Anaglecine Regulates The Microglial Phenotypic Polarization Through TLR4/MyD88/NF-?B Pathway

Objective:Ischemic stroke?ischemic stroke,IS?is the second cause of death after ischemic heart disease.At present,intravenous thrombolysis is the only clinically proven effective treatment,but its has a time window limitation and the potential risk of causing severe cerebral ischemia.The mechanism of secondary brain injury after ischemia may be due to inflammation in the brain after ischemia,which accelerates the formation of ischemic injury and affects neuron death and tissue regeneration.Neuroinflammation is characterized by microglial activation and increased secretion of pro-inflammation cytokines.More and more studies have shown that regulating microglial polarization and inhibiting microglial activation to protect neurons may be an important treatment method of ischemic brain injury.Analgecine?AGC?is a biologically active preparation extracted from rabbit skin tissue of Japanese white rabbits infected with vaccinia virus.Previous studies shown that AGC can reduce the area of cerebral infarction during middle artery embolization and improve the behavior the score of rats,but the mechanism remains unknown.The aim of the present study was to investigate the mechanism of AGC on regulating the microglial polarization and inhibiting the inflammation activation based on the TLR4/MyD88/NF-?B pathway.Methods:?1?To establish a model of microglial injury induced by deficient glucose/hypoxia and reoxygenation:Mouse microglia cells?BV-2?were added to the sugar-free medium with sodium dithionite?Na₂S₂O₄?for 1.5h and then replaced with normal medium,while giving different concentrations of AGC?0.25,0.5,1U/mL?,the cells were treated after 3h of incubation.?1?The contents of cytokines were determined by the cell supernatant,and the effect of AGC on cytokines secretion was observed.?2?The expression of M1-type microglial marker CD16+32+were observed by flow cytometry.?3?Western blot was carried out to detect the expression of TLR4,TLR9,MyD88,TRAF6,p-p65 and p65.?4?The nuclear translocation of p65 was observed by laser confocal microscope.?2?MyD88-siRNA was transfected into BV-2 cells,and the medium was replaced after 43.5h.Then the sugar-free medium with sodium dithionite?Na₂S₂O₄?was added for 1.5h and replaced with normal medium,while giving different concentrations of AGC?0.25,0.5,1U/mL?,the cells were treated after 3h of incubation.Western blot was carried out to detect the expression of TLR4,MyD88,p-p65 and p65.?3?M1-type microglia were induced by LPS+IFN-?for 18h and replaced with normal medium,while giving different concentrations of AGC?0.25,0.5,1U/mL?,the cells were treated after 24h of incubation.The expression of M1-type microglia marker CD16+32+and M2-type microglia marker CD206+were observed by flow cytometry.?4?M2-type microglia were induced by IL-4 for 24h,and LPS+IFN-?were added to convert them from M2-type to M1-type for 18h,and then replaced with normal medium,while giving different concentrations of AGC?0.25,0.5,1U/mL?,the cells were treated after 24h of incubation.The expression of M1-type microglia marker CD16+32+and M2-type microglia marker CD206+were observed by flow cytometry.?5?M1-type microglia were induced by LPS+IFN-?for 18h and replaced with normal medium,while giving different concentrations of AGC?0.25,0.5,1U/mL?for24h and replaced with normal medium.Non-contact co-culture of BV-2 cells with primary cortical neurons of C57 mice uses Transwell culture method for 24h.Then MAP-2 antibody was added for staining,and the morphological changes of neurons were observed by immunofluorescence.?6?M1-type microglia were induced by LPS+IFN-?for 18h and replaced with normal medium,while giving different concentrations of AGC?0.25,0.5,1U/mL?for24h and replaced with normal medium.With the method of contact co-culture,BV-2cells were directly exposed to primary cortical neurons of C57 mice for 24h.Then MAP-2 antibody was added for staining,and the