【作者】 徐通达；

【导师】 李东野；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2013， 博士

【摘要】 目的：本研究通过建立大鼠离体缺血/再灌注(ischemia/reperfusion, I/R)心脏和心肌细胞模型,以心肌梗死面积、心脏血流动力学变化、心肌坏死标记物、单个心肌细胞的收缩功能、凋亡相关蛋白、双特异性蛋白磷酸酶(Dual specificity protein phosphatase, DUSP)2/4/16以及ERK1/2和JNK通路上相关蛋白的表达变化为指标,观察丹酚酸A (Salvianolic acid A, SAA)对离体I/R心脏和心肌细胞收缩功能的恢复及凋亡的影响。探讨SAA对I/R损伤的保护作用及其可能的分子机制,从而为临床治疗心肌I/R损伤提供一种新的药物及治疗靶点。方法：1.清洁级成年雄性wistar大鼠,体重200-250g,分为正常对照组(CON, n=6), I/R组(I/R, n=6), SAA预处理组(SAA+I/R, n=6), ERK1/2抑制剂PD098059+I/R组(PD+I/R), ERK1/2抑制剂PD098059+SAA+I/R组(PD+SAA+I/R), JNK抑制剂SP600125+I/R组(SP+I/R)。应用离体心脏连接Langendorff装置,采用停灌、复灌方法模拟I/R过程,观察SAA(20μM)对离体I/R心脏的保护作用。CON组离体心脏持续用K-H液灌流200min; I/R组离体心脏持续灌流50min后,再缺血30min复灌120min; SAA+I/R组离体心脏持续灌流30min,进行SAA预处理20min后再缺血30min复灌120min; PD+I/R组PD预处理30min,余处理同I/R组；PD+SAA+I/R组进行SAA预处理前用PD预处理30min,余处理同SAA+I/R组；SP+I/R组SP预处理30min,余处理同I/R组。灌注过程中将一连接于压力传感器的充水水囊经肺静脉口进入左心房,经二尖瓣插入左室,以进入左室后LVEDP为5-15mmHg为充水水囊充水后标准,通过生物信号采集器测量心功能各项指标。分别于缺血前、再灌注30min、120min记录心率(HR)、左心室收缩压(LVSP).左心室舒张末压(LVEDP).左心室压变化最大速率(±dp/dtmax)。同时各组在再灌注15min时留取冠脉流出液待测乳酸脱氢酶(LDH)。再灌注结束后,冷冻心脏,TTC染色法测量各组心脏的心肌梗死面积。2.以常规酶解法分离成年大鼠心肌细胞,采用缺糖缺氧、复糖复氧的方法模拟I/R过程,观察SAA对离体I/R心肌细胞的保护作用。所得心肌细胞,分为以下各组：(1)CON组：心肌细胞正常培养18h；(2)I/R组：心肌细胞正常培养13h后,培养基更换为模拟缺血液,放入三气培养箱中进行缺氧培养3h,然后将细胞培养基更换为高糖DMEM培养基,放入C02培养箱复糖复氧模拟再灌注；(3)SAA不同浓度组+I/R组：心肌细胞正常培养1h后,加入不同浓度SAA (1,5,10,15μM)预处理12h,再进行I/R,应用心肌细胞杆状率评价细胞的存活率,根据细胞的存活率确定SAA的最佳浓度,后述实验中SAA剂量均采用最佳浓度一种剂量进行；(4) PD+SAA+I/R组：进行SAA预处理前用PD预处理1h,余处理同SAA+I/R组；(5) SP+I/R组：SP预处理1h,余处理同I/R组。再灌注时间根据不同指标分为2h或6h。处理细胞分别进行以下实验：在异丙肾上腺素刺激(isoproterenol, ISO)状态下,收缩幅度用单个心肌细胞检测技术测定；用TUNEL及DAPI法检测心肌细胞凋亡率；用免疫印迹法(Western blot)测定Bcl-2、Bax、c-Jun氨基末端激酶(c-Jun N-terminal kinases, JNK),磷酸化-JNK (p-JNK),细胞外调节蛋白激酶(extracellular regulated protein kinases, ERK1/2),磷酸化-ERK1/2(p-ERK1/2)及DUSP2, DUSP4, DUSP16蛋白表达变化。结果：1.在离体I/R心脏水平上,与CON组相比,I/R能够明显地降低心功能的各种参数(P<0.01),增加LDH释放量,心肌梗死面积及心肌细胞凋亡率(P<0.01)；与I/R组相比,SAA+I/R, SP+I/R组,PD+SAA+I/R组,均可改善心功能的各种参数,降低LDH释放量,心肌梗死面积及心肌细胞凋亡率(P<0.05-0.01),而PD+I/R组与I/R组相比,心功能的各种参数,LDH的释放量,心肌梗死面积及心肌细胞凋亡率未见明显变化(P>0.05)；各组与PD+I/R组相比,结果等同于与I/R组相比；与SAA+I/R组比较,PD+SAA+I/R组心功能参数降低,LDH释放量,心肌梗死面积及心肌细胞凋亡率均有所增加(P<0.05),而SP+I/R组与SAA+I/R组比较,心功能参数,LDH释放量,心肌梗死面积及心肌细胞凋亡率未示明显差异(P>0.05)；而与PD+SAA+I/R组相比,SP+I/R组心功能参数升高,LDH释放量,心肌梗死面积及心肌细胞凋亡率减少明显(P<0.01)。由于PD+I/R组与单存I/R组相比,心脏血流动力学指标,心肌损伤标记物LDH,以及心肌细胞凋亡率三组未见明显变化,故在心肌细胞水平中,未进一步行PD+I/R组的相关试验。2.在细胞水平上,与CON组相比,I/R后,单个心肌细胞收缩幅度、p-ERK1/2、Bcl-2及DUSP4/16蛋白表达水平明显下降,p-JNK、Bax及DUSP2蛋白表达水平明显增加(P<0.05-0.01)；用SAA预处理后,能够增加单个心肌细胞收缩幅度、p-ERK1/2、Bcl-2及DUSP4/16蛋白表达水平,降低p-JNK、Bax及DUSP2蛋白表达水平(P<0.05)；与I/R组比较,SAA+I/R, SP+I/R组,PD+SAA+I/R组,均可增加单个心肌细胞收缩幅度、p-ERK1/2、 Bcl-21及DUSP4/16蛋白表达水平,降低p-JNK、Bax及DUSP2蛋白表达水平(P<0.05)；相对于SAA+I/R组,PD+SAA+I/R组单个心肌细胞收缩幅度、p-ERK1/2、Bcl-2及DUSP4/16蛋白表达水平降低,p-JNK、Bax及DUSP2蛋白表达水平上调(P<0.05)；而SP+I/R组与SAA+I/R组比较,单个心肌细胞收缩幅度、p-ERK1/2、p-JNK、Bcl-2、Bax、DUSP2及DUSP4/16蛋白表达水平均未见明显变化(P>0.05)；与PD+SAA+I/R组相比,SP+I/R组增加了单个心肌细胞收缩幅度、p-ERK1/2、Bcl-2及DUSP4/16蛋白表达水平,降低了p-JNK、Bax及DUSP2蛋白表达水平(P<0.05)。结论：JNK可以通过DUSP2抑制ERK1/2的激活,而ERK1/2则可以通过DUSP4/16去磷酸化抑制了JNK的活性；SAA可能通过抑制DUSP2介导的JNK通路激活ERK1/2通路以及通过激活DUSP4/16介导的ERK1/2通路抑制JNK通路激活,从而发挥对缺血/再灌注心肌细胞抗凋亡的作用。更多还原

