Regulation Of Lipoxin A4 On Microglia Polarization And Its Effect On AQP4 Expression On Co-cultured Astrocytes

BackgroundAlzheimer's disease(AD)is a neurodegenerative disease,characterized by senile plaques,neurofibrillary tangles and loss of neurons.According to the Alzheimer's Disease International(ADI),it's estimated that more than 50 million people worldwide living with dementia,and the number is expected to reach 152 million by 2050.AD accounts for 50 to 60 percent of dementia.AD has become an increasingly serious problem harmful to human health worldwide.Therefore,it is of great significance to find effective measures for prevention and treatment of AD.Over the year,the pathogenesis of AD has always been controversial.In recent years,Previous studies have found that inflammatory mechanisms play an important role in the pathological process of AD,and microglia cells play a crucial role in the inflammatory response.Microglia have two polarized phenotypes:M1 and M2,M1 type cells release a variety of inflammatory factors that mediate the inflammatory damage to nerve cells and tissues,while M2 type cells inhibit inflammation and promote tissue repair.Previous studies have shown that Notch signaling pathway plays an important role in the polarization of microglia.In the course of AD,inflammatory responses can damage a variety of tissue and organ targets,including aquaporin 4(AQP4).Previous studies have shown that AQP4 plays an important role in the occurrence and development of AD.AQP4 is mainly located on the astrocyte foot process,and is involved in various pathophysiological processes such as the clearance of A?,synaptic plasticity,glutamate transport,neuroinflammation,and ion homeostasis maintenance.It can be seen from the above that it is imperative to find an appropriate drug to regulate microglia polarization through Notch signaling pathway,reduce inflammatory damage,and protect AQP4 and other tissues and organs.A large number of studies have found that Lipoxin A4(LXA4)has a good anti-inflammatory effect and promotes the regression of inflammatory response.Our team's previous studies have demonstrated that LXA4 can regulate the polarization of microglia to reduce inflammatory damage in the model of cerebral ischemia and inflammation,but it was still not clear whether LXA4 plays the same role on AD and its mechanism,and is has not been reported whether the regulation of microglia polarization by LXA4 has an effect on the expression of AQP4 on astrocytes..Therefore,this study intends to:1.AD model was constructed by microglia treated by A?1-42,the changes of microglia phenotype were detected after LXA4 intervention and the possible mechanism was explored;2.The activated microglia were co-cultured with astrocytes to detect the expression of AQP4 on astrocytes.To investigate whether LXA4 can regulate the polarization of microglia cells and whether the regulation of microglia cell polarization can affect the expression of AQP4 on astrocytes,so as to provide experimental basis for the future use of LXA4 in the treatment of AD.Objective1.To investigate the effect of LXA4 on the expression of inflammatory cytokines after A?1-42 treated microglia.2.To investigate the effect of LXA4 on the polarization of microglia after treated by A?1-42.3.To investigate the molecular mechanism of LXA4 on regulation the polarization of microglia after treated by A?1-42.4.To investigate the effect of LXA4 on microglia polarization on AQP4 expression on astrocytes.Methods1.BV2 microglia stimulated by A?1-42 was used to construct AD model,experiment group was designed as follows:(1)control group:culture medium only;(2)A?1-42 group:add A?1-42 only;(3)LXA4+A?1-42 group:A?1-42 was added after LXA4.After the treatment for 12 hours,the supernatant and cells were collected to detect the expression of the corresponding molecules of microglia polarization.The expression of inflammatory cytokines in microglia was detected by ELISA.RT-PCR was used to detect the mRNA expression of inflammatory cytokines(TNF-?,IL-1?),M1 markers(iNOS,CD32)and M2 markers(Argl,CD206)in microglia.The protein expression of M1 markers(iNOS),M2 markers(Arg-1)and signaling pathway related molecules(Notchl,Hes1 and Hes5)in microglia were detected by Western blot.Immunofluorescence assay was used to detect the protein expression of M1 markers(iNOS,CD32)and M2 markers(Argl,CD206)in microglia.2.Transwell co-culture system was established between microglia treated by A?1-42 and astrocytes,and the experiment was divided into 4 groups:(1)astrocyte group:upper chamber:no cells;lower chamber:astrocytes;(2)control group:upper chamber:untreated microglia cells,upper chamber:astrocytes;(3)A?1-42 group:upper chamber:microglia cells treated by A?1-42,lower compartment:astrocytes;(4)LXA4 +A?1-42 group:upper chamber:microglia cells treated together with LXA4 and A?1-42,upper chamber:astrocytes.The expression of AQP4 on astrocytes was detected by Western blot after 24 hours co-culture.Results1.Compared with the control group,RT-PCR and ELISA results showed that the expression of transcription and proteins of TNF-? and IL-1? in BV2 microglia cells were significantly increased after stimulation by A?1-42(P<0.05),and the expressions of TNF-? and IL-1? were decreased after LXA4 intervention(P<0.05).2.Compared with the control group,RT-PCR,Western blot and immunofluorescence results showed that after stimulation by A?1-42,the expression of M1 type markers(iNOS,CD32)(P<0.05),the expression of M2 type markers(Arg-1)increased(P<0.05),and the expression of M2 type markers(CD206)was slightly decreased(P>0.05).After LXA4 intervention,compared with A?1-42 group,the expression of M1 type markers(iNOS,CD32)decreased(P<0.05)and the expression of M2 type markers(Arg-1,CD206)increased(P<0.05).3.Western blot results showed that the expressions of Notch 1,Hes1 and Hes5 proteins related to the signaling pathway were all increased in BV2 microglia cells after A?1-42 stimulation compared with the control group(P<0.05).Compared with A?1-42 group,after LXA4 intervention,the expressions of Notch 1 and Hesl proteins were decreased(P<0.05),and the expressions of Hes5 proteins were further increased(P<0.05).4.BV2 microglia treated by A?1-42 and astrocytes were co-cultured,western blot results show the expression of astrocytes cultured alone and astrocytes cultured with untreated microglia was no significant difference(P>0.05),microglia treated by A?1-42 and astrocytes were co-cultured,AQP4 expression on astrocytes were increased significantly(P<0.05),by comparison,microglia treated by LXA4 and A?1-42 together and astrocytes were co-cultured,the expression of AQP4 on astrocytes was decreased(P<0.05).Conclusions1.LXA4 can inhibit the expression of microglial inflammatory cytokines after treated by A?1-42.2.LXA4 can regulate the polarization of microglia cells after stimulation by A?1-42,inhibit the polarization of microglia into M1 type,and promote the polarization of microglia into M2 type.3.LXA4 may regulate the polarization of microglia after stimulation by A?1-42 through Notch signaling pathway.4.LXA4 can affect the expression of AQP4 on astrocytes by regulating the polarization of microglia.