摘要
目的:探讨细胞穿透肽PEP-1介导的人过氧化氢酶(CAT)对H9c2细胞缺氧复氧损伤的保护作用及其可能的机制。方法:利用基因工程手段表达和纯化His-tag-PEP-1-CAT及His-tag-CAT融合蛋白。将H9c2细胞随机分为正常对照组(CTL组)、缺氧复氧组(H/R组)、H/R加CAT组(CAT组)、H/R加PEP-1-CAT组(PEP-1-CAT组)。分别向PEP-1-CAT组及CAT组加入2 mol/L的PEP-1-CAT及CAT蛋白500μL处理6 h,将各组细胞置于缺氧箱中缺氧21 h,再复氧6 h,通过CCK-8法监测细胞的增殖率,流式细胞仪检测细胞的凋亡水平和活性氧(ROS)的水平,DCFH-DA探针检测细胞内的ROS变化水平,JC-1试剂盒检测细胞线粒体膜电位的变化。Western blot分析凋亡蛋白Bcl-2、Bax、Caspase-3及p-p38表达量的变化。结果:与H/R组相比,PEP-1-CAT组细胞凋亡率、细胞内ROS水平、Bax、Caspase-3及p-p38表达量均明显下降(P<0.05),细胞增殖率、细胞内线粒体膜电位水平及Bcl-2的表达量升高(P<0.05)。结论:PEP-1-CAT对H9c2细胞缺氧复氧损伤有保护作用,推测机制可能与其抗氧化、抗凋亡作用有关,PEP-1-CAT可通过抑制p38MAPK信号通路阻断Bcl-2/Bax/线粒体凋亡通路。
Objective To investigate the protective effect of the PEP-1-CAT fusion protein on hypoxia/reoxygenation-in⁃duced H9c2 cells and its possible mechanisms.Methods His-tag-PEP-1-CAT and His-tag-CAT fusion proteins were ex⁃pressed and purified by genetic engineering.H9c2 cells were randomly divided into four groups:control(CTL)group,hy⁃poxia/reoxygenation(H/R)group,H/R plus CAT treatment(H/R+CAT)group,and H/R plus PEP-1-CAT treatment(H/R+PEP-1-CAT)group.The PEP-1-CAT and CAT proteins(500μL)were added at a concentration of 2 mol/L for 6 h,and the cells were then placed in a hypoxia chamber for 21 h,followed by reoxygenation for 6 h.The proliferation of H9c2 cells was monitored by the CCK-8 assay.Apoptosis and reactive oxygen species(ROS)were analyzed using flow cy⁃tometry.The changes in intracellular ROS were evaluated using DCFH-DA probes,and mitochondrial membrane potential changes were measured by the JC-1 assay kit.Western blot was performed to detect the expressions of apoptosis-related proteins Bcl-2,Bax,Caspase-3,and p-p38.Result Compared with the H/R group,the PEP-1-CAT group had de⁃creased apoptosis,intracellular ROS,Bax,Caspase-3,and p-p38(P<0.05),while the proliferation rate,mitochondrial membrane potential,and Bcl-2 were elevated(P<0.05).Conclusion The PEP-1-CAT fusion protein protects hypoxia/reoxygenation-induced H9c2 cells,and the mechanism may be related to its antioxidant and anti-apoptotic properties through the inhibition of the p38MAPK signaling pathway,thereby blocking the Bcl-2/Bax mitochondrial apoptosis path⁃way.
作者
魏双
王宣人
郑飞
王露
郭凌郧
张蕾
王家宁
WEI Shuang;WANG Xuan-ren;ZHENG Fei;WANG Lu;GUO Ling-yun;ZHANG Lei;WANG Jia-ning(Institute of Clinical Medicine,Hubei University of Medicine,Shiyan,Hubei 442000,China;Clinical Medical Research Center for Atherosclerotic Cardiovascular Diseases(ASCVD),Hubei University of Medicine,Shiyan,Hubei 442000,China;Shiyan Key Laboratory for Atherosclerotic Diseases Research,Renmin Hospital,Hubei University of Medicine,Shiyan,Hubei 442000,China)
出处
《湖北医药学院学报》
2025年第1期9-13,27,F0002,共7页
Journal of Hubei University of Medicine
基金
国家自然科学基金资助项目(81270221)
湖北省科技厅重点研发计划(2022BCE007)
十堰市科技局项目(22Y62)。
作者简介
魏双(1984-),女,湖北房县人,硕士,副主任医师,研究方向:心肌缺血再灌注损伤,心脏康复治疗;通信作者:王家宁(1967-),男,湖北房县人,博士,主任医师,教授,研究方向:心肌缺血再灌注损伤,血管生物学。
