卡维地洛在兔心肌缺血/再灌注损伤中抗炎作用的研究
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摘要
目的:心肌缺血/再灌注损伤(myocardial ischemia/ reperfusion injury, MIRI )是急性冠脉事件治疗过程中的常见现象。多项研究表明心肌缺血/再灌注损伤与中性粒细胞(polymorphonuclear neutrophil leukocytes, PMN)的激活与释放反应密切相关,而细胞因子则参与了PMN粘附、穿越和向损伤区迁移的全部过程。新的第三代β受体阻断剂——卡维地洛(carvedilol, CAR),不仅能阻断α1和β受体,而且有独立于受体阻断作用之外的抗氧化、抗炎等作用。CAR在心力衰竭中的抗炎作用已经得到了多项研究证实,但短期应用能否抑制缺血/再灌注期炎性因子的释放,能否减轻缺血/再灌注过程中PMN介导的炎性反应,国内现有报到尚少。本课题通过观察不同条件下心肌缺血/再灌注兔不同时段血清肿瘤坏死因子-α(Tumor necrosis factor-alpha TNF-α)和心肌组织中髓过氧化物酶(Myeloperoxidase MPO)、丙二醛(Malondialdehyde MDA)的动态变化,研究CAR在兔MIRI中的抗炎作用,探讨其减轻急性心肌损伤的可能机制,为CAR更广泛的临床应用提供理论依据。
材料与方法:健康新西兰白兔48只,体重2.5～3.0 kg,雌雄不拘,随机分为四组:(1)缺血再灌注组(IR组,n=12,生理盐水5ml/d灌胃1w):结扎左室支造成心肌缺血,然后按缺血40分钟、再灌40分钟、再灌120分钟三种处理方法进一步分成三个亚组,每个亚组包括成功的动物模型4只;(2)卡维地洛预处理组(CAR组,n=12,CAR 3mg/kg/d,灌胃1w):手术操作和亚组分法同IR组;(3)缺血预处理组(IPC组,n=12,生理盐水5ml/d灌胃1w):结扎左室支,缺血5分钟,再灌注5分钟,反复3次[1],然后按上述亚组要求行缺血40分钟和开放处理,最终得到缺血40分钟、再灌40分钟、再灌120分钟三个亚组;(4)假手术组(Sham组,n=12,生理盐水5ml/d灌胃1w):只于左室支处穿线而不结扎,进一步按IR组和CAR组相应时间点分成三个亚组。
成功的动物模型达实验终点后取材,分别测定血清中TNF-α和心肌组织中MDA、MPO的含量,行缺血区心肌组织HE染色,光镜下观察心肌细胞形态变化及PMN浸润情况。所有实验数据均用SPSS11.5软件包进行统计学处理,以P<0.05为有统计学差异, P<0.01为有显著统计学差异。
结果:
1心肌匀浆MDA含量在缺血/再灌注中的变化各实验组的MDA含量均随时间延长而递增。Sham组各时间段MDA含量的变化无统计学差异。IR组、CAR组与IPC组各时间段MDA的含量分别为:[(4.79±0.12)vs (9.01±0.51 )vs(22.56±1.59)]nmol/mgprot; [(4.69±0.12)vs (6.41±0.54)vs (13.92±0.53)]nmol/mgprot; ([4.71±0.12)vs(6.96±0.45)vs (14.90±0.65)] nmol/mgprot,差异均有显著统计学意义(F值分别为370.29,491.98,541.60, p值均<0.01)。同一时间段的四组间比较:IR组的MDA含量均高于Sham组、Car组和IPC组,其中再灌注40分钟和再灌注120分钟时的差异有显著统计学意义(F值分别为59.07,257.80, p值均<0.01)。再灌注40分钟和再灌注120分钟时,CAR组和IPC组的MDA含量均高于Sham组,显著有统计学意义(p值均<0.01),但IPC组和CAR组心肌组织中MDA含量的差异无统计学意义(p值分别为0.13, 0.25)。
2心肌匀浆MPO含量在缺血/再灌注中的变化
各实验组的MPO含量均随时间延长而递增。Sham组各时间段MPO含量的变化无统计学差异。IR组、CAR组与IPC组各时间段MPO的含量分别为:([2.60±0.33)vs (5.15±0.85 )vs(8.63±0.25)] mU/mgprot; [(2.54±0.19)vs (4.17±0.78)vs (6.66±0.19)]mU/mgprot; [(2.59±0.30)vs(3.80±0.23)vs (6.94±0.22)] mU/mgprot,差异均有显著统计学意义(F值分别为498.00,572.71,313.52, p值均<0.01)。同一时间段的四组比较:IR组的MPO含量均高于Sham组、CAR组和IPC组,其中再灌注40分钟和再灌注120分钟时的差异有显著统计学意义(F值分别为13.49, 540.19, p值均<0.01)。再灌注40分钟和再灌注120分钟时, CAR组和IPC组的MPO含量均高于Sham组,有显著统计学意义(p值均<0.01),但IPC组和CAR组MPO含量的差异无统计学意义(p值分别为0.39, 0.10)。
3血清TNF-α在缺血/再灌注中的变化
各实验组的TNF-α含量均随时间延长而递增。Sham组各时间段TNF-α含量的变化无统计学差异。IR组、CAR组与IPC组各时间段TNF-α的含量分别为:[(12.47±0.99)vs (23.01±0.82 )vs(45.18±1.09)] pg/ml; [(11.14±0.97)vs (15.34±1.15)vs (33.88±0.99)]pg/ml; [(11.64±0.64)vs(16.77±0.66)vs (35.30±0.95)] pg/ml,差异均有显著统计学意义(F值分别为1177.67,540.28,1063.94, p值均<0.01)。同一时间段的四组比较:IR组的TNF-α含量均高于Sham组、CAR组和IPC组,其中再灌注40分钟和再灌注120分钟时的差异有显著统计学意义(F值分别为101.83,795.40, p值均<0.01)。再灌注40分钟和再灌注120分钟时,CAR组和IPC组的TNF-α含量均高于Sham组,有显著统计学意义(p值均<0.01),但IPC组和CAR组TNF-a含量的差异无统计学意义(p值分别为0.06, 0.07)。
4各实验组的病理变化特点
正常心肌组织:心肌纤维排列整齐,细胞边界完整,心肌横纹清晰。IR组中:缺血40分钟时,心肌组织局部出现少量PMN浸润,大血管中可见PMN附壁,心肌细胞水肿,未见明显坏死灶;再灌注120分钟时,心肌细胞变性、坏死,边界不清,心肌组织中可见大量PMN浸润,心肌间质出血。CAR组和IPC组,再灌注120分钟时,心肌组织中可见PMN浸润,累及心肌层和心外膜,大小血管内外均可见PMN聚集,心肌细胞水肿、变性,心肌横纹不清楚,但程度均较IR组减轻。
结论:
1本研究通过观察IR、CAR、IPC三组缺血/再灌注兔缺血40分钟、再灌注40分钟、再灌注120分钟时的组织病理学特点及MDA、MPO、TNF-α的含量变化,发现缺血再灌注后心肌的PMN浸润程度和心肌的氧化损伤程度随时间延长而递增,二者呈正相关。
2 CAR组和IPC组中MDA、MPO、TNF-α水平在各时段均低于IR组,病理组织学显示PMN浸润程度亦较IR组减轻,而CAR组和IPC组的差别不显著,表明CAR预处理能抑制缺血/再灌注期TNF-α的释放,减少PMN浸润,减轻缺血/再灌注心肌的氧化损伤,具有确切的抗炎作用和心肌保护作用,这可能与CAR抑制NF-κB的激活和ICAM-1、TNF-α的基因表达,改善血管内皮功能,促进NO的合成等有关。
3 CAR和IPC对缺血/再灌注心肌损伤的干预效果无明显差别,提示CAR对缺血/再灌注心肌损伤具有肯定的预保护作用,为CAR更广泛地应用于临床提供了理论依据。
