心肌细胞凋亡在机械创伤致继发性心脏损伤中的作用及其可能机制
摘要
研究背景
机械性外伤是一个主要的医疗和经济问题,是一种临床常见的损伤。机械性外伤可以引起出血性休克、组织损伤和循环因子的释放,并且在伤者初步稳定后仍可能继续威胁生命。虽然临床和实验研究已经证实,损伤后器官功能衰竭是损伤晚期死亡的最主要原因,但继发性组织损伤的机理在很大程度上尚不明了。因此,鉴定外伤后器官损伤,探讨相应器官功能失调机理以寻求相应的治疗方案,从而减少一切与外伤相关的继发性器官损伤是非常关键的。
随着院前急救体系的建立和完善,原发性机械损伤已得到有效控制,但继发性损伤,因其容易漏诊,而成为威胁创伤患者的潜在杀手。临床研究表明,一些机械创伤患者在入院观察24h内未发现直接的心脏损伤,却在创伤后数天或数周出现了心肌梗死。但是,机械性创伤导致的这种继发性心脏损伤的具体机制尚不清楚。到目前为止,缺乏理想的动物模型,是制约其深入研究的主要瓶颈之一。
我们在前期研究中,选用Noble-Collip鼓制备了一种能够模拟临床继发心脏损伤的机械创伤模型,发现以40转/min的转速转动5min时,创伤大鼠在24h内心电图、平均动脉压及在体心功能均未发生明显改变,但是创伤24h时离体心脏收缩、舒张功能均显著降低。提示这种创伤模型可能引起继发性心肌损伤,只是在机体神经-体液因素的调节下,仍然能够维持正常功能。但是,这种心肌损伤的病理生理学意义仍然需要进一步的探究。第一,这种模型是能够直接引起创伤后的继发性心脏损伤,还是能够导致心脏组织对不良刺激的敏感性增高并不清楚。其次,导致这种心肌损伤的机制是什么也不清楚。
研究表明,机械创伤时大量产生的自由基(包括活性氧,一氧化氮)和炎性因子,是导致细胞死亡的主要原因。而后者的主要形式有坏死和凋亡两种,一定数量的心肌细胞丢失将导致心脏功能障碍。探讨外伤后心肌细胞丢失的机制将有利于寻找阻止外伤后心肌损伤的最佳治疗方案,并引申到外伤后其他器官损伤的防治。本研究将采用离体、在体实验模型和药理学抑制剂等方法,探寻外伤后心肌细胞丢失的可能机制,从而为寻找防止创伤后多器官功能衰竭的最佳治疗提供实验依据。
第一部分可诱发继发性心脏损伤的大鼠机械创伤模型的制备
目的
观察Noble-Collip鼓制备的机械创伤模型是否会导致继发性心肌损伤,从而为揭示继发性心脏损伤的可能机制提供研究条件,并奠定研究基础。
方法
1.机械创伤模型的制备
按照我们前期的研究选取体重约180g-220g的健康雄性SD大鼠,将其麻醉后放入直径约30.5cm的Noble-Collip鼓中,以40转/min的转速转动5min,大鼠每转动一圈跌落一次,伪创伤组大鼠用胶带黏贴固定于创伤仪中,只随创伤仪转动而不由高处跌落。
2.大鼠心电图(ECG)、平均动脉压(MABP)及在体心功能的检测
将大鼠麻醉、固定、顺序连接心电图电极针后,将连有压力换能器的聚乙烯导管沿右侧颈总动脉插入左心室,采用BL-410生物信号记录分析系统记录大鼠ECG、MABP及大鼠左心室收缩压(Left Ventricular Systolic Pressure,LVSP)、左心室舒张压(Left Ventricular Diastolic Pressure,LVDP)、左心室压力上升和下降最大变化速率(±dP/dTmax)等心功能数据。
3.大鼠离体心功能的检测
将大鼠击昏、迅速开胸取出心脏,于Krebs-Henselei(tK-H液:118mM NaCl, 4.75mM KCl, 1.19mM KH2PO4, 1.19mM MgSO4·7H2O, 2.54mM CaCl2·2H2O, 25mM NaHCO3, 0.5mM EDTA, and 11mM glucose)液中修剪后将主动脉悬挂于Langendorff灌流装置,以K-H液灌流。将起搏电极置于右心室,将一连有压力换能器的乳胶水囊经左心房插入左心室内检测大鼠离体心功能。采用BL-410生物信号记录分析系统记录大鼠LVSP、LVDP、±dP/dTmax等心功能数据。
4.大鼠心肌缺血/再灌注(Ischemia/Reperfusion,I/R)模型的制备
将大鼠麻醉固定后,做颈部正中切口,行气管插管,四肢连接心电监测电极。在心搏最明显处开胸、暴露心脏,打开心包,将6-0带针缝合线于左心耳根部下方2mm处穿过心肌表层,在肺动脉圆锥旁出针,将双线头一同穿入一塑料管,心电图稳定后按压塑料管阻断左冠状动脉前降支血流,心电图Ⅱ导联显示ST段弓背抬高,认为缺血成功。以止血钳固定塑料管,缺血30min后,放松止血钳使冠脉血流恢复再通,实现心肌再灌注,再灌注3h。
5.肌酸激酶同工酶MB(Creatine kinase isoenzyme MB,CK-MB)的检测
采用双抗体夹心ABC-ELISA法检测大鼠血清CK-MB的含量。依次加入待测血清样品、一抗工作液、酶标抗体工作液、底物工作液、终止液后,用酶标仪在450nm处测吸光度值(OD值),以OD值的大小代表大鼠血清CK-MB的含量。
6.统计处理
实验结果以均数±标准差(Mean±SD)表示,两组间均数比较采用两独立样本t检验,多组间均数比较采用单因素方差分析,使用SPSS15.0统计软件对数据进行统计分析。P<0.05认为具有统计学意义。
结果
1.机械创伤大鼠创伤后24h内ECG、MABP未出现明显改变
大鼠在Noble-Collip鼓中以40转/min的转速转动5min后,创伤后24h内各时间点ECG、MABP未出现明显变化,且大鼠内脏未出现明显的出血,24h内生存率为100%(见图3、4、5)。
2.机械创伤大鼠创伤后24h内在体心功能未出现明显改变
分别以+dP/dTmax和-dP/dTmax来反映左室心肌收缩和舒张能力。创伤后24h内各时间点±dP/dTmax分别与伪创伤组相比,均无明显差异(P>0.05)(见图6)。
3.机械创伤大鼠创伤后24h离体心功能降低
与伪创伤组相比,创伤后0h、3h、6h和12h的±dP/dTmax均无明显差异(P>0.05);而创伤后24h的+dP/dTmax和-dP/dTmax均显著降低[+dP/dTmax(:3414±208)mmHg/sec,vs.(4251±168)mmHg/sec,P<0.01;-dP/dTmax:(-3301±458)mmHg/sec vs.(-5221±488)mmHg/sec,P<0.01](见图7)。
以上结果提示以40转/min的转速转动5min所制备的创伤模型可能能够模拟临床创伤后入院观察24h内,未发现造成冠脉夹层等直接的心脏损伤,同时也未发现其它内脏直接受损,却在创伤后数天或数周出现了心脏受损的这类临床损伤情况。
4.机械创伤大鼠创伤后1周在体心功能未出现明显改变
为了观察此模型在创伤后1周(临床容易出现继发性心脏受损的时间点)心功能的变化情况,我们将创伤大鼠喂养1周后观察其±dP/dTmax,结果显示,创伤后1周的+dP/dTmax为(5580±215)mmHg/sec,与伪创伤组+dP/dTmax(5810±460)mmHg/sec相比,无显著性差别(P>0.05)(见图8);创伤后1周-dP/dTmax为(-4390±87)mmHg/sec,与伪创伤组-dP/dTmax(-4735±544)mmHg/sec相比,也无显著性差别(P>0.05)(见图8)。
5.机械创伤大鼠创伤后1周对缺血/再灌注损伤的敏感性增加
为了进一步观察创伤后心肌对缺血/再灌注损伤(一种常见的不良刺激)的敏感性,我们将喂养1周的创伤大鼠进行缺血30min、再灌注3h处理,并观察在体心功能和血清CK-MB的变化。结果显示,与伪创伤缺血/再灌注组相比,创伤缺血/再灌注组+dP/dTmax显著降低([2012±201)mmHg/sec vs(.3663±190)mmHg/sec,P<0.01(]见图9),-dP/dTmax也明显降低[(-1616±230)mmHg/sec vs. -dP/dTmax(-2602±246)mmHg/sec,P<0.01](见图9);此外,与伪创伤缺血/再灌注组相比,创伤缺血/再灌注组大鼠血清CK-MB水平显著升高[(4984±719)ng/ml vs.(2978±506)ng/ml,P<0.01)](见图10)。
小结一
结合临床出现的病例(即“一些机械创伤患者在入院观察24h内未发现直接的心脏损伤,却在创伤后数天或数周出现了心肌梗死”),同时为了符合临床只能对患者进行无创检查的实际情况,我们所制备的创伤模型应该满足:创伤后24h内无创检查(ECG、MABP检测)正常,但却存在隐匿性的心脏损伤,而这极有可能诱发创伤后的继发性心脏损伤。
通过本部分实验,我们观察到利用40转/min的转速转动5min所制备的创伤模型,其创伤后24h内ECG、MABP、在体心功能未出现明显改变,而创伤后24h离体心功能降低,尽管随后1周创伤大鼠在体心功能仍然正常,但对心脏疾病高危因素之一—缺血/再灌注损伤的敏感性却显著增加,这些结果高度提示此模型能够模拟临床创伤后入院观察24h内,未发现造成冠脉夹层等直接的心脏损伤,同时也未发现其它内脏直接受损,却在创伤后出现继发性心脏受损的这类临床损伤情况。
第二部分机械创伤致继发性心脏损伤的可能原因
目的
通过观察创伤后24h内心肌细胞死亡(坏死和/或凋亡)的发生特点,探讨机械创伤致继发性心脏损伤的可能原因。
方法
1.心肌肌钙蛋白I(cTnI)的检测
采用双抗体夹心ABC-ELISA法检测大鼠血清cTnI的含量。依次加入待测血清、一抗工作液、酶标抗体工作液、底物工作液、终止液后,用酶标仪在450nm处测吸光度值(OD值),以OD值的大小代表大鼠血清cTnI的含量。
2. DNA原位末端缺口标记法(TUNEL法)检测心肌组织的细胞凋亡
将心肌组织石蜡切片后,依次脱蜡、封闭、滴加一抗、二抗、TUNEL反应混合溶液及DAPI染液后,于激光共聚焦显微镜下观察,每张切片随机选取10个视野,每个视野计数200个细胞,蓝色代表所有细胞核,绿色代表凋亡细胞核。计算各视野范围内的全部细胞数和凋亡细胞数,凋亡细胞数除以全部细胞数即为凋亡比例,用凋亡指数表示。
3.心肌组织Caspase-3、12活性测定
Caspase-3、12活性使用Caspase-3和Caspase-12荧光检测试剂盒检测。制备心肌组织样品,用BCA蛋白定量试剂盒测定蛋白浓度,将酶标板各样品孔中依次加入样品裂解液、反应缓冲液及底物后,设定酶标仪参数(激发波长400nm,发射波长505nm)后测定吸光度值(OD值),以每孔OD值除以其蛋白浓度后的值代表大鼠心肌组织Caspase-3、12活性。
4.心肌组织Caspase-8、9活性测定
制备心肌组织样品,用BCA蛋白定量试剂盒测定蛋白浓度,将酶标板各样品孔中依次加入反应缓冲液、样品裂解液、双蒸水及底物工作液后,设定酶标仪参数(激发波长400nm,发射波长505nm)后测定吸光度值(OD值),以每孔OD值除以其蛋白浓度后的值代表大鼠心肌组织Caspase-8、9活性。
5.心肌组织蛋白浓度的测定(BCA法)
配制各浓度蛋白标准品及工作液,将酶标板各孔中加入一定体积的工作液和标准品或样品,孵育半小时后在酶标仪512nm下读数,得到吸光度值(OD值)。
6.大鼠在体心功能及大鼠血清肌酸激酶同工酶MB(Creatine kinase isoenzyme MB,CK-MB)的检测方法同第一部分。
7.统计学分析
实验结果以均数±标准差(Mean±SD)表示,两组间均数比较采用两独立样本t检验,多组间均数比较采用单因素方差分析,使用SPSS15.0统计软件对数据进行统计分析。P<0.05认为具有统计学意义。
结果
1.机械创伤未引起明显的大鼠心肌细胞坏死
(1)机械创伤大鼠创伤后24h内血清CK-MB未出现明显改变
CK-MB是反映心肌细胞损伤坏死的一个检测指标。将各个不同时间点的CK-MB含量分别与伪创伤组相比,均无明显差异(P>0.05)(见图11)。
(2)机械创伤大鼠创伤后24h内血清cTnI未出现明显改变nI是近年发展起来的一种高灵敏、高特异性的反映心肌细胞损伤坏死的血清标记物。将各个不同时间点的cTnI含量分别与伪创伤组相比,均无明显差异(P>0.05)(见图12)。
以上结果提示,创伤后24h显著降低的离体心脏收缩、舒张功能可能并非由于心肌细胞坏死所致。
2.机械创伤使大鼠心肌细胞凋亡增加
(1)机械创伤大鼠创伤后24h内心肌细胞凋亡的时间变化规律
采用TUNEL和Caspase-3活性测定两种方法检测心肌细胞凋亡情况。结果显示,与伪创伤组(Sham,0.18%±0.05%)相比,创伤后6h凋亡指数显著增加(6.71%±1.91%,P<0.01);至创伤后12h达最高(14.42%±3.46%,P<0.01 vs. Sham),创伤后24h仍维持在较高水平(8.12%±2.54%,P<0.01 vs. Sham)(见图13); Caspase-3活性也是创伤后6h显著增加(51±4),至创伤后12h达最高(69±4),创伤后24h仍维持在较高水平(46±3),与伪创伤组(16.2±2.0)相比,均具有统计学差异(P<0.01)(见图14)。
(2)给予广谱Caspase抑制剂Z-VAD-FMK对创伤后离体心功能的影响
为了观察心肌细胞凋亡在创伤所致的心功能障碍中的作用,创伤后随即给予广谱Caspase抑制剂Z-VAD-FMK,采用Langendorff离体灌流系统检测大鼠离体心功能,分别以+dP/dTmax和-dP/dTmax来反映左室心肌收缩和舒张能力。结果显示,给予广谱Caspase抑制剂Z-VAD-FMK后,创伤后24h的+dP/dTmax为(4111±189)mmHg/sec,与创伤组+dP/dTmax(3414±208)mmHg/sec相比显著升高(P<0.01);给予Z-VAD-FMK后,创伤后24h的-dP/dTmax为(-4997±351)mmHg/sec,与创伤组-dP/dTma(x-3301±458)mmHg/sec相比也明显升高(P<0.05)(见图15)。
以上结果表明给予广谱Caspase抑制剂Z-VAD-FMK后,创伤后降低的离体心功能恢复了约80%,提示心肌细胞凋亡在机械创伤所致的继发性心脏损伤中发挥着重要作用。
