程序性细胞死亡在大鼠心肌缺血/再灌注损伤中的作用
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摘要
研究背景：
心肌梗死是危害人类健康的主要疾病之一,尽早有效地恢复对缺血心肌的血液再灌注,是阻止缺血性心肌损伤的发展、减小心梗面积进而改善临床预后的根本措施。但是,缺血心肌在恢复血液灌注后可产生更为严重的损伤,即缺血／再灌注损伤,使得即使给予及时、适当的再灌注治疗,仍然有10%的心梗患者死亡,25%的心梗患者发生心力衰竭。因此,缺血／再灌注损伤成为阻碍缺血心肌从再灌注治疗中获得最佳疗效的主要难题。
研究已知,心肌细胞死亡是缺血／再灌注损伤导致心梗患者预后不佳的主要原因。关于细胞死亡的分类目前并不统一,但普遍将受基因调控、高度有序、主动的细胞死亡形式称为程序性细胞死亡。因程序性细胞死亡具可调控性,抑制程序性细胞死亡的发生可能做到,故程序性细胞死亡在心肌缺血／再灌注损伤中的作用备受关注。在心肌缺血／再灌注损伤中,程序性细胞死亡的形式有哪些、它们之间的关系如何,已经成为寻找减轻心肌缺血／再灌注损伤机制的切入点之一。
已知的程序性细胞死亡主要有三种形式：凋亡(apoptosis)、自噬(autophagy)与程序性坏死(necroptosis)。凋亡是指为维持内环境稳定,由基因控制的细胞自主的有序的死亡,天冬氨酸特异的半胱氨酸蛋白酶(caspase)在细胞凋亡的执行期发挥关键作用；自噬是亚细胞膜结构发生动态变化并经溶酶体介导对细胞内蛋白质和细胞器降解的过程,通过形成自噬体(autophagosome)降解其底物。在生理条件下,自噬主要的功能是调控长寿蛋白和细胞器的降解利用,参与细胞质的重建、维持细胞质的稳定及清除受损的细胞器,此时自噬是作为细胞存活的机制。如果自噬活性过强,破坏细胞胶质以及细胞器超过了某一界限,就会发生自噬性细胞死亡；程序性坏死是指具有坏死的细胞形态特点和自噬体生成,并且主动耗能的细胞死亡过程。
自噬与程序性坏死都有自噬体形成,但其意义却不相同。由于自噬的过度活动可以导致细胞死亡,所以在自噬过程中出现的自噬体可能与自噬引起的细胞死亡有关；但在程序性坏死过程中出现的自噬体只是一种伴随现象,与程序性坏死引起的细胞死亡无关。
虽然有研究发现,这三种程序性细胞死亡分别参与了心肌缺血／再灌注损伤,但这些研究多数是通过关注其中一种程序性细胞死亡的作用得出结论,而且已有的关于三种程序性细胞死亡形式之间的相互关系的结论,也主要来自细胞培养或其它离体模型,因此并不能很好地分析程序性细胞死亡在心肌缺血／再灌注损伤中的意义。
近年的研究发现,三种程序性细胞死亡在体内并非孤立存在,而是彼此互相联系,互相影响。例如,阻断自噬可以促进细胞凋亡的发生,而阻断细胞凋亡又可促进程序性坏死。已有的研究不能很好地反映和比较每种程序性细胞死亡在心肌缺血／再灌注损伤中的作用大小,相互之间有无关系及关系如何等。若能选用恰当的动物模型,从在体水平阐明这三种程序性细胞死亡各自的发生发展规律,以及三者之间的相互关系,将会为临床筛选治疗的最佳时间点、明确相互之间的关系,最终为临床改善心肌缺血／再灌注损伤治疗措施的制定提供重要的实验依据。
综上所述,本研究拟通过建立大鼠心肌／缺血再灌注模型进行以下研究：1.在同一个动物个体上观察三种程序性死亡在心肌／缺血再灌注过程中各自发生的时间规律；2.运用三种程序性细胞死亡的抑制剂,观察三者在心肌／缺血再灌注过程中作用的相互关系；3.分析三种程序性细胞死亡在心肌／缺血再灌注损伤中的可能的贡献。
第一部分三种程序性细胞死亡在大鼠心肌／缺血再灌注过程中各自发生的时间规律
目的：
本部分实验运用大鼠心肌缺血／再灌注模型,观察三种程序性细胞死亡形式在心肌／缺血再灌注过程中各自的动态变化规律。
材料与方法：
1.动物：
雄性Wistar大鼠,体重：230±10 g。
本实验共使用60只大鼠,死亡12只,实际用于实验研究48只。
2.分组：
(1)缺血组(n=6)：结扎冠状动脉左前降支30 min；
(2)再灌注1h组(n=6)：结扎冠状动脉左前降支30 min,再灌注1h；
(3)再灌注2h组(n=6)：结扎冠状动脉左前降支30 min,再灌注2h；
(4)再灌注3h组(n=6)：结扎冠状动脉左前降支30 min,再灌注3h；
(5)再灌注12h组(n=6)：结扎冠状动脉左前降支30 min,再灌注12h；
(6)再灌注24h组(n=6)：结扎冠状动脉左前降支30 min,再灌注24h：
(7)再灌注48h组(n=6)：结扎冠状动脉左前降支30 min,再灌注48h；
(8)伪手术组(n=6)：手术过程同缺血组但不结扎冠状动脉。
3.方法：
建立大鼠心肌缺血／再灌注模型,留取心脏,以特异性较强的caspase-3活性测定法检测心肌细胞凋亡程度；以Western blot方法检测心肌组织微管相关蛋白轻链3 (MAP LC3-Ⅱ)的蛋白表达,观测自噬体形成,从而反映自噬和程序性死亡程度。
结果：
1.心肌缺血／再灌注期间,大鼠心肌组织caspase-3活性的动态变化规律
Caspase-3是caspase依赖性细胞凋亡通路上的最后执行者,其活性可以反应细胞凋亡水平。
实验结果显示,缺血30min心肌组织Caspase-3活性与伪手术组相比无明显改变；Caspase-3活性在再灌注1h升高至峰值(1.56±0.22vs.1.0±0.21,P<0.01)；之后开始下降,再灌注24h恢复至伪手术组水平(图2)。
2.心肌缺血／再灌注期间,大鼠心肌组织LC3-Ⅱ蛋白表达的动态变化规律
LC3-Ⅱ是观察哺乳动物自噬体水平公认的生物学标记物。
实验结果显示,与伪手术组相比,心肌缺血30min后心肌组织中LC3-Ⅱ的蛋白表达开始增加(P<0.05),再灌注期间(再灌注1h、2h、3h、12h)进一步增加,在再灌注24h达到高峰(P<0.01),随后明显减少,再灌注48h逐渐回复至再灌注3h水平(图3)。
以上结果提示,心肌缺血未导致caspase依赖的心肌细胞凋亡,细胞凋亡主要发生在再灌注期间,再灌注1h达高峰,随后开始下降,再灌注24h降至正常；心肌缺血可导致心肌组织自噬体形成增加,再灌注期间自噬体形成进一步增加,再灌注24h达高峰,之后开始下降,但再灌注48h仍未恢复正常。
小结(一)
1.30min的心肌缺血未导致caspase依赖的心肌组织凋亡,凋亡发生在再灌注期间,而且以再灌注初期为主,再灌注1h即达高峰,随后开始下降,再灌注24h降至正常。
2.30min的心肌缺血可导致心肌组织自噬体形成增加,再灌注期间自噬体形成进一步增加,再灌注24h达高峰,之后开始下降,但再灌注48h仍未恢复正常。
由于自噬与程序性坏死都可以引起自噬体形成,所以在本研究中自噬体的来源、自噬与程序性坏死的变化规律有待进一步研究,将在第二部分实验中进一步阐明。
第二部分三种程序性细胞死亡在大鼠心肌／缺血再灌注过程中的作用和相互关系
目的：
依据第一部分的实验结果,分别给予Caspase阻断剂Z-VAD-fmk、自噬阻断剂三甲基腺嘌呤(3-methyladenine,3-MA)和程序性坏死阻断剂Nec-1后,观察3种程序性细胞死亡的变化,进而分析3种程序性细胞死亡在大鼠心肌／缺血再灌注过程中的作用及其相互关系。
材料与方法：
1.动物：
同第一部分。
本实验共使用动物128只,死亡26只,实际用于实验研究102只。
2.分组：
(1)I30min+DMSO组(n=6)：结扎冠状动脉左前降支30min；结扎前30min静脉注射DMSO(0.1%)0.6m1。
(2)130min+Z-VAD-fmk组(n=6)：结扎冠状动脉左前降支30min；结扎前30min静脉注射Z-VAD-fmk(1mg／kg,溶于DMSO)。
(3)130min+Nec-1组(n=6)：结扎冠状动脉左前降支30min;结扎前30min静脉注射Nec-1(0.6mg／kg,溶于DMSO)。
(4)再灌注1h+DMSO组(n=6)：结扎冠状动脉左前降支30min,再灌注1h；结扎前30min注射DMSO 0.6ml。
(5)再灌注1h+Z-VAD-fmk组(n=6)：结扎冠状动脉左前降支30min,再灌注1h;结扎前30min静脉注射Z-VAD-fmk(1mg／kg,溶于DMSO)。
(6)再灌注1h+3-MA组(n=6)：结扎冠状动脉左前降支30min,再灌注1h;结扎前30min静脉注射3-MA(15mg／kg,溶于DMSO)。
(7)再灌注1h+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注1h；结扎前30min静脉注射Nec-1 (0.6mg/kg,溶于DMSO)。
(8)再灌注3h+DMSO组(n=6)：结扎冠状动脉左前降支30min,再灌注3h；结扎前30min静脉注射DMSO 0.6ml。
(9)再灌注3h+Z-VAD-fmk组(n=6)：结扎冠状动脉左前降支30min,再灌注3h；结扎前30min静脉注射Z-VAD-fink (1mg/kg,溶于DMSO)。
(10)再灌注3h+3-MA组(n=6)：结扎冠状动脉左前降支30min,再灌注3h;结扎前30min静脉注射3-MA(15mg／kg,溶于DMSO)。
