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RISK信号转导通路对肠系膜上动脉缺血后处理的心肌保护作用


□ 纪阳 蔡尚郎 徐传金

摘 要:

目的通过在体兔心肌缺血再灌注模型,研究再灌注损伤补救酶(reperfusion injury salvage kinase,RISK)信号转导通路是否参与肠系膜上动脉缺血后处理对急性心肌缺血再灌注损伤的保护作用。方法32只兔随机分为缺血再灌注组(IR组),肠系膜上动脉缺血后处理组(MI-Post C组),缺血预处理+肠系膜上动脉缺血后处理组(IPC+MIPostC组),药物干预+肠系膜上动脉缺血后处理组(DI+MI—PostC组),每组8只。实验达终点时,取结扎血管支配部位心肌测定心肌梗死面积并观察心肌细胞超微结构改变;利用Western—blot技术检测各组动物心肌蛋白激酶B(Akt)、内皮型一氧化氮合酶(eNOS)蛋白磷酸化的表达。结果MI—PostC组和IPC+MI—PostC组坏死面积占总面积之比均明显低于其他组(F=4.72,q=3.69~4.20,P〈0.05);而二者之间相比,坏死面积占总面积之比无明显差别(P〉0.05);DI+MI—PostC组LY294002消除了缺血后处理的上述减少心肌梗死面积的作用(q=4.18,P〈0.05)。MI-PostC组Akt和eNOS蛋白表达明显高于IR和DI+MI—PostC组(F=276.34、31.8,q=9.02~29.82,P〈0.01);DI+MI—PostC组LY294002消除了缺血后处理减轻再灌注损伤作用。结论心肌缺血再灌注时立即行肠系膜上动脉缺血后处理与心肌缺血预处理共同运用并不能产生额外的心脏保护作用;RISK信号转导通路参与肠系膜上动脉缺血后处理对急性心肌缺血再灌注损伤的保护作用。

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作者单位:青岛大学医学院附属医院心内科,山东 青岛 266003

【摘要】    目的 通过在体兔心肌缺血再灌注模型, 研究再灌注损伤补救酶(reperfusion injury salvage kinase,risk)信号转导通路是否参与肠系膜上动脉缺血后处理对急性心肌缺血再灌注损伤的保护作用。方法 32只兔随机分为缺血再灌注组(ir组),肠系膜上动脉缺血后处理组(mipostc组),缺血预处理+肠系膜上动脉缺血后处理组(ipc +mipostc组),药物干预+肠系膜上动脉缺血后处理组(di+ mipostc组),每组8只。实验达终点时,取结扎血管支配部位心肌测定心肌梗死面积并观察心肌细胞超微结构改变;利用westernblot技术检测各组动物心肌蛋白激酶b(akt)、内皮型一氧化氮合酶(enos)蛋白磷酸化的表达。结果 mipostc组和ipc+mipostc组坏死面积占总面积之比均明显低于其他组(f=4.72,q=3.69~4.20,p<0.05);而二者之间相比,坏死面积占总面积之比无明显差别(p>0.05) ; di + mipostc组ly294002消除了缺血后处理的上述减少心肌梗死面积的作用(q=4.18,p<0.05)。mipostc组akt和enos蛋白表达明显高于ir和di + mipostc 组(f=276.34、31.8,q=9.02~29.82,p<0.01); di + mipostc组ly294002消除了缺血后处理减轻再灌注损伤作用。结论 心肌缺血再灌注时立即行肠系膜上动脉缺血后处理与心肌缺血预处理共同运用并不能产生额外的心脏保护作用; risk信号转导通路参与肠系膜上动脉缺血后处理对急性心肌缺血再灌注损伤的保护作用。

【关键词】  心肌 缺血后处理 risk信号转导通路 再灌注损伤 肠系膜上动脉

  [abstract] objective to study whether the reperfusion injury salvage kinase (risk) pathway take part in cardioprotection of superior mesenteric artery ischemic postconditioning on rabbit cardiac ischemiareperfusion model in vivo. methods  the rabbits were randomly divided into the following group (eight in each group): ischemiareperfusion (ir); superior mesenteric artery ischemic postconditioning (mipostc); ischemic preconditioning (ipc)+ mipostc; drug intervention (di) + mipostc. when the experiment completed,the heart in each group was harvested and dyed with nbt (nitroblue  tetrazolium), the infarct size determined. the tissue structures were observed  microscopically. myocardium akt and enos were tested by westernblot methods.  results infarct size was significantly less in the mipostc and ipc+mipostc groups than that of the other groups (f=4.72,q=3.69-4.20,p<0.05), but there were no differences between this two groups.however, in di +mipostc groups, ly294002 abrogated the myocardium infarct size reduction in mipostc group (q=4.18,p<0.05). the quantity of akt and enos in myocardium was higher in  mipostc than in the other groups (f=276.34,31.8;q=9.02-29.82;p<0.01). similarly,in di +mipostc group, ly294002 abolished this protective effect in mipostc group. conclusion when reperfusion is done for the ischemic myocardium, superior mesenteric artery ischemic postconditioning can not produce supernumerary protection on the base of ischemic preconditioning. risk pathway takes part in cardioprotection of superior mesenteric artery ischemic postconditioning.
   
