Background Nanoparticles(NPs)are a class of substances that can be loaded with therapeutic agents delivered to specific areas.In our earlier research,we identified a neuron-derived circular RNA(circRNA),circular oxogl...Background Nanoparticles(NPs)are a class of substances that can be loaded with therapeutic agents delivered to specific areas.In our earlier research,we identified a neuron-derived circular RNA(circRNA),circular oxoglutarate dehydrogenase(CircOGDH),as a promising therapeutic target for acute ischaemic stroke.This study dedicated to explore a prospective preliminary strategy of CircOGDH-based NP delivered to the ischaemic penumbra region in middle cerebral artery occlusion/reperfusion(MCAO/R)mice.Methods Immunofluorescence in primary cortex neurons and in vivo fluorescence imaging revealed endocytosis of Poly(lactide-co-glycolide)(PLGA)poly amidoamine(PAMAM)@CircOGDH small interfering RNA(siRNA)NPs.Western blotting analysis and CCK8 assay were performed to evaluate the apoptotic level in ischaemic neurons treated with PLGA–PAMAM@CircOGDH siRNA NPs.Quantitative reverse transcription PCR experiments,mice behaviour test,T2 MRI analysis,Nissl and TdT-mediated dUTP nick end labeling(TUNEL)co-staining were performed to evaluate the apoptosis level of ischaemic penumbra neurons in MCAO/R mice.Biosafety evaluation of NPs in MCAO/R mice was detected by blood routine examination,liver and kidney function examination and HE staining.Results PLGA–PAMAM@CircOGDH siRNA NPs were successfully assembled.Endocytosis of PLGA–PAMAM@CircOGDH siRNA NPs in ischaemic neurons alleviated neuronal apoptotic level in vitro and in vivo.Furthermore,mice behaviour test showed that the neurological defects of MCAO/R mice were significantly alleviated after the tail injection of PLGA–PAMAM@CircOGDH siRNA NPs,and no toxic effects were observed.Conclusion In conclusion,our results suggest that PLGA–PAMAM@CircOGDH siRNA NPs can be delivered to the ischaemic penumbra region and alleviate neuron apoptosis in MCAO/R mice and in ischaemic neurons;therefore,our study provides a desirable approach for using circRNA-based NPs for the treatment of ischaemic stroke.展开更多
Few studies have investigated the properties and protein composition of small extracellular vesicles (sEVs) derived from neurons under hypoxic conditions. Presently, the extent of the involvement of these plentiful sE...Few studies have investigated the properties and protein composition of small extracellular vesicles (sEVs) derived from neurons under hypoxic conditions. Presently, the extent of the involvement of these plentiful sEVs in the onset and progression of ischemic stroke remains an unresolved question. Our study systematically identified the characteristics of sEVs derived from neurons under hypoxic conditions (HypEVs) by physical characterization, sEV absorption, proteomics and transcriptomics analysis. The effects of HypEVs on neurites, cell survival, and neuron structure were assessed in vitro and in vivo by neural complexity tests, magnetic resonance imaging (MRI), Golgi staining, and Western blotting of synaptic plasticity-related proteins and apoptotic proteins. Knockdown of Fused in Sarcoma (FUS) small interfering RNA (siRNA) was used to validate FUS-mediated HypEV neuroprotection and mitochondrial mRNA release. Hypoxia promoted the secretion of sEVs, and HypEVs were more easily taken up and utilized by recipient cells. The MRI results illustrated that the cerebral infarction volume was reduced by 45% with the application of HypEVs, in comparison to the non- HypEV treatment group. Mechanistically, the FUS protein is necessary for the uptake and neuroprotection of HypEVs against ischemic stroke as well as carrying a large amount of mitochondrial mRNA in HypEVs. However, FUS knockdown attenuated the neuroprotective rescue capabilities of HypEVs. Our comprehensive dataset clearly illustrates that FUS-mediated HypEVs deliver exceptional neuroprotective effects against ischemic stroke, primarily through the maintenance of neurite integrity and the reduction of mitochondria-associated apoptosis.展开更多
基金supported by grants from the National Natural Science Foundation of China(81801150,81971121,82271304,82171316 and 81671167)the Science and Technology Planning Project of Guangdong Province,China(2017A020215049 and 2019A050513005)+3 种基金Natural Science Foundation of Guangdong Province(2018A0303130182,2020A1515010279 and 2022A1515012311)the Fundamental Research Funds for the Central Universities(21621102)Science and Technology Projects in Guangzhou,China(2014Y2-00505,202002020003,202201010127 and 202201020042)Clinical Frontier Technology Program of the First Affiliated Hospital of Jinan University,China(JNU1AF-CFTP-2022-a01203).