(3)机械创伤大鼠创伤后24h内心肌细胞凋亡发生的可能途径
通过检测Caspase-8、Caspase-9和Caspase-12的活性来探讨机械创伤大鼠创伤后24h内心肌细胞凋亡发生的可能途径。结果显示,与伪创伤组相比,创伤后3h Caspase-12活性显著升高(66±8),6h达到最高(89±16),与伪创伤组(27±10)相比均具有显著性差异(P<0.01),但创伤后12h又显著下降(32±8),与伪创伤组相比无显著差异(P>0.05)(见图18);而Caspase-8活性在创伤后24h明显升高(2312±648),与伪创伤组(1449±296)相比有显著性差异(P<0.01)(见图16);Caspase-9活性也是在创伤后24h明显升高(875±460),与伪创伤组(470±222)相比有显著性差异(P<0.01)(见图17)。
以上结果提示创伤早期首先通过激活Caspase-12,即内质网途径而引起心肌细胞凋亡
cT,随后通过激活Caspase-8(外源性途径)和Caspase-9(内源性途径)引起心肌细胞凋亡。
小结二
1.机械创伤后24h显著降低的离体心脏收缩、舒张功能可能与心肌细胞凋亡有关;
2.机械创伤早期通过激活Caspase-12,即内质网途径而引起心肌细胞凋亡,随后通过激活Caspase-8(外源性途径)和Caspase-9(内源性途径)引起心肌细胞凋亡。
第三部分机械创伤致心肌细胞凋亡的可能机制自由基和炎症反应的作用
目的
1.观察机械创伤时活性氧、一氧化氮(NO)的产生和炎症反应的发生特征;
2.探讨活性氧、NO因素和炎症反应在机械创伤致心肌细胞凋亡中的作用和机制。
方法
1.大鼠心肌组织总NO(NOx)含量的测定(LDH法)
将心肌组织匀浆、离心、取上清,制备硝酸盐标准品后,在酶标板各孔中依次加入双蒸水、样品、缓冲液及NADPH、硝酸还原酶混合物、辅因子溶液、LDH溶液,孵育后再加入Griess试剂R1和R2,在酶标仪540nm或550nm下测定OD值,以OD值的大小代表大鼠心肌组织NOx的含量。
2.大鼠心肌组织超氧阴离子(O_2﹒-)的测定
使用光泽精加强发光法检测心肌组织中O_2﹒-的产生量。将心肌组织放入含有光泽精(0.25mM)的PBS液中,RLU(相对发光单位)用BD公司的Monolight 2010发光计来测定。O_2﹒-用RLU/mg蛋白表示。
3.大鼠心肌组织硝基酪氨酸(NT)含量的检测
采用ELISA方法检测大鼠心肌组织NT含量。将心肌组织匀浆、离心后,用BCA法检测蛋白浓度。用抗硝基酪氨酸单克隆抗体封板后,将标准品或心肌组织样品上清、酶标二抗依次加入酶标板各孔,OPD显色,在酶标仪波长490 nm处测定吸光度值(OD值)。NT含量通过已知浓度硝化牛血清白蛋白标准曲线计算,表达为nmol/g蛋白。
4. Western blot测定一氧化氮合酶(NOS:iNOS,eNOS)含量
将心肌组织匀浆、离心后取上清,用BCA法测定蛋白浓度。配制8%凝胶,上样(上样量iNOS为30μg/孔,eNOS为60μg/孔)、电泳、转膜(半干式)、封闭、经过一抗、二抗反应后,进行化学发光,利用Image-Pro Plus5.0软件进行条带分析。用目的蛋白的灰度值除以内参β-actin的灰度值以校正误差,所得结果代表某样品的目的蛋白相对含量。
5.胸主动脉内皮功能的测定
将制备好的胸主动脉环悬挂于连有张力换能器的器官浴管中,浴管中盛有K-H液(37°C,95% O_2 + 5% CO_2)。调节最适前负荷至1.0g,待血管环稳定后,以去氧肾上腺素诱发血管收缩,分别观察创伤大鼠胸主动脉对内皮依赖性舒血管物质ACh和非内皮依赖性舒血管物质酸化NaNO_2的反应性。以血管对ACh的反应性下降而对酸化NaNO_2的反应性正常作为评判血管内皮功能障碍的标准。
6.心肌组织髓过氧化物酶(MPO)活性检测
采用比色法测定MPO活性。按重量体积比为1:19加匀浆介质制成5%的心肌组织匀浆,按照说明书依次加入各试剂后,用酶标仪于460nm处测定吸光度值(OD值),结果以每孔OD值除以其蛋白浓度后的值代表大鼠心肌组织MPO活性。
7.心肌组织MPO释放和ICAM-1表达的测定(免疫组化方法)
将心肌组织进行石蜡切片、脱蜡、抗原修复、封闭、滴加一抗、二抗及链霉素抗生物素蛋白-过氧化物酶复合物,DAB显色、复染、封固。用此法来检测心肌组织MPO释放量和ICAM-1的表达情况。
8.心肌组织蛋白浓度的测定,心肌组织Caspase-3活性测定及TUNEL等方法同第二部分。
9.统计学分析
实验结果以均数±标准差(Mean±SD)表示,两组间均数比较采用两独立样本t检验,多组间均数比较采用单因素方差分析,使用SPSS15.0统计软件对数据进行统计分析。P<0.05认为具有统计学意义。
用GrahPad Prism4.0对离体动脉血管环结果进行Logistic曲线拟合。
Western-blot结果采用Image-Pro Plus5.0软件进行图像分析。
结果
1.氧自由基和氮自由基在机械创伤致心肌细胞凋亡中的可能作用
(1)大鼠创伤后24h内心肌组织O_2﹒-水平的变化
伪创伤组大鼠心肌组织O_2﹒-含量为(12.7±1.8)RLU/mg组织,机械创伤后6h心肌
组织O_2﹒-含量显著升高为(36.4±3.5)RLU/mg组织,二者相比具有显著性差异(P<0.01)(见图19)。创伤后12h心肌组织O_2﹒-含量仍维持高水平,为(29.6±4.2)RLU/mg组织,与伪创伤组相比有显著性差异(P<0.01)(见图19)。
(2)大鼠创伤后24h内心肌组织NOx含量的变化
伪创伤组大鼠心肌组织NOx含量为(2.12±1.1)μmol/g蛋白,机械创伤后6h心肌组织NOx含量显著升高为(9.5±1.9)μmol/g蛋白,二者相比具有显著性差异(P<0.01)(见图20)。创伤后12h心肌组织NOx含量达最高,为(17.2±2.3)μmol/g蛋白,与伪创伤组相比有显著性差异(P<0.01)(见图20)。
以上结果提示机械创伤可以引起活性氧和NO的产生显著增加。
(3)机械创伤大鼠心肌组织NT含量的改变及其可能的意义
在很多病理情况下,大量生成的NO主要通过与O_2·-结合生成ONOO(-其含量通常用NT水平反映)来发挥毒性作用,为了探讨机械创伤时ONOO-是否也增加,我们检测了创伤后NT的水平。结果发现,大鼠创伤后12h心肌组织NT含量为(12.2±1.1)nmol/g蛋白,与伪创伤组(0.78±0.26)nmol/g蛋白相比,具有显著性差异(P<0.01)(见图21)。
为了探讨升高的ONOO-对心肌组织NOx、NT水平和Caspase-3活性的影响,我们在创伤后随即给予了ONOO-清除剂FeTMPyP。结果发现,给予FeTMPyP后,创伤后12h心肌组织NOx含量为(16.2±2.1)μmol/g蛋白,与创伤组(17.2±2.3)μmol/g蛋白相比无明显差别(P>0.05)(见图22);给予FeTMPyP后,创伤后12h心肌组织NT水平明显降低为(3.2±1.0)nmol/g蛋白,与创伤组(12.2±1.1)nmol/g蛋白相比有显著性差异(P<0.01)(见图23);给予FeTMPyP后,创伤后12h心肌组织Caspase-3活性为(31.2±1.9),与创伤组(68.5±3.9)相比显著降低(P<0.01)(见图24)。此外,我们意外地发现给予FeTMPyP后,创伤后心肌组织NT含量仅下降了约74%,而并未使NT含量完全恢复正常。
上述结果表明机械创伤时ONOO-含量增加,给予ONOO-清除剂FeTMPyP可以部分降低机械创伤所引起的心肌细胞凋亡,提示ONOO-是导致创伤后心肌细胞凋亡的可能原因;此外,由于给予FeTMPyP并未使NT含量完全恢复正常,提示NT升高的原因除了传统认为的由ONOO-升高所致外,可能还存在其它的机制。
(4)机械创伤大鼠心肌组织iNOS和eNOS的表达变化及其可能的意义
为了探讨机械创伤时升高NOx的酶的来源,我们检测了心肌组织iNOS和eNOS的表达。结果发现,大鼠机械创伤后6h心肌组织iNOS表达显著增加,创伤后12h达最高,24h仍维持在较高水平,与伪创伤组相比,均具有显著性差异(P<0.01)(见图25)。而创伤后各时间点eNOS水平与伪创伤组相比,无显著性差别(P>0.05)(见图26)。
为了阐明升高的iNOS对心肌组织NOx、NT水平和Caspase-3活性的影响,随后给予了iNOS抑制剂1400W处理。结果发现,1400W处理可以阻止创伤大鼠心肌组织NOx、NT水平和Caspase-3活性的升高,即给予1400W后,创伤后12h心肌组织NOx水平为(4.5±0.6)μmol/g蛋白;心肌组织NT水平为(6.3±1.5)nmol/g蛋白,Caspase-3活性为(41.7±2.6),分别与创伤组NOx、NT水平和Caspase-3活性相比,均显著降低(P<0.01)(见图27,28,29)。
以上结果提示,机械创伤有可能通过增加iNOS表达,进而引起NOx生成增加,从而导致心肌细胞凋亡。
2.炎症反应在机械创伤致心肌细胞凋亡中的作用
(1)机械创伤时大鼠胸主动脉内皮舒张功能的变化
炎症反应发生时,最先出现的是内皮功能障碍,因此首先观察了创伤后内皮功能的变化。结果发现,给予10~(-9)-10~(-5) mol/L累积浓度的ACh后,创伤组血管最大舒张程度与伪创伤组相比有显著性差异,血管最大舒张程度由85.89%±6.12%降低到61.55%±6.42%(P<0.01,见图30A);而给予10~(-9)-10~(-6) mol/L累积浓度的酸化NaNO_2后,两组间的血管舒张程度无明显差别(P>0.01,见图30B)。这一结果提示机械创伤可以导致大鼠胸主动脉内皮依赖性的舒张功能障碍。
(2)机械创伤大鼠心肌组织ICAM-1表达的变化
ICAM-1是介导炎症时中性粒细胞(PMN)黏附所必需的黏附分子。采用免疫组化方法检测创伤后6h大鼠心肌组织ICAM-1的表达。结果显示,与伪创伤组相比,创伤组心肌组织ICAM-1表达明显增加(见图31)。
(3)机械创伤大鼠心肌组织MPO的活性和释放
PMN是炎症反应的重要效应细胞,激活后可以释放MPO,通常将MPO作为PMN聚集的标志物。我们分别采用比色法和免疫组化方法检测创伤后大鼠心肌组织MPO的活性和释放。结果显示,伪创伤组心肌组织MPO活性为(0.58±0.24)U/g蛋白,创伤后6h心肌组织MPO活性显著升高为(2.91±0.36)U/g蛋白,二者相比具有统计学意义(P<0.01,见图32A)。创伤后12h MPO活性达到最大(4.41±0.72)U/g蛋白,与伪创伤组相比,有统计学意义(P<0.01,见图32A)。同时,与伪创伤组相比,创伤组大鼠创伤后6h心肌组织MPO释放量明显增加(见图32B)。
以上结果提示机械创伤可以引起炎症的基本病理改变,即血管内皮受损,心肌组织细胞粘附分子ICAM-1表达增加,MPO释放增多,活性增强。
(4)给予MPO抑制剂对心肌细胞凋亡指数的影响
为了进一步探讨炎症反应在机械创伤所致的心肌细胞凋亡中是否也发挥着一定的作用,我们给予MPO抑制剂ABAH处理。结果发现,给予MPO抑制剂ABAH后,心肌细胞凋亡程度显著下降为(7.22%±1.24%),与创伤组(14.42%±0.46%)相比有统计学意义(P<0.01)(见图33)。
以上结果提示,MPO抑制剂ABAH可以部分降低机械创伤所引起的心肌细胞凋亡。
(5) MPO参与心肌组织NT水平的增高
已有文献报道,机体除了ONOO-外,还存在非ONOO-蛋白质硝基化(选择性硝化酪氨酸残基)途径,其中最常见的是MPO导致的蛋白质硝基化。为了观察创伤后升高的NT是否与MPO有关,因此给予了MPO抑制剂ABAH处理。结果发现,给予ABAH后,心肌组织NT水平显著下降为(5.15±1.15)nmol/g蛋白,与创伤组(12.2±1.1)nmol/g蛋白相比有统计学意义(P<0.01)(见图34)。
这一结果提示机械创伤时MPO可能通过增加NT水平,进而导致心肌细胞凋亡。
小结三
1.机械创伤可以使心肌组织O2﹒-、NO、ONOO-及iNOS水平显著增高,清除ONOO-及抑制iNOS均可以显著降低机械创伤所引起的心肌细胞凋亡;
2.机械创伤可以导致心脏发生炎症反应,抑制MPO可以显著降低机械创伤所引起的心肌细胞凋亡;
3. ONOO-和MPO均可能通过引起NT增加,进而导致创伤时心肌细胞凋亡。
Background
Mechanical trauma is a major medical and economic problem and a common injury in clinics. It can induce hemorrhagic shock, tissue injury and the release of circulating factors. After initial stabilization in the injured people, mechanical trauma still might be life-threatening. Though it has been proved that organ failure after trauma is a main cause of death in late injury patients, the mechanisms of the secondary tissue injuy is unclear. So it is critical to identify the organ injury after trauma and investigate the possible mechanisms of organ dysfunction, which would be helpful for seeking appropriate treatment to decrease the incidence of the secondary organ injuy associated with trauma.
