Objective:Neuropathic pain(NP)is one of the most common forms of chronic pain,yet current treatment options are limited in effectiveness.Peripheral nerve injury activates spinal microglia,altering their inflammatory r...Objective:Neuropathic pain(NP)is one of the most common forms of chronic pain,yet current treatment options are limited in effectiveness.Peripheral nerve injury activates spinal microglia,altering their inflammatory response and phagocytic functions,which contributes to the progression of NP.Most current research on NP focuses on microglial inflammation,with relatively little attention to their phagocytic function.Early growth response factor 2(EGR2)has been shown to regulate microglial phagocytosis,but its specific role in NP remains unclear.This study aims to investigate how EGR2 modulates microglial phagocytosis and its involvement in NP,with the goal of identifying potential therapeutic targets.Methods:Adult male Sprague-Dawley(SD)rats were used to establish a chronic constriction injury(CCI)model of the sciatic nerve.Pain behaviors were assessed on days 1,3,7,10,and 14 post-surgery to confirm successful model induction.The temporal and spatial expression of EGR2 in the spinal cord was examined using real-time quantitative PCR(RT-qPCR),Western blotting,and immunofluorescence staining.Adeno-associated virus(AAV)was used to overexpress EGR2 in the spinal cord,and behavioral assessments were performed to evaluate the effects of EGR2 modulation of NP.CCI and lipopolysaccharide(LPS)models were established in animals and microglial cell lines,respectively,and changes in phagocytic activity were measured using RT-qPCR and fluorescent latex bead uptake assays.After confirming the involvement of microglial phagocytosis in NP,AAV was used to overexpress EGR2 in both in vivo and in vitro models,and phagocytic activity was further evaluated.Finally,eukaryotic transcriptome sequencing was conducted to screen differentially expressed mRNAs,followed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses to identify potential downstream effectors of EGR2.Results:The CCI model successfully induced NP.Following CCI,EGR2 expression in the spinal cord was upregulated in parallel with NP development.Overexpression of EGR2 via spinal AAV injection enhanced microglial phagocytic activity and increased pain hypersensitivity in rats.Both animal and cellular models showed that CCI or LPS stimulation enhanced microglial phagocytosis,which was further amplified by EGR2 overexpression.Transcriptomic analysis of spinal cord tissues from CCI rats overexpressing EGR2 revealed upregulation of numerous genes associated with microglial phagocytosis and pain regulation.Among them,Lag3 emerged as a potential downstream target of EGR2.Conclusion:EGR2 contributes to the maintenance of NP by enhancing microglial phagocytosis in the spinal dorsal horn.展开更多
The purpose of this study was to explore the effects of recombinant human intestinal alkaline phosphatase(recIAP) on human neutrophils in vitro, and the migration, phagocytosis, apoptosis in presence and absence of LP...The purpose of this study was to explore the effects of recombinant human intestinal alkaline phosphatase(recIAP) on human neutrophils in vitro, and the migration, phagocytosis, apoptosis in presence and absence of LPS. In this study, freshly extracted human neutrophils were used to establish an inflammatory cell model, and the control group, recIAP group, LPS group and recIAP +LPS group were set up to stimulate the model. The migration of neutrophils was detected by agarose gel drop method. Fluorescent particles and fluorescent probes were added to different treatment groups, and the phagocytic rate of neutrophils and the release of reactive oxygen species(ROS) from neutrophils were detected by flow cytometry. The apoptosis rate of neutrophils was detected by flow cytometry according to Annexin V-FITC apoptosis detection kit. The results showed that regardless of the presence or absence of LPS, recIAP could inhibit the migration of neutrophils, phagocytosis and the release of ROS. In addition, recIAP could weaken the inhibitory effect of LPS on neutrophils apoptosis.展开更多