Objective: At present, it shows that myocardial ischemia/ reperfusion injury has a close correlation with the activation of polymorphonuclear neutrophilic leukocytes, however, many cytokines play important role in this complicated process. Carvedilol, a novel beta adrenoceptor antagonist, not only can blockadeα1-adrenoceptor andβ-adrenoceptor, but also have other usefulness which is independent of the above-mentioned contribution. By now it remains unclear if a short period’s using of carvedilol can also have anti-inflammatory effects on myocardial ischemia/reperfusion injury. To investigate this question, we developed different rabbit model in vivo to observe successive change of TNF-αin blood-serum and MDA、MPO in myocardium, furthermore, histopathologic feature of each group was depicted to reflect the degree of polymorphonuclear neutrophilic leukocytes infiltrating in myocardium directly.
Materials and Methods: 48 New Zealand White rabbits(2.5-3.0kg) were randomly divided into four groups: (1) Ischemia and Reperfusion group (IR group, n=12, normal saline 5m1/d): The group was divided into three subunits furthermore according to the different ways of the following treatments, the left ventricular branches of coronary were ligated and obstructed for 40 minutes, obstructed for 40 minutes and reperfused for 40 minutes, obstructed for 40 minutes and reperfused for 120 minutes, moreover, each subunit was composed by four rabbit models; (2) Carvedilol precondition group (CAR group, n=12, carvedilol 3mg/kg/d): The same model as IR group; (3) Ischemic precondition group (IPC group, n=12, normal saline 5m1/d): The left ventricular branches of coronary were obstructed for 5 minutes and reperfused for 5 minutes thrice, then the subunits were completed in the same ways as IR group and CAR group; (4) Sham operated group (Sham group, n=12, normal saline 5m1/d): The left ventricular branches of coronary artery were falsely ligated and three subunits were obtained at the same points of IR group and CAR group. After surgical procedures, we detected the concentration of TNF-αin blood-serum and MDA、MPO in myocardium separately. We also chose some models for histological examination (stained with hematoxylin and eosin) of the ischemic myocardial tissue. All data were presented as mean士SD. The statistical significance of the changes in different models were analyzed by SPSS11.5 statistics software. A value of P<0.05 was accepted as statistically significant.
Results:
1 The concentration changes of MDA in myocardium
Over time, the concentration of MDA in each group increased. But statistical differences occurred only in IR group, CAR group and IPC group: [(4.79±0.12)vs (9.01±0.51 )vs(22.56±1.59)]nmol/mgprot; [(4.69±0.12)vs (6.41±0.54)vs (13.92±0.53)]nmol/mgprot; [(4.71±0.12)vs(6.96±0.45)vs (14.90±0.65)] nmol/mgprot,(F=370.29,491.98,541.60, P<0.01). At the same point, compared with the Sham, CAR and IPC groups, the concentration of MDA in IR group was the highest, and statistical differences were found at reperfusion for 40 minutes and 120 minutes points ( F=59.07,257.80, p<0.01). At the points of reperfusion for 40 minutes and 120 minutes, statistical significances of MDA were found between CAR group and Sham group, IPC group and Sham group (p<0.01), not found between CAR group and IPC group(p=0.13, 0.25).
2 The concentration changes of MPO in myocardium
Over time, the concentration of MPO in each group increased. But statistical differences occurred only in IR group, CAR group and IPC group: [(2.60±0.33)vs (5.15±0.85 )vs(8.63±0.25)] mU/mgprot; [(2.54±0.19)vs (4.17±0.78)vs (6.66±0.19)]mU/mgprot; [(2.59±0.30)vs(3.80±0.23)vs (6.94±0.22)] mU/mgprot,(F=498.00,572.71,313.52, p<0.01). At the same point, compared with the Sham, CAR and IPC groups, the concentration of MPO in IR group was the highest, and statistical differences were found at reperfusion for 40 minutes and 120 minutes points(F=13.49, 540.19, p<0.01). At the points of reperfusion for 40 minutes and 120 minutes, statistical significances of MPO were found between CAR group and Sham group, IPC group and Sham group (p<0.01), not found between CAR group and IPC group(p=0.39, 0.10).