Due to the establishment and improvement of pre-hospital emergency care systems, the primary injury has been effectively controlled. However, the secondary injuy is becoming a potential killer threatened traumatic patient, because it is easily missed. Recent clinical reports have suggested that mechnical trauma induces myocardial infarction several days after trauma even in the absence of direct heart injury within 24h after trauma. However, the mechanisms responsible for this trauma-induced secondary heart injury have not been identified. So far, the lack of ideal animal models is one of the major problems that limiting deep study on the secondary heart injury.
In our previous studies, we used Noble-Collip drum to establish trauma-induced secondary heart injury. The results showed that Electrocardiograph (ECG), mean arterial blood pressure (MABP) and cardiac function in vivo within 24h after trauma did not change significantly when the rats were subjected to a total of 200 revolutions at a rate of 40r/min, but cardiac function in vitro 24h after trauma obviously deceased. The results suggested that the trauma models directly caused the heart injury, but the heart still maintained normal function under the regulation of nerve and humoral factors. However, the pathophysiological significance of the direct heart injury need to be further explored. Firstly, it is unclear whether the mechanical trauma model could result in secondary heart injury induced by mechanical trauma, or lead to the increased sensitivity of heart tissue to negative stimulus. Secondly, it is uncertain that what the reason that causes the direct heart injury is.
Many studies have shown that the factors led to cell death primaryly mainly include reactive oxygen species (ROS), nitric oxide (NO) and inflammatory factors in mechanical trauma. Necrosis and apoptosis are two major forms of cell death. A certain number of myocardial cell losses will cause cardiac dysfunction. To explore the mechanisms of myocardial cell loss after trauma would benefit for searching for the best treatment to prevent myocardial injury, and be helpful for the prevention and treatment of other organ damage. In the present study, we observed the possible mechanisms of myocardial cell loss after trauma through the use of in vitro and in vivo experimental models and pharmacological inhibitors treatment, which would supply experimental evidence for seeking the best treatment to prevent multiple organ failure after trauma.
SECTION 1
The establishment of mechanical trauma model induced secondary heart injury
Objective:
To observe whether the established mechanical trauma models with Noble-Collip drum could result in secondary heart injury, which would provide research conditions for revealing the possible mechanisms of secondary heart injury.
Methods:
1. The preparation of mechanical trauma model According to our previous studies, adult healthy male Sprague Dawley rats weighing 180g-220g were randomly divided into sham trauma group and trauma group. The rats were anesthetized with chloral hydrate (400 mg/kg, i.p), placed into a Noble-Collip drum (diameter: 30.5cm) and were subjected to a total of 200 revolutions at a rate of 40 rpm. As the wheel was rotated, trauma rats were traumatized, while sham trauma rats were fixed on the drum with tape and could not fall from a height.
2. The detection of rat ECG, MABP and cardiac function in vivo The rats were reanesthetized, fixed and connected with ECG. Then a PE-10 catheter filled with heparinized 0.9% NaCl solution was inserted into the left ventricle along the right carotid artery for recording ECG, MABP, LVSP, LVDP,±dP/dTmax with BL-410 biological signal processing system.
3. The detection of rat cardiac function in vitro The rat was stunned and a midsternal thoracotomy was performed. Then the heart was excised immediately, trimmed in ice-cold Krebs-Henseleit and mounted onto a Langendorff heart perfusion apparatus. The pacing lead was placed into the right ventricle, and a latex balloon was inserted into the left ventricular cavity through the left atrium and connected to a pressure transducer. LVSP, LVDP,±dP/dTmax were recorded with BL-410 biological signal processing system.
4.The establishment of rat myocardial ischemia/reperfusion (I/R) model The rats were reanesthetized and a mid neck incision was performed. After a tracheostomy was carried out, the animals were intubated and connected with ECG. Left thoracotomy was performed and the heart exposed through the fifth intercostal space. The pericardium was incised and a 6-0 silk suture was placed around the proximal portion of the left coronary artery, beneath the left atrial appendage. The ligature ends were passed through a small length of plastic tube to form a snare. For coronary artery occlusion, the snare was pressed onto the surface of the heart directly above the coronary artery and hemostat was applied to the snare. Ischemia was confirmed by the camponotus upward elevation of the ST segment. After 30 min of occlusion, the hemostat was removed and snare released for reperfusion, with the ligature left loose on the surface of the heart. Successful reperfusion was indicated by the restoration of normal rubor. In sham-operated rats, the same procedure was executed, without tightening the snare.
5. The detection of creatine kinase isoenzyme MB (CK-MB) Creatine kinase isoenzyme MB (CK-MB) in rat serum was determined by ABC-ELISA method. After adding serum samples and other reagents according to the manual, set the wavelength of the microplate reader at 450nm to determine the optical density (OD) value. The levels of CK-MB were indicated by OD value of the well.
6. Statistical analysis All calculations and the statistical analysis were performed using the statistical software program SPSS 15.0. All values are presented as means±SEM. Differences between two means were compared by Student’s t test. All of the other values were analyzed by ANOVA followed by a Fisher least significant difference (post hoc) test for multiple comparisons. Probabilities of P<0.05 were considered statistically significant.
Results:
1. There was no obvious change in ECG, MABP within 24h after mechanic trauma When the rats were subjected to a total of 200 revolutions at a rate of 40r/min, there was no
obvious change in ECG, MABP within 24h after mechanic trauma. And there was no obvious bleeding in rat visceral. The 24h survival rate was 100% (Fig. 3, 4, 5).
2. There was no obvious change in cardiac function in vivo within 24h after mechanic trauma
+dP/dT_(max) and -dP/dT_(max) reflect the left ventricular systolic function and diastolic function, respectively. Compared the cardiac function in vivo at all time points in the trauma group with the sham trauma group, there was no significant difference (P>0.05) (Fig. 6).
3. The isolated cardiac function decreased 24h after mechanic trauma in rats
Compared with the sham trauma group, +dP/dT_(max) 24h after trauma was significant decreased [(3414±208) mmHg/sec vs. (4251±168) mmHg/sec, P<0.01], and -dP/dT_(max) was also markedly decreased [(-3301±458) mmHg/sec vs. (-5221±488) mmHg/sec, P<0.01] 24h after trauma (Fig. 7). While compared the isolated cardiac function at other time points in the trauma group with the sham trauma group, there was no significant difference (P>0.05) (Fig. 7).
These results suggested that the established trauma models might be able to simulate mechnical trauma resulted in secondary heart injury.
4. No obvious change was observed in cardiac function in vivo at 1week after mechanic trauma
To further observe the change of cardiac function at 1week after trauma in established traumatic models,±dP/dT_(max) was detected 1week after trauma (the common time point of secondary heart injury in clinic). The results showed that +dP/dT_(max) 1week after trauma was (5580±215) mmHg/sec. Compared with the sham trauma group, (5810±460) mmHg/sec, there was no significant difference (P>0.05). -dP/dT_(max) 1week after trauma was (-4390±87) mmHg/sec. Compared with the sham trauma group, (-4735±544) mmHg/sec, there was also no significant difference (P>0.05) (Fig. 8).
5. Mechanical trauma increased the sensitivity of rat hearts to I/R injury at 1week after trauma
To further observe the susceptibility of the traumatic myocardium to I/R injury, the rats were subjected to I30min/R3h treatment 1week after trauma. And then cardiac function in vivo and the serum CK-MB level were detected. The results showed that compared with the sham traumatic I/R group, +dP/dT_(max) in traumatic I/R group was significant decreased [(2012±201) mmHg/sec vs. (3663±190) mmHg/sec, P<0.01], and -dP/dT_(max) in traumatic I/R group was also obviously decreased [(-1616±230) mmHg/sec vs. (-2602±246) mmHg/sec, P<0.01] (Fig. 9).
Moreover, compared with the sham traumatic I/R group, the levels of serum CK-MB in traumatic I/R group was markedly increased [(4984±719) ng/ml vs. (2978±506) ng/ml, P<0.01)] (Fig. 10).
Summary:
In order to simulate trauma-induced secdondary heart injury and be consistent with the situation that only non-invasive examination can be used on clinic, the established trauma models should meet the following specification: non-invasive examination within 24h after trauma was normal, but there was occult cardiac injury that likely induced post-traumatic secdondary heart injury.
In our previous studies, we found that ECG, MABP and cardiac function in vivo within 24h after trauma did not change significantly when the rats were subjected to a total of 200 revolutions at a rate of 40r/min, but cardiac function in vitro 24h after trauma obviously deceased. In the present study we firstly demonstrated mechanical trauma increased the sensitivity of rat heart to I/R injury 1week after trauma, even though cardiac function in vivo remained nomal. These results strongly suggested that this mechanical trauma models could simulate trauma-induced secdondary heart injury.
SECTION 2
The possible reasons responsible for mechanical trauma-induced secondary heart injury
Objective:
To explore the possible reasons responsible for mechanical trauma-induced secondary heart injury through observation of the post-traumatic necrosis and apoptosis in myocardium within 24h.
Methods:
1. The detection of cardiac troponin I (cTnI) cTnI in rat serum was determined by ABC-ELISA method. After adding serum samples and other reagents according to the manual, set the wavelength of the microplate reader at 450nm to determine the optical density (OD) value. The levels of CK-MB were indicated by OD value of the well.
2. The detection of myocardium apoptosis by terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay Myocardial tissues were embedded in a paraffin block and two slides at 4- to 5-μm thickness were cut from each tissue block. Immunohistochemical procedures for detecting apoptotic cardiomyocytes were performed by using an apoptosis detection kit according to the manufacturer’s instructions. An additional staining was performed with monoclonal anti-αsarcomeric actin. This staining enables the identification of myocytes and, therefore, a distinction between myocyte nuclei and nuclei of other cells in cardiac tissue. After being rinsed with PBS, slides were coverslipped with mounting medium containing DAPI to permit total nuclei counting. Then the tissue slide was observed by laser scanning confocal microscopy (LSCM). 10 fields were selected randomly in each slide, and 200 cells were counted in one field. Total nuclei (DAPI staining, blue) and TUNEL positive nuclei (green) in each field were counted. The index of apoptosis (number of TUNEL-positive nuclei/total number of nuclei) was automatically calculated and exported to Microsoft Excel for further analysis. Results from different fields taken from the same animal were averaged and counted as one sample.
3. The determination of myocardial Caspase-3, 8, 9, 12 activity In brief, after myocardial tissue was homogenized, centrifuged, the supernatants were collected, and protein concentrations were measured by the bicinchoninic acid (BCA) method. To each well of a 96-well plate, supernatant containing 200μg of protein was loaded and incubated with 25μg substrate. AFC was cleaved from DEVD and the free AFC was quantified by using microplate reader with a 400nm excitation filter and 505nm emission filter. The activities of Caspase-3, 8, 9, 12 were indicated by OD value of the well normalized with total protein concentration.
4. The determination of protein concentration in myocardial tissue (BCA method) In brief, after adding the prepared working solutions, protein standards or the myocardial tissue supernatants into a 96-well plate, set the wavelength of the microplate reader at 512nm to determine the optical density (OD) value. The protein concentration of myocardial tissue was indicated by OD value of the well.
5. The detection of creatine kinase isoenzyme MB (CK-MB) and cardiac function in vivo The method was the same as partⅠ.
6. Statistical analysis All calculations and the statistical analysis were performed using the statistical software program SPSS 15.0. All values are presented as means±SEM. Differences between two means were compared by Student’s t test. All of the other values were analyzed by ANOVA followed by Fisher least significant difference (post hoc) test for multiple comparisons. Probabilities of P<0.05 were considered statistically significant.
Results:
1. Mechanical injury did not cause obvious myocardial necrosis in rats
(1) There was no obvious change in serum CK-MB within 24h after mechanic trauma
CK-MB is an index refelected myocardial necrosis. Compared the serum CK-MB at all time points in the trauma group with the sham trauma group, there was no significant difference (P>0.05) (Fig. 11).
(2) No obvious change was observed in serum cTnI within 24h after mechanic trauma
cTnI is a highly sensitive and specific biomarker of myocardial necrosis. Compared the serum cTnI at all time points in the trauma group with the sham trauma group, there was no significant difference (P>0.05) (Fig. 12).
These results suggested that the significantly reduced systolic and diastolic function 24h after trauma in isolated heart might not be the result of myocardial necrosis.
2. Mechanical trauma increased cardiomyocytes apoptosis in rats.
(1) The time course of cardiomyocytes apoptosis within 24h after mechanical trauma in rats
The cardiomyocytes apoptosis was measured by TUNEL and Caspase-3 activity. The results showed that the myocardial apoptosis index in sham trauma group was (0.18%±0.05%), while it significantly increased 6h after trauma (6.71%±1.91%, P<0.01 vs. Sham) (Fig. 13). 12h after trauma it reached the peak, (14.42%±3.46%, P<0.01 vs. Sham), and it still remained at high level (8.12%±2.54%, P<0.01 vs. Sham) 24h after trauma. Caspase-3 activity markedly increased 6h after trauma [(51±4) vs. Sham (16.2±2.0), P<0.01]. 12h after trauma it reached the peak, (69±4, P<0.01 vs. Sham), and it still remained at high level (46±3, P<0.01 vs. Sham) 24h after trauma (Fig. 14).
(2) The effect of Z-VAD-FMK (the broad-spectrum caspase inhibitor) on isolated cardiac function of traumatic rats
In order to observe the role of myocardial apoptosis in isolated cardiac dysfunction induced by mechanical trauma, the trauma rats were treated with Z-VAD-FMK immediately after trauma. Cardiac function of traumatic rats was evaluated with isolated Langendorff heart perfusion. The left ventricular systolic function and diastolic function were assessed by +dP/dT_(max) and -dP/dT_(max), respectively. The results showed that after Z-VAD-FMK treatment, +dP/dT_(max) 24h
after trauma obviously increased compared with the trauma group [(4111±189) mmHg/sec vs. (3414±208) mmHg/sec, P<0.01]. After Z-VAD-FMK treatment, -dP/dT_(max) 24h after trauma was (-4997±351) mmHg/sec. Compared with the trauma group, (-3301±458) mmHg/sec, it was also significantly elevated (P<0.05) (Fig. 15). The results showed that Z-VAD-FMK could markedly improve the reduced cardiac function in vitro induced by trauma, which suggested that myocardial apoptosis played a very important role in the secondary heart injury induced by trauma.