(11)再灌注3h+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注3h；结扎前30min静脉注射Nec-1(0.6mg／kg,溶于DMSO)。
(12)再灌注12h+DMSO组(n=6)：结扎冠状动脉左前降支30min,再灌注12h;结扎前30min静脉注射DMSO 0.6ml。
(12)再灌注12h+Z-VAD-fmk组(n=6)：结扎冠状动脉左前降支30min,再灌注12h；结扎前30min,再灌注8h(据Z-VAD-fmk半衰期确定药物追加时间)分别静脉注射Z-VAD-fmk(1mg／kg,溶于DMSO)。
(14)再灌注12h+3-MA组(n=6)：结扎冠状动脉左前降支30min,再灌注12h；结扎前30min静脉注射3-MA (15mg/kg,溶于DMSO).
(15)再灌注12h+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注12h；结扎前30min静脉注射Nec-1 (0.6mg/kg,溶于DMSO)。
(16)再灌注24h+DMSO组(n=6)：结扎冠状动脉左前降支30min,再灌注24h；再灌注前30min静脉注射DMSO 0.6ml。
(17)再灌注24h+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注24h；结扎前30min静脉注射Nec-1(0.6mg／kg,溶于DMSO)。
3.方法：
同第一部分
结果：
第一部分结果显示：LC3-Ⅱ蛋白表达在缺血期已增高,再灌注期间进一步增高,再灌注24h达高峰。故选择缺血30min、再灌注1h、2h、3h、12h和24h观察LC3-Ⅱ蛋白表达。
2.1分别给予Nec-1、3-MA和Z-VAD-fink后,大鼠缺血／再灌注心肌组织中LC3-Ⅱ蛋白表达的变化。
2.1.1给予Nec-1后,大鼠缺血／再灌注心肌组织中LC3-Ⅱ蛋白表达的变化。
给予Nec-1后,与相应时间点DMSO组相比,缺血30min、再灌注1h、2h和3h心肌组织中LC3-Ⅱ表达无明显改变；而再灌注12h和24h心肌组织中LC3-Ⅱ蛋白表达明显减少(12h：1.13±0.15 vs.0.65±0.08,P<0.01；24h：1.415±0.08vs.0.82±0.20,P<0.01)(图4,5,6)。以上结果提示,再灌注3h之前的自噬体形成可能是由自噬引起的,而再灌注12h至24h的自噬体形成可能是由程序性坏死引起,但不能排除自噬也参与12h至24h的自噬体形成。同时也说明抑制程序性坏死可能对自噬无影响。
2.1.2给予3-MA后,观察缺血／再灌注大鼠心肌组织中LC3-Ⅱ蛋白表达变化。
给予3-MA后,与DMSO组相比,再灌注3h、12h心肌组织中LC3-Ⅱ蛋白表达均明显减少(3h：0.61±0.07vs.0.43±0.03,P<0.01；12h:1.125±0.15 vs.0.84±0.04,P<0.05)(图7,8,9)。这一结果提示,3-MA对自噬及程序性坏死的自噬体形成可都有抑制作用,但阻断自噬体的形成是否影响程序性坏死引起的细胞死亡有待进一步研究,将在第三部分进一步探讨。
2.1.3给予z-VAD-fmk后,观察缺血／再灌注心肌组织中LC3-Ⅱ蛋白表达的变化
有研究证实,应用广谱的Caspase抑制剂z-VAD-fmk阻断凋亡后,诱发程序性坏死,导致自噬体生成增多。因此,本实验中,我们在应用z-VAD-fmk后,观察缺血／再灌注过程中不同时间点心肌组织中LC3-Ⅱ的表达情况。根据研究结果2.1.1,选用再灌注3h为观察自噬时间点,再灌注12h观察程序性坏死的时间点。
给予z-VAD-fmk后,观察心肌缺血30min、再灌注3h,12h心肌组织LC3-Ⅱ的蛋白表达。结果显示,与DMSO组相比,给予Z-VAD-fmk后,缺血30min心肌组织LC3-Ⅱ蛋白无改变,而心肌缺血再灌注3h,12h心肌组织中LC3-Ⅱ蛋白表达均明显增加(3h：0.61±0.07 vs.0.74±0.08,P<0.05；12h：1.125±0.15 vs.1.255±0.04,P<0.05)(图10,11,12)。第一部分结果提示缺血30min心肌组织中只有自噬体的改变而无Caspase-3活性的变化,此时给予z-VAD-fink后LC3-Ⅱ蛋白无改变,而再灌注3h、12h Caspase-3活性升高,给予z-VAD-fmk后LC3-Ⅱ蛋白表达增加。提示,z-VAD-fmk本身不影响LC3-Ⅱ蛋白表达,其使得再灌注3h、12h LC3-Ⅱ蛋白表达增加是通过阻断凋亡而起作用。如前结果所述再灌注3h自噬体的生成可能主要来源于自噬,再灌注12h自噬体的生成可能主要来源于程序性坏死,故提示阻断凋亡可能促进自噬与程序性坏死。
2.2分别观察Caspase广谱抑制剂、细胞自噬特异性抑制剂以及程序性坏死特异性抑制剂对大鼠心肌缺血／再灌注后Caspase-3活性的影响
因Caspase-3活性在再灌注1h达高峰,再灌注12h的自噬体形成有程序性坏死参与,故选择再灌注1h观察Z-VAD-fmk及3-MA对caspase-3活性的影响；再灌注12h观察Nec-1对caspase-3活性的影响。
2.2.1给予Caspase广谱抑制剂(Z-VAD-fmk)后,再灌注1h大鼠心肌组织Caspase-3活性降低
与DMSO组相比,给予Z-VAD-fmk后再灌注1h大鼠心肌组织Caspase-3活性明显下降(1.59±0.23vs.1.11±0.03,P<0.01)(图13)。
2.2.2给予细胞自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3-MA)后,再灌注1h大鼠心肌组织Caspase-3活性增加。
与DMSO组相比,给予3-MA后再灌注1h大鼠心肌组织Caspase-3活性显著升高(1.58±0.49vs.1.81±0.06,P<0.01)(图14)。以上结果提示,心肌缺血／再灌注期间,抑制心肌细胞自噬反而会增加心肌细胞凋亡。
2.2.3给予程序性坏死特异性抑制剂(Necrostatin-1, Nec-1)后,再灌注1h大鼠心肌组织Caspase-3活性无影响。
与相应DMSO组相比,给予Nec-1后再灌注1h、12h大鼠心肌组织Caspase-3活性无变化(图15)。这一结果提示,缺血／再灌注期间,抑制细胞程序性坏死对心肌细胞凋亡的影响不明显。
小结(二)
1.在大鼠心肌缺血／再灌注引起的三种程序性死亡中自噬发生的最早,缺血期即增高,再灌注期间进一步升高；之后细胞凋亡开始发生,在再灌注1h达高峰,随后开始下降；程序性坏死发生的最晚,大概在再灌注12h左右发生,24h达高峰,再灌注48h仍未恢复正常。
2.在大鼠心肌缺血／再灌注中抑制凋亡可能促进自噬与程序性坏死,抑制自噬则可能促进凋亡,但抑制程序性坏死可能对另两种程序性死亡无明显影响。
第三部分三种程序性细胞死亡在心肌／缺血再灌注损伤中的贡献
目的：
根据第一、二部分结果的提示,本实验给予三种程序性细胞死亡阻断剂(分别单独使用一种,两两联合应用,以及同时应用三种),检测三种程序性细胞死亡的水平及心梗面积、心功能改变,以观察三种程序性死亡在心肌／缺血再灌注损伤中的可能贡献。
材料与方法：
1.动物：
同第一部分
本实验共使用动物68只,死亡14只,实际用于实验研究54只。
2.分组：
(1) R48h+DMSO组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；结扎前30min静脉注射DMSO 0.6ml。
(2) R48h+Z-VAD-fmk组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；结扎前30min、再灌注8h、16h、24h各静脉注射Z-VAD-fmk(1mg／kg,溶于DMSO)。
(3)R48h+3-MA组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；结扎前30min静脉注射3-MA (15mg/kg,溶于DMSO)。
(4)R48h+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；结扎前30min静脉注射Nec-1 (0.6mg/kg,溶于DMSO)。
(5) R48h+Z-VAD-fmk+3-MA组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；静脉注射Z-VAD-fink如第2组,3-MA如第3组。
(6) R48h+Z-VAD-fmk+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；静脉注射Z-VAD-fmk如第2组,Nec-1如第4组。
(7) R48h+3-MA+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；静脉注射3-MA如第3组,Nec-1如第4组。
