We previously reported that postsynaptic density-93 mediates neuron-microglia crosstalk by interacting with amino acids 357–395 of C-X3-C motif chemokine ligand 1(CX3 CL1) to induce microglia polarization. More impor...We previously reported that postsynaptic density-93 mediates neuron-microglia crosstalk by interacting with amino acids 357–395 of C-X3-C motif chemokine ligand 1(CX3 CL1) to induce microglia polarization. More importantly, the peptide Tat-CX3 CL1(comprising amino acids 357–395 of CX3 CL1) disrupts the interaction between postsynaptic density-93 and CX3 CL1, reducing neurological impairment and exerting a protective effect in the context of acute ischemic stroke. However, the mechanism underlying these effects remains unclear. In the current study, we found that the pro-inflammatory M1 phenotype increased and the anti-inflammatory M2 phenotype decreased at different time points. The M1 phenotype increased at 6 hours after stroke and peaked at 24 hours after perfusion, whereas the M2 phenotype decreased at 6 and 24 hours following reperfusion. We found that the peptide Tat-CX3 CL1(357–395 aa) facilitates microglial polarization from M1 to M2 by reducing the production of soluble CX3 CL1. Furthermore, the a disintegrin and metalloprotease domain 17(ADAM17) inhibitor GW280264 x, which inhibits metalloprotease activity and prevents CX3 CL1 from being sheared into its soluble form, facilitated microglial polarization from M1 to M2 by inhibiting soluble CX3 CL1 formation. Additionally, Tat-CX3 CL1(357–395 aa) attenuated long-term cognitive deficits and improved white matter integrity as determined by the Morris water maze test at 31–34 days following surgery and immunofluorescence staining at 35 days after stroke, respectively. In conclusion, Tat-CX3 CL1(357–395 aa) facilitates functional recovery after ischemic stroke by promoting microglial polarization from M1 to M2. Therefore, the Tat-CX3 CL1(357–395 aa) is a potential therapeutic agent for ischemic stroke.展开更多
Background:Macrophages play an important role in renal ischemia reperfusion injury,but the functional changes of macrophages under hypoxia/reoxygenation and the related mechanism are unclear and need to be further cla...Background:Macrophages play an important role in renal ischemia reperfusion injury,but the functional changes of macrophages under hypoxia/reoxygenation and the related mechanism are unclear and need to be further clarified.Methods:The effects of hypoxia/reoxygenation on functional characteristics of RAW264.7 macrophages were analyzed through the protein expression detection of pro-inflammatory factors TNF-αand CD80,anti-inflammatory factors ARG-1 and CD206.The functional implications of C-X3-C motif chemokine receptor 1(CX3CR1)down-regulation in hypoxic macrophages were explored using small interfering RNA technology.Significance was assessed by the parametrict-test or nonparametric Mann-Whitney test for two group comparisons,and a one-way ANOVA or the Kruskal-Wallis test for multiple group comparisons.Results:Hypoxia/reoxygenation significantly increased the protein expression of M1-related pro-inflammatory factors TNF-α,CD80 and chemokine C-X3-C motif chemokine ligand 1(CX3CL1)/CX3CR1 and inhibited the protein expression of M2-related anti-inflammatory factors ARG-1 and CD206 in a time-dependent manner in RAW264.7 cells.However,the silencing of CX3CR1 in RAW264.7 cells using specific CX3CR1-siRNA,significantly attenuated the increase in protein expression of TNF-α(P<0.05)and CD80(P<0.01)and the inhibition of ARG-1(P<0.01)and CD206(P<0.01)induced by hypoxia/reoxygenation.In addition,we also found that hypoxia/reoxygenation could significantly enhance the migration(2.2-fold,P<0.01)and adhesion capacity(1.5-fold,P<0.01)of RAW264.7 macrophages compared with the control group,and CX3CR1-siRNA had an inhibitory role(40%and 20%reduction,respectively).For elucidating the mechanism,we showed that the phosphorylation levels of ERK(P<0.01)and the p65 subunit of NF-κB(P<0.01)of the RAW264.7 cells in the hypoxic/reoxygenation group were