(3) The possible apoptotic pathways in cardiomyocytes within 24h after mechanical trauma
To observe the possible apoptotic pathways in rat cardiomyocytes within 24h after trauma, the activity of Caspase-8, Caspase-9 and Caspase-12 were detected. The results showed that Caspase-12 activity significantly increased 3h after trauma (66±8). Compared with the sham group (27±10), there was significant difference (P<0.01). 6h after trauma it reached the peak, (89±16, P<0.01 vs. Sham) , but 12h after trauma it significant decreased (P>0.05 vs. Sham)(Fig. 18). Caspase-8 activity significantly increased 24h after trauma (2312±648). Compared with the sham group (1449±296), there was significant difference (P<0.01)(Fig. 16). Caspase-9 activity also significantly increased 24h after trauma (875±460). Compared with the sham group (470±222), there was significant difference (P<0.01)(Fig. 17). The results suggested that in the early period of trauma, cardiomyocyte apoptosis was induced by activation the Caspase-12, an endoplasmic reticulum (ER)-specific caspase, following by activation of Caspase-8 (extrinsic pathway) and Caspase-9 (intrinsic pathway).
Summary:
1. The significantly reduced isolated systolic and diastolic function 24h after trauma might be related with cardiomyocyte apoptosis.
2. In the early period of trauma, cardiomyocyte apoptosis was induced by activation the Caspase-12, an endoplasmic reticulum (ER)-specific caspase, following by activation of Caspase-8 (extrinsic pathway) and Caspase-9 (intrinsic pathway).
SECTION 3
The possible mechanisms of cardiomyocyte apoptosis induced by mechanical trauma.—The role of free radicals and inflammation
Objective:
1. To observe the production of reactive oxygen species, NO and the occurrence of inflammatory response after mechanical trauma.
2. To explore the role of reactive oxygen species, NO and inflammation in cardiomyocyte apoptosis induced by mechanical trauma.
Methods:
1. The detection of totla NO (NOx) content in myocardial tissue (LDH method) After myocardial tissue was homogenized, centrifuged, the supernatants were collected. Then nitrate standards were prepared. After adding double distilled water, cardiac tissue supernatants, buffer and NADPH, nitrate reductase mixture, cofactor solution, LDH solution and Griess reagent R1 and R2 into a 96-well plate, set the wavelength of the microplate reader at 540nm or 550nm to determine the optical density (OD) value. The totla NO (NOx) content of myocardial tissue was indicated by OD value of the well.
2. The detection of superoxide anion (O_2﹒-) in myocardial tissue The generation of O_2﹒- in myocardial tissues was detected by lucigenin-enhanced luminescence. In brief, cardiac tissues were placed into PBS solution containing lucigenin (0.25mM). RLU (relative light units) were determined by Monolight 2010 (BD, USA). The amount of O_2﹒- in tissue samples was expressed as RLU per milligram of protein.
3. The determination of nitrotyrosine (NT) content in rat myocardial tissue. Cardiac nitrotyrosine content was determined using an enzyme linked immunosorbent assay (ELISA) method. In brief, after the myocardial tissues were homogenized, centrifuged, the supernatants were collected and protein concentrations were determined by the BCA method. A nitrated protein solution was prepared for use as a standard. These standard samples, along with tissue samples from hearts, were applied to disposable sterile ELISA plates and incubated overnight with primary antibody. The secondary antibody was added, and the peroxidase reaction product was generated by using O-phenylenediamine dihydrochloride (OPD) solution. The optical density was measured at 490 nm with a microplate reader. The amount of nitrotyrosine content in tissue samples was expressed as nanomoles per gram of protein.
4. The determination of nitric oxide synthase (NOS: iNOS, eNOS) expression by Western blot analysis
In brief, after the myocardial tissues were homogenized, centrifuged, the supernatants were collected and protein concentrations were determined by the BCA method. Equal amounts of protein (iNOS: 30μg protein/lane, eNOS: 60μg protein/lane) were electrophoresed on the 8% SDS-polyacrylamide gel and then electrophoretically transferred to a polyvinylidene di?uoride membrane. After blocking with blocking solution, the membrane was incubated with monoclonal antibody against either iNOS or eNOS. Following incubation with secondary antibody, the blot was developed with an ECL chemiluminescent detection kit and visualized with a Kodak Image Station 400. The blot densities were analyzed with Image-Pro Plus5.0 software.
5. The determination of endothelial function of thoracic aorta Briefly, aortic rings were mounted onto hooks, suspended in organ chambers filled with K-H buffer and aerated with 95% O_2 and 5% CO_2 at 37°C, and connected to force transducers to record changes via a Powerlab data acquisition system. After equilibration for 60min at a preload of 1g, the rings were precontracted with norepinephrine. Once a stable contraction was achieved, the rings were exposed to cumulative concentrations of endothelium-dependent vasodilator (acetylcholine, ACh). After the cumulative response stabilized, the rings were washed and allowed to equilibrate to baseline. The procedure was then repeated with an endothelium-independent vasodilator (acidified NaNO_2). A successful denudation of endothelial cells was confirmed by observing a lack of vasorelaxation to ACh, but a normal vasodilatation response to acidified NaNO_2.
6. The determination of myeloperoxidase (MPO) activity in myocardial tissue MPO activity was detected by colorimetric method. Myocardial tissues were homogenized (5% wt/vol) in homogenate media. In accordance with the instructions, once the proper reagents have been added to a 96-well plate, the OD values were recorded at 460 nm using a microplate reader.
7. Immunohistochemical detection of MPO and intercellular adhesion molecule-1 (ICAM-1) Briefly, optimal cutting temperature compound-embedded tissues were cut into 6mm thickness and stained with primary antibody, secondary antibody. MPO was detected with alkaline phosphatase kit and ICAM-1 expression was detected with horseradish-peroxidase kit.
8. The determination of protein concentration, Caspase-3 activity and TUNEL assay in myocardial tissue The method was the same as partⅡ.
9. Statistical analysis
All calculations and the statistical analysis were performed using the statistical software program SPSS 15.0. All values are presented as means±SEM. Differences between two means were compared by Student’s t test. All of the other values were analyzed by ANOVA followed by a Fisher least significant difference (post hoc) test for multiple comparisons. Probabilities of P<0.05 were considered statistically significant. Vascular ring results were analyzed with a non-linear curve fitting by a Logistic equation with GrahPad Prism4.0 software.
Results:
1. The possible role of oxygen free radicals and nitrogen radicals in mechanical trauma-induced myocardial apoptosis
(1) The change of myocardial O_2﹒- levels within 24h after mechanical trauma
Compared with the sham trauma group, the myocardial O_2﹒- levels 6h after trauma obviously increased [(36.4±3.5) RLU/mg tissue vs. (12.7±1.8) RLU/mg tissue, P<0.01] (Fig. 19). 12h after trauma it still remained at high level, (29.6±4.2) RLU/mg tissue. Compared with the sham trauma group, there was significant difference (P<0.01) (Fig. 19).
(2) The change of myocardial NOx content within 24h after mechanical trauma
The myocardial NOx content in sham trauma group was (2.12±1.1)μmol/g protein, while it increased to (9.5±1.9)μmol/g protein 6h after trauma. There was significant difference between the two groups (P<0.01) (Fig. 20). 12h after trauma it reached the peak, (17.2±2.3)μmol/g protein. Compared with the sham trauma group, there was significant difference (P<0.01) (Fig. 20).
These results suggested that mechanical trauma resulted in the overproduction of O_2﹒- and NO.
(3) The change of post-traumatic myocardial NT content and the possible significance
In many pathological situations, NO plays its toxic role by reacting with O_2﹒- to generate peroxynitrite (ONOO~-). ONOO~- levels usually can be expressed by NT content. To investigate whether mechanical trauma could increase ONOO~- content, NT levels were detected by ELISA method. The results showed that the myocardial NT content in sham trauma group was (0.78±0.26) nmol/g protein, while it increased to (12.2±1.1) nmol/g protein 12h after trauma. There was significant difference between the two groups (P<0.01) (Fig. 21).
To explore the effects of ONOO~- on NOx, NT and Caspase-3 activity in myocardial tissues, the trauma rats were treated with FeTMPyP (ONOO~- decomposition catalyst) immediately after trauma. The results showed that treatment with FeTMPyP did not significantly alter the NOx content at 12h after trauma (Fig. 22). However, after FeTMPyP treatment, the myocardial NT content decreased to (3.2±1.0) nmol/g protein. Compared with trauma group (veichle control), there was significant difference (P<0.01) (Fig. 23). In addition, after FeTMPyP treatment, the myocardial Caspase-3 activity significantly decreased to (31.2±1.9). Compared with trauma group (veichle control), there was significant difference (P<0.01) (Fig. 24). Moreover, we accidentally discovered after FeTMPyP treatment, the myocardial NT content after trauma decreased about 70 percent.
These results suggested that mechanical trauma increased ONOO~- content, and FeTMPyP treatment could partially reduced cardiomyocyte apoptosis induced by mechanical trauma. Moreover, the result that FeTMPyP treatment could not completely recover NT content suggested there were other mechanisms that caused the overproduction of NT, in addition to the traditional view that increased ONOO~- caused elevated NT.
(4) The change of post-traumatic myocardial iNOS and eNOS and the possible significance
To investigate the enzyme source of elevated NOx, iNOS and eNOS expression in myocardial tissue were detected by Western blot. The results showed that iNOS expression significantly increased 6h after trauma. Compared with the sham trauma group, there was significant difference (P<0.01) (Fig. 25). At 12h after trauma iNOS expression reached its peak, and it still remained at high level 24h after trauma. The myocafdial eNOS expression at each time point in trauma group had no significant difference compared with trauma group (P>0.05) (Fig. 26).
To elaborate the effects of increased iNOS on NOx, NT and Caspase-3 activity in myocardial tissues, the trauma rats were treated with 1400W (a selective iNOS inhibitor) immediately after trauma. The results showed that administration of 1400W prevented the increase of the myocardial NOx, NT content and Caspase-3 activity. Respectively compared with NOx, NT and Caspase-3 activity of trauma group (veichle control), there was significant difference (P<0.01) (Fig. 27, 28, 29).
These results suggested that mechanical trauma might increase NO production by upregulating iNOS expression, and then induced myocardial apoptosis.
2. The role of inflammation response in mechanical trauma-induced myocardial apoptosis
(1) The change of endothelial function of rat thoracic aorta after trauma
The inflammatory response is triggered by an early endothelial dysfunction. So firsty we observed the endothelial function of traumatized rat. The results showed that the maximal relaxation to cumulative ACh (10~(-9)-10~(-5) mol/L) decreased from 85.89%±6.12% in sham trauma group to 61.55%±6.42% in trauma group (P<0.01) (Fig. 30A). There was no significant difference in acidified NaNO_2-induced maximal relaxation between the two groups (P>0.01, Fig. 30B). The results suggested that mechanical trauma induced endothelial-dependent vasodilation dysfunction.
(2) The change of myocardial ICAM-1 expression of traumatized rat
ICAM-1, as one of the most critical adhesion molecules, plays an important role in polymorphonuclear neutrophils (PMNs) adherence to the endothelium. The expression of ICAM-1 was determined by immunohistochemical staining. The results showed that ICAM-1 expression was markedly upregulated 6h after trauma in myocardial tissue obtained from traumatized rats (Fig. 31).
(3) The activity and release of myocardial MPO in traumatized rat
MPO, an enzyme occurring virtually exclusively in PMNs, was an index that has been shown to correlate closely with PMNs accumulation and infiltration in the heart. The activity and release of myocardial MPO in traumatized rats were detected by colorimetry and immunohistochemistry, respectively. The results showed that the myocardial MPO activity in sham trauma group was (0.58±0.24) U/g protein, while it increased to (2.91±0.36) U/g protein 6h after trauma. There was significant difference between the two groups (P<0.01) (Fig. 32A). 12h after trauma it reached the peak [(4.41±0.72) U/g protein vs. sham, P<0.01] (Fig. 32A). Moreover, compared with the sham trauma, MPO immunostaining was markedly increased in cardiac tissue from rats subjected to mechanical traumatic injury (Fig. 32B).
These results suggested that mechanical trauma resulted in the basic pathologic changes of inflammation, including the damage of vascular endothelial, the increasing of ICAM-1 expression and MPO release, the raising of MPO activity.
(4) The effect of MPO inhibitor on cardiomyocyte apoptosis
To further study the role of inflammation response in myocardial apoptosis induced by trauma, the trauma rats were treated with ABAH (a specific MPO inhibitor) immediately after trauma. The results showed that treatment with ABAH prevented the increase of the myocardial apoptosis index [(7.22%±1.24%) vs. trauma (veichle control, 14.42%±0.46%), P<0.01] (Fig. 33).
The results suggested that ABAH could partially reduced mechanical trauma-induced cardiomyocyte apoptosis.
(5) MPO was involved in the increased myocardial NT content
It has been reported that there is ONOO~- -independent protein nitration pathway besides ONOO~-, in which MPO induced protein nitration is the most common. To observe whether the increased NT was related with MPO, the trauma rats were treated with ABAH. The results showed that after ABAH treatment, the myocardial NT content obviously decreased to (5.15±1.15) nmol/g protein. Compared with the trauma group (veichle control), there was significant difference (P<0.01) (Fig. 34).
The results suggested that MPO might induce cardiomyocyte apoptosis by increasing NT content.
