血红素加氧酶-1在肢体缺血后处理对脑缺血再灌注损伤的保护作用
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摘要
目的探讨血红素加氧酶-1(HO-1)在肢体缺血后处理(LPostC)对脑缺血再灌注损伤的保护作用及机制。方法 80只雄性SD大鼠随机分为假手术组(sham组)、缺血再灌注组(I/R组)、肢体缺血后处理组(LPostC组)和血红素加氧酶抑制剂锌原卟啉组(ZnPP组),每组20只。采用石蜡线栓法建立大脑中动脉缺血再灌注模型。夹闭双侧股动脉5 min,松开5 min,反复循环3次,制备LPostC模型。再灌注24 h后,采用2,3,5-氯化三苯基四氮唑(TTC)法测脑梗死体积;末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定(TUNEL)法检测神经细胞凋亡;用免疫组化和Western blotting方法测定HO-1的表达;分光光度计法测脑组织中丙二醛(MDA)和超氧化物歧化酶(SOD)的水平。结果与sham组相比,I/R组HO-1表达减少,SOD活性降低,MDA含量增加(均P<0.05)。与I/R组相比,LpostC组脑梗死体积缩小,神经细胞凋亡数量显著减少,HO-1表达明显增加,SOD活性升高且MDA含量降低(均P<0.05)。与LPostC组相比,ZnPP组脑梗死体积扩大,神经细胞凋亡数量增多,HO-1表达明显减少,SOD活性降低,MDA含量升高(均P<0.05)。结论 LPostC对脑缺血再灌注损伤具有保护作用,其作用机制可能与HO-1的表达增加有关。
Objective To investigate the protection of heme oxygenase-1(HO-1) in limb ischemic postconditioning(LPostC) on cerebral ischemia reperfusion injury. Methods Eighty male SD rats were randomly divided into sham-operation group(sham group), ischemia reperfusion group(I/R group), limb ischemia postconditioning group(LPostC group) and inhibitor of HO-1 group(ZnPP group), 20 rats in each group. The ischemia and reperfusion injury model was established by line embolism with paraffin to block the middle cerebral artery. It includes three cycles of 5 min to occlusion and 5 min to loosen the bilateral femoral artery to make the LPostC model. After 24 h reperfusion, adopted 2,3,5-Triphenyltetrazolium chloride(TTC) staining to measure cerebral infarction volume. TdT-mediated dUTP Nick-End Labeling(TUNEL) staining was used to detected neuronal apoptosis. Immunohistochemistry and Western blotting method were employed to detect the expression of HO-1. Malondialdehyde(MDA) content and Superoxide Dismutase(SOD) activity in brain tissue were measured by spectrophotometer.Results The expression of HO-1 and SOD activity were lower whereas MDA content was higher in I/R group than in sham group(all P<0.05). When compared with those in I/R group, the cerebral infarction volume diminished, and the number of nerve cell apoptosis significantly reduced, and the expression of HO-1 sharply increased, meanwhile SOD activity rose and MDA content decreased in LPostC group(all P<0.05). Cerebral infarction volume, nerve cell apoptosis and MDA content stronger, whereas the expression of HO-1 and SOD activity markedly lower in ZnPP group than in LPostC group(all P<0.05). Conclusion Limb ischemia postconditioning plays the role to protect cerebral ischemia reperfusion injury, and the mechanism may be related to the upregulation of HO-1.
Objective To investigate the protection of heme oxygenase-1(HO-1) in limb ischemic postconditioning(LPostC) on cerebral ischemia reperfusion injury. Methods Eighty male SD rats were randomly divided into sham-operation group(sham group), ischemia reperfusion group(I/R group), limb ischemia postconditioning group(LPostC group) and inhibitor of HO-1 group(ZnPP group), 20 rats in each group. The ischemia and reperfusion injury model was established by line embolism with paraffin to block the middle cerebral artery. It includes three cycles of 5 min to occlusion and 5 min to loosen the bilateral femoral artery to make the LPostC model. After 24 h reperfusion, adopted 2,3,5-Triphenyltetrazolium chloride(TTC) staining to measure cerebral infarction volume. TdT-mediated dUTP Nick-End Labeling(TUNEL) staining was used to detected neuronal apoptosis. Immunohistochemistry and Western blotting method were employed to detect the expression of HO-1. Malondialdehyde(MDA) content and Superoxide Dismutase(SOD) activity in brain tissue were measured by spectrophotometer.Results The expression of HO-1 and SOD activity were lower whereas MDA content was higher in I/R group than in sham group(all P<0.05). When compared with those in I/R group, the cerebral infarction volume diminished, and the number of nerve cell apoptosis significantly reduced, and the expression of HO-1 sharply increased, meanwhile SOD activity rose and MDA content decreased in LPostC group(all P<0.05). Cerebral infarction volume, nerve cell apoptosis and MDA content stronger, whereas the expression of HO-1 and SOD activity markedly lower in ZnPP group than in LPostC group(all P<0.05). Conclusion Limb ischemia postconditioning plays the role to protect cerebral ischemia reperfusion injury, and the mechanism may be related to the upregulation of HO-1.