(8) R48h+Z-VAD-fmk+Nec-1+3-MA组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；静脉注射Z-VAD-fmk如第2组,3-MA如第3组；Nec-1如如第4组。
(9)伪手术组(n=6)：手术过程同手术组但不结扎冠状动脉。
3.方法：
建立大鼠心肌缺血／再灌注模型,在体监测各项心功能指标、以Evanse blue和TTC复染法检测缺血／再灌注后大鼠心肌梗死面积。
结果：
由第一、二部分结果可知在大鼠心肌缺血／再灌注中,三种程序性细胞死亡发生最晚的程序性坏死于再灌注24h达高峰,随后下降。再灌注48h,三种程序性死亡基本达到稳态,故选择再灌注48h观测在体心功能和心梗面积。
3.1心肌缺血再灌注48h心肌细胞凋亡、细胞自噬以及程序性坏死对心梗面积的影响。
3.1.1观察分别单独应用z-VAD-fmk、3-N(?)A和Nec-1对心梗面积的影响。
经测定,各组大鼠危险区面积与左心室面积比值(AAR/LV)无统计学意义,说明模型建立手术手法一致,排除了由此而造成的误差。
与DMSO组比较,单独给予Z-VAD-fmk后,心梗面积明显缩小(44.08±2.84 vs.28.13±2.45,P<0.01)(图16,17,22,23);单独给予3-MA后,心梗面积明显缩小(44.08±2.84vs.32.23±1.90,P<0.01)(图16,17,22,23)；单独给予Nec-1后,心梗面积明显缩小(44.08±2.84vs.30.07±2.63,P<0.01)(图16,17,22,23)。以上实验结果显示,单独阻断任一种程序性坏死,均可有效地减小心梗面积,提示每一种程序性坏死在缺血／再灌注中均发挥其致损伤作用。
本实验结果还显示,单独给予z-VAD-fmk心梗面积较单独给予3-MA明显缩小(P<0.01)；但单独给予z-VAD和单独给予Nec-1组比,以及单独给予3-MA与单独给予Nec-1相比,心梗面积无统计学差异(P>0.05)。如第二部分结果所示,给予z-VAD-fmk阻断凋亡可促进自噬与程序性坏死,但仍以给予z-VAD-fmk后心梗面积缩小最为明显,这一结果提示,在心肌缺血／再灌注过程中凋亡的致心肌损伤作用较自噬可能更明显一些。三种阻断剂中单独给予3-MA缩小心梗面积的作用最差,一方面可能是由于阻断自噬可能促进凋亡的缘故,另一方面说明阻断自噬体的形成可能并不影响程序性坏死导致的细胞死亡。
3.1.2观察两两联合使用z-VAD-fmk、3-MA和Nec-1对心梗面积的影响。
与DMSO组相比,联合使用z-VAD-fmk与3-MA、联合使用z-VAD-fmk与Nec-1以及联合使用3-MA与Nec-1均使再灌注48h的心梗面积显著减小(z-VAD-fmk+3-MA: 44.08±2.84 vs.25.41±3.04, P<0.01; z-VAD-fmk+Nec-1:44.08±2.84vs.25.833.04, P<0.01; 3-MA+Nec-1:44.08±2.84 vs.26.90±2.90, P＜0.01);但联用两种抑制剂各组心梗面积两两之间比较无统计学差异；而联用两种抑制剂各组心梗面积与单独使用3-MA相比均明显减小(P<0.01),与单独给予Nec-1相比心梗面积均明显缩小(P<0.05)(图18,19,22,23)。
3.1.3观察同时联合使用z-VAD-fmk、3-MA和Nec-1对心梗面积的影响。
联合使用三种抑制剂(z-VAD-fmk+3-MA+Nec-1,23.28±3.10)(图20,21,22,23),与单独使用z-VAD-fmk (P＜0.05)、3-MA (P<0.01)及Nec-1 (P<0.01)相比,心梗面积明显减小；与联用3-MA和Nec-1组相比,心梗面积也明显缩小(P<0.05,图23)。
以上实验结果提示,心肌缺血／再灌注后心梗面积的大小受到心肌细胞凋亡、自噬以及程序性坏死的共同影响。其中心肌细胞凋亡可能是影响心梗面积最主要的因素,而心肌细胞自噬和程序性坏死可能只发挥了相对较小的作用。由于单独阻断一种程序性细胞死亡可能促进另一种程序性细胞死亡,故同时阻断三种程序性细胞死亡可更明显地减小心梗面积。
3.2 z-VAD-fmk、3-MA和Nec-1对心肌缺血再灌注48h心功能的影响。
3.2.1 z-VAD-fmk、3-MA和Nec-1对心肌缺血／再灌注48h+dp/dtmax的影响。
+dp/dtmax为左心室压力上升最大变化速率,可反映在体心脏收缩功能,该指标数值越小,心脏收缩功能越差。
与伪手术组相比,心肌缺血／再灌注48h+DMSO组+dp/dtmax明显降低(878.00±57.12vs.416.00±27.517,P<0.01)；与DMSO组相比,联用三种抑制剂可以明显改善心肌缺血／再灌注48h在体心脏收缩功能(416.00±27.517vs.527.83±12.73,P<0.01)；与DMSO组相比,单独给予Z-VAD-fmk、3-MA以及Nec-1均对心功能损伤的改善不明显,虽有好转趋势,。但无统计学意义(图24,25,26,27)。
3.2.2 z-VAD-fmk、3-MA和Nec-1对心肌缺血再灌注48h-dp/dtmax的影响。
-dp/dtmax为左心室压力下降最大变化速率,可反映在体心脏舒张功能,该指标数值越小,心功能越差。
实验结果显示,与伪手术组相比,心肌缺血／再灌注48h+DMSO组的-dp/dtmax明显降低(744.17±34.61vs.408.00±22.8,P<0.01)。与DMSO组相比,联用三种抑制剂(Z-VAD-fmk +3-MA+Nec-1)可以明显改善心肌缺血再灌注48h在体心脏舒张功能(408.00±22.80vs496.00±36.25,P<0.01)；单独给予Z-VAD-fmk、3-MA以及Nec-1均对心功能损伤的改善影响不明显,虽有好转趋势,但无统计学意义(图28,29,30,31)。
小结(三)
1.单用三种阻断剂均可明显减小心梗面积,提示三种程序性细胞死亡在大鼠心肌／缺血再灌注损伤中均发挥重要作用；其中给予z-VAD-fink缩小心梗面积最为明显,提示在大鼠心肌／缺血再灌注损伤中细胞凋亡的作用可能最大,自噬与程序性坏死的作用大小则无明显区别。
2.分别联合阻断两种程序性死亡后心梗面积较单独阻断自噬或程序性坏死均明显缩小。
3.联合阻断细胞凋亡、自噬及程序性坏死则可明显减小心梗面积。
4.只有联合阻断凋亡、自噬及程序性坏死三种程序性死亡可使大鼠心肌／缺血再灌注后48h心功能明显改善；但两两联合或单独应用一种阻断剂仅有改善心功能损伤的趋势。
结论：
1.在大鼠心肌缺血／再灌注过程中发生的三种程序性细胞死亡中,自噬发生得最早,随后为细胞凋亡的发生,程序性坏死发生得最晚；
2.在大鼠心肌缺血／再灌注过程中,抑制细胞凋亡有可能促进自噬与程序性坏死的发生；抑制自噬可能促进细胞凋亡的发生；抑制程序性坏死则对另两种程序性细胞死亡形式无明显影响;
3.三种程序性细胞死亡在大鼠心肌缺血／再灌注损伤中可能均发挥重要作用,其中细胞凋亡的作用可能最大；
4.大鼠心肌缺血／再灌注过程中联合阻断细胞凋亡、自噬及程序性坏死三种程序性细胞死亡较单独阻断一种程序性细胞死亡可更明显地减小心梗面积并改善心功能。
Background
The ischemic heart disease is the leading cause of death worldwide, and 3.8 million men and 3.4 million women die of the disease each year. After an acute myocardial infarction, early and successful myocardial reperfusion is the most effective strategy for reducing the size of a myocardial infarct and improving the clinical outcome. However, numerous studies have shown that reperfusion itself may enhance the injury, resulting in extension of infarct size after ischemia (ie, ischemia-reperfusion [IR] injury). Despite optimal myocardial reperfusion, the rate of death after an acute myocardial infarction approaches to 10%, and the incidence of cardiac failure after an acute myocardial infarction is almost 25% of myocardial reperfusion. So,the most difficult problem is how to gain the best efficacy from reperfusion in ischemic myocardium.