Since the 1 960 s,many successful space missions have highlighted the advantages and necessity of humans in the exploration of space,but scientists have long worried about the adverse effects of spaceflight on Astrona...Since the 1 960 s,many successful space missions have highlighted the advantages and necessity of humans in the exploration of space,but scientists have long worried about the adverse effects of spaceflight on Astronaut.Space flight and models that create conditions similar to those that occur during space flight have been shown to deleteriously affect a variety of immunological responses.The mechanisms and biomedical consequences of these changes remain to be established.Conducting experiments in an environment of true microgravity requires a roundtrip ticket into space,a feat that is both expensive and challenging.Simulated microgravity(SMG)models allow scientists to gather preliminary data without the cost and logistical challenges of spaceflight.The objective of the present study was to evaluate the effects of SMG on immunity function of macrophages that exposed to RPM and RCCS separately.While many studies have demonstrated that alterations occur in the immune system as a result of space travel,the level at which these mechanisms exert their effect,at the level of the mature immune cell or earlier at the progenitor or stem cell stage is not known.In particular,macrophages,as one of the most important immune cells and play a key role in both specific and non-specific immunity,did not have received much attention.Therefore,in our study,we mainly study the influence of microgravity on the immune function of macrophages.In this study,we evaluated the immune dysfunction of macrophages under SMG.Firstly,we found that the morphology and structure of the macrophages were changed,specifically,we observed that there were more protrusions on cell surface and the cells were shrinking significantly after exposure to SMG.Secondly,we demonstrated that under simulated microgravity(SMG)conditions,the phagocytic and proliferative functions of macrophages were significantly reduced.Thirdly,several processes,including surface receptor expression,cytoskeleton,and cytokines secreted were investigated in macrophages under SMG.Phagocytosis is one of the important means for macrophages to exert immune function,and cell surface phagocytosis-related receptors play an important role.Here,we selected four common receptors(TLR2,FcyR1,CD11b and CD 18)to detect.The results indicate that SMG(RPM and RCCS)have a great influence on the expression of surface phagocytosis-related receptors,which may be one of the main reasons for the decline of immune function ofmacrophages.Macrophages exert immune function through phagocytosis,and the cytoskeleton plays an important role in the process of phagocytosis.The results indicate that SMG(RPM and RCCS)have a great influence on the expression of cytoskeleton-related proteins,which provides me with a new idea that SMG may regulate immunity of macrophage by affecting the cytoskeleton.Immune-related cytokines play an important role in macrophage immune process.Here,we selected four common immunocytokine(TNF-α,IL-1β,IL-6 and IL-10)to detect.The change of these four immunocytokine further demonstrate that SMG significantly decline the immunity of macrophage,we must pay enough attention to the impact of SMG on macrophage.The above factors such as the changes of morphology and structure of the macrophages and the decreased expression of Arp2/3 complex related proteins,cytokine secretion,and cell surface receptors may be responsible for the immune dysfunction of macrophages under SMG.展开更多
OBJECTIVE To establish an in vitro cel model based on patient-specific human microglia to study the pathological mechanism of Alzheimer disease(AD) and to screen candidate drugs.METHODS First,the induced pluripotent s...OBJECTIVE To establish an in vitro cel model based on patient-specific human microglia to study the pathological mechanism of Alzheimer disease(AD) and to screen candidate drugs.METHODS First,the induced pluripotent stem cells(iPSCs) of AD patients and cognitive normal controls(CNC) were induced to hematopoietic progenitor cells(HPCs),and then HPCs were further induced with IL-34,M-CSF,GM-CSF and TGF-β1 for 20 d to obtain microglialike cells(MGLCs).HPCs were isolated by flow cytometry and MGLCs were identified by immunofluorescence.Cell phagocytosis was determined by phagocytosis neutral red experiusing Luminex assay kits,and the cell growth curve during the experiment was recorded by IncuCyte ZOOM.The phagocytic ability and secretion of cytokines of MGLCs were observed under the stimulation of LPS.RESULTS MGLCs from AD patients(AD-MGLCs) and CNC expressed microglia markers IBA1,TMEM119,P2 RY12,TREM2 and CD11 B.The results of phagocytosis neutral red experiment showed that under normal conditions,AD-MGLCs had stronger phagocytic ability(P<0.01).Stimulation by LPS resulted in increased phagocytosis of cel s,and the increase in phagocytosis of CNC-MGLCs was higher than AD-MGLCs(P<0.01).Experiments showed that high concentrations of LPS(>2 mg·L^(-1)) resulted in CNC-microglia death(P<0.01),whereas ADMGLCs did not show significant death.The cytokine assay showed that under normal conditions,the concentrations of IFN-γ and IL-2 secreted by AD patients were slightly higher than those of CNC.After LPS stimulation,the secretion of TNF-α,IL-6 and IL-10 was significantly increased.The increased secretion of AD-MGLCs was greater than that CNC-MGLCs(P<0.01).CONCLUSION AD-iPSCs derived MGLCs exhibit significant inflammatory characteristics and are more active than CNC,which may be associated with chronic inflammatory responses caused by microglia in AD,thus may provide valuable new tools to screen candidate drugs for the disease and to discover the mechanisms underlying AD pathogenesis.展开更多