Summary:
1. Mechanical trauma significantly increased the levels of O_2. -, NO, ONOO~- and iNOS in myocardial tissues, and the cleavage of ONOO~- and inhibition of iNOS significantly reduced cardiomyocyte apoptosis induced by mechanical trauma;
2. Mechanical trauma resulted in the occurrence of cardiac inflammation response, and inhibition of MPO also significantly decreased cardiomyocyte apoptosis induced by mechanical trauma;
3. Both ONOO~- and MPO might result in myocardial apoptosis after trauma by increasing NT content.
机械性外伤是一个主要的医疗和经济问题,是一种临床常见的损伤。机械性外伤可以引起出血性休克、组织损伤和循环因子的释放,并且在伤者初步稳定后仍可能继续威胁生命。虽然临床和实验研究已经证实,损伤后器官功能衰竭是损伤晚期死亡的最主要原因,但继发性组织损伤的机理在很大程度上尚不明了。因此,鉴定外伤后器官损伤,探讨相应器官功能失调机理以寻求相应的治疗方案,从而减少一切与外伤相关的继发性器官损伤是非常关键的。
随着院前急救体系的建立和完善,原发性机械损伤已得到有效控制,但继发性损伤,因其容易漏诊,而成为威胁创伤患者的潜在杀手。临床研究表明,一些机械创伤患者在入院观察24h内未发现直接的心脏损伤,却在创伤后数天或数周出现了心肌梗死。但是,机械性创伤导致的这种继发性心脏损伤的具体机制尚不清楚。到目前为止,缺乏理想的动物模型,是制约其深入研究的主要瓶颈之一。
我们在前期研究中,选用Noble-Collip鼓制备了一种能够模拟临床继发心脏损伤的机械创伤模型,发现以40转/min的转速转动5min时,创伤大鼠在24h内心电图、平均动脉压及在体心功能均未发生明显改变,但是创伤24h时离体心脏收缩、舒张功能均显著降低。提示这种创伤模型可能引起继发性心肌损伤,只是在机体神经-体液因素的调节下,仍然能够维持正常功能。但是,这种心肌损伤的病理生理学意义仍然需要进一步的探究。第一,这种模型是能够直接引起创伤后的继发性心脏损伤,还是能够导致心脏组织对不良刺激的敏感性增高并不清楚。其次,导致这种心肌损伤的机制是什么也不清楚。
研究表明,机械创伤时大量产生的自由基(包括活性氧,一氧化氮)和炎性因子,是导致细胞死亡的主要原因。而后者的主要形式有坏死和凋亡两种,一定数量的心肌细胞丢失将导致心脏功能障碍。探讨外伤后心肌细胞丢失的机制将有利于寻找阻止外伤后心肌损伤的最佳治疗方案,并引申到外伤后其他器官损伤的防治。本研究将采用离体、在体实验模型和药理学抑制剂等方法,探寻外伤后心肌细胞丢失的可能机制,从而为寻找防止创伤后多器官功能衰竭的最佳治疗提供实验依据。
第一部分可诱发继发性心脏损伤的大鼠机械创伤模型的制备
目的
观察Noble-Collip鼓制备的机械创伤模型是否会导致继发性心肌损伤,从而为揭示继发性心脏损伤的可能机制提供研究条件,并奠定研究基础。
方法
1.机械创伤模型的制备
按照我们前期的研究选取体重约180g-220g的健康雄性SD大鼠,将其麻醉后放入直径约30.5cm的Noble-Collip鼓中,以40转/min的转速转动5min,大鼠每转动一圈跌落一次,伪创伤组大鼠用胶带黏贴固定于创伤仪中,只随创伤仪转动而不由高处跌落。
2.大鼠心电图(ECG)、平均动脉压(MABP)及在体心功能的检测
将大鼠麻醉、固定、顺序连接心电图电极针后,将连有压力换能器的聚乙烯导管沿右侧颈总动脉插入左心室,采用BL-410生物信号记录分析系统记录大鼠ECG、MABP及大鼠左心室收缩压(Left Ventricular Systolic Pressure,LVSP)、左心室舒张压(Left Ventricular Diastolic Pressure,LVDP)、左心室压力上升和下降最大变化速率(±dP/dTmax)等心功能数据。
3.大鼠离体心功能的检测
将大鼠击昏、迅速开胸取出心脏,于Krebs-Henselei(tK-H液:118mM NaCl, 4.75mM KCl, 1.19mM KH2PO4, 1.19mM MgSO4·7H2O, 2.54mM CaCl2·2H2O, 25mM NaHCO3, 0.5mM EDTA, and 11mM glucose)液中修剪后将主动脉悬挂于Langendorff灌流装置,以K-H液灌流。将起搏电极置于右心室,将一连有压力换能器的乳胶水囊经左心房插入左心室内检测大鼠离体心功能。采用BL-410生物信号记录分析系统记录大鼠LVSP、LVDP、±dP/dTmax等心功能数据。
4.大鼠心肌缺血/再灌注(Ischemia/Reperfusion,I/R)模型的制备
将大鼠麻醉固定后,做颈部正中切口,行气管插管,四肢连接心电监测电极。在心搏最明显处开胸、暴露心脏,打开心包,将6-0带针缝合线于左心耳根部下方2mm处穿过心肌表层,在肺动脉圆锥旁出针,将双线头一同穿入一塑料管,心电图稳定后按压塑料管阻断左冠状动脉前降支血流,心电图Ⅱ导联显示ST段弓背抬高,认为缺血成功。以止血钳固定塑料管,缺血30min后,放松止血钳使冠脉血流恢复再通,实现心肌再灌注,再灌注3h。
5.肌酸激酶同工酶MB(Creatine kinase isoenzyme MB,CK-MB)的检测
采用双抗体夹心ABC-ELISA法检测大鼠血清CK-MB的含量。依次加入待测血清样品、一抗工作液、酶标抗体工作液、底物工作液、终止液后,用酶标仪在450nm处测吸光度值(OD值),以OD值的大小代表大鼠血清CK-MB的含量。
6.统计处理
实验结果以均数±标准差(Mean±SD)表示,两组间均数比较采用两独立样本t检验,多组间均数比较采用单因素方差分析,使用SPSS15.0统计软件对数据进行统计分析。P<0.05认为具有统计学意义。
结果
1.机械创伤大鼠创伤后24h内ECG、MABP未出现明显改变
大鼠在Noble-Collip鼓中以40转/min的转速转动5min后,创伤后24h内各时间点ECG、MABP未出现明显变化,且大鼠内脏未出现明显的出血,24h内生存率为100%(见图3、4、5)。
2.机械创伤大鼠创伤后24h内在体心功能未出现明显改变
分别以+dP/dTmax和-dP/dTmax来反映左室心肌收缩和舒张能力。创伤后24h内各时间点±dP/dTmax分别与伪创伤组相比,均无明显差异(P>0.05)(见图6)。
3.机械创伤大鼠创伤后24h离体心功能降低
与伪创伤组相比,创伤后0h、3h、6h和12h的±dP/dTmax均无明显差异(P>0.05);而创伤后24h的+dP/dTmax和-dP/dTmax均显著降低[+dP/dTmax(:3414±208)mmHg/sec,vs.(4251±168)mmHg/sec,P<0.01;-dP/dTmax:(-3301±458)mmHg/sec vs.(-5221±488)mmHg/sec,P<0.01](见图7)。
以上结果提示以40转/min的转速转动5min所制备的创伤模型可能能够模拟临床创伤后入院观察24h内,未发现造成冠脉夹层等直接的心脏损伤,同时也未发现其它内脏直接受损,却在创伤后数天或数周出现了心脏受损的这类临床损伤情况。
4.机械创伤大鼠创伤后1周在体心功能未出现明显改变
为了观察此模型在创伤后1周(临床容易出现继发性心脏受损的时间点)心功能的变化情况,我们将创伤大鼠喂养1周后观察其±dP/dTmax,结果显示,创伤后1周的+dP/dTmax为(5580±215)mmHg/sec,与伪创伤组+dP/dTmax(5810±460)mmHg/sec相比,无显著性差别(P>0.05)(见图8);创伤后1周-dP/dTmax为(-4390±87)mmHg/sec,与伪创伤组-dP/dTmax(-4735±544)mmHg/sec相比,也无显著性差别(P>0.05)(见图8)。
5.机械创伤大鼠创伤后1周对缺血/再灌注损伤的敏感性增加
为了进一步观察创伤后心肌对缺血/再灌注损伤(一种常见的不良刺激)的敏感性,我们将喂养1周的创伤大鼠进行缺血30min、再灌注3h处理,并观察在体心功能和血清CK-MB的变化。结果显示,与伪创伤缺血/再灌注组相比,创伤缺血/再灌注组+dP/dTmax显著降低([2012±201)mmHg/sec vs(.3663±190)mmHg/sec,P<0.01(]见图9),-dP/dTmax也明显降低[(-1616±230)mmHg/sec vs. -dP/dTmax(-2602±246)mmHg/sec,P<0.01](见图9);此外,与伪创伤缺血/再灌注组相比,创伤缺血/再灌注组大鼠血清CK-MB水平显著升高[(4984±719)ng/ml vs.(2978±506)ng/ml,P<0.01)](见图10)。
小结一
结合临床出现的病例(即“一些机械创伤患者在入院观察24h内未发现直接的心脏损伤,却在创伤后数天或数周出现了心肌梗死”),同时为了符合临床只能对患者进行无创检查的实际情况,我们所制备的创伤模型应该满足:创伤后24h内无创检查(ECG、MABP检测)正常,但却存在隐匿性的心脏损伤,而这极有可能诱发创伤后的继发性心脏损伤。
通过本部分实验,我们观察到利用40转/min的转速转动5min所制备的创伤模型,其创伤后24h内ECG、MABP、在体心功能未出现明显改变,而创伤后24h离体心功能降低,尽管随后1周创伤大鼠在体心功能仍然正常,但对心脏疾病高危因素之一—缺血/再灌注损伤的敏感性却显著增加,这些结果高度提示此模型能够模拟临床创伤后入院观察24h内,未发现造成冠脉夹层等直接的心脏损伤,同时也未发现其它内脏直接受损,却在创伤后出现继发性心脏受损的这类临床损伤情况。
第二部分机械创伤致继发性心脏损伤的可能原因
目的
通过观察创伤后24h内心肌细胞死亡(坏死和/或凋亡)的发生特点,探讨机械创伤致继发性心脏损伤的可能原因。
方法
1.心肌肌钙蛋白I(cTnI)的检测
采用双抗体夹心ABC-ELISA法检测大鼠血清cTnI的含量。依次加入待测血清、一抗工作液、酶标抗体工作液、底物工作液、终止液后,用酶标仪在450nm处测吸光度值(OD值),以OD值的大小代表大鼠血清cTnI的含量。
2. DNA原位末端缺口标记法(TUNEL法)检测心肌组织的细胞凋亡
将心肌组织石蜡切片后,依次脱蜡、封闭、滴加一抗、二抗、TUNEL反应混合溶液及DAPI染液后,于激光共聚焦显微镜下观察,每张切片随机选取10个视野,每个视野计数200个细胞,蓝色代表所有细胞核,绿色代表凋亡细胞核。计算各视野范围内的全部细胞数和凋亡细胞数,凋亡细胞数除以全部细胞数即为凋亡比例,用凋亡指数表示。
3.心肌组织Caspase-3、12活性测定
Caspase-3、12活性使用Caspase-3和Caspase-12荧光检测试剂盒检测。制备心肌组织样品,用BCA蛋白定量试剂盒测定蛋白浓度,将酶标板各样品孔中依次加入样品裂解液、反应缓冲液及底物后,设定酶标仪参数(激发波长400nm,发射波长505nm)后测定吸光度值(OD值),以每孔OD值除以其蛋白浓度后的值代表大鼠心肌组织Caspase-3、12活性。
4.心肌组织Caspase-8、9活性测定
制备心肌组织样品,用BCA蛋白定量试剂盒测定蛋白浓度,将酶标板各样品孔中依次加入反应缓冲液、样品裂解液、双蒸水及底物工作液后,设定酶标仪参数(激发波长400nm,发射波长505nm)后测定吸光度值(OD值),以每孔OD值除以其蛋白浓度后的值代表大鼠心肌组织Caspase-8、9活性。
5.心肌组织蛋白浓度的测定(BCA法)
配制各浓度蛋白标准品及工作液,将酶标板各孔中加入一定体积的工作液和标准品或样品,孵育半小时后在酶标仪512nm下读数,得到吸光度值(OD值)。
6.大鼠在体心功能及大鼠血清肌酸激酶同工酶MB(Creatine kinase isoenzyme MB,CK-MB)的检测方法同第一部分。
7.统计学分析
实验结果以均数±标准差(Mean±SD)表示,两组间均数比较采用两独立样本t检验,多组间均数比较采用单因素方差分析,使用SPSS15.0统计软件对数据进行统计分析。P<0.05认为具有统计学意义。
结果
1.机械创伤未引起明显的大鼠心肌细胞坏死
(1)机械创伤大鼠创伤后24h内血清CK-MB未出现明显改变
CK-MB是反映心肌细胞损伤坏死的一个检测指标。将各个不同时间点的CK-MB含量分别与伪创伤组相比,均无明显差异(P>0.05)(见图11)。
(2)机械创伤大鼠创伤后24h内血清cTnI未出现明显改变nI是近年发展起来的一种高灵敏、高特异性的反映心肌细胞损伤坏死的血清标记物。将各个不同时间点的cTnI含量分别与伪创伤组相比,均无明显差异(P>0.05)(见图12)。
以上结果提示,创伤后24h显著降低的离体心脏收缩、舒张功能可能并非由于心肌细胞坏死所致。
2.机械创伤使大鼠心肌细胞凋亡增加
(1)机械创伤大鼠创伤后24h内心肌细胞凋亡的时间变化规律
采用TUNEL和Caspase-3活性测定两种方法检测心肌细胞凋亡情况。结果显示,与伪创伤组(Sham,0.18%±0.05%)相比,创伤后6h凋亡指数显著增加(6.71%±1.91%,P<0.01);至创伤后12h达最高(14.42%±3.46%,P<0.01 vs. Sham),创伤后24h仍维持在较高水平(8.12%±2.54%,P<0.01 vs. Sham)(见图13); Caspase-3活性也是创伤后6h显著增加(51±4),至创伤后12h达最高(69±4),创伤后24h仍维持在较高水平(46±3),与伪创伤组(16.2±2.0)相比,均具有统计学差异(P<0.01)(见图14)。
(2)给予广谱Caspase抑制剂Z-VAD-FMK对创伤后离体心功能的影响
为了观察心肌细胞凋亡在创伤所致的心功能障碍中的作用,创伤后随即给予广谱Caspase抑制剂Z-VAD-FMK,采用Langendorff离体灌流系统检测大鼠离体心功能,分别以+dP/dTmax和-dP/dTmax来反映左室心肌收缩和舒张能力。结果显示,给予广谱Caspase抑制剂Z-VAD-FMK后,创伤后24h的+dP/dTmax为(4111±189)mmHg/sec,与创伤组+dP/dTmax(3414±208)mmHg/sec相比显著升高(P<0.01);给予Z-VAD-FMK后,创伤后24h的-dP/dTmax为(-4997±351)mmHg/sec,与创伤组-dP/dTma(x-3301±458)mmHg/sec相比也明显升高(P<0.05)(见图15)。
