Blocking postsynaptic density-93 binding to C-X3-C motif chemokine ligand 1 promotes microglial phenotypic transformation during acute ischemic stroke

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.

C-X3-C motif chemokine ligand 1/receptor 1 regulates the M1 polarization and chemotaxis of macrophages after hypoxia/reoxygenation injury

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.

G1254023X对白细胞介素6受体、趋化因子CX3CL1的抑制作用及其对冠心病的影响

目的通过检测G1254023X干预的冠心病（CHD）小鼠主动脉血管直径及白细胞介素6受体（IL-6R）、趋化因子Chemokine（C-X3-Cmotif）ligand1（CX3CL1）的表达，初步探究去整合素和金属蛋白酶10（ADAM10）影响CHD的机制。方法用高脂高糖饲料饲养Ap0E基因敲除小鼠制备CHD小鼠模型；采用B超法测量G1254023X组、模型组及空白对照组小鼠的主动脉血管直径；采用酶联免疫吸附实验（ELISA）分别检测三组小鼠外周血IL-6R、CX3CL1的浓度；采用苏木素一伊红染色法（HE）检测小鼠主动脉组织的病理变化；分别用蛋白免疫印记法（WB），免疫组织化学法（IHC）检测三组小鼠主动脉组织中IL-6R、CX3CL1的表达差异。测量G1254023X组、模型组及空白对照组小鼠的主动脉血管直径。结果模型组小鼠主动脉血管直径显著大于G1254023X组；模型组小鼠主动脉组织病理学变化大于G1254023X组；外周血样本和主动脉组织中IL-6R、CX3CL1浓度G1254023X组显著低于模型组；各项指标G1254023X组与空白对照组无显著差异。结论特异性ADAM10抑制剂G1254023X可以有效缓解CHD小鼠主动脉血管直径狭窄，抑制IL-6R、CX3CL1的表达，延缓冠心病发展。