Myocardial IR injury causes four types of cardiac dysfunction.①Myocardial stunning:the myocardium usually recovers from this reversible form of injury after several days or weeks.②No-reflow phenomenon:while the recovery of the blood perfusion of the organs, but the perfusion area organizations have emerged the phenomenon of non-reperfusion.③Reperfusion arrhythmias:refers to that the blood flow in acute ischemic myocardium can be restored once again appeared in a few seconds which may lead to a brief acceleration of idioventricular rhythm, ventricular tachycardia, atrioventricular or bundle branch block suddenly disappeared or a transient sinus bradycardia, sinoatrial block, etc. These arrhythmias, especially ventricular arrhythmias, can often lead to patient's hemodynamic change which is one of the causes of death broken and is potentially harmful, but effective treatments are available.④Lethal reperfusion injury:refers to reperfusion leading the survival of cardiac cell to death. Lethal reperfusion injury independently mediated cardiac cell death, and increased the size of myocardial infarction,which was the most serious type in myocardial ischemia/reperfusion injury, so it prompted us to study the myocardial ischemia/reperfusion injury-related cell death.
The programmed cell death is a kind of gene regulation, highly ordered, active cell death process and thus programmed cell death is considered controllable. So, programmed cell death may be inhibited. Therefore, programmed cell death in myocardial ischemia/reperfusion injury attracts everyone's attention.
1. Single-use blocking agents against each of the three kinds of programmed cell death can be significantly reduced infarct area. This shows that all three kinds of programmed cell death in rat myocardium/ischemia-reperfusion injury play an important role. Among them, using z-VAD-fmk can most obviously reduced infarct size of myocardial infarction, indicating that apoptosis may play the most an important role, roles of autophagy and necroptosis have no significant difference.
2. There was no statistical difference of the myocardial infarct area between respectively combined blocking two kinds of programmed cell death and separated blocking apoptosis, but there was a trend to further reduce myocardial infarction size.
3. Joint blocking apoptosis, autophagy and necroptosis can significantly reduce infarct size.
4. Joint blocking apoptosis, autophagy and necroptosis can significantly improve the cardiac function in 48 hours after rat myocardium ischemia/reperfusion, but the remaining blocker group had improved cardiac function trend.
Conclusion:
1. The occurring of autophagy is the earliest of the three types of programmed cell death caused by rat myocardial ischemia/reperfusion injury. In the period of ischemia it increased, and further increased during reperfusion; in the early period of reperfusion, apoptosis occurred. It reached the peak at 1 hour after reperfusion, then began to decline. Necroptosis is the latest, and it happened about 12 hours after reperfusion,24 hours later reached the peak,48 hours after the reperfusion still did not return to the normal.
2.In myocardial ischemia/reperfusion of rats, the inhibition of apoptosis may contribute to autophagy and necroptosis; inhibiting autophagy may promote apoptosis; inhibiting necroptosis might had no significant effect on the other two kinds of programmed cell death.
3. Three kinds of programmed cell death all play important role in rat myocardium/ ischemia-reperfusion injury, and the role of apoptosis may be the most important. The roles of autophagy and necroptosis have no significant difference.
4. Jointing blocking of apoptosis, autophagy and necroptosis can reduce infarct size and improve cardiac function in the rat myocardium/ischemia-reperfusion more effectively than the single blocking of one programmed cell death of them.