3 The concentration changes of TNF-αin blood-serum
Over time, the concentration of TNF-αin each group increased. But statistical differences occurred only in IR group, CAR group and IPC group: ([12.47±0.99)vs (23.01±0.82 )vs(45.18±1.09)] pg/ml; [(11.14±0.97)vs (15.34±1.15)vs (33.88±0.99)]pg/ml; [(11.64±0.64)vs(16.77±0.66)vs (35.30±0.95)] pg/ml,(F=1177.67,540.28,1063.94, p<0.01). At the same point, compared with the Sham, CAR and IPC groups, the concentration of TNF-αin IR group was the highest, and statistical differences were found at reperfusion for 40 minutes and 120 minutes points(F=101.83,795.40, P<0.01). At the points of reperfusion for 40 minutes and 120 minutes, statistical significances of TNF-αwere found between CAR group and Sham group, between IPC group and Sham group (p<0.01), not found between CAR group and IPC group(p=0.06, 0.07).
4 Histopathologic feature of each group HE stained(10×40) Normal myocardium: myocardial fibre arranged in order, with regular bouncary. I/R group: At ischemia for 40 minutes point, few polymorphonuclear neutrophilic leukocytes infiltrated in myocardial interstitium and adhered to vascular endothelial cells of big blood vessel. Myocardial edema could be observed in ischemic region; At reperfusion for 120 minutes point, myocardial interstitium bleeding, sarcoplasm liquefaction and necrosis could be observed, lots of polymorphonuclear neutrophilic leukocytes infiltrated in myocardial interstitium. CAR and IPC group: At reperfusion for 120 minutes point, myocardial edema, degeneration and some of sarcoplasm liquefaction could also be observed, few of polymorphonuclear neutrophilic leukocytes infiltrated in myocardial interstitium and adhered to vascular endothelial cells of blood vessel.
Conclusion:
1 From this experiment, we could see that over time, the concentrations of MDA, MPO and TNF-αin IR, CAR and IPC groups were increasing. It showed that myocardium injury increased with the accelerating of myocardial inflammatory activity.
2 At different points, the concentrations of MDA, MPO and TNF-αin CAR and IPC groups were lower than that in IR group. The degrees of polymorphonuclear neutrophilic leukocytes infiltration were also depressed. It showed that the therapy of carvedilol and ischemic precondition could effectively inhibit TNF-αexpression, diminish polymorphonuclear neutrophilic leukocytes infiltration, which proved the strong anti-inflammatory effects of carvedilol on ischemia/ reperfusion injury, partly explaining its mechanisms of myocardium protection. Its anti-inflammatory effects perhaps were related to the inhibition of NF-κB, reducting the expression of ICAM-1 and TNF-α, protection of vascular endothelial cell function and improvement of NO etc.
3 No statistical difference was found between CAR and IPC groups in short period. It implied that carvedilol could prevent myocardial ischemia/reperfusion injury effectively, expected to be a theoretical support for its applying on clinic more widely.
材料与方法:健康新西兰白兔48只,体重2.5～3.0 kg,雌雄不拘,随机分为四组:(1)缺血再灌注组(IR组,n=12,生理盐水5ml/d灌胃1w):结扎左室支造成心肌缺血,然后按缺血40分钟、再灌40分钟、再灌120分钟三种处理方法进一步分成三个亚组,每个亚组包括成功的动物模型4只;(2)卡维地洛预处理组(CAR组,n=12,CAR 3mg/kg/d,灌胃1w):手术操作和亚组分法同IR组;(3)缺血预处理组(IPC组,n=12,生理盐水5ml/d灌胃1w):结扎左室支,缺血5分钟,再灌注5分钟,反复3次[1],然后按上述亚组要求行缺血40分钟和开放处理,最终得到缺血40分钟、再灌40分钟、再灌120分钟三个亚组;(4)假手术组(Sham组,n=12,生理盐水5ml/d灌胃1w):只于左室支处穿线而不结扎,进一步按IR组和CAR组相应时间点分成三个亚组。
成功的动物模型达实验终点后取材,分别测定血清中TNF-α和心肌组织中MDA、MPO的含量,行缺血区心肌组织HE染色,光镜下观察心肌细胞形态变化及PMN浸润情况。所有实验数据均用SPSS11.5软件包进行统计学处理,以P<0.05为有统计学差异, P<0.01为有显著统计学差异。
结果:
1心肌匀浆MDA含量在缺血/再灌注中的变化各实验组的MDA含量均随时间延长而递增。Sham组各时间段MDA含量的变化无统计学差异。IR组、CAR组与IPC组各时间段MDA的含量分别为:[(4.79±0.12)vs (9.01±0.51 )vs(22.56±1.59)]nmol/mgprot; [(4.69±0.12)vs (6.41±0.54)vs (13.92±0.53)]nmol/mgprot; ([4.71±0.12)vs(6.96±0.45)vs (14.90±0.65)] nmol/mgprot,差异均有显著统计学意义(F值分别为370.29,491.98,541.60, p值均<0.01)。同一时间段的四组间比较:IR组的MDA含量均高于Sham组、Car组和IPC组,其中再灌注40分钟和再灌注120分钟时的差异有显著统计学意义(F值分别为59.07,257.80, p值均<0.01)。再灌注40分钟和再灌注120分钟时,CAR组和IPC组的MDA含量均高于Sham组,显著有统计学意义(p值均<0.01),但IPC组和CAR组心肌组织中MDA含量的差异无统计学意义(p值分别为0.13, 0.25)。
2心肌匀浆MPO含量在缺血/再灌注中的变化
各实验组的MPO含量均随时间延长而递增。Sham组各时间段MPO含量的变化无统计学差异。IR组、CAR组与IPC组各时间段MPO的含量分别为:([2.60±0.33)vs (5.15±0.85 )vs(8.63±0.25)] mU/mgprot; [(2.54±0.19)vs (4.17±0.78)vs (6.66±0.19)]mU/mgprot; [(2.59±0.30)vs(3.80±0.23)vs (6.94±0.22)] mU/mgprot,差异均有显著统计学意义(F值分别为498.00,572.71,313.52, p值均<0.01)。同一时间段的四组比较:IR组的MPO含量均高于Sham组、CAR组和IPC组,其中再灌注40分钟和再灌注120分钟时的差异有显著统计学意义(F值分别为13.49, 540.19, p值均<0.01)。再灌注40分钟和再灌注120分钟时, CAR组和IPC组的MPO含量均高于Sham组,有显著统计学意义(p值均<0.01),但IPC组和CAR组MPO含量的差异无统计学意义(p值分别为0.39, 0.10)。