引文
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[12] Wang P,Liang X,Lu Y,et al.MicroRNA-93 downregulation ameliorates cerebral ischemic injury through the Nrf2/HO-1 defense pathway[J].Neurochem Res,2016,41(10):1-9.
[13] Chao G,Wang S,Duan J,et al.Protocatechualdehyde protects against cerebral ischemia-reperfusion-induced oxidative injury via protein kinase Cε/Nrf2/HO-1 pathway[J].Mol Neurobiol,2017,54(2):833-845.
[14] Wang X,Zhang L,Zhao W,et al.The protective effects of hydrogen on HO-1 expression in the brainafter focal cerebral ischemia reperfusion in rats[J].Turk J Med Sci,2016,46(5):1534-1539.
[15] 薛芬芬,李浩.肢体缺血后处理脑保护作用的研究进展[J].内科,2015,10(2):254-256.
[16] Zhang X,Jizhang Y,Xu X,et al.Protective effects of remote ischemic conditioning against ischemia/reperfusion-induced retinal injury in rats[J].Vis Neurosci,2014,31(3):245-252.
[17] Kim YH,Yoon DW,Kim JH,et al.Effect of remote ischemic post-conditioning on systemic inflammatory response and survival rate in lipopolysaccharide-induced systemic inflammation model[J].J Inflamm,2014,11(1):16-16.
[18] 郭继芳,游陆,李作武.肢体缺血后处理对心肌缺血/再灌注大鼠血红素加氧酶表达的影响[J].牡丹江医学院学报,2009,30(3):27-29.
[2] 刘芳怡,刘开祥,李浩,等.氯化锂在肢体缺血后处理中对脑缺血再灌注大鼠的糖原合酶激酶3β/微管相关蛋白2a/b通路的影响[J].临床神经病学杂志,2014,27(6):442-445.
[3] Li H,Zhou S,Wu L,et al.The role of p38MAPK signal pathway in the neuroprotective mechanism of limb postconditioning against rat cerebral ischemia/reperfusion injury [J].J Neurol Sci,2015,357(1-2):270-275.
[4] 李浩,石胜良,刘开祥,等.青藤碱对大鼠局灶性脑缺血再灌注损伤保护作用及机制[J].重庆医学,2010,39(19):2558-2560.
[5] 李浩,刘开祥,俸军林,等.环磷酰胺预处理对鼠脑缺血再灌注损伤炎症反应和自由基的影响[J].中国现代医学杂志,2008,18(18):2605-2608.
[6] 李浩,张多斌,吴岚,等.丹参酮ⅡA对脑缺血再灌注损伤大鼠磷酸化p38MAPK和MMP-9表达及细胞凋亡的影响[J].中风与神经疾病,2013,30(3):229-233.
[7] Xia DY,Li W,Qian HR,et al.Ischemia preconditioning is neuroprotective in a rat cerebral ischemic injury model through autophagy activation and apoptosis inhibition.[J].Braz J Med Biol Res,2013,46(7):580-588.
[8] Liu P,Zhao H,Wang R,et al.MicroRNA-424 protects against focal cerebral ischemia and reperfusion injury in mice by suppressing oxidative stress[J].Stroke,2015,46(2):513-519.
[9] Le WD,Xie WJ,Appel SH.Protective role of heme oxygenase-1 in oxidative stress-induced neuronal injury[J].J Neurosci Res,2015,56(6):652-658.
[10] Gozzelino R,Jeney V,Soares MP.Mechanisms of cell protection by heme oxygenase-1[J].Annu Rev Pharmacol Toxicol,2015,50(1):323-354.
[11] Aztatzi-Santillán E,Nares-López FE,Márquez-Valadez B,et al.The protective role of heme oxygenase-1 in cerebral ischemia[J].Cent Nerv Syst Agents Med Chem,2010,10(4):310-316.
[12] Wang P,Liang X,Lu Y,et al.MicroRNA-93 downregulation ameliorates cerebral ischemic injury through the Nrf2/HO-1 defense pathway[J].Neurochem Res,2016,41(10):1-9.
[13] Chao G,Wang S,Duan J,et al.Protocatechualdehyde protects against cerebral ischemia-reperfusion-induced oxidative injury via protein kinase Cε/Nrf2/HO-1 pathway[J].Mol Neurobiol,2017,54(2):833-845.
[14] Wang X,Zhang L,Zhao W,et al.The protective effects of hydrogen on HO-1 expression in the brainafter focal cerebral ischemia reperfusion in rats[J].Turk J Med Sci,2016,46(5):1534-1539.
[15] 薛芬芬,李浩.肢体缺血后处理脑保护作用的研究进展[J].内科,2015,10(2):254-256.
[16] Zhang X,Jizhang Y,Xu X,et al.Protective effects of remote ischemic conditioning against ischemia/reperfusion-induced retinal injury in rats[J].Vis Neurosci,2014,31(3):245-252.
[17] Kim YH,Yoon DW,Kim JH,et al.Effect of remote ischemic post-conditioning on systemic inflammatory response and survival rate in lipopolysaccharide-induced systemic inflammation model[J].J Inflamm,2014,11(1):16-16.
[18] 郭继芳,游陆,李作武.肢体缺血后处理对心肌缺血/再灌注大鼠血红素加氧酶表达的影响[J].牡丹江医学院学报,2009,30(3):27-29.