【Abstract】 Objective:The purpose of this study is to establish an rat ischemia/reperfusion (I/R) heart and cardiomyocyte model, and to observe the effects of Salvianolic acid A (SAA) pretreatment on the I/R myocardium in vitro and cardiomyocyte contraction function and apoptosis with these indexes for myocardium infarction area, cardiac hemodynamics, myocardium necrosis markers, single cardiomyocytes contractile function, apoptosis related proteins, dual specificity protein phosphatase (DUSP)2/4/16and ERK1/2/JNK pathways related protein expression. Meanwhile, the cardioprotection against I/R injury and underlying molecular mechanisms are able to be discussed, thus a kind of new drugs and therapeutic targets can be provides for clinical treatment of myocardium I/R.Methods:1. Clean grade adult male Wistar rats, weight220-260g, were divided into normal control group (CON group, n=6), I/R group (I/R, n=6), SAA pretreatment group (SAA+I/R, n=6), ERK1/2inhibitors PD098059+I/R group (PD+I/R), ERK1/2inhibitors PD098059+SAA+I/R group (PD+SAA+I/R), JNK inhibitors SP600125+I/R group (SP+I/R). The cardioprotection of SAA (20μM) on I/R heart in vitro was observed by application of Langendorff heart connection device in vitro and I/R process was simulated by stopping perfusion following reperfusion. The heart in vitro in CON group was continuously perfused K-H solution perfusion for160min; the heart in vitro in I/R group was perfused for10min, then ischemia for30min following reperfusion for120min; In SAA+I/R group, the heart in vitro was perfused for10min, then SAA pretreatment for20min, ischemia for30min following reperfusion for120min; In PD+I/R group, the heart in vitro was perfused for10min, then PD pretreatment for20min, the following procedure was the same as I/R group; In PD+SAA+I/R group, PD was pretreated for30min, the following process was the same as SAA+I/R group; In SP+SP group, SP was pretreated for20min, the following procedure was the same as I/R group; During the course of reperfusion, A filling water sac connecting pressure sensor was inserted into left ventricle via pulmonary vein orifice, left atrium, and mitral valve, the filling water sac was located in LVEDP for5-15mmHg. heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and maximum rate of left ventricular pressure (+dp/dtmax) were recorded for30min prior to I/R and120min after I/R. At the same time, lactate dehydrogenase (LDH) from coronary effluent for reperfusion15min was assayed. After reperfusion, heart was frozen, myocardial infarction areas were measured by TTC staining method.Adult rat cardiomyocytes were isolated in conventional enzymolysis approach, then I/R process was simulated by lacking of oxygen, sugar following recovering sugar, oxygen to observe cardioprotection of SAA for I/R cardiomyocytes in vitro.In cells level, there were five groups.(1) CON group:cardiomyocytes were cultivated for18h;(2) I/R group:cardiomyocytes were cultivated for13h, then were placed in three gas incubator for3h, next, cardiomyocyt es were cultured in high glucose DMEM medium and CO2incubator to simulate reperfusion.(3) SAA+I/R group:cardiomyocytes were cultivated for1h, then I/R was performed after different concentrations of SAA (1,5,10,15^M) pretreatment for12h. The optimal concentration of SAA was determined according to cell survival rate by observing the rod rate of cardiomyocytes and then was performed in following experiments.