心肌梗死是危害人类健康的主要疾病之一,尽早有效地恢复对缺血心肌的血液再灌注,是阻止缺血性心肌损伤的发展、减小心梗面积进而改善临床预后的根本措施。但是,缺血心肌在恢复血液灌注后可产生更为严重的损伤,即缺血／再灌注损伤,使得即使给予及时、适当的再灌注治疗,仍然有10%的心梗患者死亡,25%的心梗患者发生心力衰竭。因此,缺血／再灌注损伤成为阻碍缺血心肌从再灌注治疗中获得最佳疗效的主要难题。
研究已知,心肌细胞死亡是缺血／再灌注损伤导致心梗患者预后不佳的主要原因。关于细胞死亡的分类目前并不统一,但普遍将受基因调控、高度有序、主动的细胞死亡形式称为程序性细胞死亡。因程序性细胞死亡具可调控性,抑制程序性细胞死亡的发生可能做到,故程序性细胞死亡在心肌缺血／再灌注损伤中的作用备受关注。在心肌缺血／再灌注损伤中,程序性细胞死亡的形式有哪些、它们之间的关系如何,已经成为寻找减轻心肌缺血／再灌注损伤机制的切入点之一。
已知的程序性细胞死亡主要有三种形式：凋亡(apoptosis)、自噬(autophagy)与程序性坏死(necroptosis)。凋亡是指为维持内环境稳定,由基因控制的细胞自主的有序的死亡,天冬氨酸特异的半胱氨酸蛋白酶(caspase)在细胞凋亡的执行期发挥关键作用；自噬是亚细胞膜结构发生动态变化并经溶酶体介导对细胞内蛋白质和细胞器降解的过程,通过形成自噬体(autophagosome)降解其底物。在生理条件下,自噬主要的功能是调控长寿蛋白和细胞器的降解利用,参与细胞质的重建、维持细胞质的稳定及清除受损的细胞器,此时自噬是作为细胞存活的机制。如果自噬活性过强,破坏细胞胶质以及细胞器超过了某一界限,就会发生自噬性细胞死亡；程序性坏死是指具有坏死的细胞形态特点和自噬体生成,并且主动耗能的细胞死亡过程。
自噬与程序性坏死都有自噬体形成,但其意义却不相同。由于自噬的过度活动可以导致细胞死亡,所以在自噬过程中出现的自噬体可能与自噬引起的细胞死亡有关；但在程序性坏死过程中出现的自噬体只是一种伴随现象,与程序性坏死引起的细胞死亡无关。
虽然有研究发现,这三种程序性细胞死亡分别参与了心肌缺血／再灌注损伤,但这些研究多数是通过关注其中一种程序性细胞死亡的作用得出结论,而且已有的关于三种程序性细胞死亡形式之间的相互关系的结论,也主要来自细胞培养或其它离体模型,因此并不能很好地分析程序性细胞死亡在心肌缺血／再灌注损伤中的意义。
近年的研究发现,三种程序性细胞死亡在体内并非孤立存在,而是彼此互相联系,互相影响。例如,阻断自噬可以促进细胞凋亡的发生,而阻断细胞凋亡又可促进程序性坏死。已有的研究不能很好地反映和比较每种程序性细胞死亡在心肌缺血／再灌注损伤中的作用大小,相互之间有无关系及关系如何等。若能选用恰当的动物模型,从在体水平阐明这三种程序性细胞死亡各自的发生发展规律,以及三者之间的相互关系,将会为临床筛选治疗的最佳时间点、明确相互之间的关系,最终为临床改善心肌缺血／再灌注损伤治疗措施的制定提供重要的实验依据。
综上所述,本研究拟通过建立大鼠心肌／缺血再灌注模型进行以下研究：1.在同一个动物个体上观察三种程序性死亡在心肌／缺血再灌注过程中各自发生的时间规律；2.运用三种程序性细胞死亡的抑制剂,观察三者在心肌／缺血再灌注过程中作用的相互关系；3.分析三种程序性细胞死亡在心肌／缺血再灌注损伤中的可能的贡献。
第一部分三种程序性细胞死亡在大鼠心肌／缺血再灌注过程中各自发生的时间规律
目的：
本部分实验运用大鼠心肌缺血／再灌注模型,观察三种程序性细胞死亡形式在心肌／缺血再灌注过程中各自的动态变化规律。
材料与方法：
1.动物：
雄性Wistar大鼠,体重：230±10 g。
本实验共使用60只大鼠,死亡12只,实际用于实验研究48只。
2.分组：
(1)缺血组(n=6)：结扎冠状动脉左前降支30 min；
(2)再灌注1h组(n=6)：结扎冠状动脉左前降支30 min,再灌注1h；
(3)再灌注2h组(n=6)：结扎冠状动脉左前降支30 min,再灌注2h；
(4)再灌注3h组(n=6)：结扎冠状动脉左前降支30 min,再灌注3h；
(5)再灌注12h组(n=6)：结扎冠状动脉左前降支30 min,再灌注12h；
(6)再灌注24h组(n=6)：结扎冠状动脉左前降支30 min,再灌注24h：
(7)再灌注48h组(n=6)：结扎冠状动脉左前降支30 min,再灌注48h；
(8)伪手术组(n=6)：手术过程同缺血组但不结扎冠状动脉。
3.方法：
建立大鼠心肌缺血／再灌注模型,留取心脏,以特异性较强的caspase-3活性测定法检测心肌细胞凋亡程度；以Western blot方法检测心肌组织微管相关蛋白轻链3 (MAP LC3-Ⅱ)的蛋白表达,观测自噬体形成,从而反映自噬和程序性死亡程度。
结果：
1.心肌缺血／再灌注期间,大鼠心肌组织caspase-3活性的动态变化规律
Caspase-3是caspase依赖性细胞凋亡通路上的最后执行者,其活性可以反应细胞凋亡水平。
实验结果显示,缺血30min心肌组织Caspase-3活性与伪手术组相比无明显改变；Caspase-3活性在再灌注1h升高至峰值(1.56±0.22vs.1.0±0.21,P<0.01)；之后开始下降,再灌注24h恢复至伪手术组水平(图2)。
2.心肌缺血／再灌注期间,大鼠心肌组织LC3-Ⅱ蛋白表达的动态变化规律
LC3-Ⅱ是观察哺乳动物自噬体水平公认的生物学标记物。
实验结果显示,与伪手术组相比,心肌缺血30min后心肌组织中LC3-Ⅱ的蛋白表达开始增加(P<0.05),再灌注期间(再灌注1h、2h、3h、12h)进一步增加,在再灌注24h达到高峰(P<0.01),随后明显减少,再灌注48h逐渐回复至再灌注3h水平(图3)。
以上结果提示,心肌缺血未导致caspase依赖的心肌细胞凋亡,细胞凋亡主要发生在再灌注期间,再灌注1h达高峰,随后开始下降,再灌注24h降至正常；心肌缺血可导致心肌组织自噬体形成增加,再灌注期间自噬体形成进一步增加,再灌注24h达高峰,之后开始下降,但再灌注48h仍未恢复正常。
小结(一)
1.30min的心肌缺血未导致caspase依赖的心肌组织凋亡,凋亡发生在再灌注期间,而且以再灌注初期为主,再灌注1h即达高峰,随后开始下降,再灌注24h降至正常。
2.30min的心肌缺血可导致心肌组织自噬体形成增加,再灌注期间自噬体形成进一步增加,再灌注24h达高峰,之后开始下降,但再灌注48h仍未恢复正常。
由于自噬与程序性坏死都可以引起自噬体形成,所以在本研究中自噬体的来源、自噬与程序性坏死的变化规律有待进一步研究,将在第二部分实验中进一步阐明。
第二部分三种程序性细胞死亡在大鼠心肌／缺血再灌注过程中的作用和相互关系
目的：
依据第一部分的实验结果,分别给予Caspase阻断剂Z-VAD-fmk、自噬阻断剂三甲基腺嘌呤(3-methyladenine,3-MA)和程序性坏死阻断剂Nec-1后,观察3种程序性细胞死亡的变化,进而分析3种程序性细胞死亡在大鼠心肌／缺血再灌注过程中的作用及其相互关系。
材料与方法：
1.动物：
同第一部分。
本实验共使用动物128只,死亡26只,实际用于实验研究102只。
2.分组：
(1)I30min+DMSO组(n=6)：结扎冠状动脉左前降支30min；结扎前30min静脉注射DMSO(0.1%)0.6m1。
(2)130min+Z-VAD-fmk组(n=6)：结扎冠状动脉左前降支30min；结扎前30min静脉注射Z-VAD-fmk(1mg／kg,溶于DMSO)。
(3)130min+Nec-1组(n=6)：结扎冠状动脉左前降支30min;结扎前30min静脉注射Nec-1(0.6mg／kg,溶于DMSO)。
(4)再灌注1h+DMSO组(n=6)：结扎冠状动脉左前降支30min,再灌注1h；结扎前30min注射DMSO 0.6ml。
(5)再灌注1h+Z-VAD-fmk组(n=6)：结扎冠状动脉左前降支30min,再灌注1h;结扎前30min静脉注射Z-VAD-fmk(1mg／kg,溶于DMSO)。
(6)再灌注1h+3-MA组(n=6)：结扎冠状动脉左前降支30min,再灌注1h;结扎前30min静脉注射3-MA(15mg／kg,溶于DMSO)。
(7)再灌注1h+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注1h；结扎前30min静脉注射Nec-1 (0.6mg/kg,溶于DMSO)。
(8)再灌注3h+DMSO组(n=6)：结扎冠状动脉左前降支30min,再灌注3h；结扎前30min静脉注射DMSO 0.6ml。
(9)再灌注3h+Z-VAD-fmk组(n=6)：结扎冠状动脉左前降支30min,再灌注3h；结扎前30min静脉注射Z-VAD-fink (1mg/kg,溶于DMSO)。
(10)再灌注3h+3-MA组(n=6)：结扎冠状动脉左前降支30min,再灌注3h;结扎前30min静脉注射3-MA(15mg／kg,溶于DMSO)。
(11)再灌注3h+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注3h；结扎前30min静脉注射Nec-1(0.6mg／kg,溶于DMSO)。
