PEP-1-CAT预处理对H9c2细胞缺氧复氧损伤的保护作用

The protective effect of PEP-1-CAT fusion protein on hypoxia-reoxygenation injury in rat H9c2 cells

目的:探讨细胞穿透肽PEP-1介导的人过氧化氢酶(catalase,CAT)对H9c2细胞缺氧复氧损伤的保护作用。方法:利用基因工程方法表达和纯化His-tag-PEP-l-CAT融合蛋白,将H9c2细胞随机分为正常对照组(CTL)、缺氧复氧组(H/R)、H/R加CAT组(H/R+CAT)、H/R加PEP-1-CAT组(H/R+PEP-1-CAT)。分别向H/R+PEP-1-CAT及H/R+CAT组加入2 mol/L的PEP-1-CAT及CAT500μl处理6 h,将各组细胞置于缺氧箱中缺氧21 h,再复氧6 h。采用试剂盒检测乳酸脱氢酶(LDH)及丙二醛(MDA)水平;通过流式细胞仪检测细胞的凋亡水平;通过Western blot检测凋亡蛋白Bcl-2、Bax的表达。结果:与H/R组相比,H/R+PEP-1-CAT组的细胞的LDH、MDA、细胞的凋亡率及Bax的表达量均明显下降,Bcl-2的表达量升高(P<0.05)。结论:PEP-1-CAT预处理通过抗氧化和抗凋亡的作用发挥对H9c2细胞缺氧复氧损伤的保护作用。

Objective：To investigate the protective effect of PEP-I-CAT fusion protein on hypoxia/ reoxygenation injury in rat H9c2 cells. Methods： Cultured rat H9c2 cells were randomly divided into four groups： control group （CTL）, hypoxia/reoxygenation group （H/R）, hypoxia/reoxygenation with PEP-I-CAT treatment group （H/R ＋ PEP-I-CAT） and hypoxia/reoxygenation with CAT treatment group （H/R＋ CAT）. After pretreatment with PEP-I-CAT for 6 hours, the H9c2 cells were put into humidified hypoxia chamber for 21 hours and re-oxygenated for 6 hours. The changes of apoptosis in H9c2 cells were quantitatively assayed with Annexin V and PI double staining by flow cytometry. LDH and MDA were simultaneously measured. The expressions of Bcl-2 and Bax in H9e2 cells were detected by Western blotting. Results~ Compared with H/R group, the release of LDH and the content of MDA, the apoptosis rates and Bax expression were significantly decreased in H/R ＋ PEP-I-CAT group （P^0.05）. The expression of Bcl-2 was correspondingly increased in the PEP-l-CAT group （P^0.05） compared with H/R group. Conclusion： PEP-I-CAT fusion protein could obviously protect hypoxia/ reoxygenated cardiomyocytes, which may be related to its ant-oxidant and ant- apoptosis effects.