DNA2,a multifunctional enzyme with structure-specific nuclease,5'-to-3'helicase,and DNA-dependent ATPase activities,plays a pivotal role in the cellular response to DNA damage.However,its involvement in cerebr...DNA2,a multifunctional enzyme with structure-specific nuclease,5'-to-3'helicase,and DNA-dependent ATPase activities,plays a pivotal role in the cellular response to DNA damage.However,its involvement in cerebral ischemia/reperfusion(I/R)injury remains to be elucidated.This study investigated the involvement of DNA2 in cerebral I/R injury using conditional knockout(cKO)mice(Nestin-Cre)subjected to middle cerebral artery occlusion(MCAO),an established model of cerebral I/R.Results demonstrated a gradual up-regulation of DNA2 expression,peaking at 72 h post-MCAO.Notably,DNA2 cKO mice exhibited more pronounced brain injury,neurological deficits,and neuronal apoptosis within the penumbra following MCAO.Additionally,DNA2 expression was elevated in an oxygen-glucose deprivation/reoxygenation(OGD/R)cell culture model,and DNA2 knockdown(KD)exacerbated neuronal apoptosis and oxidative stress.Transcriptome analysis of ischemic penumbra tissues via RNA sequencing revealed significant down-regulation of Homer1 in DNA2 cKO mice.Furthermore,in vitro experiments demonstrated that overexpression of Homer1a ameliorated DNA2 KD-induced neuronal apoptosis.Collectively,these findings demonstrate that DNA2 deficiency exacerbates cerebral I/R injury through the down-regulation of Homer1a,highlighting a novel regulatory axis in ischemic neuroprotection.展开更多
Background Liver ischemia/reperfusion(I/R)injury is usually caused by hepatic inflow occlusion during liver surgery,and is frequently observed during war wounds and trauma.Hepatocyte ferroptosis plays a critical role ...Background Liver ischemia/reperfusion(I/R)injury is usually caused by hepatic inflow occlusion during liver surgery,and is frequently observed during war wounds and trauma.Hepatocyte ferroptosis plays a critical role in liver I/R injury,however,it remains unclear whether this process is controlled or regulated by members of the DEAD/DExH-box helicase(DDX/DHX)family.Methods The expression of DDX/DHX family members during liver I/R injury was screened using transcriptome analysis.Hepatocyte-specific Dhx58 knockout mice were constructed,and a partial liver I/R operation was performed.Single-cell RNA sequencing(scRNA-seq)in the liver post I/R suggested enhanced ferroptosis by Dhx58hep−/−.The mRNAs and proteins associated with DExH-box helicase 58(DHX58)were screened using RNA immunoprecipitation-sequencing(RIP-seq)and IP-mass spectrometry(IP-MS).Results Excessive production of reactive oxygen species(ROS)decreased the expression of the IFN-stimulated gene Dhx58 in hepatocytes and promoted hepatic ferroptosis,while treatment using IFN-αincreased DHX58 expression and prevented ferroptosis during liver I/R injury.Mechanistically,DHX58 with RNA-binding activity constitutively associates with the mRNA of glutathione peroxidase 4(GPX4),a central ferroptosis suppressor,and recruits the m6A reader YT521-B homology domain containing 2(YTHDC2)to promote the translation of Gpx4 mRNA in an m6A-dependent manner,thus enhancing GPX4 protein levels and preventing hepatic ferroptosis.Conclusions This study provides mechanistic evidence that IFN-αstimulates DHX58 to promote the translation of m6A-modified Gpx4 mRNA,suggesting the potential clinical application of IFN-αin the prevention of hepatic ferroptosis during liver I/R injury.展开更多
基金supported by the National Natural Science Foundation of China (32070979)Shenzhen Science and Technology Program (JCYJ20220530161604009,JCYJ20240813150734043)+3 种基金Key Research and Development Program of Shaanxi (2024SF,YBXM,050)Fundamental Research Funds for the Central Universities (31020190QD004,3102019YX01001)Double First-Class Project of China Pharmaceutical University (CPUQNJC22_02)Global Pharmaceutical Development Alliance Plan of China Pharmaceutical University (1302090024-05)。
文摘DNA2,a multifunctional enzyme with structure-specific nuclease,5'-to-3'helicase,and DNA-dependent ATPase activities,plays a pivotal role in the cellular response to DNA damage.However,its involvement in cerebral ischemia/reperfusion(I/R)injury remains to be elucidated.This study investigated the involvement of DNA2 in cerebral I/R injury using conditional knockout(cKO)mice(Nestin-Cre)subjected to middle cerebral artery occlusion(MCAO),an established model of cerebral I/R.Results demonstrated a gradual up-regulation of DNA2 expression,peaking at 72 h post-MCAO.Notably,DNA2 cKO mice exhibited more pronounced brain injury,neurological deficits,and neuronal apoptosis within the penumbra following MCAO.Additionally,DNA2 expression was elevated in an oxygen-glucose deprivation/reoxygenation(OGD/R)cell culture model,and DNA2 knockdown(KD)exacerbated neuronal apoptosis and oxidative stress.Transcriptome analysis of ischemic penumbra tissues via RNA sequencing revealed significant down-regulation of Homer1 in DNA2 cKO mice.Furthermore,in vitro experiments demonstrated that overexpression of Homer1a ameliorated DNA2 KD-induced neuronal apoptosis.Collectively,these findings demonstrate that DNA2 deficiency exacerbates cerebral I/R injury through the down-regulation of Homer1a,highlighting a novel regulatory axis in ischemic neuroprotection.
基金National Key Research and Development Program of China(2023YFC2505900)National Natural Science Foundation of China(92269204,82171755,92369106,82171749,82171811,82073184)+1 种基金Military Outstanding Youth Program(2020QN06119,01-SWK JYCJJ07,23SWAQ53)Program of Leading Talents in Shanghai,and Shanghai Shuguang Program(20SG39)。
文摘Background Liver ischemia/reperfusion(I/R)injury is usually caused by hepatic inflow occlusion during liver surgery,and is frequently observed during war wounds and trauma.Hepatocyte ferroptosis plays a critical role in liver I/R injury,however,it remains unclear whether this process is controlled or regulated by members of the DEAD/DExH-box helicase(DDX/DHX)family.Methods The expression of DDX/DHX family members during liver I/R injury was screened using transcriptome analysis.Hepatocyte-specific Dhx58 knockout mice were constructed,and a partial liver I/R operation was performed.Single-cell RNA sequencing(scRNA-seq)in the liver post I/R suggested enhanced ferroptosis by Dhx58hep−/−.The mRNAs and proteins associated with DExH-box helicase 58(DHX58)were screened using RNA immunoprecipitation-sequencing(RIP-seq)and IP-mass spectrometry(IP-MS).Results Excessive production of reactive oxygen species(ROS)decreased the expression of the IFN-stimulated gene Dhx58 in hepatocytes and promoted hepatic ferroptosis,while treatment using IFN-αincreased DHX58 expression and prevented ferroptosis during liver I/R injury.Mechanistically,DHX58 with RNA-binding activity constitutively associates with the mRNA of glutathione peroxidase 4(GPX4),a central ferroptosis suppressor,and recruits the m6A reader YT521-B homology domain containing 2(YTHDC2)to promote the translation of Gpx4 mRNA in an m6A-dependent manner,thus enhancing GPX4 protein levels and preventing hepatic ferroptosis.Conclusions This study provides mechanistic evidence that IFN-αstimulates DHX58 to promote the translation of m6A-modified Gpx4 mRNA,suggesting the potential clinical application of IFN-αin the prevention of hepatic ferroptosis during liver I/R injury.
