比心肌I/R前后血液动力学参数、凋亡和梗死面积,探讨七氟醚后处理对大鼠心肌缺血再灌注损伤的保护作用。方法建立大鼠心脏Langendorff离体心脏灌流模型,取制备离体模型成功的心脏32个,分为4组(n=8):假手术组(SHAM组),单纯七氟醚组(S组),单纯缺血再灌注组(I/R组),七氟醚后处理组(SEVOP组)。(1)SHAM组:K-H液持续灌注180 min;(2)S组:K-H液持续灌注60 min,且于60min时给予含2.5%七氟醚(批号:2217,丸石制药株氏会社,日本)的K-H液灌注15 min,继续K-H液灌注105 min;(3)I/R组:平衡30 min,缺血30 min,再灌注120 min;(4)SEV0P组:于再灌注初给予含2.5%七氟醚的K-H液灌注15 min;记录各组血液动力学参数;TUNEL法检测细胞凋亡部分;TTC法计算心肌梗死面积;Western blot测Bcl-2、Caspase-3的蛋白表达。结果与T0比,I/R和SEVOP组在T1、T2、T3、T4时点LVSP、HR、±dp/dtmax、降低(P0.05),LVEDP升高(P0.05);在T1、T2、T3、T4时刻,与SHAM组比较,I/R和SEVOP组LVSP、HR、±dp/dtma降低,LVEDP升高(P0.05);与I/R组比较,SEVOP组LVSP、HR、±dp/dtma升高,LVEDP降低(P0.05)。与I/R组(51±5)%相比,SEVOP组(31±4)%的心肌梗死范围缩小(P0.05)。TUNEL法染色结果示,与SHAM组相比,I/R组及SEVOP组心肌细胞TUNEL染色阳性细胞增多,TUNEL凋亡指数增高(P0.05);与I/R组比,SEVOP组心肌细胞TUNEL染色阳性细胞较少,凋亡指数较低(P0.05)。与SHAM组相比,I/R组及SEVOP组心肌Bcl-2蛋白表达下降,Caspase-3表达增加,差别有统计学意义。(P0.05);与I/R组比,SEVOP组心肌Bcl-2蛋白表达升高,Caspase-3表达下降,差别有统计学意义。(P0.05)结论七氟醚后处理后大鼠左室舒张功能增加,心肌收缩力增强,心功能改善明显;七氟醚后处理能抑制心肌细胞凋亡,减小梗死面积,减轻大鼠心肌缺血再灌注损伤。第二部分七氟醚后处理减轻离体大鼠心肌缺血再灌注损伤的分子机制:NOS/NO-NHE1-MPTP途径目的通过心肌I/R后,七氟醚后处理,用或不用L-NAME后NOS/NO、NHE1/p-NHE1、和NAD+含量变化,探讨七氟醚后处理离体大鼠心肌缺血再灌注损伤的保护作用的分子机制。方法Langendorff灌流模型成功的雄性SD大鼠离体心脏72个,采用随机数字表法,将其分为6组(n=12):假手术组(S组):连续用K-H液灌注180 min;七氟醚组(Sev组):K-H液灌注60 min后,用含2.5%七氟醚的K-H液灌注15 min,再用K-H液灌注105 min;缺血再灌注组(I/R组):K-H液灌注30 min后,停灌30 min,再灌注120min,制备心肌缺血再灌注损伤模型;七氟醚后处理组(SEVOP组):K-H液灌注30 min后,停灌30 min,于再灌注开始即刻用含2.5%七氟醚的K-H液灌注15 min,随后再用K-H液灌注105 min;七氟醚后处理+NOS抑制剂旋硝基精氨酸甲基酯(L-NAME)组(SEVOP+L-NAME组):再灌注开始用含浓度为100μmol/L L-NAME的K-H液灌注60 min(再灌注开始同时注入K-H液2.5%七氟醚并维持15分钟),随后再K-H液灌注60 min;NOS抑制剂旋硝基精氨酸甲基酯(L-NAME)组(L组):再灌注开始即刻用含浓度为100μmol/L L-NAME的K-H液灌注60 min,再换用上述单纯K-H液灌注60 min;于再灌注2 h时取心肌组织,采用分光光度法检测NO、NAD+含量和NOS活性,Western Blot法测定总NHE1(t-NHE1)和磷酸化NHE1(p-NHE1)的表达,RT-PCR法测定NHE1 m RNA的表达水平,并测定心肌梗死体积,观察心肌超微结构。结果与S组比较,I/R组、SEVOP组、SEVOP+L-NAME组和L组心肌梗死体积增大,心肌组织NO、NOS和NAD+水平降低,p-NHE1及NHE1 m RNA表达上调(P0.05),Sev组上述指标差异无统计学意义(P0.05);与I/R组比较,SEVOP组心肌梗死体积减小,心肌组织NO、NOS和NAD+水平升高,p-NHE1及NHE1 m RNA表达下调(P0.05),SEVOP+L-NAME组和L组上述指标差异无统计学意义(P0.05);与SEVOP组比较,SEVOP+L-NAME组和L组心肌梗死体积增大,心肌组织NO、NOS和NAD+水平降低,p-NHE1及NHE1 m RNA表达上调(P0.05)。SEVOP组心肌病理学损伤较I/R组和SEVOP+L-NAME组减轻。结论七氟醚后处理可能通过增强心肌NOS活性,促进NO合成,抑制NHE1功能,减少MPTP开放,从而减轻大鼠离体心脏缺血再灌注损伤。第三部分七氟醚后处理对离体大鼠缺血再灌注心肌胀亡和自噬的影响目的观察七氟醚后处理对大鼠心肌胀亡和自噬活动的影响。方法建立大鼠心脏Langendorff离体心脏灌流模型,取制备离体模型成功的心脏32个,分为4组(n=8):假手术组(SHAM组),单纯七氟醚组(S组),单纯缺血再灌注组(I/R组),七氟醚后处理组(SEVOP组),各组处理方法同第一部分。透射电镜观察细胞胀亡和自噬;Western blot测自噬相关分子LC3I、LC3II,Beclin-1和胀亡相关分子porimin的蛋白表达。结果Sham组自噬小体数目是1.2±0.7,胀亡细胞数是3.2±1.5;S组自噬小体的数目是1.0±0.6,胀亡细胞数是3.0±1.3,两组对比,无统计学差异。而在缺血再灌注组,I/R组自噬小体数目是10.4±2.4,胀亡细胞数是15.5±4.2;七氟醚后处理组SEVOP组自噬小体的数目是6.8±1.8,胀亡细胞数是11.7±3.5表明缺血再灌注后自噬小体和胀亡细胞数目显著增加,而七氟醚后处理能显著减少噬小体和胀亡细胞生成的数目。结论单独使用七氟醚不影响自噬小体和胀亡细胞数目,缺血再灌注后自噬小体和胀亡细胞数目显著增加,而七氟醚后处理能显著减少自噬小体和胀亡细胞生成的数目。
