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.
Abstract
Objective To investigate the molecular mechanism of oxidative stress damage induced by hy-poxia/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-Keapl 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 sur-vival 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-Keapl,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.
基金项目
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)
国家科技期刊平台
NSTL
万方数据