Alzheimer disease(AD)is a common neurodegenerative disease in the elderly,but nowadays the pathogenesis of AD is unclear.Myeloid cell 2 trigger receptor(TREM2)is one of the most famous and most common rare mutations i...Alzheimer disease(AD)is a common neurodegenerative disease in the elderly,but nowadays the pathogenesis of AD is unclear.Myeloid cell 2 trigger receptor(TREM2)is one of the most famous and most common rare mutations in neurodegenerative disease research,and its functional site mutation can significantly increase the incidence of AD.In this paper,we summary the structure,localization,and function and related signaling pathways of TREM2,review the latest epidemiological findings of TREM2 associated with the pathogenesis of AD,and speculate on the possible role of TREM2 in the progression of this disease,as well as the expression of TREM2 and the role of soluble TREM2 in AD brain are further elucidated.Based on the potential protective effect of TREM2 in the pathogenesis of AD,Therefore,targeting TREM2 may provide new opportunities and a reference for AD treatment.As the TREM2 variant appears to be widely involved in neurodegenerative diseases,there is an urgent need to further study the function of TREM2 in the brain and to find its ligands involved in TREM2-mediated signaling transduction and its specific role in AD pathogenesis.展开更多
基金supported by the National Natural Science Foundation of China(82071249 and 81771207).
文摘Objective:Neuropathic pain(NP)is one of the most common forms of chronic pain,yet current treatment options are limited in effectiveness.Peripheral nerve injury activates spinal microglia,altering their inflammatory response and phagocytic functions,which contributes to the progression of NP.Most current research on NP focuses on microglial inflammation,with relatively little attention to their phagocytic function.Early growth response factor 2(EGR2)has been shown to regulate microglial phagocytosis,but its specific role in NP remains unclear.This study aims to investigate how EGR2 modulates microglial phagocytosis and its involvement in NP,with the goal of identifying potential therapeutic targets.Methods:Adult male Sprague-Dawley(SD)rats were used to establish a chronic constriction injury(CCI)model of the sciatic nerve.Pain behaviors were assessed on days 1,3,7,10,and 14 post-surgery to confirm successful model induction.The temporal and spatial expression of EGR2 in the spinal cord was examined using real-time quantitative PCR(RT-qPCR),Western blotting,and immunofluorescence staining.Adeno-associated virus(AAV)was used to overexpress EGR2 in the spinal cord,and behavioral assessments were performed to evaluate the effects of EGR2 modulation of NP.CCI and lipopolysaccharide(LPS)models were established in animals and microglial cell lines,respectively,and changes in phagocytic activity were measured using RT-qPCR and fluorescent latex bead uptake assays.After confirming the involvement of microglial phagocytosis in NP,AAV was used to overexpress EGR2 in both in vivo and in vitro models,and phagocytic activity was further evaluated.Finally,eukaryotic transcriptome sequencing was conducted to screen differentially expressed mRNAs,followed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses to identify potential downstream effectors of EGR2.Results:The CCI model successfully induced NP.Following CCI,EGR2 expression in the spinal cord was upregulated in parallel with NP development.Overexpression of EGR2 via spinal AAV injection enhanced microglial phagocytic activity and increased pain hypersensitivity in rats.Both animal and cellular models showed that CCI or LPS stimulation enhanced microglial phagocytosis,which was further amplified by EGR2 overexpression.Transcriptomic analysis of spinal cord tissues from CCI rats overexpressing EGR2 revealed upregulation of numerous genes associated with microglial phagocytosis and pain regulation.Among them,Lag3 emerged as a potential downstream target of EGR2.Conclusion:EGR2 contributes to the maintenance of NP by enhancing microglial phagocytosis in the spinal dorsal horn.
