Recombinant PTD-Cu/Zn SOD attenuates hypoxia-reoxygenation injury in cardiomyocytes
Recombinant PTD-Cu/Zn SOD attenuates hypoxia-reoxygenation injury in cardiomyocytes
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重组 PTD-Cu/Zn SOD 减轻心肌细胞缺氧-复氧损伤
DOI：
10.3109/10715762.2013.780286
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发表时间：
2013-05-01
影响因子：
3.3
通讯作者：
Xia, Z.
中科院分区：
文献类型：
--
作者：
Liu, J.;Hou, J.;Xia, Z.
背景氧化应激在心肌缺血再灌注损伤中起着关键作用。提高细胞内主要的内源性抗氧化酶Cu/Zn SOD的蛋白表达，可减轻或防止培养心肌细胞缺氧-复氧损伤（HRI）。然而，外源性Cu/Zn-SOD很难进入细胞发挥生物学效应。本研究构建了具有蛋白转导结构域（Protein transduction domain，PTD）的PTD-Cu/Zn SOD质粒，并在缺氧/复氧损伤（HRI）的H9 c2大鼠心肌细胞中检测其跨膜能力和抗氧化作用。方法.构建了pET-PTD-Cu/Zn SOD（CDs）原核表达载体，并将其插入E. coli BL 21诱导表达PTD-Cu/Zn SOD。H9 c2心肌细胞HRI是通过将心肌细胞暴露于12 h缺氧然后2 h复氧来实现的。Western blot检测心肌细胞PTD-Cu/Zn SOD蛋白表达，免疫组化和流式细胞术检测心肌细胞PTD-Cu/Zn SOD酶活性。结果在培养的心肌细胞缺氧复氧损伤模型中，外源性PTD-Cu/Zn SOD可穿透细胞膜清除HRI后H9 c2心肌细胞内的超氧阴离子，降低HRI后H9 c2心肌细胞内的过氧化氢水平。PTD-Cu/Zn SOD可使心肌细胞线粒体膜电位恢复正常，细胞凋亡减少。结论重组PTD-Cu/Zn SOD能抑制培养心肌细胞内超氧阴离子自由基的释放，保护线粒体免受损伤，减轻心肌细胞缺氧/复氧损伤。
Background. Oxidative stress plays a pivotal role in myocardial ischemia-reperfusion injury. Increasing the protein expression of intracellular Cu/Zn SOD, which is the major endogenous antioxidant enzyme, may attenuate or prevent hypoxia-reoxygenation injury (HRI) in cultured cardiomyocytes. However, ectogenic Cu/Zn-SOD can hardly be transferred into cells to exert biological effects. In this study, we constructed PTD-Cu/Zn SOD plasmid with a kind of translocation structure-Protein transduction domain (PTD) and detected its transmembrane ability and antioxidant effects in H9c2 rat cardiomyocytes subjected to hypoxia/reoxygenation injury (HRI). Methods. We constructed the pET-PTD-Cu/Zn SOD (CDs) prokaryotic expression vectors in plasmid that were inserted into E. coli BL21 to induce the protein expression of PTD-Cu/Zn SOD. H9c2 cardiomyocyte HRI was achieved by exposing cardiomyocytes to 12 h hypoxia followed by 2 h reoxygenation. Protein expression of PTD-Cu/Zn SOD in cardiomyocytes was assayed by Western blot and their enzyme activities were investigated by immunohistochemistry and flow cytometry. Results. In cultured cardiomyocytes hypoxia reoxygenation injury model, exogenous PTD-Cu/Zn SOD could penetrate cell membrane to clear superoxide anion and decrease hydrogen peroxide level in H9c2 cardiomyocytes subjected to HRI. The level of mitochondrial membrane potential was restored to normal, and the cell apoptosis was reduced in cardiomyocytes with PTD-Cu/Zn SOD treatment during HRI. Conclusion. Recombinant PTD-Cu/Zn SOD could scavenge intracellular-free superoxide anion, protect mitochondria from damages, and attenuate the hypoxia reoxygenation injury in cultured cardiomyocytes.