3血清TNF-α在缺血/再灌注中的变化
各实验组的TNF-α含量均随时间延长而递增。Sham组各时间段TNF-α含量的变化无统计学差异。IR组、CAR组与IPC组各时间段TNF-α的含量分别为:[(12.47±0.99)vs (23.01±0.82 )vs(45.18±1.09)] pg/ml; [(11.14±0.97)vs (15.34±1.15)vs (33.88±0.99)]pg/ml; [(11.64±0.64)vs(16.77±0.66)vs (35.30±0.95)] pg/ml,差异均有显著统计学意义(F值分别为1177.67,540.28,1063.94, p值均<0.01)。同一时间段的四组比较:IR组的TNF-α含量均高于Sham组、CAR组和IPC组,其中再灌注40分钟和再灌注120分钟时的差异有显著统计学意义(F值分别为101.83,795.40, p值均<0.01)。再灌注40分钟和再灌注120分钟时,CAR组和IPC组的TNF-α含量均高于Sham组,有显著统计学意义(p值均<0.01),但IPC组和CAR组TNF-a含量的差异无统计学意义(p值分别为0.06, 0.07)。
4各实验组的病理变化特点
正常心肌组织:心肌纤维排列整齐,细胞边界完整,心肌横纹清晰。IR组中:缺血40分钟时,心肌组织局部出现少量PMN浸润,大血管中可见PMN附壁,心肌细胞水肿,未见明显坏死灶;再灌注120分钟时,心肌细胞变性、坏死,边界不清,心肌组织中可见大量PMN浸润,心肌间质出血。CAR组和IPC组,再灌注120分钟时,心肌组织中可见PMN浸润,累及心肌层和心外膜,大小血管内外均可见PMN聚集,心肌细胞水肿、变性,心肌横纹不清楚,但程度均较IR组减轻。
结论:
1本研究通过观察IR、CAR、IPC三组缺血/再灌注兔缺血40分钟、再灌注40分钟、再灌注120分钟时的组织病理学特点及MDA、MPO、TNF-α的含量变化,发现缺血再灌注后心肌的PMN浸润程度和心肌的氧化损伤程度随时间延长而递增,二者呈正相关。
2 CAR组和IPC组中MDA、MPO、TNF-α水平在各时段均低于IR组,病理组织学显示PMN浸润程度亦较IR组减轻,而CAR组和IPC组的差别不显著,表明CAR预处理能抑制缺血/再灌注期TNF-α的释放,减少PMN浸润,减轻缺血/再灌注心肌的氧化损伤,具有确切的抗炎作用和心肌保护作用,这可能与CAR抑制NF-κB的激活和ICAM-1、TNF-α的基因表达,改善血管内皮功能,促进NO的合成等有关。
3 CAR和IPC对缺血/再灌注心肌损伤的干预效果无明显差别,提示CAR对缺血/再灌注心肌损伤具有肯定的预保护作用,为CAR更广泛地应用于临床提供了理论依据。
Objective: At present, it shows that myocardial ischemia/ reperfusion injury has a close correlation with the activation of polymorphonuclear neutrophilic leukocytes, however, many cytokines play important role in this complicated process. Carvedilol, a novel beta adrenoceptor antagonist, not only can blockadeα1-adrenoceptor andβ-adrenoceptor, but also have other usefulness which is independent of the above-mentioned contribution. By now it remains unclear if a short period’s using of carvedilol can also have anti-inflammatory effects on myocardial ischemia/reperfusion injury. To investigate this question, we developed different rabbit model in vivo to observe successive change of TNF-αin blood-serum and MDA、MPO in myocardium, furthermore, histopathologic feature of each group was depicted to reflect the degree of polymorphonuclear neutrophilic leukocytes infiltrating in myocardium directly.
Materials and Methods: 48 New Zealand White rabbits(2.5-3.0kg) were randomly divided into four groups: (1) Ischemia and Reperfusion group (IR group, n=12, normal saline 5m1/d): The group was divided into three subunits furthermore according to the different ways of the following treatments, the left ventricular branches of coronary were ligated and obstructed for 40 minutes, obstructed for 40 minutes and reperfused for 40 minutes, obstructed for 40 minutes and reperfused for 120 minutes, moreover, each subunit was composed by four rabbit models; (2) Carvedilol precondition group (CAR group, n=12, carvedilol 3mg/kg/d): The same model as IR group; (3) Ischemic precondition group (IPC group, n=12, normal saline 5m1/d): The left ventricular branches of coronary were obstructed for 5 minutes and reperfused for 5 minutes thrice, then the subunits were completed in the same ways as IR group and CAR group; (4) Sham operated group (Sham group, n=12, normal saline 5m1/d): The left ventricular branches of coronary artery were falsely ligated and three subunits were obtained at the same points of IR group and CAR group. After surgical procedures, we detected the concentration of TNF-αin blood-serum and MDA、MPO in myocardium separately. We also chose some models for histological examination (stained with hematoxylin and eosin) of the ischemic myocardial tissue. All data were presented as mean士SD. The statistical significance of the changes in different models were analyzed by SPSS11.5 statistics software. A value of P<0.05 was accepted as statistically significant.
Results:
1 The concentration changes of MDA in myocardium
Over time, the concentration of MDA in each group increased. But statistical differences occurred only in IR group, CAR group and IPC group: [(4.79±0.12)vs (9.01±0.51 )vs(22.56±1.59)]nmol/mgprot; [(4.69±0.12)vs (6.41±0.54)vs (13.92±0.53)]nmol/mgprot; [(4.71±0.12)vs(6.96±0.45)vs (14.90±0.65)] nmol/mgprot,(F=370.29,491.98,541.60, P<0.01). At the same point, compared with the Sham, CAR and IPC groups, the concentration of MDA in IR group was the highest, and statistical differences were found at reperfusion for 40 minutes and 120 minutes points ( F=59.07,257.80, p<0.01). At the points of reperfusion for 40 minutes and 120 minutes, statistical significances of MDA were found between CAR group and Sham group, IPC group and Sham group (p<0.01), not found between CAR group and IPC group(p=0.13, 0.25).