以上结果表明给予广谱Caspase抑制剂Z-VAD-FMK后,创伤后降低的离体心功能恢复了约80%,提示心肌细胞凋亡在机械创伤所致的继发性心脏损伤中发挥着重要作用。
(3)机械创伤大鼠创伤后24h内心肌细胞凋亡发生的可能途径
通过检测Caspase-8、Caspase-9和Caspase-12的活性来探讨机械创伤大鼠创伤后24h内心肌细胞凋亡发生的可能途径。结果显示,与伪创伤组相比,创伤后3h Caspase-12活性显著升高(66±8),6h达到最高(89±16),与伪创伤组(27±10)相比均具有显著性差异(P<0.01),但创伤后12h又显著下降(32±8),与伪创伤组相比无显著差异(P>0.05)(见图18);而Caspase-8活性在创伤后24h明显升高(2312±648),与伪创伤组(1449±296)相比有显著性差异(P<0.01)(见图16);Caspase-9活性也是在创伤后24h明显升高(875±460),与伪创伤组(470±222)相比有显著性差异(P<0.01)(见图17)。
以上结果提示创伤早期首先通过激活Caspase-12,即内质网途径而引起心肌细胞凋亡
cT,随后通过激活Caspase-8(外源性途径)和Caspase-9(内源性途径)引起心肌细胞凋亡。
小结二
1.机械创伤后24h显著降低的离体心脏收缩、舒张功能可能与心肌细胞凋亡有关;
2.机械创伤早期通过激活Caspase-12,即内质网途径而引起心肌细胞凋亡,随后通过激活Caspase-8(外源性途径)和Caspase-9(内源性途径)引起心肌细胞凋亡。
第三部分机械创伤致心肌细胞凋亡的可能机制自由基和炎症反应的作用
目的
1.观察机械创伤时活性氧、一氧化氮(NO)的产生和炎症反应的发生特征;
2.探讨活性氧、NO因素和炎症反应在机械创伤致心肌细胞凋亡中的作用和机制。
方法
1.大鼠心肌组织总NO(NOx)含量的测定(LDH法)
将心肌组织匀浆、离心、取上清,制备硝酸盐标准品后,在酶标板各孔中依次加入双蒸水、样品、缓冲液及NADPH、硝酸还原酶混合物、辅因子溶液、LDH溶液,孵育后再加入Griess试剂R1和R2,在酶标仪540nm或550nm下测定OD值,以OD值的大小代表大鼠心肌组织NOx的含量。
2.大鼠心肌组织超氧阴离子(O_2﹒-)的测定
使用光泽精加强发光法检测心肌组织中O_2﹒-的产生量。将心肌组织放入含有光泽精(0.25mM)的PBS液中,RLU(相对发光单位)用BD公司的Monolight 2010发光计来测定。O_2﹒-用RLU/mg蛋白表示。
3.大鼠心肌组织硝基酪氨酸(NT)含量的检测
采用ELISA方法检测大鼠心肌组织NT含量。将心肌组织匀浆、离心后,用BCA法检测蛋白浓度。用抗硝基酪氨酸单克隆抗体封板后,将标准品或心肌组织样品上清、酶标二抗依次加入酶标板各孔,OPD显色,在酶标仪波长490 nm处测定吸光度值(OD值)。NT含量通过已知浓度硝化牛血清白蛋白标准曲线计算,表达为nmol/g蛋白。
4. Western blot测定一氧化氮合酶(NOS:iNOS,eNOS)含量
将心肌组织匀浆、离心后取上清,用BCA法测定蛋白浓度。配制8%凝胶,上样(上样量iNOS为30μg/孔,eNOS为60μg/孔)、电泳、转膜(半干式)、封闭、经过一抗、二抗反应后,进行化学发光,利用Image-Pro Plus5.0软件进行条带分析。用目的蛋白的灰度值除以内参β-actin的灰度值以校正误差,所得结果代表某样品的目的蛋白相对含量。
5.胸主动脉内皮功能的测定
将制备好的胸主动脉环悬挂于连有张力换能器的器官浴管中,浴管中盛有K-H液(37°C,95% O_2 + 5% CO_2)。调节最适前负荷至1.0g,待血管环稳定后,以去氧肾上腺素诱发血管收缩,分别观察创伤大鼠胸主动脉对内皮依赖性舒血管物质ACh和非内皮依赖性舒血管物质酸化NaNO_2的反应性。以血管对ACh的反应性下降而对酸化NaNO_2的反应性正常作为评判血管内皮功能障碍的标准。
6.心肌组织髓过氧化物酶(MPO)活性检测
采用比色法测定MPO活性。按重量体积比为1:19加匀浆介质制成5%的心肌组织匀浆,按照说明书依次加入各试剂后,用酶标仪于460nm处测定吸光度值(OD值),结果以每孔OD值除以其蛋白浓度后的值代表大鼠心肌组织MPO活性。
7.心肌组织MPO释放和ICAM-1表达的测定(免疫组化方法)
将心肌组织进行石蜡切片、脱蜡、抗原修复、封闭、滴加一抗、二抗及链霉素抗生物素蛋白-过氧化物酶复合物,DAB显色、复染、封固。用此法来检测心肌组织MPO释放量和ICAM-1的表达情况。
8.心肌组织蛋白浓度的测定,心肌组织Caspase-3活性测定及TUNEL等方法同第二部分。
9.统计学分析
实验结果以均数±标准差(Mean±SD)表示,两组间均数比较采用两独立样本t检验,多组间均数比较采用单因素方差分析,使用SPSS15.0统计软件对数据进行统计分析。P<0.05认为具有统计学意义。
用GrahPad Prism4.0对离体动脉血管环结果进行Logistic曲线拟合。
Western-blot结果采用Image-Pro Plus5.0软件进行图像分析。
结果
1.氧自由基和氮自由基在机械创伤致心肌细胞凋亡中的可能作用
(1)大鼠创伤后24h内心肌组织O_2﹒-水平的变化
伪创伤组大鼠心肌组织O_2﹒-含量为(12.7±1.8)RLU/mg组织,机械创伤后6h心肌
组织O_2﹒-含量显著升高为(36.4±3.5)RLU/mg组织,二者相比具有显著性差异(P<0.01)(见图19)。创伤后12h心肌组织O_2﹒-含量仍维持高水平,为(29.6±4.2)RLU/mg组织,与伪创伤组相比有显著性差异(P<0.01)(见图19)。
(2)大鼠创伤后24h内心肌组织NOx含量的变化
伪创伤组大鼠心肌组织NOx含量为(2.12±1.1)μmol/g蛋白,机械创伤后6h心肌组织NOx含量显著升高为(9.5±1.9)μmol/g蛋白,二者相比具有显著性差异(P<0.01)(见图20)。创伤后12h心肌组织NOx含量达最高,为(17.2±2.3)μmol/g蛋白,与伪创伤组相比有显著性差异(P<0.01)(见图20)。
以上结果提示机械创伤可以引起活性氧和NO的产生显著增加。
(3)机械创伤大鼠心肌组织NT含量的改变及其可能的意义
在很多病理情况下,大量生成的NO主要通过与O_2·-结合生成ONOO(-其含量通常用NT水平反映)来发挥毒性作用,为了探讨机械创伤时ONOO-是否也增加,我们检测了创伤后NT的水平。结果发现,大鼠创伤后12h心肌组织NT含量为(12.2±1.1)nmol/g蛋白,与伪创伤组(0.78±0.26)nmol/g蛋白相比,具有显著性差异(P<0.01)(见图21)。
为了探讨升高的ONOO-对心肌组织NOx、NT水平和Caspase-3活性的影响,我们在创伤后随即给予了ONOO-清除剂FeTMPyP。结果发现,给予FeTMPyP后,创伤后12h心肌组织NOx含量为(16.2±2.1)μmol/g蛋白,与创伤组(17.2±2.3)μmol/g蛋白相比无明显差别(P>0.05)(见图22);给予FeTMPyP后,创伤后12h心肌组织NT水平明显降低为(3.2±1.0)nmol/g蛋白,与创伤组(12.2±1.1)nmol/g蛋白相比有显著性差异(P<0.01)(见图23);给予FeTMPyP后,创伤后12h心肌组织Caspase-3活性为(31.2±1.9),与创伤组(68.5±3.9)相比显著降低(P<0.01)(见图24)。此外,我们意外地发现给予FeTMPyP后,创伤后心肌组织NT含量仅下降了约74%,而并未使NT含量完全恢复正常。
上述结果表明机械创伤时ONOO-含量增加,给予ONOO-清除剂FeTMPyP可以部分降低机械创伤所引起的心肌细胞凋亡,提示ONOO-是导致创伤后心肌细胞凋亡的可能原因;此外,由于给予FeTMPyP并未使NT含量完全恢复正常,提示NT升高的原因除了传统认为的由ONOO-升高所致外,可能还存在其它的机制。
(4)机械创伤大鼠心肌组织iNOS和eNOS的表达变化及其可能的意义
为了探讨机械创伤时升高NOx的酶的来源,我们检测了心肌组织iNOS和eNOS的表达。结果发现,大鼠机械创伤后6h心肌组织iNOS表达显著增加,创伤后12h达最高,24h仍维持在较高水平,与伪创伤组相比,均具有显著性差异(P<0.01)(见图25)。而创伤后各时间点eNOS水平与伪创伤组相比,无显著性差别(P>0.05)(见图26)。
为了阐明升高的iNOS对心肌组织NOx、NT水平和Caspase-3活性的影响,随后给予了iNOS抑制剂1400W处理。结果发现,1400W处理可以阻止创伤大鼠心肌组织NOx、NT水平和Caspase-3活性的升高,即给予1400W后,创伤后12h心肌组织NOx水平为(4.5±0.6)μmol/g蛋白;心肌组织NT水平为(6.3±1.5)nmol/g蛋白,Caspase-3活性为(41.7±2.6),分别与创伤组NOx、NT水平和Caspase-3活性相比,均显著降低(P<0.01)(见图27,28,29)。
以上结果提示,机械创伤有可能通过增加iNOS表达,进而引起NOx生成增加,从而导致心肌细胞凋亡。
2.炎症反应在机械创伤致心肌细胞凋亡中的作用
(1)机械创伤时大鼠胸主动脉内皮舒张功能的变化
炎症反应发生时,最先出现的是内皮功能障碍,因此首先观察了创伤后内皮功能的变化。结果发现,给予10~(-9)-10~(-5) mol/L累积浓度的ACh后,创伤组血管最大舒张程度与伪创伤组相比有显著性差异,血管最大舒张程度由85.89%±6.12%降低到61.55%±6.42%(P<0.01,见图30A);而给予10~(-9)-10~(-6) mol/L累积浓度的酸化NaNO_2后,两组间的血管舒张程度无明显差别(P>0.01,见图30B)。这一结果提示机械创伤可以导致大鼠胸主动脉内皮依赖性的舒张功能障碍。
(2)机械创伤大鼠心肌组织ICAM-1表达的变化
ICAM-1是介导炎症时中性粒细胞(PMN)黏附所必需的黏附分子。采用免疫组化方法检测创伤后6h大鼠心肌组织ICAM-1的表达。结果显示,与伪创伤组相比,创伤组心肌组织ICAM-1表达明显增加(见图31)。
(3)机械创伤大鼠心肌组织MPO的活性和释放
PMN是炎症反应的重要效应细胞,激活后可以释放MPO,通常将MPO作为PMN聚集的标志物。我们分别采用比色法和免疫组化方法检测创伤后大鼠心肌组织MPO的活性和释放。结果显示,伪创伤组心肌组织MPO活性为(0.58±0.24)U/g蛋白,创伤后6h心肌组织MPO活性显著升高为(2.91±0.36)U/g蛋白,二者相比具有统计学意义(P<0.01,见图32A)。创伤后12h MPO活性达到最大(4.41±0.72)U/g蛋白,与伪创伤组相比,有统计学意义(P<0.01,见图32A)。同时,与伪创伤组相比,创伤组大鼠创伤后6h心肌组织MPO释放量明显增加(见图32B)。
以上结果提示机械创伤可以引起炎症的基本病理改变,即血管内皮受损,心肌组织细胞粘附分子ICAM-1表达增加,MPO释放增多,活性增强。
(4)给予MPO抑制剂对心肌细胞凋亡指数的影响
为了进一步探讨炎症反应在机械创伤所致的心肌细胞凋亡中是否也发挥着一定的作用,我们给予MPO抑制剂ABAH处理。结果发现,给予MPO抑制剂ABAH后,心肌细胞凋亡程度显著下降为(7.22%±1.24%),与创伤组(14.42%±0.46%)相比有统计学意义(P<0.01)(见图33)。
以上结果提示,MPO抑制剂ABAH可以部分降低机械创伤所引起的心肌细胞凋亡。
(5) MPO参与心肌组织NT水平的增高
已有文献报道,机体除了ONOO-外,还存在非ONOO-蛋白质硝基化(选择性硝化酪氨酸残基)途径,其中最常见的是MPO导致的蛋白质硝基化。为了观察创伤后升高的NT是否与MPO有关,因此给予了MPO抑制剂ABAH处理。结果发现,给予ABAH后,心肌组织NT水平显著下降为(5.15±1.15)nmol/g蛋白,与创伤组(12.2±1.1)nmol/g蛋白相比有统计学意义(P<0.01)(见图34)。
这一结果提示机械创伤时MPO可能通过增加NT水平,进而导致心肌细胞凋亡。
小结三
1.机械创伤可以使心肌组织O2﹒-、NO、ONOO-及iNOS水平显著增高,清除ONOO-及抑制iNOS均可以显著降低机械创伤所引起的心肌细胞凋亡;
2.机械创伤可以导致心脏发生炎症反应,抑制MPO可以显著降低机械创伤所引起的心肌细胞凋亡;
3. ONOO-和MPO均可能通过引起NT增加,进而导致创伤时心肌细胞凋亡。
Background
Mechanical trauma is a major medical and economic problem and a common injury in clinics. It can induce hemorrhagic shock, tissue injury and the release of circulating factors. After initial stabilization in the injured people, mechanical trauma still might be life-threatening. Though it has been proved that organ failure after trauma is a main cause of death in late injury patients, the mechanisms of the secondary tissue injuy is unclear. So it is critical to identify the organ injury after trauma and investigate the possible mechanisms of organ dysfunction, which would be helpful for seeking appropriate treatment to decrease the incidence of the secondary organ injuy associated with trauma.