significantly increased,which could be attenuated by down-regulation of CX3CR1 expression(P<0.01,both).ERK inhibitors also significantly blocked the effects of hypoxic/reoxygenation on the protein expression of M1-related pro-inflammatory factors TNF-α,CD80 and M2-related anti-inflammatory factors ARG-1 and CD206.Moreover,we found that conditioned medium from polarized M1 macrophages induced by hypoxia/reoxygenation,notably increased the degree of apoptosis of hypoxia/reoxygenation-induced TCMK-1 cells,and promoted the protein expression of pro-apoptotic proteins bax(P<0.01)and cleaved-caspase 3(P<0.01)and inhibited the expression of anti-apoptotic protein bcl-2(P<0.01),but silencing CX3CR1 in macrophages had a protective role.Finally,we also found that the secretion of soluble CX3CL1 in RAW264.7 macrophages under hypoxia/reoxygenation was significantly increased.Conclusions:The findings suggest that hypoxia/reoxygenation could promote M1 polarization,cell migration,and adhesion of macrophages,and that polarized macrophages induce further apoptosis of hypoxic renal tubular epithelial cells by regulating of CX3CL1/CX3CR1 signaling pathway.展开更多
目的:通过外源性注射趋化因子(C-X3-C基序)配体1(Fractalkine,CX3CL1)探索其对阿尔茨海默病(Alzheimer’sdisease,AD)小鼠神经元功能的影响及机制。方法:采用APP/PS1双转基因小鼠建立AD模型。将小鼠随机分为4组:对照组、AD模型组、AD+PB...目的:通过外源性注射趋化因子(C-X3-C基序)配体1(Fractalkine,CX3CL1)探索其对阿尔茨海默病(Alzheimer’sdisease,AD)小鼠神经元功能的影响及机制。方法:采用APP/PS1双转基因小鼠建立AD模型。将小鼠随机分为4组:对照组、AD模型组、AD+PBS溶剂组、AD+CX3CL1组。AD+CX3CL1组颅内给予CX3CL1。AD+PBS溶剂组注射等容积的PBS溶剂。采用旷场实验和Morris水迷宫测试小鼠认知行为,免疫荧光染色、TUNEL染色、尼氏染色、蛋白质印迹和RT-PCR技术对小鼠海马组织进行分析。结果:①AD小鼠接受CX3CL1注射后增加了移动距离,但没有显著差异(P=0.189);在水迷宫测试中,AD+CX3CL1组寻找水下平台的移动距离显著缩短(P=0.001);②AD模型组中GSDMD(Gasdermin D)蛋白和炎症相关蛋白表达显著升高(P<0.05),而CX3CL1注射能显著降低这些蛋白的表达水平(P<0.05);③AD模型组的尼氏体数量、突触相关蛋白25 kDa(synaptosomal-associated protein of 25 kDa,SNAP25)、突触后致密区蛋白95(post synaptic density protein 95,PSD95)和囊泡相关膜蛋白1(vesicle-associated membrane protein 1,VAMP1)表达水平显著降低,而CX3CL1组尼氏体数量(P=0.001)和SNAP25、PSD95、VAMP1蛋白的表达增加(P=0.043,P=0.026,P=0.003)。结论:外源性CX3CL1注射能够通过调节炎症因子和减少细胞焦亡来改善AD小鼠的神经元功能,保护神经元免受损伤。展开更多
基金supported by the National Natural Science Foundation of China,Nos. 82071304 (to QXZ), 81671149 (to QXZ),and 81971179 (to XML)the Natural Science Foundation of Jiangsu Province,Nos. BK20191463 (to XML) and BK20161167 (to QXZ)。
文摘We previously reported that postsynaptic density-93 mediates neuron-microglia crosstalk by interacting with amino acids 357–395 of C-X3-C motif chemokine ligand 1(CX3 CL1) to induce microglia polarization. More importantly, the peptide Tat-CX3 CL1(comprising amino acids 357–395 of CX3 CL1) disrupts the interaction between postsynaptic density-93 and CX3 CL1, reducing neurological impairment and exerting a protective effect in the context of acute ischemic stroke. However, the mechanism underlying these effects remains unclear. In the current study, we found that the pro-inflammatory M1 phenotype increased and the anti-inflammatory M2 phenotype decreased at different time points. The M1 phenotype increased at 6 hours after stroke and peaked at 24 hours after perfusion, whereas the M2 phenotype decreased at 6 and 24 hours following reperfusion. We found that the peptide Tat-CX3 CL1(357–395 aa) facilitates microglial polarization from M1 to M2 by reducing the production of soluble CX3 CL1. Furthermore, the a disintegrin and metalloprotease domain 17(ADAM17) inhibitor GW280264 x, which inhibits metalloprotease activity and prevents CX3 CL1 from being sheared into its soluble form, facilitated microglial polarization from M1 to M2 by inhibiting soluble CX3 CL1 formation. Additionally, Tat-CX3 CL1(357–395 aa) attenuated long-term cognitive deficits and improved white matter integrity as determined by the Morris water maze test at 31–34 days following surgery and immunofluorescence staining at 35 days after stroke, respectively. In conclusion, Tat-CX3 CL1(357–395 aa) facilitates functional recovery after ischemic stroke by promoting microglial polarization from M1 to M2. Therefore, the Tat-CX3 CL1(357–395 aa) is a potential therapeutic agent for ischemic stroke.