2 The concentration changes of MPO in myocardium
Over time, the concentration of MPO in each group increased. But statistical differences occurred only in IR group, CAR group and IPC group: [(2.60±0.33)vs (5.15±0.85 )vs(8.63±0.25)] mU/mgprot; [(2.54±0.19)vs (4.17±0.78)vs (6.66±0.19)]mU/mgprot; [(2.59±0.30)vs(3.80±0.23)vs (6.94±0.22)] mU/mgprot,(F=498.00,572.71,313.52, p<0.01). At the same point, compared with the Sham, CAR and IPC groups, the concentration of MPO in IR group was the highest, and statistical differences were found at reperfusion for 40 minutes and 120 minutes points(F=13.49, 540.19, p<0.01). At the points of reperfusion for 40 minutes and 120 minutes, statistical significances of MPO were found between CAR group and Sham group, IPC group and Sham group (p<0.01), not found between CAR group and IPC group(p=0.39, 0.10).
3 The concentration changes of TNF-αin blood-serum
Over time, the concentration of TNF-αin each group increased. But statistical differences occurred only in IR group, CAR group and IPC group: ([12.47±0.99)vs (23.01±0.82 )vs(45.18±1.09)] pg/ml; [(11.14±0.97)vs (15.34±1.15)vs (33.88±0.99)]pg/ml; [(11.64±0.64)vs(16.77±0.66)vs (35.30±0.95)] pg/ml,(F=1177.67,540.28,1063.94, p<0.01). At the same point, compared with the Sham, CAR and IPC groups, the concentration of TNF-αin IR group was the highest, and statistical differences were found at reperfusion for 40 minutes and 120 minutes points(F=101.83,795.40, P<0.01). At the points of reperfusion for 40 minutes and 120 minutes, statistical significances of TNF-αwere found between CAR group and Sham group, between IPC group and Sham group (p<0.01), not found between CAR group and IPC group(p=0.06, 0.07).
4 Histopathologic feature of each group HE stained(10×40) Normal myocardium: myocardial fibre arranged in order, with regular bouncary. I/R group: At ischemia for 40 minutes point, few polymorphonuclear neutrophilic leukocytes infiltrated in myocardial interstitium and adhered to vascular endothelial cells of big blood vessel. Myocardial edema could be observed in ischemic region; At reperfusion for 120 minutes point, myocardial interstitium bleeding, sarcoplasm liquefaction and necrosis could be observed, lots of polymorphonuclear neutrophilic leukocytes infiltrated in myocardial interstitium. CAR and IPC group: At reperfusion for 120 minutes point, myocardial edema, degeneration and some of sarcoplasm liquefaction could also be observed, few of polymorphonuclear neutrophilic leukocytes infiltrated in myocardial interstitium and adhered to vascular endothelial cells of blood vessel.
Conclusion:
1 From this experiment, we could see that over time, the concentrations of MDA, MPO and TNF-αin IR, CAR and IPC groups were increasing. It showed that myocardium injury increased with the accelerating of myocardial inflammatory activity.
2 At different points, the concentrations of MDA, MPO and TNF-αin CAR and IPC groups were lower than that in IR group. The degrees of polymorphonuclear neutrophilic leukocytes infiltration were also depressed. It showed that the therapy of carvedilol and ischemic precondition could effectively inhibit TNF-αexpression, diminish polymorphonuclear neutrophilic leukocytes infiltration, which proved the strong anti-inflammatory effects of carvedilol on ischemia/ reperfusion injury, partly explaining its mechanisms of myocardium protection. Its anti-inflammatory effects perhaps were related to the inhibition of NF-κB, reducting the expression of ICAM-1 and TNF-α, protection of vascular endothelial cell function and improvement of NO etc.
3 No statistical difference was found between CAR and IPC groups in short period. It implied that carvedilol could prevent myocardial ischemia/reperfusion injury effectively, expected to be a theoretical support for its applying on clinic more widely.
引文
1 Murry CE,Tennings RB,Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium[J]. Circulation, 1986, 74(5):1124-1136
2 金惠铭,王建枝主编. 缺血再灌注损伤. 病理生理学. 第6 版. 北京:人民卫生出版社, 2003, 204~208
3 Kametani R, Miura T, Harada N,et al. Carvedilol inhibits mitochondrial oxygen consumption and superoxide production during calcium overload in isolated heart mitochondria. J Circ, 2006, 70(3):321~326
4 Oliveira PJ, Gon?alves L, Monteiro P,et al. Are the antioxidant properties of carvedilol important for the protection of cardiac mitochondria? Curr Vasc Pharmacol, 2005, 3(2):147~158
5 Butler S, Wang R, Wunder SL,et al. Perturbing effects of carvedilol on a model membrane system: role of lipophilicity and chemical structure. Biophys Chem, 2006, 119 (3):307~315
6 Drábiková K,Jancinová V,Nosál' R,et al. On the antioxidant activity of carvedilol in human polymorphonuclear leukocytes in vitro and ex vivo. Neuro Endocrinol Lett, 2006, 27 Suppl 2:138~140
7 Kastratovi? DA, Vasiljevi? ZM, Spasi? MB,et al. Carvedilol increases copper-zinc superoxide dismutase activity in patients with acute myocardial infarction. Basic Clin Pharmacol Toxicol, 2007, 101(2):138~142.
8 Rossig L,Haendeler J,Mallat Z,et al. Congestive heart failure induces endothelial cell apoptosis: protective role of carvedilol[J]. J Am Coll Cardiol, 2000, 36(7):2081~2089
9 Cargnoni A,Ceconi C,Bernocchi P,et al. Reduction of oxidative stress by carvedilol: role in maintenance of ischemic myocardium viability[J]. Cardiovasc Res, 2000, 47(3):556~566
10 Sack M. Tumor necrosis factor-alpha in cardiovascular biology and the potential role for anti-tumor necrosis factor-alpha therapy in heart disease. Pharmacol Ther,2002 ,94(1-2):123-135
11 Smith SC,Allen PM. Expression of tumor necrosis factor-alpha in myocardium and dorsal root ganglion of coronary artery occlusion in rats. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, 2007, 19(11):657~661.
12 Osmala W,Przewlocka-Kosmala M,Mazurek W. Proinflammatory cytokines and myocardial viability in patients after acute myocardial infarction. Int J Cardiol. 2005 101(3):449~456.
