OBJECTIVE To investigate the effects of IMM-H004 on permanent focal cerebral ischemia injury and associated cardiopulmonary complications,further elucidating the molecular mechanisms.METHODS The effects of IMM-H004 we...OBJECTIVE To investigate the effects of IMM-H004 on permanent focal cerebral ischemia injury and associated cardiopulmonary complications,further elucidating the molecular mechanisms.METHODS The effects of IMM-H004 were investigated in wild-type(WT) and CKLF1-/-rats.The effects of IMM-H004 on ischemic stroke injury and its cardiopulmonary complications were determined using 2,3,5-triphenyltetrazolium chloride(TTC) staining,behavior tests,magnetic resonance imaging(MRI)scans,enzyme-linked immunosorbent assay(ELISA),Nissl staining,and histo-pathological examination.Multiple molecular experiments including immunohistological staining,immunofluorescence staining,quantitative RT-PCR,Western blotting,and co-immunoprecipitation assays were used to elucidate the underlying mechanisms.RESULTS IMM-H004 treatment provided significant protection against ischemic stroke-induced brain injury and associated cardiopulmonary complications,through CKLF1-depedent-anti-inflammation pathway in rats.IMM-H004 downregulated the amount of CKLF1 and disturbed the combination between CKLF1 and C-C chemokine receptor type 4,suppressing the inflammatory response and protecting the damaged organs in ischemic setting.CONCLUSION This preclinical study established efficacy of IMM-H004 as a potential therapeutic medicine for ischemic stroke and associated cardiopulmonary complications.The protective effects of IMM-H004 may due to its specific mechanism through CKLF1.These results support further efforts to develop IMM-H004 for human clinical trials in acute cerebral ischemia,especially for patients who are not suitable for reperfusion therapy.展开更多
OBJECTIVE To investigate the CKLF1 mediated expression of microglia/macrophage phenotypes in vitro and in vivo,discussing the involved pathway.METHODS In vitro,primary microglia isolated from mice cortex were used to ...OBJECTIVE To investigate the CKLF1 mediated expression of microglia/macrophage phenotypes in vitro and in vivo,discussing the involved pathway.METHODS In vitro,primary microglia isolated from mice cortex were used to study the effects of CKLF1 by qPCR analysis and immunofluorescence staining.In vivo,WT C57 and CKLF1 deficient mice were used to explore the effects of CKLF1.TTC staining,MRI and Nissl staining were applied to examine the infarction or neuron loss.Zea longa test was used to evaluate the neurological deficit of mice.Western blotting was used to investigate the changes of specific protein and discuss the involved pathway.We also used qPCR analysis and immunofluorescence staining for polarization markers to determine the effects of CKLF1.RESULTS CKLF1 could drive primary microglia to M1 phenotype for 24 h stimulation in primary microglia.In mice transient ischemic stroke model,CKLF1 attenuated ischemic injury,and accompanied by promoting microglia/macrophage toward M1 polarization.Increased expression of pro-inflammatory cytokines and decreased expression of neurotropic factors and anti-inflammatory cytokines were observed in mice subjected to cerebral ischemia with C27.Moreover,NF-κB activation enhancement was detected in C27 modulated M1 polarization effects.CONCLUSION CKLF1 is an important mediator of driving M1 phenotype of microglia/macrophage at early stage of cerebral ischemic injury,contributing to aggravation of cerebral ischemia injury,which closely related to microglia/macrophage M1 polarization guided inflammatory response.Targeting CKLF1 has the potential to treat ischemic stroke.展开更多
基金The project supported by ZYBZH-Y-HUN-24National Natural Science Foundation of China(81730096+6 种基金U1402221)National Mega-project for Innovative Drugs(2018ZX09711001-002-0072018ZX09711001-003-0052018ZX09711001-009-013)CAMS Innovation Fund for Medical Sciences(2016-I2M-1-004)Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study(BZ0150)PUMC Graduate Educationand Teaching Reform Project(10023201600801)
文摘OBJECTIVE To investigate the effects of IMM-H004 on permanent focal cerebral ischemia injury and associated cardiopulmonary complications,further elucidating the molecular mechanisms.METHODS The effects of IMM-H004 were investigated in wild-type(WT) and CKLF1-/-rats.The effects of IMM-H004 on ischemic stroke injury and its cardiopulmonary complications were determined using 2,3,5-triphenyltetrazolium chloride(TTC) staining,behavior tests,magnetic resonance imaging(MRI)scans,enzyme-linked immunosorbent assay(ELISA),Nissl staining,and histo-pathological examination.Multiple molecular experiments including immunohistological staining,immunofluorescence staining,quantitative RT-PCR,Western blotting,and co-immunoprecipitation assays were used to elucidate the underlying mechanisms.RESULTS IMM-H004 treatment provided significant protection against ischemic stroke-induced brain injury and associated cardiopulmonary complications,through CKLF1-depedent-anti-inflammation pathway in rats.IMM-H004 downregulated the amount of CKLF1 and disturbed the combination between CKLF1 and C-C chemokine receptor type 4,suppressing the inflammatory response and protecting the damaged organs in ischemic setting.CONCLUSION This preclinical study established efficacy of IMM-H004 as a potential therapeutic medicine for ischemic stroke and associated cardiopulmonary complications.The protective effects of IMM-H004 may due to its specific mechanism through CKLF1.These results support further efforts to develop IMM-H004 for human clinical trials in acute cerebral ischemia,especially for patients who are not suitable for reperfusion therapy.
基金National Natural Science Foundation of China(81730096U1402221)+4 种基金National Mega-projectfor Innovative Drugs (2018ZX09711001-002-0072018ZX09711001-003-0052018ZX09711001-009-013)CAMS Innovation Fund for MedicalSciences (CIFMS) (2016-I2M-1-004)Beijing KeyL
文摘OBJECTIVE To investigate the CKLF1 mediated expression of microglia/macrophage phenotypes in vitro and in vivo,discussing the involved pathway.METHODS In vitro,primary microglia isolated from mice cortex were used to study the effects of CKLF1 by qPCR analysis and immunofluorescence staining.In vivo,WT C57 and CKLF1 deficient mice were used to explore the effects of CKLF1.TTC staining,MRI and Nissl staining were applied to examine the infarction or neuron loss.Zea longa test was used to evaluate the neurological deficit of mice.Western blotting was used to investigate the changes of specific protein and discuss the involved pathway.We also used qPCR analysis and immunofluorescence staining for polarization markers to determine the effects of CKLF1.RESULTS CKLF1 could drive primary microglia to M1 phenotype for 24 h stimulation in primary microglia.In mice transient ischemic stroke model,CKLF1 attenuated ischemic injury,and accompanied by promoting microglia/macrophage toward M1 polarization.Increased expression of pro-inflammatory cytokines and decreased expression of neurotropic factors and anti-inflammatory cytokines were observed in mice subjected to cerebral ischemia with C27.Moreover,NF-κB activation enhancement was detected in C27 modulated M1 polarization effects.CONCLUSION CKLF1 is an important mediator of driving M1 phenotype of microglia/macrophage at early stage of cerebral ischemic injury,contributing to aggravation of cerebral ischemia injury,which closely related to microglia/macrophage M1 polarization guided inflammatory response.Targeting CKLF1 has the potential to treat ischemic stroke.