  [key words] myocardium; ischemic postconditioning; risk pathway; reperfusion injury; the superior mesenteric artery
   
  ischemic reperfusion injury is a common pathophysiologic phenomenon clinically, which refers to aggravated disorder of function and injury of structure happened to ischemic tissues after reperfusion. it often takes place during the processes of thrombolysis therapy, coronary artery bypass graft surgery, cardiopulmonary resuscitation, limb replantation, organ transplantation, and so on[1]. in 1980s, murry et al[2] proposed that repeated temporal ischemiareperfusion could lessen the myocardial damage induced by  the following longplaying ischemia. this effect was called ischemic preconditioning (ip) and was confirmed by kinds of animal models. however, its clinical application was limited because postconditioning had to be done before ischemia. feng et al[3]  used riperat as an animal model and found that the survival rate of ischemic cadiocytes could be obviously increased when repeated temporal oxygen supply was given to them. this was socalled hypoxic postconditioning (hp). in the past few yeas, many overseas and domestic scholars have confirmed the protective effect of ip against ischemic injury[4], but its exact mechanism has not been get clearly elucidated. so our experiment were aimed to validate the following questions:if the hp of superior mesenteric artery could protect the ischemicreperfused myocardium?whether or not this kind of protective effect was carried out through the reperfusion injury salvage kinase (risk) signal transduction pathway.

  1  materials and methods

  1.1  materials
   
  thirtytwo healthy new zealand rabbits weighing 2.0-2.5 kg were bought from the animal experiment centre of henan medical college. grouping:the animals were randomly divided into four groups, eight cases in each group.

  1.2  methods

  1.2.1  modeling establishment and grouping ischemiareperfusion modeling  the chest of rabbits was opened  and the left ventricular branch of coronary artery ligated. superior mesenteric artery ischemic postconditioning modeling: the abdominal cavity of rabbits was opened along a 5-6 cm longitudinal incision in midand upperabdomen. then superior mesenteric artery was separated and marked by suture silk that blood vessel could be easily occluded by an artery clamp.

  1.2.2  grouping ir group (as control)  left coronary artery occlusion (lado) for one hour and reperfusion lasted six hours. mipostc group: after 60 minutes of lado, thesuperiormesentericarterywasoccludedfor30secondsand  released for 30 seconds for three cycles, then the coronary artery was reperfused for six hours; ipc+mipostc group: three cycles of myocardial ischemia (5 min) and reperfusion (10 min) preceded for 60 minutes of lado, executing the manipulation of mipostc group, then the coronary artery was reperfused for six hours; di+ mipostc group: after 60 minutes of lado, rabbits received the pi3k antagonist ly294002 (0.3 mg/kg), then the other steps followed mipostc group.

  1.3  index and measurement

  1.3.1  myocardium infarct size estimation  at the end of each experiment the estimated risk zone at the apex of the heart was rapidly frozen in liquid nitrogen. the hearts were then cut into 2 mm thick slices parallel to the atrioventricular groove. the slices were then thawed and incubated in 1% nbt (nitroblue tetrazolium) phosphatebuffered solution at 37 ℃ for 15 min. the area of the left ventricle (stained blue), and the infarcted area (unstained) were traced onto acetate transparencies, and with the use of computerized planimetry program, the respective volumes were calculated. infarct size was expressed as a percentage of the risk zone.

  1.3.2  spectroscopy observing  when the reperfusion was finished, we observed the myocardium morphology changing by he dying ischemic necrosis myocardium.

  1.3.3  the expression of akt and enos  hearts were assigned randomly to treatment groups 1, 2, and 4 as described previously. at the end of reperfusion, the ventricular tissue at risk was excised and freezeclamped in liquid nitrogen before being stored at -80 ℃. phosphorylation states of akt (phosphoakt, serine[ser] 473) (1∶1 000 dilution) and endothelial no synthase (phosphoenos, ser 1177) (1∶1 000 dilution) proteins were analyzed by sdspage. we detected the expression of myocardium related gene akt (1∶200 dilution),enos (1∶200 dilution),βactin (1∶200 dilution) and goat antimouse igg/hrp (1∶5 000 dilution) by western blot technology. we undertook quantitative analysis by gel analysis softwarequantity one and undertook singlefactor analysis of variance by softwareorigin 75. immunoelectrophoresis using antibodies obtained from cell signaling company and beijing boisynthesis biotechnology limited company.

  1.3.4  data statistical analysis  experimental data was expressed with x-±s and analyzed by statistics softwarespss 12.0, data was compared with singlefactor analysis of variance in the same group and compared with q test in two groups (p<0.05 was defined as a statistical significance).

  2  results

  2.1  myocardium infarct size estimation
   
  when the experiment was finished, the heart in each group was quickly taken and dyed by nbt, it was found that the ratio between infarct size and total size had a significant decrease in mipostc group (9.45±5.78)% and ipc+mipostc group (9.39±6.12)% compared with ir group (17.58±6.71)% (f=4.72,q=3.69-4.20,p<0.05). there was no significant difference between ir group and di+mipostc group. however, in the di +mipostc groups (18.64±6.92)%, ly294002 abrogated the myocardium infarct size reduction in mipostc group (9.45±5.78)% (q=4.18,p<0.05). the analysis also showed there was no significant difference between other groups (p>0.05). the protection induced by the combination of both ipc and mipostcond did not differ compared with mipostc group alone [ipc+mipostc (9.39±6.12)% versus (9.45±5.78)% with mipostc; p>0.05].

  table 1  the comparison of myocardium infarct size(略)

  2.2  spectroscopy observing ......(未完,请点击下方“在线阅读”)

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