13 Abacilar F, Dogan OF, Duman U,et al. The changes and effects of the plasma levels of tumor necrosis factor after coronary artery bypass surgery with cardiopulmonary bypass. Heart Surg Forum, 2006, 9(4):E703~709
14 Onimaru S,Nakamura K,Kariyazono H,et al. Inhibitory effects of edaravone on the production of tumor necrosis factor-alpha in the isolated heart undergoing ischemia and reperfusion. Heart Vessels, 2006, 21(2):108~115
15 Meldrum DR,Cleveland JC Jr,Cain BS,et al. Increased myocardial tumor necrosis factor in a crystalloid-perfusion model of cardiac ischemia-reperfusion injury. Ann Thorac Surg, 1998, 65(2):439~441
16 牛丽丽,祝善俊,史亚非等. 心肌缺血再灌注中 TNF 动态变化及药物干预. 实用医药杂志, 2003, 20 (9): 671~672
17 Elizabeth N,Morgan MD,Edward M,et al. An essential role of NF-κB in the cardioadaptive response to ischemia[J]. Ann Thorac Surg, 1999, 68(2):377~382
18 Li C,Browder W,Kao RL. Early activation of transcription factor NF-kappaB during ischemia in perfused rat heart. Am J Physiol, 1999, 276:H543~H552
19 Arnhold J. Properties, functions, and secretion of human myeloperoxidase[J]. Biochemistry (Mosc), 2004, 69:4~9
20 Vienman CE,Ma XL,Lefer AM,et al. Time course of endothelial dysfunction and myocardial injury during coronary arterial occlusion. AM J Physiol, 1991, 261: H874~H876
21 Byrne IG,Karavas AN,Ehalabi A,et al. Myocardial neutrophil sequestration during reperfusion of the transplanted rabbit heart.J Heart Lung Transplant, 2000, 19(8):786~788
22 吴志峰,余伯阳,朱丹尼等. 大鼠心肌缺血再灌注模型中中性粒细胞与心肌损伤程度的相关性研究[J]. 中国药科大学学报, 2002, 33(3) :231~233
23 Bril A,Slivjak MJ,Dimartino MJ,Feuerstein GZ,et a1. Cardioprotective effects of carvedilol. A novel beta adrenoceptor antagonist with vasodilating properties, in anaesthetised minipigs: comparison with propranolo[J]. Cadiovasc Res, 2002, 26:518~525
24 Ma XL,Yue TL,Lopez BL,et a1. Carvedilol, a new adrenoceptor antagonist and free radical scavenger, attenuates myocardial ischemia-reperfusion injury in hypercholesterolemic rabbits[J]. J Pharmacol Exp Ther Apeut, 2005, 277:128~136
25 Durieu TO,Chaverot N,Cazaubon S,et al. Intercellular adhesion molecule-1 activation induces tyrosine phosphorylation of the cytoskeleton-associated protein cortactin in brain microvessel endothelil cells[J]. J Biol Chem, 2003, 269( 17):12536~12540
26 吴强,李隆贵,蔡运昌等.卡维地洛对心衰大鼠心肌细胞间粘附分子-1 表达的影响[J].中国分子心脏病学杂志, 2002, 2( 5):21~24
27 Pabla R,Buda AJ,Flynn DM,et al. Nitric oxide attenuates neutrophil-mediated myocardial contractile dysfunction after ischemia and reperfusion. Cir Res, 1996, 78: 65~72
28 Zhao T,Xi L,Che11iah J,et al. Inducible nitric oxide synthase mediates delayed myocardial protection induced by activation of adenosin A(1) receptors: evidence from gene-knockout mice. Circulation, 2000, 102(8):902~907
29 Pabla R,Buda AJ,Flynn DM,et al. Nitric oxide attenuates neutrophil-mediated myocardial contractile dysfunction after ischemia and reperfusion. Cir Re, 2003, 78:65~72
30 Szajerski P, Zielonka J, Sikora A,et a1. Radical scavenging and NO-releasing properties of selected beta-adrenoreceptor antagonists. Free Radic Res, 2006, 40(7):741~752
31 Afonso RA,Patarr?o RS,Macedo MP,et a1. Carvedilol's actions are largely mediated by endogenous nitric oxide. Rev Port Cardiol, 2006, 25(10):911~917
1 Hong YJ,Jeong MH,Lee SH,et al. J Interv Cardio1, 2003, 16(3):209~216
2 Starkopf J,Hegna S,Johansen OE,et al. In vivo chronic carvedilol treatment in rats attenuates ex vivo regional infarction of the heart. J Scand Cardiovasc, 2006, 40(4): 240~247
3 Drábiková K,Jancinová V,Nosál' R,et al. On the antioxidant activity of carvedilol in human polymorphonuclear leukocytes in vitro and ex vivo. Neuro Endocrinol Lett, 2006, 27 Suppl 2:138~140
4 Oliveira PJ, Gon?alves L, Monteiro P,et al. Are the antioxidant properties of carvedilol important for the protection of cardiac mitochondria? Curr Vasc Pharmacol, 2005, 3(2):147~158
5 Kastratovi? DA,Vasiljevi? ZM,Spasi? MB,et al. Carvedilol increases copper-zinc superoxide dismutase activity in patients with acute myocardial infarction. Basic Clin Pharmacol Toxicol, 2007, 101(2):138~142.