Due to the establishment and improvement of pre-hospital emergency care systems, the primary injury has been effectively controlled. However, the secondary injuy is becoming a potential killer threatened traumatic patient, because it is easily missed. Recent clinical reports have suggested that mechnical trauma induces myocardial infarction several days after trauma even in the absence of direct heart injury within 24h after trauma. However, the mechanisms responsible for this trauma-induced secondary heart injury have not been identified. So far, the lack of ideal animal models is one of the major problems that limiting deep study on the secondary heart injury.
In our previous studies, we used Noble-Collip drum to establish trauma-induced secondary heart injury. The results showed that Electrocardiograph (ECG), mean arterial blood pressure (MABP) and cardiac function in vivo within 24h after trauma did not change significantly when the rats were subjected to a total of 200 revolutions at a rate of 40r/min, but cardiac function in vitro 24h after trauma obviously deceased. The results suggested that the trauma models directly caused the heart injury, but the heart still maintained normal function under the regulation of nerve and humoral factors. However, the pathophysiological significance of the direct heart injury need to be further explored. Firstly, it is unclear whether the mechanical trauma model could result in secondary heart injury induced by mechanical trauma, or lead to the increased sensitivity of heart tissue to negative stimulus. Secondly, it is uncertain that what the reason that causes the direct heart injury is.
Many studies have shown that the factors led to cell death primaryly mainly include reactive oxygen species (ROS), nitric oxide (NO) and inflammatory factors in mechanical trauma. Necrosis and apoptosis are two major forms of cell death. A certain number of myocardial cell losses will cause cardiac dysfunction. To explore the mechanisms of myocardial cell loss after trauma would benefit for searching for the best treatment to prevent myocardial injury, and be helpful for the prevention and treatment of other organ damage. In the present study, we observed the possible mechanisms of myocardial cell loss after trauma through the use of in vitro and in vivo experimental models and pharmacological inhibitors treatment, which would supply experimental evidence for seeking the best treatment to prevent multiple organ failure after trauma.
SECTION 1
The establishment of mechanical trauma model induced secondary heart injury
Objective:
To observe whether the established mechanical trauma models with Noble-Collip drum could result in secondary heart injury, which would provide research conditions for revealing the possible mechanisms of secondary heart injury.
Methods:
1. The preparation of mechanical trauma model According to our previous studies, adult healthy male Sprague Dawley rats weighing 180g-220g were randomly divided into sham trauma group and trauma group. The rats were anesthetized with chloral hydrate (400 mg/kg, i.p), placed into a Noble-Collip drum (diameter: 30.5cm) and were subjected to a total of 200 revolutions at a rate of 40 rpm. As the wheel was rotated, trauma rats were traumatized, while sham trauma rats were fixed on the drum with tape and could not fall from a height.
2. The detection of rat ECG, MABP and cardiac function in vivo The rats were reanesthetized, fixed and connected with ECG. Then a PE-10 catheter filled with heparinized 0.9% NaCl solution was inserted into the left ventricle along the right carotid artery for recording ECG, MABP, LVSP, LVDP,±dP/dTmax with BL-410 biological signal processing system.
3. The detection of rat cardiac function in vitro The rat was stunned and a midsternal thoracotomy was performed. Then the heart was excised immediately, trimmed in ice-cold Krebs-Henseleit and mounted onto a Langendorff heart perfusion apparatus. The pacing lead was placed into the right ventricle, and a latex balloon was inserted into the left ventricular cavity through the left atrium and connected to a pressure transducer. LVSP, LVDP,±dP/dTmax were recorded with BL-410 biological signal processing system.
4.The establishment of rat myocardial ischemia/reperfusion (I/R) model The rats were reanesthetized and a mid neck incision was performed. After a tracheostomy was carried out, the animals were intubated and connected with ECG. Left thoracotomy was performed and the heart exposed through the fifth intercostal space. The pericardium was incised and a 6-0 silk suture was placed around the proximal portion of the left coronary artery, beneath the left atrial appendage. The ligature ends were passed through a small length of plastic tube to form a snare. For coronary artery occlusion, the snare was pressed onto the surface of the heart directly above the coronary artery and hemostat was applied to the snare. Ischemia was confirmed by the camponotus upward elevation of the ST segment. After 30 min of occlusion, the hemostat was removed and snare released for reperfusion, with the ligature left loose on the surface of the heart. Successful reperfusion was indicated by the restoration of normal rubor. In sham-operated rats, the same procedure was executed, without tightening the snare.
5. The detection of creatine kinase isoenzyme MB (CK-MB) Creatine kinase isoenzyme MB (CK-MB) in rat serum was determined by ABC-ELISA method. After adding serum samples and other reagents according to the manual, set the wavelength of the microplate reader at 450nm to determine the optical density (OD) value. The levels of CK-MB were indicated by OD value of the well.
6. Statistical analysis All calculations and the statistical analysis were performed using the statistical software program SPSS 15.0. All values are presented as means±SEM. Differences between two means were compared by Student’s t test. All of the other values were analyzed by ANOVA followed by a Fisher least significant difference (post hoc) test for multiple comparisons. Probabilities of P<0.05 were considered statistically significant.
Results:
1. There was no obvious change in ECG, MABP within 24h after mechanic trauma When the rats were subjected to a total of 200 revolutions at a rate of 40r/min, there was no
obvious change in ECG, MABP within 24h after mechanic trauma. And there was no obvious bleeding in rat visceral. The 24h survival rate was 100% (Fig. 3, 4, 5).
2. There was no obvious change in cardiac function in vivo within 24h after mechanic trauma
+dP/dT_(max) and -dP/dT_(max) reflect the left ventricular systolic function and diastolic function, respectively. Compared the cardiac function in vivo at all time points in the trauma group with the sham trauma group, there was no significant difference (P>0.05) (Fig. 6).
3. The isolated cardiac function decreased 24h after mechanic trauma in rats
Compared with the sham trauma group, +dP/dT_(max) 24h after trauma was significant decreased [(3414±208) mmHg/sec vs. (4251±168) mmHg/sec, P<0.01], and -dP/dT_(max) was also markedly decreased [(-3301±458) mmHg/sec vs. (-5221±488) mmHg/sec, P<0.01] 24h after trauma (Fig. 7). While compared the isolated cardiac function at other time points in the trauma group with the sham trauma group, there was no significant difference (P>0.05) (Fig. 7).
These results suggested that the established trauma models might be able to simulate mechnical trauma resulted in secondary heart injury.
4. No obvious change was observed in cardiac function in vivo at 1week after mechanic trauma
To further observe the change of cardiac function at 1week after trauma in established traumatic models,±dP/dT_(max) was detected 1week after trauma (the common time point of secondary heart injury in clinic). The results showed that +dP/dT_(max) 1week after trauma was (5580±215) mmHg/sec. Compared with the sham trauma group, (5810±460) mmHg/sec, there was no significant difference (P>0.05). -dP/dT_(max) 1week after trauma was (-4390±87) mmHg/sec. Compared with the sham trauma group, (-4735±544) mmHg/sec, there was also no significant difference (P>0.05) (Fig. 8).
5. Mechanical trauma increased the sensitivity of rat hearts to I/R injury at 1week after trauma
To further observe the susceptibility of the traumatic myocardium to I/R injury, the rats were subjected to I30min/R3h treatment 1week after trauma. And then cardiac function in vivo and the serum CK-MB level were detected. The results showed that compared with the sham traumatic I/R group, +dP/dT_(max) in traumatic I/R group was significant decreased [(2012±201) mmHg/sec vs. (3663±190) mmHg/sec, P<0.01], and -dP/dT_(max) in traumatic I/R group was also obviously decreased [(-1616±230) mmHg/sec vs. (-2602±246) mmHg/sec, P<0.01] (Fig. 9).
Moreover, compared with the sham traumatic I/R group, the levels of serum CK-MB in traumatic I/R group was markedly increased [(4984±719) ng/ml vs. (2978±506) ng/ml, P<0.01)] (Fig. 10).
Summary:
In order to simulate trauma-induced secdondary heart injury and be consistent with the situation that only non-invasive examination can be used on clinic, the established trauma models should meet the following specification: non-invasive examination within 24h after trauma was normal, but there was occult cardiac injury that likely induced post-traumatic secdondary heart injury.
In our previous studies, we found that ECG, MABP and cardiac function in vivo within 24h after trauma did not change significantly when the rats were subjected to a total of 200 revolutions at a rate of 40r/min, but cardiac function in vitro 24h after trauma obviously deceased. In the present study we firstly demonstrated mechanical trauma increased the sensitivity of rat heart to I/R injury 1week after trauma, even though cardiac function in vivo remained nomal. These results strongly suggested that this mechanical trauma models could simulate trauma-induced secdondary heart injury.
SECTION 2
The possible reasons responsible for mechanical trauma-induced secondary heart injury
Objective:
To explore the possible reasons responsible for mechanical trauma-induced secondary heart injury through observation of the post-traumatic necrosis and apoptosis in myocardium within 24h.
Methods:
1. The detection of cardiac troponin I (cTnI) cTnI in rat serum was determined by ABC-ELISA method. After adding serum samples and other reagents according to the manual, set the wavelength of the microplate reader at 450nm to determine the optical density (OD) value. The levels of CK-MB were indicated by OD value of the well.
2. The detection of myocardium apoptosis by terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay Myocardial tissues were embedded in a paraffin block and two slides at 4- to 5-μm thickness were cut from each tissue block. Immunohistochemical procedures for detecting apoptotic cardiomyocytes were performed by using an apoptosis detection kit according to the manufacturer’s instructions. An additional staining was performed with monoclonal anti-αsarcomeric actin. This staining enables the identification of myocytes and, therefore, a distinction between myocyte nuclei and nuclei of other cells in cardiac tissue. After being rinsed with PBS, slides were coverslipped with mounting medium containing DAPI to permit total nuclei counting. Then the tissue slide was observed by laser scanning confocal microscopy (LSCM). 10 fields were selected randomly in each slide, and 200 cells were counted in one field. Total nuclei (DAPI staining, blue) and TUNEL positive nuclei (green) in each field were counted. The index of apoptosis (number of TUNEL-positive nuclei/total number of nuclei) was automatically calculated and exported to Microsoft Excel for further analysis. Results from different fields taken from the same animal were averaged and counted as one sample.
3. The determination of myocardial Caspase-3, 8, 9, 12 activity In brief, after myocardial tissue was homogenized, centrifuged, the supernatants were collected, and protein concentrations were measured by the bicinchoninic acid (BCA) method. To each well of a 96-well plate, supernatant containing 200μg of protein was loaded and incubated with 25μg substrate. AFC was cleaved from DEVD and the free AFC was quantified by using microplate reader with a 400nm excitation filter and 505nm emission filter. The activities of Caspase-3, 8, 9, 12 were indicated by OD value of the well normalized with total protein concentration.
4. The determination of protein concentration in myocardial tissue (BCA method) In brief, after adding the prepared working solutions, protein standards or the myocardial tissue supernatants into a 96-well plate, set the wavelength of the microplate reader at 512nm to determine the optical density (OD) value. The protein concentration of myocardial tissue was indicated by OD value of the well.
5. The detection of creatine kinase isoenzyme MB (CK-MB) and cardiac function in vivo The method was the same as partⅠ.
6. Statistical analysis All calculations and the statistical analysis were performed using the statistical software program SPSS 15.0. All values are presented as means±SEM. Differences between two means were compared by Student’s t test. All of the other values were analyzed by ANOVA followed by Fisher least significant difference (post hoc) test for multiple comparisons. Probabilities of P<0.05 were considered statistically significant.
Results:
1. Mechanical injury did not cause obvious myocardial necrosis in rats
(1) There was no obvious change in serum CK-MB within 24h after mechanic trauma
CK-MB is an index refelected myocardial necrosis. Compared the serum CK-MB at all time points in the trauma group with the sham trauma group, there was no significant difference (P>0.05) (Fig. 11).
(2) No obvious change was observed in serum cTnI within 24h after mechanic trauma
cTnI is a highly sensitive and specific biomarker of myocardial necrosis. Compared the serum cTnI at all time points in the trauma group with the sham trauma group, there was no significant difference (P>0.05) (Fig. 12).
These results suggested that the significantly reduced systolic and diastolic function 24h after trauma in isolated heart might not be the result of myocardial necrosis.
2. Mechanical trauma increased cardiomyocytes apoptosis in rats.
(1) The time course of cardiomyocytes apoptosis within 24h after mechanical trauma in rats
The cardiomyocytes apoptosis was measured by TUNEL and Caspase-3 activity. The results showed that the myocardial apoptosis index in sham trauma group was (0.18%±0.05%), while it significantly increased 6h after trauma (6.71%±1.91%, P<0.01 vs. Sham) (Fig. 13). 12h after trauma it reached the peak, (14.42%±3.46%, P<0.01 vs. Sham), and it still remained at high level (8.12%±2.54%, P<0.01 vs. Sham) 24h after trauma. Caspase-3 activity markedly increased 6h after trauma [(51±4) vs. Sham (16.2±2.0), P<0.01]. 12h after trauma it reached the peak, (69±4, P<0.01 vs. Sham), and it still remained at high level (46±3, P<0.01 vs. Sham) 24h after trauma (Fig. 14).
(2) The effect of Z-VAD-FMK (the broad-spectrum caspase inhibitor) on isolated cardiac function of traumatic rats
In order to observe the role of myocardial apoptosis in isolated cardiac dysfunction induced by mechanical trauma, the trauma rats were treated with Z-VAD-FMK immediately after trauma. Cardiac function of traumatic rats was evaluated with isolated Langendorff heart perfusion. The left ventricular systolic function and diastolic function were assessed by +dP/dT_(max) and -dP/dT_(max), respectively. The results showed that after Z-VAD-FMK treatment, +dP/dT_(max) 24h
after trauma obviously increased compared with the trauma group [(4111±189) mmHg/sec vs. (3414±208) mmHg/sec, P<0.01]. After Z-VAD-FMK treatment, -dP/dT_(max) 24h after trauma was (-4997±351) mmHg/sec. Compared with the trauma group, (-3301±458) mmHg/sec, it was also significantly elevated (P<0.05) (Fig. 15). The results showed that Z-VAD-FMK could markedly improve the reduced cardiac function in vitro induced by trauma, which suggested that myocardial apoptosis played a very important role in the secondary heart injury induced by trauma.