[Abstract]:The first part of sevoflurane relieves myocardial ischemia and reperfusion injury in isolated rat myocardium by comparing the hemodynamic parameters, apoptosis and infarct area before and after I/R, and exploring the protective effect of sevoflurane postconditioning on myocardial ischemia reperfusion injury in rats. Methods to establish a rat heart perfusion model of cardiac Langendorff in vitro, and prepare the preparation of the rat heart perfusion model. 32 successful hearts in the isolated model were divided into 4 groups (n=8): sham operation group (group SHAM), simple sevoflurane group (group S), simple ischemia reperfusion group (group I/R), sevoflurane group (group SEVOP). (1) SHAM group: K-H solution continuous perfusion 180 min; (2) S group: K-H liquid continuous perfusion 60 min, and 2.5% sevoflurane in 60min (batch number: 2217, pill stone pharmaceutical strain) The K-H solution in the society, Japan, was perfused 15 min, continued the K-H solution of 105 min, and (3) I/R group: balance 30 min, ischemia 30 min, and reperfusion 120 min; (4) SEV0P group: 2.5% sevoflurane containing K-H solution was given at first, and the hemodynamic parameters of each group were recorded, the apoptotic part of the cells was detected by TUNEL method. Bcl-2, Caspase-3 protein expression. Results compared with T0, I/R and SEVOP group in T1, T2, T3, T4 time point LVSP, HR, dp/dtmax, decreased. Compared with group R (51 + 5)%, SEVOP group (31 + 4)% of myocardial infarct scope reduced (P0.05).TUNEL staining results, compared with group SHAM, TUNEL staining positive cells in group I/R and SEVOP group increased and TUNEL apoptosis index increased (P0.05), and the number of apoptotic cells in SEVOP group was less and the apoptosis index was lower than that in I/R group. Compared with group I/R and group SEVOP, the expression of Bcl-2 protein decreased and the expression of Caspase-3 increased. (P0.05). Compared with group I/R, the expression of Bcl-2 protein in SEVOP group increased and Caspase-3 expression decreased, and the difference was statistically significant. (P0.05) conclusion the left ventricular diastolic function of rats after sevoflurane after treatment was increased and the contractility of myocardium was enhanced. The treatment of sevoflurane can inhibit cardiomyocyte apoptosis, reduce infarct size and reduce myocardial ischemia reperfusion injury in rats. The second part of sevoflurane relieves the molecular mechanism of myocardial ischemia reperfusion injury in isolated rat myocardium: after the NOS/NO-NHE1-MPTP pathway, after I/R, sevoflurane is treated with or without L-N After AME NOS/NO, NHE1/p-NHE1, and NAD+ content changes, the protective effects of sevoflurane on myocardial ischemia and reperfusion injury in isolated rat myocardium were investigated. Methods 72 isolated male SD rats were successfully treated with Langendorff perfusion model, and they were divided into 6 groups (n=12): the sham operation group (S group): perfusion of K-H solution of 18. 0 min, sevoflurane group (group Sev): after K-H solution was perfused 60 min, 15 min was perfused with K-H solution containing 2.5% sevoflurane, and 105 min was perfused with K-H solution; ischemia reperfusion group (I/R group): after K-H solution was perfused 30 min, 30 min was stopped and reperfusion injury model of myocardial ischemia and reperfusion was prepared. N, infusion of 15 min with 2.5% sevoflurane solution at the beginning of reperfusion, followed by 105 min with K-H solution, and after sevoflurane