morphological changes of neurons were observed by immunofluorescence.Results:?1?Cytokine secretion in supernatant after oxygen-glucose deprivation/reperfusion?OGD/R?by Luminex.The results shown that,compared with the control group,Eotaxin,GM-CSF,IFN-?,IL-1?,IL-6,IL-9,IL-12P40,IL-12P70,IL-17?,MIP-1?and TNF-?level in OGD/R group were increased,IL-40 and IL-10 level in OGD/R group were decreased.Compared with OGD/R group,Eotaxin,GM-CSF,IFN-?,IL-1?,IL-6,IL-9,IL-12P40,IL-12P70,IL-17?,MIP-1?and TNF-?level in each AGC dose groups,peptide 1 group and peptide 5 group were decreased,IL-40and IL-10 level in each AGC dose groups,peptide 1 group and peptide 5 group were increased.?2?Expression of M1-type phenotypic marker after OGD/R was observed by flow cytometry.The results shown that,compared with the control group,the expression of CD16+CD32+in OGD/R group was increased.Compared with OGD/R group,the expression of CD16+CD32+in each AGC dose groups was decreased.?3?Western blot was used to observe the expression of related proteins in TLR4 signaling pathway.The results shown that,compared with the control group,the expression of TLR4,MyD88,TRAF6 and p-p65 in OGD/R group was increased,and the expression of TLR9 had no significant change.Western blot and immunofluorescence results showed that the expression of p65 in nuclear protein was increased,the expression of p65 in cytoplasmic protein was decreased,and p65 shown nuclear translocation.Compared with OGD/R group,the expression of TLR4,MyD88,TRAF6 and p-p65 in each AGC dose groups was decreased.The expression of p65 in microglial nuclear protein was decreased,the expression of p65 in cytoplasmic protein was increased,and the phenomenon of p65 nuclear translocation was inhibited.?4?In order to validate whether AGC regulates microglia through regulating the MyD88-dependent pathway,we transfected the MyD88-siRNA into BV-2 cells.The results shown that,compared with the control group,the expression of MyD88 was decreased in MyD88-siRNA group.Compared with MyD88-siRNA group,the expression of MyD88 and p-p65/p65 were increased slightly after OGD/R treatment.Compared with MyD88-siRNA+OGD/R group,after AGC treatment,the expression of MyD88 was significant decreased in the high-dose AGC group,and AGC had no effect on the expression of p-p65/p65 and TLR4.The results indicate that AGC may regulate microglial polarization through the MyD88-dependent and MyD88-independent pathway.?5?Flow cytometry was used to observed the role of AGC in regulating microglial polarization.The results shown that,compared with the control group,the expression of CD16+CD32+in LPS+IFN-?group was increased,the expression of CD206+was decreased.Compared with the LPS+IFN-?group,the expression of CD16+CD32+in each AGC dose groups was decreased,the expression of CD206+was increased.Compared with the IL-4 group,the IL-4+LPS+IFN-?group shown an increasing trend of CDCD16+CD32+and a decreasing trend of CD206.Compared with the IL-4+LPS+IFN-?group,the each AGC dose groups shown a decreasing trend of CDCD16+CD32+and an increasing trend of CD206+.?6?Neurons were co-cultured with LPS+IFN-?-injured microglia by Transwell and contact co-culture,and the morphology of neurons was observed by immunofluorescence.The results shown that,the neuronal processes in normal culture were intact and continuous.After co-culture with normal BV-2 cells,there was no significant change in neuron morphology.After co-culture with LPS+IFN-?-injured microglia,the neuronal processes became short and discontinuous.However,after co-culture with M1-type microglia treated with AGC,the neuronal processes were relatively complete and continuous.Conclusion:AGC can regulate the polarization of microglia from M1-type to M2-type,inhibit the secretion of inflammation cytokines,increase the secretion of anti-inflammation cytokines,and protect neuron.The mechanism of AGC regulating microglial polarization may be related to AGC inhibiting the activation of TLR4/MyD88/NF-?B signaling pathway.