文摘Background Nanoparticles(NPs)are a class of substances that can be loaded with therapeutic agents delivered to specific areas.In our earlier research,we identified a neuron-derived circular RNA(circRNA),circular oxoglutarate dehydrogenase(CircOGDH),as a promising therapeutic target for acute ischaemic stroke.This study dedicated to explore a prospective preliminary strategy of CircOGDH-based NP delivered to the ischaemic penumbra region in middle cerebral artery occlusion/reperfusion(MCAO/R)mice.Methods Immunofluorescence in primary cortex neurons and in vivo fluorescence imaging revealed endocytosis of Poly(lactide-co-glycolide)(PLGA)poly amidoamine(PAMAM)@CircOGDH small interfering RNA(siRNA)NPs.Western blotting analysis and CCK8 assay were performed to evaluate the apoptotic level in ischaemic neurons treated with PLGA–PAMAM@CircOGDH siRNA NPs.Quantitative reverse transcription PCR experiments,mice behaviour test,T2 MRI analysis,Nissl and TdT-mediated dUTP nick end labeling(TUNEL)co-staining were performed to evaluate the apoptosis level of ischaemic penumbra neurons in MCAO/R mice.Biosafety evaluation of NPs in MCAO/R mice was detected by blood routine examination,liver and kidney function examination and HE staining.Results PLGA–PAMAM@CircOGDH siRNA NPs were successfully assembled.Endocytosis of PLGA–PAMAM@CircOGDH siRNA NPs in ischaemic neurons alleviated neuronal apoptotic level in vitro and in vivo.Furthermore,mice behaviour test showed that the neurological defects of MCAO/R mice were significantly alleviated after the tail injection of PLGA–PAMAM@CircOGDH siRNA NPs,and no toxic effects were observed.Conclusion In conclusion,our results suggest that PLGA–PAMAM@CircOGDH siRNA NPs can be delivered to the ischaemic penumbra region and alleviate neuron apoptosis in MCAO/R mice and in ischaemic neurons;therefore,our study provides a desirable approach for using circRNA-based NPs for the treatment of ischaemic stroke.
基金the National Natural Science Foundation of China(82271304,81801150,81971121,82171316 and 81671167)the Science and Technology Planning Project of Guangdong Province,China(2017A020215049,2019A050513005)+6 种基金Natural Science Foundation of Guangdong Province(2018A0303130182,2020A1515010279 and 2022A1515012311)the Fundamental Research Funds for the Central Universities(21621102)Science and Technology Projects in Guangzhou,China(2014Y2-00505,202002020003,202201010127,and SL2023A03J01214)Science and Technology Program of Guangzhou:Key Lab of Guangzhou Basic and Translational Research of Pan-vascular Diseases(202201020042)Young Talent Support Project of Guangzhou Association for Science and Technology(QT-2023-024)Guangdong Basic and Applied Basic Research Foundation(2021A1515111226)China Postdoctoral Science Foundation(2022M710058).
文摘Few studies have investigated the properties and protein composition of small extracellular vesicles (sEVs) derived from neurons under hypoxic conditions. Presently, the extent of the involvement of these plentiful sEVs in the onset and progression of ischemic stroke remains an unresolved question. Our study systematically identified the characteristics of sEVs derived from neurons under hypoxic conditions (HypEVs) by physical characterization, sEV absorption, proteomics and transcriptomics analysis. The effects of HypEVs on neurites, cell survival, and neuron structure were assessed in vitro and in vivo by neural complexity tests, magnetic resonance imaging (MRI), Golgi staining, and Western blotting of synaptic plasticity-related proteins and apoptotic proteins. Knockdown of Fused in Sarcoma (FUS) small interfering RNA (siRNA) was used to validate FUS-mediated HypEV neuroprotection and mitochondrial mRNA release. Hypoxia promoted the secretion of sEVs, and HypEVs were more easily taken up and utilized by recipient cells. The MRI results illustrated that the cerebral infarction volume was reduced by 45% with the application of HypEVs, in comparison to the non- HypEV treatment group. Mechanistically, the FUS protein is necessary for the uptake and neuroprotection of HypEVs against ischemic stroke as well as carrying a large amount of mitochondrial mRNA in HypEVs. However, FUS knockdown attenuated the neuroprotective rescue capabilities of HypEVs. Our comprehensive dataset clearly illustrates that FUS-mediated HypEVs deliver exceptional neuroprotective effects against ischemic stroke, primarily through the maintenance of neurite integrity and the reduction of mitochondria-associated apoptosis.