treatment of +NOS inhibitor, +NOS inhibitor, group SEVOP+L-NAME (SEVOP+L-NAME group): reperfusion began to pour 60 min (2.5% seven fluorine at the beginning of reperfusion) with a K-H liquid containing a concentration of 100 micron L-NAME. The ether was maintained for 15 minutes, and then the K-H solution was followed by 60 min, and the NOS inhibitor, the group of L-NAME (group L), was injected with the K-H solution containing 100 mu mol/L L-NAME, and then perfused 60 min, and then changed to 60 min by the pure K-H solution. NOS activity, Western Blot method was used to determine the expression of total NHE1 (t-NHE1) and phosphorylated NHE1 (p-NHE1). RT-PCR assay was used to determine the expression level of NHE1 m RNA, and the volume of myocardial infarction was measured and the myocardial ultrastructure was observed. The expression of E1 and NHE1 m RNA was up-regulated (P0.05), and there was no significant difference in the above indexes in group Sev (P0.05). Compared with group I/R, the volume of myocardial infarction decreased in SEVOP group, NO, NOS and NAD+ levels in the myocardium. Myocardial infarction volume in group P+L-NAME and group L increased, myocardial tissue NO, NOS and NAD+ decreased, p-NHE1 and NHE1 m RNA expression was up regulation (P0.05).SEVOP group. The effect of third part of sevoflurane on myocardial atolis and autophagy in rats in vitro. Objective To observe the effect of sevoflurane on cardiac atolum and autophagy in rats. Methods a rat heart perfusion model of Langendorff in vitro was established. 32 hearts were divided into 4 groups (n=8): sham operation group (group SHAM), simple sevoflurane group (group S), simple ischemia-reperfusion group (group I/R), and sevoflurane group (group SEVOP). The treatment methods of each group were the same as the first part. Transmission electron microscopy was used to observe the cell atoly and autophagy; Western blot was used to detect autophagic related molecules LC3I, LC3II, Beclin-1, and bulging associated molecules. Results the number of autophagic corpuscles in group Sham was 1.2 + 0.7, the number of bulging cells was 3.2 + 1.5, the number of autophagic bodies in group S was 1 + 0.6, the number of bulging cells was 3 + 1.3, and there was no statistical difference between the two groups. In the ischemia reperfusion group, the number of autophagic corpuscles in the group I/R was 10.4 + 2.4, the number of the bulging cells was 15.5 + 4.2, and the SEVOP group of the sevoflurane post treatment group. The number of autophagic bodies was 6.8 + 1.8, and the number of bulging cells was 11.7 + 3.5. The number of autophagic corpuscles and bulging cells increased significantly after ischemia-reperfusion, while sevoflurane reprocessing could significantly reduce the number of phagocytic and bulging cells. The number of phagocytic bodies and apoptotic cells increased significantly, while sevoflurane postconditioning significantly reduced the number of autophagic bodies and the number of apoptotic cells.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R614

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