(3) The possible apoptotic pathways in cardiomyocytes within 24h after mechanical trauma
To observe the possible apoptotic pathways in rat cardiomyocytes within 24h after trauma, the activity of Caspase-8, Caspase-9 and Caspase-12 were detected. The results showed that Caspase-12 activity significantly increased 3h after trauma (66±8). Compared with the sham group (27±10), there was significant difference (P<0.01). 6h after trauma it reached the peak, (89±16, P<0.01 vs. Sham) , but 12h after trauma it significant decreased (P>0.05 vs. Sham)(Fig. 18). Caspase-8 activity significantly increased 24h after trauma (2312±648). Compared with the sham group (1449±296), there was significant difference (P<0.01)(Fig. 16). Caspase-9 activity also significantly increased 24h after trauma (875±460). Compared with the sham group (470±222), there was significant difference (P<0.01)(Fig. 17). The results suggested that in the early period of trauma, cardiomyocyte apoptosis was induced by activation the Caspase-12, an endoplasmic reticulum (ER)-specific caspase, following by activation of Caspase-8 (extrinsic pathway) and Caspase-9 (intrinsic pathway).
Summary:
1. The significantly reduced isolated systolic and diastolic function 24h after trauma might be related with cardiomyocyte apoptosis.
2. In the early period of trauma, cardiomyocyte apoptosis was induced by activation the Caspase-12, an endoplasmic reticulum (ER)-specific caspase, following by activation of Caspase-8 (extrinsic pathway) and Caspase-9 (intrinsic pathway).
SECTION 3
The possible mechanisms of cardiomyocyte apoptosis induced by mechanical trauma.—The role of free radicals and inflammation
Objective:
1. To observe the production of reactive oxygen species, NO and the occurrence of inflammatory response after mechanical trauma.
2. To explore the role of reactive oxygen species, NO and inflammation in cardiomyocyte apoptosis induced by mechanical trauma.
Methods:
1. The detection of totla NO (NOx) content in myocardial tissue (LDH method) After myocardial tissue was homogenized, centrifuged, the supernatants were collected. Then nitrate standards were prepared. After adding double distilled water, cardiac tissue supernatants, buffer and NADPH, nitrate reductase mixture, cofactor solution, LDH solution and Griess reagent R1 and R2 into a 96-well plate, set the wavelength of the microplate reader at 540nm or 550nm to determine the optical density (OD) value. The totla NO (NOx) content of myocardial tissue was indicated by OD value of the well.
2. The detection of superoxide anion (O_2﹒-) in myocardial tissue The generation of O_2﹒- in myocardial tissues was detected by lucigenin-enhanced luminescence. In brief, cardiac tissues were placed into PBS solution containing lucigenin (0.25mM). RLU (relative light units) were determined by Monolight 2010 (BD, USA). The amount of O_2﹒- in tissue samples was expressed as RLU per milligram of protein.
3. The determination of nitrotyrosine (NT) content in rat myocardial tissue. Cardiac nitrotyrosine content was determined using an enzyme linked immunosorbent assay (ELISA) method. In brief, after the myocardial tissues were homogenized, centrifuged, the supernatants were collected and protein concentrations were determined by the BCA method. A nitrated protein solution was prepared for use as a standard. These standard samples, along with tissue samples from hearts, were applied to disposable sterile ELISA plates and incubated overnight with primary antibody. The secondary antibody was added, and the peroxidase reaction product was generated by using O-phenylenediamine dihydrochloride (OPD) solution. The optical density was measured at 490 nm with a microplate reader. The amount of nitrotyrosine content in tissue samples was expressed as nanomoles per gram of protein.
4. The determination of nitric oxide synthase (NOS: iNOS, eNOS) expression by Western blot analysis
In brief, after the myocardial tissues were homogenized, centrifuged, the supernatants were collected and protein concentrations were determined by the BCA method. Equal amounts of protein (iNOS: 30μg protein/lane, eNOS: 60μg protein/lane) were electrophoresed on the 8% SDS-polyacrylamide gel and then electrophoretically transferred to a polyvinylidene di?uoride membrane. After blocking with blocking solution, the membrane was incubated with monoclonal antibody against either iNOS or eNOS. Following incubation with secondary antibody, the blot was developed with an ECL chemiluminescent detection kit and visualized with a Kodak Image Station 400. The blot densities were analyzed with Image-Pro Plus5.0 software.
5. The determination of endothelial function of thoracic aorta Briefly, aortic rings were mounted onto hooks, suspended in organ chambers filled with K-H buffer and aerated with 95% O_2 and 5% CO_2 at 37°C, and connected to force transducers to record changes via a Powerlab data acquisition system. After equilibration for 60min at a preload of 1g, the rings were precontracted with norepinephrine. Once a stable contraction was achieved, the rings were exposed to cumulative concentrations of endothelium-dependent vasodilator (acetylcholine, ACh). After the cumulative response stabilized, the rings were washed and allowed to equilibrate to baseline. The procedure was then repeated with an endothelium-independent vasodilator (acidified NaNO_2). A successful denudation of endothelial cells was confirmed by observing a lack of vasorelaxation to ACh, but a normal vasodilatation response to acidified NaNO_2.
6. The determination of myeloperoxidase (MPO) activity in myocardial tissue MPO activity was detected by colorimetric method. Myocardial tissues were homogenized (5% wt/vol) in homogenate media. In accordance with the instructions, once the proper reagents have been added to a 96-well plate, the OD values were recorded at 460 nm using a microplate reader.
7. Immunohistochemical detection of MPO and intercellular adhesion molecule-1 (ICAM-1) Briefly, optimal cutting temperature compound-embedded tissues were cut into 6mm thickness and stained with primary antibody, secondary antibody. MPO was detected with alkaline phosphatase kit and ICAM-1 expression was detected with horseradish-peroxidase kit.
8. The determination of protein concentration, Caspase-3 activity and TUNEL assay in myocardial tissue The method was the same as partⅡ.
9. Statistical analysis
All calculations and the statistical analysis were performed using the statistical software program SPSS 15.0. All values are presented as means±SEM. Differences between two means were compared by Student’s t test. All of the other values were analyzed by ANOVA followed by a Fisher least significant difference (post hoc) test for multiple comparisons. Probabilities of P<0.05 were considered statistically significant. Vascular ring results were analyzed with a non-linear curve fitting by a Logistic equation with GrahPad Prism4.0 software.
Results:
1. The possible role of oxygen free radicals and nitrogen radicals in mechanical trauma-induced myocardial apoptosis
(1) The change of myocardial O_2﹒- levels within 24h after mechanical trauma
Compared with the sham trauma group, the myocardial O_2﹒- levels 6h after trauma obviously increased [(36.4±3.5) RLU/mg tissue vs. (12.7±1.8) RLU/mg tissue, P<0.01] (Fig. 19). 12h after trauma it still remained at high level, (29.6±4.2) RLU/mg tissue. Compared with the sham trauma group, there was significant difference (P<0.01) (Fig. 19).
(2) The change of myocardial NOx content within 24h after mechanical trauma
The myocardial NOx content in sham trauma group was (2.12±1.1)μmol/g protein, while it increased to (9.5±1.9)μmol/g protein 6h after trauma. There was significant difference between the two groups (P<0.01) (Fig. 20). 12h after trauma it reached the peak, (17.2±2.3)μmol/g protein. Compared with the sham trauma group, there was significant difference (P<0.01) (Fig. 20).
These results suggested that mechanical trauma resulted in the overproduction of O_2﹒- and NO.
(3) The change of post-traumatic myocardial NT content and the possible significance
In many pathological situations, NO plays its toxic role by reacting with O_2﹒- to generate peroxynitrite (ONOO~-). ONOO~- levels usually can be expressed by NT content. To investigate whether mechanical trauma could increase ONOO~- content, NT levels were detected by ELISA method. The results showed that the myocardial NT content in sham trauma group was (0.78±0.26) nmol/g protein, while it increased to (12.2±1.1) nmol/g protein 12h after trauma. There was significant difference between the two groups (P<0.01) (Fig. 21).
To explore the effects of ONOO~- on NOx, NT and Caspase-3 activity in myocardial tissues, the trauma rats were treated with FeTMPyP (ONOO~- decomposition catalyst) immediately after trauma. The results showed that treatment with FeTMPyP did not significantly alter the NOx content at 12h after trauma (Fig. 22). However, after FeTMPyP treatment, the myocardial NT content decreased to (3.2±1.0) nmol/g protein. Compared with trauma group (veichle control), there was significant difference (P<0.01) (Fig. 23). In addition, after FeTMPyP treatment, the myocardial Caspase-3 activity significantly decreased to (31.2±1.9). Compared with trauma group (veichle control), there was significant difference (P<0.01) (Fig. 24). Moreover, we accidentally discovered after FeTMPyP treatment, the myocardial NT content after trauma decreased about 70 percent.
These results suggested that mechanical trauma increased ONOO~- content, and FeTMPyP treatment could partially reduced cardiomyocyte apoptosis induced by mechanical trauma. Moreover, the result that FeTMPyP treatment could not completely recover NT content suggested there were other mechanisms that caused the overproduction of NT, in addition to the traditional view that increased ONOO~- caused elevated NT.
(4) The change of post-traumatic myocardial iNOS and eNOS and the possible significance
To investigate the enzyme source of elevated NOx, iNOS and eNOS expression in myocardial tissue were detected by Western blot. The results showed that iNOS expression significantly increased 6h after trauma. Compared with the sham trauma group, there was significant difference (P<0.01) (Fig. 25). At 12h after trauma iNOS expression reached its peak, and it still remained at high level 24h after trauma. The myocafdial eNOS expression at each time point in trauma group had no significant difference compared with trauma group (P>0.05) (Fig. 26).
To elaborate the effects of increased iNOS on NOx, NT and Caspase-3 activity in myocardial tissues, the trauma rats were treated with 1400W (a selective iNOS inhibitor) immediately after trauma. The results showed that administration of 1400W prevented the increase of the myocardial NOx, NT content and Caspase-3 activity. Respectively compared with NOx, NT and Caspase-3 activity of trauma group (veichle control), there was significant difference (P<0.01) (Fig. 27, 28, 29).
These results suggested that mechanical trauma might increase NO production by upregulating iNOS expression, and then induced myocardial apoptosis.
2. The role of inflammation response in mechanical trauma-induced myocardial apoptosis
(1) The change of endothelial function of rat thoracic aorta after trauma
The inflammatory response is triggered by an early endothelial dysfunction. So firsty we observed the endothelial function of traumatized rat. The results showed that the maximal relaxation to cumulative ACh (10~(-9)-10~(-5) mol/L) decreased from 85.89%±6.12% in sham trauma group to 61.55%±6.42% in trauma group (P<0.01) (Fig. 30A). There was no significant difference in acidified NaNO_2-induced maximal relaxation between the two groups (P>0.01, Fig. 30B). The results suggested that mechanical trauma induced endothelial-dependent vasodilation dysfunction.
(2) The change of myocardial ICAM-1 expression of traumatized rat
ICAM-1, as one of the most critical adhesion molecules, plays an important role in polymorphonuclear neutrophils (PMNs) adherence to the endothelium. The expression of ICAM-1 was determined by immunohistochemical staining. The results showed that ICAM-1 expression was markedly upregulated 6h after trauma in myocardial tissue obtained from traumatized rats (Fig. 31).
(3) The activity and release of myocardial MPO in traumatized rat
MPO, an enzyme occurring virtually exclusively in PMNs, was an index that has been shown to correlate closely with PMNs accumulation and infiltration in the heart. The activity and release of myocardial MPO in traumatized rats were detected by colorimetry and immunohistochemistry, respectively. The results showed that the myocardial MPO activity in sham trauma group was (0.58±0.24) U/g protein, while it increased to (2.91±0.36) U/g protein 6h after trauma. There was significant difference between the two groups (P<0.01) (Fig. 32A). 12h after trauma it reached the peak [(4.41±0.72) U/g protein vs. sham, P<0.01] (Fig. 32A). Moreover, compared with the sham trauma, MPO immunostaining was markedly increased in cardiac tissue from rats subjected to mechanical traumatic injury (Fig. 32B).
These results suggested that mechanical trauma resulted in the basic pathologic changes of inflammation, including the damage of vascular endothelial, the increasing of ICAM-1 expression and MPO release, the raising of MPO activity.
(4) The effect of MPO inhibitor on cardiomyocyte apoptosis
To further study the role of inflammation response in myocardial apoptosis induced by trauma, the trauma rats were treated with ABAH (a specific MPO inhibitor) immediately after trauma. The results showed that treatment with ABAH prevented the increase of the myocardial apoptosis index [(7.22%±1.24%) vs. trauma (veichle control, 14.42%±0.46%), P<0.01] (Fig. 33).
The results suggested that ABAH could partially reduced mechanical trauma-induced cardiomyocyte apoptosis.
(5) MPO was involved in the increased myocardial NT content
It has been reported that there is ONOO~- -independent protein nitration pathway besides ONOO~-, in which MPO induced protein nitration is the most common. To observe whether the increased NT was related with MPO, the trauma rats were treated with ABAH. The results showed that after ABAH treatment, the myocardial NT content obviously decreased to (5.15±1.15) nmol/g protein. Compared with the trauma group (veichle control), there was significant difference (P<0.01) (Fig. 34).
The results suggested that MPO might induce cardiomyocyte apoptosis by increasing NT content.
Summary:
1. Mechanical trauma significantly increased the levels of O_2. -, NO, ONOO~- and iNOS in myocardial tissues, and the cleavage of ONOO~- and inhibition of iNOS significantly reduced cardiomyocyte apoptosis induced by mechanical trauma;
2. Mechanical trauma resulted in the occurrence of cardiac inflammation response, and inhibition of MPO also significantly decreased cardiomyocyte apoptosis induced by mechanical trauma;
3. Both ONOO~- and MPO might result in myocardial apoptosis after trauma by increasing NT content.
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