Liraglutide Regulates Activation Of Primary Microglia Via Notch Signaling Pathway

Microglia,a macrophage in the brain,is one of the key players in the central nervous system(CNS)immunity.They are distributed throughout the central nervous system,accounting for about 10%to 20%of the total glial cells in the brain.It was first proposed by Piodel Rio Hortega in 1932.Since its discovery,its origin,characteristics,function,physiology,and pathological state have been tirelessly debated by researchers.However,there is a general consensus that microglia are derived from primitive yolk sac macrophages that migrate to the brain and become fixed cells during early embryonic development.In a healthy mature brain and under normal physiological conditions,neuroglia exhibit a monitoring behavior whose morphology changes as the microenvironment changes.Microglia are divided into two states,resting state and activated state.The activated state can be divided into M1 type and M2 type.Morphologically,it is "branched" and "analytic insect".M1 type microglia can promote the release of inflammatory factors and produce cytotoxic effects on nerve cells.M2 type microglia can release anti-inflammatory factors and protect nerve cells.These cells are sensitive to changes in the brain's microenvironment.Once the homeostasis changes,the cells become activated,which ultimately leads to changes in cell function,gene and protein expression,as well as the release of a range of factors,including cytokines(Interleukin,tumor necrosis factor,chemokine(MCP-1),excitatory neurotransmitter(glutamate),complement factor,prostaglandin,reactive oxygen species and nitrogen.Therefore,inhibiting the excessive activation of neuroglia(M1 type)and reducing the production of cellular inflammatory factors have become an important part of alleviating neuroinflammatory reactions.Glucagon-like peptide-1(GLP-1)is a polypeptide hormone secreted by intestinal L cells.It is not only a commonly used hypoglycemic agent,but also has a nutritional and protective effect on nerve cells.GLP-1 receptor(GLP-1R)is present on microglia,and GLP-1 analogues act by binding to GLP-1R on the cell membrane surface.however.The specific mechanism of the neuroprotective effect of the current GLP-1 analogue liraglutide is unclear.Therefore,the mechanism is the premise and research focus of liraglutide as a neuroprotective drug.Notch signaling pathway is an important pathway regulating embryonic development,which can regulate the polarization of mononuclear macrophages.Excessive activation of mononuclear macrophages leads to the growth of M1 cells,while microglia function similar to mononuclear macrophages.It can also be regulated by the Notch signaling pathway.Therefore,we conclude that the GLP-1 analogue liraglutide regulates the activation of primary microglia via the Notch signaling pathway.In this study,primary microglia cells extracted from newborn 24 h SD rats were used as cell models to investigate the mechanism by which liraglutide regulates hypothalamic microglial activation via Notch signaling pathway.I will introduce this topic from three parts:Part ?:Preliminary study on primary culture of microglia.METHODS:The cerebral cortex tissues of SD rats were taken out for 24 h,digested into individual cells by trypsinization,and cultured.After the cells were overgrown,the flasks were vigorously tapped for 3 min,centrifuged and collected,and new medium was added for further culture.The purity was identified by immunofluorescence.RESULTS:Confocal microscopy confirmed the purity of microglia to 96.85%.Part ?:the cytotoxic effect of lipopolysaccharide,liraglutide and the activation model of establishing cells.METHODS:Lipopolysaccharide(LPS)-treated microglia were divided into 5 groups:control group,1 ng/ml LPS,10 ng/ml LPS,100 ng/ml LPS,and 1000 ng/ml LPS.Liraglutide-treated LPS-induced microglia were divided into 5 groups:control group,100 nM Lira,10 ng/ml LPS+1 nM Lira,10 ng/ml LPS+10 nM Lira,10 ng/ml LPS+100 nM Lira.The toxicity of LPS and liraglutide to cells was examined by a microplate reader using CCK-8 reagent.RESULTS:In the LPS cytotoxicity experiment,the cell viability was(49.13%±9.515%),(40.60%±6.638%),and the cell viability was significantly decreased after treatment with 100 ng/mL and 1000 ng/ml lipopolysaccharide(LPS)for 24 h.After treatment with 1 ng/ml LPS and 10 ng/mL LPS,the viability was(113.79%±4.933%)and(89.24%±2.597%),indicating that the concentration had no significant effect on cell viability.In the liraglutide cytotoxicity assay,cell viability after treatment with 100 nM liraglutide was(87.62%± 5.650%).At the same concentration of 10 ng/ml LPS,the cell viabilities after treatment with different concentrations of liraglutide were:1 nM(83.17%±5..719%),10 nM(92.36%±6.609%),100 nM(99.72%± 5.806%),indicating that there was no significant decrease in cell viability after treatment with 10 ng/ml lipopolysaccharide and different concentrations of liraglutide.Therefore,a cell model induced by LPS(10 ng/ml)and 100 nM liraglutide were used in the following experiments.Part III:Mechanism of Liraglutide Regulating Microglia Activation via Notch Pathway.-METHODS:After purification,the cells were divided into 5 groups(control group,LPS group,liraglutide group,LPS+liraglutide group,LPS+DAPT group).The enzyme-linked immunosorbent assay(Elisa)measures the concentration of inflammatory factors(TNF-?,IL-6).Immunofluorescence was used for the quantitative and semi-quantitative detection of M1 marker(iNOS)and M2 marker(Argl).The expressions of Notch-1,Hes-1,CYLD,NF-kB/p65 and p38MAPK protein concentrations and gene levels were analyzed by RT-PCR and ELISA,respectively.RESULTS:In the microglia treated by immunofluorescence,the expression of M1 marker(iNOS)was significantly increased in LPS-induced microglia,and the expression of M2 marker(Argl)was significantly decreased.Liraglutide and DAPT treatment of LPS-induced microglia inhibited microglia activation.In Elisa experiments,LPS-induced microglia inflammatory factors TNF-? and IL-6 production were increased,and liraglutide and DAPT treatment decreased LPS-induced microglia production of TNF-? and IL-6.In immuno-imprinting experiments,LPS-induced microglia increased levels of Notch-1,Hes-1,NF-kB/p65,and p38MAPK proteins,and there was no significant difference in protein expression of CYLD,liraglutide and DAPT-treated LPS-induced microglia reduced the expression of Notch-1 and Hes-1 protein levels,and the levels of NF-kB/p65 and p38MAPK proteins were also significantly decreased,while CYLD protein levels were increased.Real-time PCR analysis showed that the results were consistent with immunoblotting experiments.Conclusion:This study demonstrates that the expression of Notch signaling pathway is enhanced by LPS-induced activation of primary microglia in vitro,and confirmed that the GLP-1 analogue liraglutide can mediate the Notch signaling pathway.The activation state of microglia(transformation between M1 and M2 types)and the release of inflammatory factors are regulated.Therefore,liraglutide can inhibit the activation of microglia and the inflammatory response by blocking the Notch signaling pathway.