6 Andona P,Karne R,Ghanim H,et al. Carvedilol inhibits reaction oxygen species generation by leukocytes and oxidative damage to amino acids[J]. Circulation, 2000, 101: 122~124
7 Abreu RMV,Santos DJSL,Moreno AJM. Effects of Carvediol and its analog BM-2910228 on mitochondrialfunction and oxidative stress[J]. J Pharmacol Exp Therapeut, 2000, 295: 1022~1030
8 Butler S, Wang R, Wunder SL,et al. Perturbing effects of carvedilol on a model membrane system: role of lipophilicity and chemical structure. Biophys Chem, 2006, 119 (3):307~315
9 Rossig L,Haendeler J,Mallat Z,et al. Congestive heart failure induces endothelial cell apoptosis: protective role of carvedilol[J]. J Am Coll Cardiol, 2000, 36(7): 2081~2089
10 Cargnoni A,Ceconi C,Bernocchi P,et al. Reduction of oxidative stress by carvedilol: role in maintenance of ischemic myocardium viability[J]. Cardiovasc Res, 2000, 47(3):556~566
11 Vienman CE, Ma XL, Lefer AM,et al. Time course of endothelial dysfunction and myocardial injury during coronary arterial occlusion. AM J Physiol, 1991, 261:H874
12 Feuerstein GZ,Shusterman NH,Roffolo RR Jr. Carvedilol update Ⅳ : Prevention of oxidative stress, cardiac remodeling and progression of congestive heart failure[J]. Drugs Today, 2004, 33:453~473
13 Bril A,Slivjak MJ,Dimartino MJ,Feuerstein GZ,et a1. Cardioprotective effects of carvedilol. A novel beta adrenoceptor antagonist with vasodilating properties, in anaesthetised minipigs: comparison with propranolo[J]. Cadiovasc Res, 2002, 26:518~525
14 Ma XL,Yue TL,Lopez BL,et a1. Carvedilol, a newadrenoceptor antagonist and free radical scavenger, attenuates myocardial ischemia-reperfusion injury in hypercholesterolemic rabbits[J]. J Pharmacol Exp Ther Apeut, 2005, 277:128~136
15 Macicková T, Pecivová J, Nosál' R,et a1. Influence of carvedilol on superoxide generation and enzyme release from stimulated human neutrophils. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 2005, 149(2):389~392.
16 Pabla R,Buda AJ,Flynn DM,et al. Nitric oxide attenuates neutrophil-mediated myocardial contractile dysfunction after ischemia and reperfusion. Cir Res, 2003, 78:65~72
17 Zhao T,Xi L,Che11iah J,et al. Inducible nitric oxide synthase mediates delayed myocardial protection induced by activation of adenosin A(1) receptors: evidence from gene-knockout mice. Circulation, 2000, 102(8):902~907
18 Szajerski P, Zielonka J, Sikora A,et a1. Radical scavenging and NO-releasing properties of selected beta-adrenoreceptor antagonists. Free Radic Res, 2006, 40(7):741~752
19 Afonso RA,Patarr?o RS,Macedo MP,et a1. Carvedilol's actions are largely mediated by endogenous nitric oxide. Rev Port Cardiol, 2006, 25(10):911~917
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28 Brunvand H,Kvitting PM,Rynniong SE,et al. Carvedilol protects against lethal reperfusion injury through antiadrenergic mechanisms[J]. J Cardiovasc Res, 2002, 26:518~525
29 Feuerstein GZ,Ruffolo RR Jr,Yue TL. Apoptosis and heart failure[J]. Trends Cardiovasc Med, 2002, 7:249~255
30 Krown KA,Page MT,Nguyen C. Tumor necrosis factor-alpha induced apoptosis in cardiac myocytes[J]. J Clin Invest, 2005, 98:2854~2865
31 Yue TL,Ma XL,et al. Possible involvement of sress-activated protein kinase signaling pathway and Fas receptor expression in prevention of ischaemia/reperfusion induced cardiomyocyte apoptosis by carvedilol[J]. Circ Res, 2005, 82: 166~174
32 Teiger E,Than VD,Richard L,et al. Apoptosis in pressure overload-induced heart hypertrophy in the rat[J]. J Clin Invest, 2004, 97:2891~2897
33 Huang H, Shan J, Pan XH,et al. Carvedilol protected diabetic rat hearts via reducing oxidative stress. J Zhejiang Univ Sci B,2006,7(9):725~731
34 Yang YJ, Chen YF, Ruan YM,et al. Beneficial effects of carvedilol on cardiomyocyte apoptosis and bcl-2/bax expression after acute myocardial infarction an experiment with rats. Zhonghua Yi Xue Za Zhi, 2006, 86(13):919~92
2 金惠铭,王建枝主编. 缺血再灌注损伤. 病理生理学. 第6 版. 北京:人民卫生出版社, 2003, 204~208
3 Kametani R, Miura T, Harada N,et al. Carvedilol inhibits mitochondrial oxygen consumption and superoxide production during calcium overload in isolated heart mitochondria. J Circ, 2006, 70(3):321~326
4 Oliveira PJ, Gon?alves L, Monteiro P,et al. Are the antioxidant properties of carvedilol important for the protection of cardiac mitochondria? Curr Vasc Pharmacol, 2005, 3(2):147~158
5 Butler S, Wang R, Wunder SL,et al. Perturbing effects of carvedilol on a model membrane system: role of lipophilicity and chemical structure. Biophys Chem, 2006, 119 (3):307~315
6 Drábiková K,Jancinová V,Nosál' R,et al. On the antioxidant activity of carvedilol in human polymorphonuclear leukocytes in vitro and ex vivo. Neuro Endocrinol Lett, 2006, 27 Suppl 2:138~140
7 Kastratovi? DA, Vasiljevi? ZM, Spasi? MB,et al. Carvedilol increases copper-zinc superoxide dismutase activity in patients with acute myocardial infarction. Basic Clin Pharmacol Toxicol, 2007, 101(2):138~142.
8 Rossig L,Haendeler J,Mallat Z,et al. Congestive heart failure induces endothelial cell apoptosis: protective role of carvedilol[J]. J Am Coll Cardiol, 2000, 36(7):2081~2089
9 Cargnoni A,Ceconi C,Bernocchi P,et al. Reduction of oxidative stress by carvedilol: role in maintenance of ischemic myocardium viability[J]. Cardiovasc Res, 2000, 47(3):556~566
10 Sack M. Tumor necrosis factor-alpha in cardiovascular biology and the potential role for anti-tumor necrosis factor-alpha therapy in heart disease. Pharmacol Ther,2002 ,94(1-2):123-135
11 Smith SC,Allen PM. Expression of tumor necrosis factor-alpha in myocardium and dorsal root ganglion of coronary artery occlusion in rats. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, 2007, 19(11):657~661.
12 Osmala W,Przewlocka-Kosmala M,Mazurek W. Proinflammatory cytokines and myocardial viability in patients after acute myocardial infarction. Int J Cardiol. 2005 101(3):449~456.