(12)再灌注12h+DMSO组(n=6)：结扎冠状动脉左前降支30min,再灌注12h;结扎前30min静脉注射DMSO 0.6ml。
(12)再灌注12h+Z-VAD-fmk组(n=6)：结扎冠状动脉左前降支30min,再灌注12h；结扎前30min,再灌注8h(据Z-VAD-fmk半衰期确定药物追加时间)分别静脉注射Z-VAD-fmk(1mg／kg,溶于DMSO)。
(14)再灌注12h+3-MA组(n=6)：结扎冠状动脉左前降支30min,再灌注12h；结扎前30min静脉注射3-MA (15mg/kg,溶于DMSO).
(15)再灌注12h+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注12h；结扎前30min静脉注射Nec-1 (0.6mg/kg,溶于DMSO)。
(16)再灌注24h+DMSO组(n=6)：结扎冠状动脉左前降支30min,再灌注24h；再灌注前30min静脉注射DMSO 0.6ml。
(17)再灌注24h+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注24h；结扎前30min静脉注射Nec-1(0.6mg／kg,溶于DMSO)。
3.方法：
同第一部分
结果：
第一部分结果显示：LC3-Ⅱ蛋白表达在缺血期已增高,再灌注期间进一步增高,再灌注24h达高峰。故选择缺血30min、再灌注1h、2h、3h、12h和24h观察LC3-Ⅱ蛋白表达。
2.1分别给予Nec-1、3-MA和Z-VAD-fink后,大鼠缺血／再灌注心肌组织中LC3-Ⅱ蛋白表达的变化。
2.1.1给予Nec-1后,大鼠缺血／再灌注心肌组织中LC3-Ⅱ蛋白表达的变化。
给予Nec-1后,与相应时间点DMSO组相比,缺血30min、再灌注1h、2h和3h心肌组织中LC3-Ⅱ表达无明显改变；而再灌注12h和24h心肌组织中LC3-Ⅱ蛋白表达明显减少(12h：1.13±0.15 vs.0.65±0.08,P<0.01；24h：1.415±0.08vs.0.82±0.20,P<0.01)(图4,5,6)。以上结果提示,再灌注3h之前的自噬体形成可能是由自噬引起的,而再灌注12h至24h的自噬体形成可能是由程序性坏死引起,但不能排除自噬也参与12h至24h的自噬体形成。同时也说明抑制程序性坏死可能对自噬无影响。
2.1.2给予3-MA后,观察缺血／再灌注大鼠心肌组织中LC3-Ⅱ蛋白表达变化。
给予3-MA后,与DMSO组相比,再灌注3h、12h心肌组织中LC3-Ⅱ蛋白表达均明显减少(3h：0.61±0.07vs.0.43±0.03,P<0.01；12h:1.125±0.15 vs.0.84±0.04,P<0.05)(图7,8,9)。这一结果提示,3-MA对自噬及程序性坏死的自噬体形成可都有抑制作用,但阻断自噬体的形成是否影响程序性坏死引起的细胞死亡有待进一步研究,将在第三部分进一步探讨。
2.1.3给予z-VAD-fmk后,观察缺血／再灌注心肌组织中LC3-Ⅱ蛋白表达的变化
有研究证实,应用广谱的Caspase抑制剂z-VAD-fmk阻断凋亡后,诱发程序性坏死,导致自噬体生成增多。因此,本实验中,我们在应用z-VAD-fmk后,观察缺血／再灌注过程中不同时间点心肌组织中LC3-Ⅱ的表达情况。根据研究结果2.1.1,选用再灌注3h为观察自噬时间点,再灌注12h观察程序性坏死的时间点。
给予z-VAD-fmk后,观察心肌缺血30min、再灌注3h,12h心肌组织LC3-Ⅱ的蛋白表达。结果显示,与DMSO组相比,给予Z-VAD-fmk后,缺血30min心肌组织LC3-Ⅱ蛋白无改变,而心肌缺血再灌注3h,12h心肌组织中LC3-Ⅱ蛋白表达均明显增加(3h：0.61±0.07 vs.0.74±0.08,P<0.05；12h：1.125±0.15 vs.1.255±0.04,P<0.05)(图10,11,12)。第一部分结果提示缺血30min心肌组织中只有自噬体的改变而无Caspase-3活性的变化,此时给予z-VAD-fink后LC3-Ⅱ蛋白无改变,而再灌注3h、12h Caspase-3活性升高,给予z-VAD-fmk后LC3-Ⅱ蛋白表达增加。提示,z-VAD-fmk本身不影响LC3-Ⅱ蛋白表达,其使得再灌注3h、12h LC3-Ⅱ蛋白表达增加是通过阻断凋亡而起作用。如前结果所述再灌注3h自噬体的生成可能主要来源于自噬,再灌注12h自噬体的生成可能主要来源于程序性坏死,故提示阻断凋亡可能促进自噬与程序性坏死。
2.2分别观察Caspase广谱抑制剂、细胞自噬特异性抑制剂以及程序性坏死特异性抑制剂对大鼠心肌缺血／再灌注后Caspase-3活性的影响
因Caspase-3活性在再灌注1h达高峰,再灌注12h的自噬体形成有程序性坏死参与,故选择再灌注1h观察Z-VAD-fmk及3-MA对caspase-3活性的影响；再灌注12h观察Nec-1对caspase-3活性的影响。
2.2.1给予Caspase广谱抑制剂(Z-VAD-fmk)后,再灌注1h大鼠心肌组织Caspase-3活性降低
与DMSO组相比,给予Z-VAD-fmk后再灌注1h大鼠心肌组织Caspase-3活性明显下降(1.59±0.23vs.1.11±0.03,P<0.01)(图13)。
2.2.2给予细胞自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3-MA)后,再灌注1h大鼠心肌组织Caspase-3活性增加。
与DMSO组相比,给予3-MA后再灌注1h大鼠心肌组织Caspase-3活性显著升高(1.58±0.49vs.1.81±0.06,P<0.01)(图14)。以上结果提示,心肌缺血／再灌注期间,抑制心肌细胞自噬反而会增加心肌细胞凋亡。
2.2.3给予程序性坏死特异性抑制剂(Necrostatin-1, Nec-1)后,再灌注1h大鼠心肌组织Caspase-3活性无影响。
与相应DMSO组相比,给予Nec-1后再灌注1h、12h大鼠心肌组织Caspase-3活性无变化(图15)。这一结果提示,缺血／再灌注期间,抑制细胞程序性坏死对心肌细胞凋亡的影响不明显。
小结(二)
1.在大鼠心肌缺血／再灌注引起的三种程序性死亡中自噬发生的最早,缺血期即增高,再灌注期间进一步升高；之后细胞凋亡开始发生,在再灌注1h达高峰,随后开始下降；程序性坏死发生的最晚,大概在再灌注12h左右发生,24h达高峰,再灌注48h仍未恢复正常。
2.在大鼠心肌缺血／再灌注中抑制凋亡可能促进自噬与程序性坏死,抑制自噬则可能促进凋亡,但抑制程序性坏死可能对另两种程序性死亡无明显影响。
第三部分三种程序性细胞死亡在心肌／缺血再灌注损伤中的贡献
目的：
根据第一、二部分结果的提示,本实验给予三种程序性细胞死亡阻断剂(分别单独使用一种,两两联合应用,以及同时应用三种),检测三种程序性细胞死亡的水平及心梗面积、心功能改变,以观察三种程序性死亡在心肌／缺血再灌注损伤中的可能贡献。
材料与方法：
1.动物：
同第一部分
本实验共使用动物68只,死亡14只,实际用于实验研究54只。
2.分组：
(1) R48h+DMSO组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；结扎前30min静脉注射DMSO 0.6ml。
(2) R48h+Z-VAD-fmk组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；结扎前30min、再灌注8h、16h、24h各静脉注射Z-VAD-fmk(1mg／kg,溶于DMSO)。
(3)R48h+3-MA组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；结扎前30min静脉注射3-MA (15mg/kg,溶于DMSO)。
(4)R48h+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；结扎前30min静脉注射Nec-1 (0.6mg/kg,溶于DMSO)。
(5) R48h+Z-VAD-fmk+3-MA组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；静脉注射Z-VAD-fink如第2组,3-MA如第3组。
(6) R48h+Z-VAD-fmk+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；静脉注射Z-VAD-fmk如第2组,Nec-1如第4组。
(7) R48h+3-MA+Nec-1组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；静脉注射3-MA如第3组,Nec-1如第4组。
(8) R48h+Z-VAD-fmk+Nec-1+3-MA组(n=6)：结扎冠状动脉左前降支30min,再灌注48h；静脉注射Z-VAD-fmk如第2组,3-MA如第3组；Nec-1如如第4组。
