| BackgroundRecent studies have shown that inflammatory responses play an important role in the pathogenesis of neurological diseases such as ischemic stroke,Alzheimer's disease,Parkinson's disease,and multiple sclerosis,and microglia(Microglia,MG).It also plays an important role in the inflammatory response of the central nervous system,as immune cells in the brain,is rapidly activated and mainly and mainly polar into M1 and M2 states after brain injury.After activating of M1(pro-inflammatory)cells,they can produce a large number of inflammatory factors,cytotoxic related factors,etc,which can accelerate the nerve injury.After activation,M2 type(anti-inflammatory)cells release brain-derived neurotrophic factor,vascular endothelial growth factor,and anti-inflammatory mediators,which inhibit inflammatory responses and promote tissue repair.Previous studies have found that the Notch signaling pathway affects the polarization of microglia.Therefore,the aim of the study is to search for safe and effective drugs which can act on the Notch signaling pathway will help regulate the polarization of microglia and avoid harm.It might be a great significance to improve the prognosis of various inflammatory-related nervous system diseases.Lipoxin A4(Lipoxin A4)is a metabolite of arachidonic acid,an endogenous lipid mediator with anti-inflammatory and pro-inflammatory effects,which can play an important role in the process of nervous system inflammation.In vivo studies have shown that LXA4 can reduce the neuritis and neuralgia after spinal cord injury,relieve the brain edema caused by activation of microglia after subarachnoid hemorrhage,and improve neurological function.In addition,LXA4 protects neurodegenerative diseases including vascular dementia,Alzheimer's disease(AD)and Parkinson's disease(PD).However,whether the Notch signaling pathway is the target of LXA4 to regulate the polarization of microglia has not been reported.In this study,BV2 microglia induced by lipopolysaccharide(LPS)and Lipoxin A4(LXA4)were used to clarify the anti-inflammatory effects of LXA4 to elucidate the role of LXA4 in regulating the polarization of microglia in the inflammatory response and to explore its possible mechanism.The purpose of this study is to provide a theoretical basis for LXA4 in the treatment of inflammatory diseases of the central nervous system.ObjectiveWe investigate the effect of LXA4 on the expression of inflammatory mediators after activation of BV2 microglia induced by LPS,to elucidate the regulatory effect of LXA4 on the polarization of microglia in the inflammatory reaction,and to explore the possible mechanism of the effect of LXA4 on the polarization of microglia.Methods1.Material: murine BV2 microglia cell lines were purchased from the typical culture preservation center of Wuhan University.2.Construction of cellular inflammatory model: BV2 microglia were treated with 200ng/ml LPS;3.Cell grouping:Part one:(1)Control group: culture medium only;(2)LXA4 group: add LXA4;(3)LPS group: add LPS;(4)LPS LXA4 group: LXA4 pretreatment and then LPS.The second part:(1)Control group: culture medium only;(2)LPS group: adding LPS;(3)DAPT LPS group: after adding DAPT pretreatment,adding LPS culture;(4)LXA4 LPS group: adding LXA4 pretreatment,adding LPS treatment;(5)DAPT LXA4 LPS group: after DAPT pretreatment,LXA4 pretreatment was added,then LPS treatment was added.4.Detective methods4.1 The levels of TNF-?,IL-1 ?,IL-10 in the supernatant of microglial cells treated with LPS were determined by ELISA method.4.2 RT-PCR was used to detect the expression of iNOS,CD32,TNF-?,IL-1 ? and Arg-1,CD206,IL-10 mRNA in M1 microglia after LPS treatment.4.3.The protein expression of M1 microglia marker(iNOS)and M2 microglial marker(Arg-1)was detected by Western blot Western-blot.4.4.The expression of M 1 microglia marker(iNOS,CD32)and M 2 microglia marker(Arg-1,CD206)was detected by immunofluorescence assay.4.5.The protein expression of CD32,CD206 was detected by flow cytometry.4.6.Notch1,Hes1,Hes5 mRNA expression related to Notch signaling pathway was detected by-PCR.4.7.The expression of Notch1,Hes1,Hes5 protein was detected by Western blot Western-blot.Results1.After LPS treatment on BV2 microglia,the results of RT-PCR and ELISA showed that the synthesis and secretion of inflammatory cytokines TNF-? and IL-1? was increased,while the synthesis and secretion of TNF-? and IL-1? were decreased significantly after LXA4 treatment.The synthesis and secretion of anti-inflammatory cytokines IL-10 were significantly increased.2.After LPS treatment of BV2 microglia,RT-PCR,cellular immunofluorescence and flow cytometry detection results showed that the expression levels of iNOS and CD32,the M1-type molecular markers,were significantly increased,while the expression levels of Arg1 and CD206,the M2-type molecular markers were not significantly increased.However,after LXA4 treatment,the expression levels of M2-type molecular markers were significantly increased.3.After LPS treated BV2 microglia,RT-PCR and Western blot results showed that the Notch effector molecules Notch1 and Hes1 were significantly increased,while the expression of Hes5 was not significantly changed.Compared with the LPS group,the expression of Notch1 and Hes1 were significantly decreased and the expression of Hes5 was significantly increased after the treatment with LXA4.4.The inhibitory effect of LXA4 on inflammatory factors was blocked after giving the specific blocker DAPT,a ?-secretase inhibitor of the Notch signaling pathway.Meanwhile,the regulation of LXA4 on microglia polarization was blocked.ConclusionsLXA4 can inhibit the secretion of pro-inflammatory cytokines in BV2 microglia after LPS and regulate the polarization of microglia,which may be achieved through the Notch signaling pathway. |
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