13 Abacilar F, Dogan OF, Duman U,et al. The changes and effects of the plasma levels of tumor necrosis factor after coronary artery bypass surgery with cardiopulmonary bypass. Heart Surg Forum, 2006, 9(4):E703~709
14 Onimaru S,Nakamura K,Kariyazono H,et al. Inhibitory effects of edaravone on the production of tumor necrosis factor-alpha in the isolated heart undergoing ischemia and reperfusion. Heart Vessels, 2006, 21(2):108~115
15 Meldrum DR,Cleveland JC Jr,Cain BS,et al. Increased myocardial tumor necrosis factor in a crystalloid-perfusion model of cardiac ischemia-reperfusion injury. Ann Thorac Surg, 1998, 65(2):439~441
16 牛丽丽,祝善俊,史亚非等. 心肌缺血再灌注中 TNF 动态变化及药物干预. 实用医药杂志, 2003, 20 (9): 671~672
17 Elizabeth N,Morgan MD,Edward M,et al. An essential role of NF-κB in the cardioadaptive response to ischemia[J]. Ann Thorac Surg, 1999, 68(2):377~382
18 Li C,Browder W,Kao RL. Early activation of transcription factor NF-kappaB during ischemia in perfused rat heart. Am J Physiol, 1999, 276:H543~H552
19 Arnhold J. Properties, functions, and secretion of human myeloperoxidase[J]. Biochemistry (Mosc), 2004, 69:4~9
20 Vienman CE,Ma XL,Lefer AM,et al. Time course of endothelial dysfunction and myocardial injury during coronary arterial occlusion. AM J Physiol, 1991, 261: H874~H876
21 Byrne IG,Karavas AN,Ehalabi A,et al. Myocardial neutrophil sequestration during reperfusion of the transplanted rabbit heart.J Heart Lung Transplant, 2000, 19(8):786~788
22 吴志峰,余伯阳,朱丹尼等. 大鼠心肌缺血再灌注模型中中性粒细胞与心肌损伤程度的相关性研究[J]. 中国药科大学学报, 2002, 33(3) :231~233
23 Bril A,Slivjak MJ,Dimartino MJ,Feuerstein GZ,et a1. Cardioprotective effects of carvedilol. A novel beta adrenoceptor antagonist with vasodilating properties, in anaesthetised minipigs: comparison with propranolo[J]. Cadiovasc Res, 2002, 26:518~525
24 Ma XL,Yue TL,Lopez BL,et a1. Carvedilol, a new adrenoceptor antagonist and free radical scavenger, attenuates myocardial ischemia-reperfusion injury in hypercholesterolemic rabbits[J]. J Pharmacol Exp Ther Apeut, 2005, 277:128~136
25 Durieu TO,Chaverot N,Cazaubon S,et al. Intercellular adhesion molecule-1 activation induces tyrosine phosphorylation of the cytoskeleton-associated protein cortactin in brain microvessel endothelil cells[J]. J Biol Chem, 2003, 269( 17):12536~12540
26 吴强,李隆贵,蔡运昌等.卡维地洛对心衰大鼠心肌细胞间粘附分子-1 表达的影响[J].中国分子心脏病学杂志, 2002, 2( 5):21~24
27 Pabla R,Buda AJ,Flynn DM,et al. Nitric oxide attenuates neutrophil-mediated myocardial contractile dysfunction after ischemia and reperfusion. Cir Res, 1996, 78: 65~72
28 Zhao T,Xi L,Che11iah J,et al. Inducible nitric oxide synthase mediates delayed myocardial protection induced by activation of adenosin A(1) receptors: evidence from gene-knockout mice. Circulation, 2000, 102(8):902~907
29 Pabla R,Buda AJ,Flynn DM,et al. Nitric oxide attenuates neutrophil-mediated myocardial contractile dysfunction after ischemia and reperfusion. Cir Re, 2003, 78:65~72
30 Szajerski P, Zielonka J, Sikora A,et a1. Radical scavenging and NO-releasing properties of selected beta-adrenoreceptor antagonists. Free Radic Res, 2006, 40(7):741~752
31 Afonso RA,Patarr?o RS,Macedo MP,et a1. Carvedilol's actions are largely mediated by endogenous nitric oxide. Rev Port Cardiol, 2006, 25(10):911~917
1 Hong YJ,Jeong MH,Lee SH,et al. J Interv Cardio1, 2003, 16(3):209~216
2 Starkopf J,Hegna S,Johansen OE,et al. In vivo chronic carvedilol treatment in rats attenuates ex vivo regional infarction of the heart. J Scand Cardiovasc, 2006, 40(4): 240~247
3 Drábiková K,Jancinová V,Nosál' R,et al. On the antioxidant activity of carvedilol in human polymorphonuclear leukocytes in vitro and ex vivo. Neuro Endocrinol Lett, 2006, 27 Suppl 2:138~140
4 Oliveira PJ, Gon?alves L, Monteiro P,et al. Are the antioxidant properties of carvedilol important for the protection of cardiac mitochondria? Curr Vasc Pharmacol, 2005, 3(2):147~158
5 Kastratovi? DA,Vasiljevi? ZM,Spasi? MB,et al. Carvedilol increases copper-zinc superoxide dismutase activity in patients with acute myocardial infarction. Basic Clin Pharmacol Toxicol, 2007, 101(2):138~142.
6 Andona P,Karne R,Ghanim H,et al. Carvedilol inhibits reaction oxygen species generation by leukocytes and oxidative damage to amino acids[J]. Circulation, 2000, 101: 122~124
7 Abreu RMV,Santos DJSL,Moreno AJM. Effects of Carvediol and its analog BM-2910228 on mitochondrialfunction and oxidative stress[J]. J Pharmacol Exp Therapeut, 2000, 295: 1022~1030
8 Butler S, Wang R, Wunder SL,et al. Perturbing effects of carvedilol on a model membrane system: role of lipophilicity and chemical structure. Biophys Chem, 2006, 119 (3):307~315
9 Rossig L,Haendeler J,Mallat Z,et al. Congestive heart failure induces endothelial cell apoptosis: protective role of carvedilol[J]. J Am Coll Cardiol, 2000, 36(7): 2081~2089
10 Cargnoni A,Ceconi C,Bernocchi P,et al. Reduction of oxidative stress by carvedilol: role in maintenance of ischemic myocardium viability[J]. Cardiovasc Res, 2000, 47(3):556~566
11 Vienman CE, Ma XL, Lefer AM,et al. Time course of endothelial dysfunction and myocardial injury during coronary arterial occlusion. AM J Physiol, 1991, 261:H874
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