(4) PD+SAA+I/R group:PD was added for1h prior to SAA pretreatment, then the following process was as the same as SAA+I/R group;(5) SP+I/R group:pretreatment with SP for1h, I/R was performed. The contraction amplitude of cardiomyocytes was determined with a single detection technology by isopropyl adrenaline stimulating; Cardiomyocytes apoptosis was detected by TUNEL and DAPI; The protein expression levels for Bcl-2, Bax, JNK, phosphorylation-JNK (p-JNK), ERK1/2, phosphorylation-ERK1/2(p-ERK1/2), DUSP2/4/16were assayed by Western blot.Results:1. In cardiac I/R level in vitro, compared with CON group, various parameters of heart function were reduced significantly(P<0.01) and LDH release, area of myocardial infarction, cell apoptosis were increased significantly following I/R (P<0.01); Compared with I/R group, various parameters of heart function were improved significantly ((P<0.05)) and LDH release, area of myocardial infarction, cell apoptosis were reduced significantly among SAA+I/R group, SP+I/R group, PD+SAA+I/R group (P＜0.01); The results, PD+I/R compared with different groups, were as the same as I/R group; In PD+SAA+I/R group, heart function parameters were reduced and LDH release, area of myocardial infarction, cell apoptosis rate were increased (P<0.05) by comparsion with SAA+I/R group; However, SP+I/R group compared with SAA+I/R group, the indexes mentioned above were not significant different (P>0.05); Compared with PD+SAA+I/R group, various parameters of heart function were elevated significantly (P＜0.05)) and LDH release, area of myocardial infarction, cell apoptosis were reduced significantly in SP+I/R group (P＜0.01). With regard to the values of hemodynamic indicators, LDH and apoptosis rate between PD+I/R group and I/R group were not statistically different (P>0.05), so the related test in PD+I/R group was not performed at cell level.2. At cellular level, compared with CON group, contraction amplitude of single cardiomyocyte, protein expression level for p-ERK1/2, Bcl-2and DUSP4/16were decreased significantly(P<0.01), however, p-JNK, Bax and DUSP2protein expressions were increased significantly (P＜0.05-0.01) following I/R; With SAA pretreatment, contraction amplitude of single cardiomyocyte. protein expression level for p-ERK1/2, Bcl-2, DUSP4/16were increased and p-JNK, Bax, DUSP2protein levels were reduced significantly (P＜0.05); Compared with I/R group, contraction amplitude of single cardiomyocyte, protein expression level for p-ERK1/2, Bcl-2, DUSP4/16were increased significantly and p-JNK, Bax, DUSP2protein expressions were reduced significantly (P＜0.05) among SAA+I/R group, SP+I/R group, PD+SAA+I/R group (P<0.01); In PD+SAA+I/R group, contraction amplitude of single cardiomyocyte, protein expression level for p-ERK1/2, Bcl-2, DUSP4/16were reduced significantly and p-JNK, Bax, DUSP2protein expressions were activated significantly (P<0.05) by comparsion with SAA+I/R group; However, SP+I/R group compared with SAA+I/R group, the indexes mentioned above were not significant different (P>0.05); Compared with PD+SAA+I/R group, the protein expression levels for p-ERK1/2, Bcl-2, DUSP4/16and single cardiomyocyte contraction amplitude were increased significantly and p-JNK, Bax, DUSP2protein levels were lowered in SP+I/R group (P<0.05).Conclusion:During the course of I/R, JNK pathway can inhibit the ERK1/2activation via DUSP2and ERK1/2inhibit ERK1/2activation via DUSP4/16.SAA can inhibit DUSP2-mediated JNK pathway for activation of ERK1/2and activate DUSP4/16-mediated ERK1/2by JNK dephosphorylation to play anti-apoptosis role in I/R cardiomyocytes.更多还原