摘要
目的基于Nrf2-ARE信号途径探讨DJ-1抗心肌细胞缺氧/复氧所诱发氧化应激损伤的分子机制。方法H9c2心肌样细胞分别转染DJ-1 siRNA、NC siRNA、pFlag-DJ-1、pFlag,通过Western blot检测DJ-1、锰超氧化物歧化酶(Manganese Superoxide Dismutase,MnSOD)、过氧化氢酶(Catalase,CAT)、谷胱甘肽过氧化物酶(Glutathione Peroxidase,GPx)的表达;建立心肌细胞缺氧/复氧(Hypoxia/Reoxygenation,H/R)模型,检测各组心肌细胞存活率、乳酸脱氢酶((Lactate Dehydrogenase,LDH)的活性、丙二醛(Malondialdehyde,MDA)和活性氧(Reactive Oxygen Species,ROS)的含量;观察DJ-1对Nrf2-Keap1的解离、Nrf2核转位、Nrf2与MnSOD、CAT、GPx–ARE的结合、Nrf2转录活性的影响,进而观察DJ-1对Nrf2信号通路的影响;H9c2细胞先转染pFlag-DJ-1/pFlag后,再分别转染Nrf2 siRNA/NC siRNA,采用Western Blot检测MnSOD、CAT、GPx表达的变化;建立H/R损伤模型,检测各组心肌细胞存活率、LDH活性、MDA和ROS的含量,观察抑制Nrf2信号通路后对DJ-1抗氧化应激影响。结果H9c2转染pFlag-DJ-1后,DJ-1、MnSOD、CAT、GPx的表达水平均显著上调。H9c2转染pFlag-DJ-1后,H/R损伤心肌细胞的生存率显著提高了,同时显著降低了LDH的活性、ROS和MDA的产生;而当转染DJ-1 siRNA后,上述效应被逆转。H9c2细胞转染pFLAG-DJ-1后,显著促进了Nrf2-Keap1的解离,Nrf2的入核、Nrf2在核内与ARE的结合及转录的活性;当使DJ-1沉默后,上述效应被逆转。当细胞内Nrf2信号通路被抑制后,DJ-1过表达上调MnSOD、CAT、GPX表达的作用被逆转。DJ-1过表达显著提高了H/R损伤细胞的生存率,抑制LDH的活性以及ROS和MDA的产生;当沉默Nrf2后,上述效应均被明显抑制。结论DJ-1可能通过激活Nrf2-ARE信号通路,诱导抗氧化酶的表达,从而发挥抗心肌细胞H/R所诱发的氧化应激损伤。
Objective To investigate the molecular mechanism of oxidative stress damage induced by hypoxia/reoxygenation of anti-cardiomyocytes by DJ-1 based on the Nrf2-ARE signaling pathway.Methods H9c2 cardiomyocytes were transfected with DJ-1 siRNA,NC siRNA,pFlag-DJ-1,and pFlag,and the expressions of DJ-1 and antioxidant enzymes(MnSOD,CAT and GPx)were detected by Western blot.The H/R model in cardiomyocytes was established,and the cell viability,LDH activity,MDA levels,and ROS content in each group were accessed.The DJ-1’s effect on Nrf2-Keap1 dissociation,the Nrf2 nuclear translocation,the binding of Nrf2 to MnSOD,CAT,and GPx-ARE,and the influence of transcriptional activity of Nrf2 were observed,in order to observe the effect of DJ-1 on the Nrf2 signaling pathway.H9c2 cells were transfected with pFlag-DJ-1/pFlag and then transfected with Nrf2 siRNA/NC siRNA,and the changes in the expression of MnSOD,CAT,and GPx were detected by Western blot;H/R damage model was established to detect the survival rate of cardiomyocytes,LDH activity,and MDA and ROS contents in each group,and observe the effect of inhibition of Nrf2 signaling pathway on antioxidant stress in DJ-1.Results The expression levels of DJ-1,MnSOD,CAT,and GPx were significantly increased after transfection with pFlag-DJ-1 with H9c2.The survival rate of H/R-damaged cardiomyocytes was significantly im-proved after H9c2 transfection with pFlag-DJ-1,but the LDH activity,and ROS and MDA production were signifi-cantly reduced,while the above effects were reversed when transfected with DJ-1 siRNA.After transfection of H9c2 cells with pFLAG-DJ-1,the dissociation of Nrf2-Keap1,the entry of Nrf2 into the nucleus,the binding of Nrf2 to ARE in the nucleus were improved,while the above effects were reversed when DJ-1 is silenced..When the intracel-lular Nrf2 signaling pathway was inhibited,the effect of DJ-1 overexpression which up-regulated the expression of MnSOD,CAT,and GPX was reversed.DJ-1 overexpression significantly improved the survival rate of H/R-damaged cells,inhibited LDH activity,and the production of ROS and MDA,but these effects were significantly inhibited by Nrf2 silencing.Conclusion DJ-1 may induce the expression of antioxidant enzymes by activating the Nrf2-ARE signaling pathway,thereby exerting anti-cardiomyocyte H/R-induced oxidative stress damage.
作者
钱汝平
马建军
顾维民
QIAN Ruping;MA Jianjun;GU Weimin(The First People's Hospital of Aksu RegionCardiology Department II,Aksu 843000,China)
出处
《中华灾害救援医学》
2024年第4期381-385,共5页
Chinese Journal of Disaster Medicine
基金
省部共建中亚高发病成因与防治国家重点实验室开放课题资助项目(SKL-HIDCA-2023-AY2)。
关键词
细胞
信号传导
氧化性应激
cells
signal transduction
oxidative stress