(9)伪手术组(n=6)：手术过程同手术组但不结扎冠状动脉。
3.方法：
建立大鼠心肌缺血／再灌注模型,在体监测各项心功能指标、以Evanse blue和TTC复染法检测缺血／再灌注后大鼠心肌梗死面积。
结果：
由第一、二部分结果可知在大鼠心肌缺血／再灌注中,三种程序性细胞死亡发生最晚的程序性坏死于再灌注24h达高峰,随后下降。再灌注48h,三种程序性死亡基本达到稳态,故选择再灌注48h观测在体心功能和心梗面积。
3.1心肌缺血再灌注48h心肌细胞凋亡、细胞自噬以及程序性坏死对心梗面积的影响。
3.1.1观察分别单独应用z-VAD-fmk、3-N(?)A和Nec-1对心梗面积的影响。
经测定,各组大鼠危险区面积与左心室面积比值(AAR/LV)无统计学意义,说明模型建立手术手法一致,排除了由此而造成的误差。
与DMSO组比较,单独给予Z-VAD-fmk后,心梗面积明显缩小(44.08±2.84 vs.28.13±2.45,P<0.01)(图16,17,22,23);单独给予3-MA后,心梗面积明显缩小(44.08±2.84vs.32.23±1.90,P<0.01)(图16,17,22,23)；单独给予Nec-1后,心梗面积明显缩小(44.08±2.84vs.30.07±2.63,P<0.01)(图16,17,22,23)。以上实验结果显示,单独阻断任一种程序性坏死,均可有效地减小心梗面积,提示每一种程序性坏死在缺血／再灌注中均发挥其致损伤作用。
本实验结果还显示,单独给予z-VAD-fmk心梗面积较单独给予3-MA明显缩小(P<0.01)；但单独给予z-VAD和单独给予Nec-1组比,以及单独给予3-MA与单独给予Nec-1相比,心梗面积无统计学差异(P>0.05)。如第二部分结果所示,给予z-VAD-fmk阻断凋亡可促进自噬与程序性坏死,但仍以给予z-VAD-fmk后心梗面积缩小最为明显,这一结果提示,在心肌缺血／再灌注过程中凋亡的致心肌损伤作用较自噬可能更明显一些。三种阻断剂中单独给予3-MA缩小心梗面积的作用最差,一方面可能是由于阻断自噬可能促进凋亡的缘故,另一方面说明阻断自噬体的形成可能并不影响程序性坏死导致的细胞死亡。
3.1.2观察两两联合使用z-VAD-fmk、3-MA和Nec-1对心梗面积的影响。
与DMSO组相比,联合使用z-VAD-fmk与3-MA、联合使用z-VAD-fmk与Nec-1以及联合使用3-MA与Nec-1均使再灌注48h的心梗面积显著减小(z-VAD-fmk+3-MA: 44.08±2.84 vs.25.41±3.04, P<0.01; z-VAD-fmk+Nec-1:44.08±2.84vs.25.833.04, P<0.01; 3-MA+Nec-1:44.08±2.84 vs.26.90±2.90, P＜0.01);但联用两种抑制剂各组心梗面积两两之间比较无统计学差异；而联用两种抑制剂各组心梗面积与单独使用3-MA相比均明显减小(P<0.01),与单独给予Nec-1相比心梗面积均明显缩小(P<0.05)(图18,19,22,23)。
3.1.3观察同时联合使用z-VAD-fmk、3-MA和Nec-1对心梗面积的影响。
联合使用三种抑制剂(z-VAD-fmk+3-MA+Nec-1,23.28±3.10)(图20,21,22,23),与单独使用z-VAD-fmk (P＜0.05)、3-MA (P<0.01)及Nec-1 (P<0.01)相比,心梗面积明显减小；与联用3-MA和Nec-1组相比,心梗面积也明显缩小(P<0.05,图23)。
以上实验结果提示,心肌缺血／再灌注后心梗面积的大小受到心肌细胞凋亡、自噬以及程序性坏死的共同影响。其中心肌细胞凋亡可能是影响心梗面积最主要的因素,而心肌细胞自噬和程序性坏死可能只发挥了相对较小的作用。由于单独阻断一种程序性细胞死亡可能促进另一种程序性细胞死亡,故同时阻断三种程序性细胞死亡可更明显地减小心梗面积。
3.2 z-VAD-fmk、3-MA和Nec-1对心肌缺血再灌注48h心功能的影响。
3.2.1 z-VAD-fmk、3-MA和Nec-1对心肌缺血／再灌注48h+dp/dtmax的影响。
+dp/dtmax为左心室压力上升最大变化速率,可反映在体心脏收缩功能,该指标数值越小,心脏收缩功能越差。
与伪手术组相比,心肌缺血／再灌注48h+DMSO组+dp/dtmax明显降低(878.00±57.12vs.416.00±27.517,P<0.01)；与DMSO组相比,联用三种抑制剂可以明显改善心肌缺血／再灌注48h在体心脏收缩功能(416.00±27.517vs.527.83±12.73,P<0.01)；与DMSO组相比,单独给予Z-VAD-fmk、3-MA以及Nec-1均对心功能损伤的改善不明显,虽有好转趋势,。但无统计学意义(图24,25,26,27)。
3.2.2 z-VAD-fmk、3-MA和Nec-1对心肌缺血再灌注48h-dp/dtmax的影响。
-dp/dtmax为左心室压力下降最大变化速率,可反映在体心脏舒张功能,该指标数值越小,心功能越差。
实验结果显示,与伪手术组相比,心肌缺血／再灌注48h+DMSO组的-dp/dtmax明显降低(744.17±34.61vs.408.00±22.8,P<0.01)。与DMSO组相比,联用三种抑制剂(Z-VAD-fmk +3-MA+Nec-1)可以明显改善心肌缺血再灌注48h在体心脏舒张功能(408.00±22.80vs496.00±36.25,P<0.01)；单独给予Z-VAD-fmk、3-MA以及Nec-1均对心功能损伤的改善影响不明显,虽有好转趋势,但无统计学意义(图28,29,30,31)。
小结(三)
1.单用三种阻断剂均可明显减小心梗面积,提示三种程序性细胞死亡在大鼠心肌／缺血再灌注损伤中均发挥重要作用；其中给予z-VAD-fink缩小心梗面积最为明显,提示在大鼠心肌／缺血再灌注损伤中细胞凋亡的作用可能最大,自噬与程序性坏死的作用大小则无明显区别。
2.分别联合阻断两种程序性死亡后心梗面积较单独阻断自噬或程序性坏死均明显缩小。
3.联合阻断细胞凋亡、自噬及程序性坏死则可明显减小心梗面积。
4.只有联合阻断凋亡、自噬及程序性坏死三种程序性死亡可使大鼠心肌／缺血再灌注后48h心功能明显改善；但两两联合或单独应用一种阻断剂仅有改善心功能损伤的趋势。
结论：
1.在大鼠心肌缺血／再灌注过程中发生的三种程序性细胞死亡中,自噬发生得最早,随后为细胞凋亡的发生,程序性坏死发生得最晚；
2.在大鼠心肌缺血／再灌注过程中,抑制细胞凋亡有可能促进自噬与程序性坏死的发生；抑制自噬可能促进细胞凋亡的发生；抑制程序性坏死则对另两种程序性细胞死亡形式无明显影响;
3.三种程序性细胞死亡在大鼠心肌缺血／再灌注损伤中可能均发挥重要作用,其中细胞凋亡的作用可能最大；
4.大鼠心肌缺血／再灌注过程中联合阻断细胞凋亡、自噬及程序性坏死三种程序性细胞死亡较单独阻断一种程序性细胞死亡可更明显地减小心梗面积并改善心功能。
Background
The ischemic heart disease is the leading cause of death worldwide, and 3.8 million men and 3.4 million women die of the disease each year. After an acute myocardial infarction, early and successful myocardial reperfusion is the most effective strategy for reducing the size of a myocardial infarct and improving the clinical outcome. However, numerous studies have shown that reperfusion itself may enhance the injury, resulting in extension of infarct size after ischemia (ie, ischemia-reperfusion [IR] injury). Despite optimal myocardial reperfusion, the rate of death after an acute myocardial infarction approaches to 10%, and the incidence of cardiac failure after an acute myocardial infarction is almost 25% of myocardial reperfusion. So,the most difficult problem is how to gain the best efficacy from reperfusion in ischemic myocardium.