基金supported by Scientific Research Starting Foundation of Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology(No.2014KYQD01,2018YJJA04)。
文摘Background:Macrophages play an important role in renal ischemia reperfusion injury,but the functional changes of macrophages under hypoxia/reoxygenation and the related mechanism are unclear and need to be further clarified.Methods:The effects of hypoxia/reoxygenation on functional characteristics of RAW264.7 macrophages were analyzed through the protein expression detection of pro-inflammatory factors TNF-αand CD80,anti-inflammatory factors ARG-1 and CD206.The functional implications of C-X3-C motif chemokine receptor 1(CX3CR1)down-regulation in hypoxic macrophages were explored using small interfering RNA technology.Significance was assessed by the parametrict-test or nonparametric Mann-Whitney test for two group comparisons,and a one-way ANOVA or the Kruskal-Wallis test for multiple group comparisons.Results:Hypoxia/reoxygenation significantly increased the protein expression of M1-related pro-inflammatory factors TNF-α,CD80 and chemokine C-X3-C motif chemokine ligand 1(CX3CL1)/CX3CR1 and inhibited the protein expression of M2-related anti-inflammatory factors ARG-1 and CD206 in a time-dependent manner in RAW264.7 cells.However,the silencing of CX3CR1 in RAW264.7 cells using specific CX3CR1-siRNA,significantly attenuated the increase in protein expression of TNF-α(P<0.05)and CD80(P<0.01)and the inhibition of ARG-1(P<0.01)and CD206(P<0.01)induced by hypoxia/reoxygenation.In addition,we also found that hypoxia/reoxygenation could significantly enhance the migration(2.2-fold,P<0.01)and adhesion capacity(1.5-fold,P<0.01)of RAW264.7 macrophages compared with the control group,and CX3CR1-siRNA had an inhibitory role(40%and 20%reduction,respectively).For elucidating the mechanism,we showed that the phosphorylation levels of ERK(P<0.01)and the p65 subunit of NF-κB(P<0.01)of the RAW264.7 cells in the hypoxic/reoxygenation group were significantly increased,which could be attenuated by down-regulation of CX3CR1 expression(P<0.01,both).ERK inhibitors also significantly blocked the effects of hypoxic/reoxygenation on the protein expression of M1-related pro-inflammatory factors TNF-α,CD80 and M2-related anti-inflammatory factors ARG-1 and CD206.Moreover,we found that conditioned medium from polarized M1 macrophages induced by hypoxia/reoxygenation,notably increased the degree of apoptosis of hypoxia/reoxygenation-induced TCMK-1 cells,and promoted the protein expression of pro-apoptotic proteins bax(P<0.01)and cleaved-caspase 3(P<0.01)and inhibited the expression of anti-apoptotic protein bcl-2(P<0.01),but silencing CX3CR1 in macrophages had a protective role.Finally,we also found that the secretion of soluble CX3CL1 in RAW264.7 macrophages under hypoxia/reoxygenation was significantly increased.Conclusions:The findings suggest that hypoxia/reoxygenation could promote M1 polarization,cell migration,and adhesion of macrophages,and that polarized macrophages induce further apoptosis of hypoxic renal tubular epithelial cells by regulating of CX3CL1/CX3CR1 signaling pathway.
文摘目的:通过外源性注射趋化因子(C-X3-C基序)配体1(Fractalkine,CX3CL1)探索其对阿尔茨海默病(Alzheimer’sdisease,AD)小鼠神经元功能的影响及机制。方法:采用APP/PS1双转基因小鼠建立AD模型。将小鼠随机分为4组:对照组、AD模型组、AD+PBS溶剂组、AD+CX3CL1组。AD+CX3CL1组颅内给予CX3CL1。AD+PBS溶剂组注射等容积的PBS溶剂。采用旷场实验和Morris水迷宫测试小鼠认知行为,免疫荧光染色、TUNEL染色、尼氏染色、蛋白质印迹和RT-PCR技术对小鼠海马组织进行分析。结果:①AD小鼠接受CX3CL1注射后增加了移动距离,但没有显著差异(P=0.189);在水迷宫测试中,AD+CX3CL1组寻找水下平台的移动距离显著缩短(P=0.001);②AD模型组中GSDMD(Gasdermin D)蛋白和炎症相关蛋白表达显著升高(P<0.05),而CX3CL1注射能显著降低这些蛋白的表达水平(P<0.05);③AD模型组的尼氏体数量、突触相关蛋白25 kDa(synaptosomal-associated protein of 25 kDa,SNAP25)、突触后致密区蛋白95(post synaptic density protein 95,PSD95)和囊泡相关膜蛋白1(vesicle-associated membrane protein 1,VAMP1)表达水平显著降低,而CX3CL1组尼氏体数量(P=0.001)和SNAP25、PSD95、VAMP1蛋白的表达增加(P=0.043,P=0.026,P=0.003)。结论:外源性CX3CL1注射能够通过调节炎症因子和减少细胞焦亡来改善AD小鼠的神经元功能,保护神经元免受损伤。