基金the Heilongjiang Natural Science Fund Project (C2017035)。
文摘The purpose of this study was to explore the effects of recombinant human intestinal alkaline phosphatase(recIAP) on human neutrophils in vitro, and the migration, phagocytosis, apoptosis in presence and absence of LPS. In this study, freshly extracted human neutrophils were used to establish an inflammatory cell model, and the control group, recIAP group, LPS group and recIAP +LPS group were set up to stimulate the model. The migration of neutrophils was detected by agarose gel drop method. Fluorescent particles and fluorescent probes were added to different treatment groups, and the phagocytic rate of neutrophils and the release of reactive oxygen species(ROS) from neutrophils were detected by flow cytometry. The apoptosis rate of neutrophils was detected by flow cytometry according to Annexin V-FITC apoptosis detection kit. The results showed that regardless of the presence or absence of LPS, recIAP could inhibit the migration of neutrophils, phagocytosis and the release of ROS. In addition, recIAP could weaken the inhibitory effect of LPS on neutrophils apoptosis.
文摘Since the 1 960 s,many successful space missions have highlighted the advantages and necessity of humans in the exploration of space,but scientists have long worried about the adverse effects of spaceflight on Astronaut.Space flight and models that create conditions similar to those that occur during space flight have been shown to deleteriously affect a variety of immunological responses.The mechanisms and biomedical consequences of these changes remain to be established.Conducting experiments in an environment of true microgravity requires a roundtrip ticket into space,a feat that is both expensive and challenging.Simulated microgravity(SMG)models allow scientists to gather preliminary data without the cost and logistical challenges of spaceflight.The objective of the present study was to evaluate the effects of SMG on immunity function of macrophages that exposed to RPM and RCCS separately.While many studies have demonstrated that alterations occur in the immune system as a result of space travel,the level at which these mechanisms exert their effect,at the level of the mature immune cell or earlier at the progenitor or stem cell stage is not known.In particular,macrophages,as one of the most important immune cells and play a key role in both specific and non-specific immunity,did not have received much attention.Therefore,in our study,we mainly study the influence of microgravity on the immune function of macrophages.In this study,we evaluated the immune dysfunction of macrophages under SMG.Firstly,we found that the morphology and structure of the macrophages were changed,specifically,we observed that there were more protrusions on cell surface and the cells were shrinking significantly after exposure to SMG.Secondly,we demonstrated that under simulated microgravity(SMG)conditions,the phagocytic and proliferative functions of macrophages were significantly reduced.Thirdly,several processes,including surface receptor expression,cytoskeleton,and cytokines secreted were investigated in macrophages under SMG.Phagocytosis is one of the important means for macrophages to exert immune function,and cell surface phagocytosis-related receptors play an important role.Here,we selected four common receptors(TLR2,FcyR1,CD11b and CD 18)to detect.The results indicate that SMG(RPM and RCCS)have a great influence on the expression of surface phagocytosis-related receptors,which may be one of the main reasons for the decline of immune function ofmacrophages.Macrophages exert immune function through phagocytosis,and the cytoskeleton plays an important role in the process of phagocytosis.The results indicate that SMG(RPM and RCCS)have a great influence on the expression of cytoskeleton-related proteins,which provides me with a new idea that SMG may regulate immunity of macrophage by affecting the cytoskeleton.Immune-related cytokines play an important role in macrophage immune process.Here,we selected four common immunocytokine(TNF-α,IL-1β,IL-6 and IL-10)to detect.The change of these four immunocytokine further demonstrate that SMG significantly decline the immunity of macrophage,we must pay enough attention to the impact of SMG on macrophage.The above factors such as the changes of morphology and structure of the macrophages and the decreased expression of Arp2/3 complex related proteins,cytokine secretion,and cell surface receptors may be responsible for the immune dysfunction of macrophages under SMG.