Myocardial IR injury causes four types of cardiac dysfunction.①Myocardial stunning:the myocardium usually recovers from this reversible form of injury after several days or weeks.②No-reflow phenomenon:while the recovery of the blood perfusion of the organs, but the perfusion area organizations have emerged the phenomenon of non-reperfusion.③Reperfusion arrhythmias:refers to that the blood flow in acute ischemic myocardium can be restored once again appeared in a few seconds which may lead to a brief acceleration of idioventricular rhythm, ventricular tachycardia, atrioventricular or bundle branch block suddenly disappeared or a transient sinus bradycardia, sinoatrial block, etc. These arrhythmias, especially ventricular arrhythmias, can often lead to patient's hemodynamic change which is one of the causes of death broken and is potentially harmful, but effective treatments are available.④Lethal reperfusion injury:refers to reperfusion leading the survival of cardiac cell to death. Lethal reperfusion injury independently mediated cardiac cell death, and increased the size of myocardial infarction,which was the most serious type in myocardial ischemia/reperfusion injury, so it prompted us to study the myocardial ischemia/reperfusion injury-related cell death.
The programmed cell death is a kind of gene regulation, highly ordered, active cell death process and thus programmed cell death is considered controllable. So, programmed cell death may be inhibited. Therefore, programmed cell death in myocardial ischemia/reperfusion injury attracts everyone's attention.
1. Single-use blocking agents against each of the three kinds of programmed cell death can be significantly reduced infarct area. This shows that all three kinds of programmed cell death in rat myocardium/ischemia-reperfusion injury play an important role. Among them, using z-VAD-fmk can most obviously reduced infarct size of myocardial infarction, indicating that apoptosis may play the most an important role, roles of autophagy and necroptosis have no significant difference.
2. There was no statistical difference of the myocardial infarct area between respectively combined blocking two kinds of programmed cell death and separated blocking apoptosis, but there was a trend to further reduce myocardial infarction size.
3. Joint blocking apoptosis, autophagy and necroptosis can significantly reduce infarct size.
4. Joint blocking apoptosis, autophagy and necroptosis can significantly improve the cardiac function in 48 hours after rat myocardium ischemia/reperfusion, but the remaining blocker group had improved cardiac function trend.
Conclusion:
1. The occurring of autophagy is the earliest of the three types of programmed cell death caused by rat myocardial ischemia/reperfusion injury. In the period of ischemia it increased, and further increased during reperfusion; in the early period of reperfusion, apoptosis occurred. It reached the peak at 1 hour after reperfusion, then began to decline. Necroptosis is the latest, and it happened about 12 hours after reperfusion,24 hours later reached the peak,48 hours after the reperfusion still did not return to the normal.
2.In myocardial ischemia/reperfusion of rats, the inhibition of apoptosis may contribute to autophagy and necroptosis; inhibiting autophagy may promote apoptosis; inhibiting necroptosis might had no significant effect on the other two kinds of programmed cell death.
3. Three kinds of programmed cell death all play important role in rat myocardium/ ischemia-reperfusion injury, and the role of apoptosis may be the most important. The roles of autophagy and necroptosis have no significant difference.
4. Jointing blocking of apoptosis, autophagy and necroptosis can reduce infarct size and improve cardiac function in the rat myocardium/ischemia-reperfusion more effectively than the single blocking of one programmed cell death of them.
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