基金National Key Research and Development Program(2016YFC1306301).
文摘OBJECTIVE To establish an in vitro cel model based on patient-specific human microglia to study the pathological mechanism of Alzheimer disease(AD) and to screen candidate drugs.METHODS First,the induced pluripotent stem cells(iPSCs) of AD patients and cognitive normal controls(CNC) were induced to hematopoietic progenitor cells(HPCs),and then HPCs were further induced with IL-34,M-CSF,GM-CSF and TGF-β1 for 20 d to obtain microglialike cells(MGLCs).HPCs were isolated by flow cytometry and MGLCs were identified by immunofluorescence.Cell phagocytosis was determined by phagocytosis neutral red experiusing Luminex assay kits,and the cell growth curve during the experiment was recorded by IncuCyte ZOOM.The phagocytic ability and secretion of cytokines of MGLCs were observed under the stimulation of LPS.RESULTS MGLCs from AD patients(AD-MGLCs) and CNC expressed microglia markers IBA1,TMEM119,P2 RY12,TREM2 and CD11 B.The results of phagocytosis neutral red experiment showed that under normal conditions,AD-MGLCs had stronger phagocytic ability(P<0.01).Stimulation by LPS resulted in increased phagocytosis of cel s,and the increase in phagocytosis of CNC-MGLCs was higher than AD-MGLCs(P<0.01).Experiments showed that high concentrations of LPS(>2 mg·L^(-1)) resulted in CNC-microglia death(P<0.01),whereas ADMGLCs did not show significant death.The cytokine assay showed that under normal conditions,the concentrations of IFN-γ and IL-2 secreted by AD patients were slightly higher than those of CNC.After LPS stimulation,the secretion of TNF-α,IL-6 and IL-10 was significantly increased.The increased secretion of AD-MGLCs was greater than that CNC-MGLCs(P<0.01).CONCLUSION AD-iPSCs derived MGLCs exhibit significant inflammatory characteristics and are more active than CNC,which may be associated with chronic inflammatory responses caused by microglia in AD,thus may provide valuable new tools to screen candidate drugs for the disease and to discover the mechanisms underlying AD pathogenesis.
基金supported by National Natural Science Foundation of China(81473549)
文摘Alzheimer disease(AD)is a common neurodegenerative disease in the elderly,but nowadays the pathogenesis of AD is unclear.Myeloid cell 2 trigger receptor(TREM2)is one of the most famous and most common rare mutations in neurodegenerative disease research,and its functional site mutation can significantly increase the incidence of AD.In this paper,we summary the structure,localization,and function and related signaling pathways of TREM2,review the latest epidemiological findings of TREM2 associated with the pathogenesis of AD,and speculate on the possible role of TREM2 in the progression of this disease,as well as the expression of TREM2 and the role of soluble TREM2 in AD brain are further elucidated.Based on the potential protective effect of TREM2 in the pathogenesis of AD,Therefore,targeting TREM2 may provide new opportunities and a reference for AD treatment.As the TREM2 variant appears to be widely involved in neurodegenerative diseases,there is an urgent need to further study the function of TREM2 in the brain and to find its ligands involved in TREM2-mediated signaling transduction and its specific role in AD pathogenesis.
