| Background:Inflammation is an important pathological process after cerebral infarction,and microglia play a major role in it.Microglia can be polarized into two phenotypes,including M1 type that aggravates inflammation and M2 type that suppresses inflammation.Inflammation and microglial phenotypes are regulated by PPARγpathway.Netrin-1 has brain protective effect in animal models of cerebral ischemia,while its mechanisms remain to be studied,and it is unclear whether it has a regulatory effect on microglial phenotypes.Methods:In in vivo experiments,we constructed rat models of middle cerebral artery occlusion(MCAO)and treated rats with tail vein injection of recombinant human Netrin-1 protein,dimethyl sulfoxide(DMSO),and PPARγpathway antagonist GW9662.Western-blot was used to detect i NOS and Arg1,q RT-PCR was used to detect IL-1β,TNF-α,IL-4,IL-10,and immunofluorescence was used to detect Iba1~+CD16~+,Iba1~+CD206~+,Neu N~+cell numbers,Fluore Jade C staining was used to detect degenerated neurons,TTC staining was used to evaluate cerebral infarct volume,modified neurological deficiency scoring,rotarod test and beam walk test were used to evaluate neurological funtions.In in vitro experiments,we extracted primary microglia,constructed OGD models and then treated microglia with Netrin-1,DMSO,and GW9662.CCK-8 assay was used to detect microglial viability,Western-blot was used to detect i NOS,Arg1 and PPARγ,q RT-PCR was used to detect IL-1β,TNF-α,IL-4,IL-10,flow cytometry was used to detect proportion of CD16~+and CD206~+cells.We also extracted rat primary neurons,construected neuron-microglial co-culture systems with transwell chambers,treated them with OGD and above drugs.CCK-8assay was then used to detect neuronal viability,flow cytometry was used to detect percentage of apoptotic neurons.Results:(1)In in vivo experiments,the M1 markers i NOS,IL-1β,and TNF-αincreased continuously within 72 hours after MCAO,while the M2markers Arg1,IL-4,and IL-10 increased briefly within 24 hours after the surgery and then continued to decline.In in vitro experiments,M1 type markers increased continuously within 24 hours after OGD treatment,while M2 type markers increased briefly within 6 hours and then continued to decrease.(2)Ischemia and hypoxia can cause the expression of M1 markers i NOS,IL-1β,TNF-α,CD16 and M2 markers Arg1,IL-4,IL-10,and CD206 to rise at different degrees.After Netrin-1 treatment,the expression of M1markers decreased significantly,while the expression of M2 markers increased significantly.(3)In in vivo experiments,cerebral ischemia resulted in a significant increase in the number of degenerated neurons and a significant decrease in the number of survived neurons.Netrin-1 treatment reduced the number of degenerated neurons and increased the number of survived neurons.In vitro experiments,OGD treatment led to a significant decrease in neuronal viability and a significant increase in percentage of apoptotic neurons in the neuron-microglia co-culture system.Netrin-1treatment enhanced neuronal viability and decreased percentage of apoptotic neurons.(4)Netrin-1 treatment reduced infarct volume in rats and improved neurological deficit symptoms.(5)After blocking PPARγwith GW9662,the above effects of Netrin-1were reversed.(6)OGD treatment decreased cytoplasmic PPARγlevel and increased nucleus PPARγlevel in microglia.Netrin-1 treatment enhanced cytoplasmic PPARγlevel and decreased nucleus PPARγlevel.GW9662decreased the levels of PPARγboth in cytoplasm and nucleus.Conclusions:(1)Ischemia and hypoxia promotes polarization of microglia to M1 type.(2)Netrin-1 can induce microglia to polarize to M2 type via PPARγpathway.(3)Netrin-1 can reduce neuronal damage caused by ischemia and hypoxia,which is related to the induction of microglial polarization to M2 type.(4)Netrin-1 can reduce the infarct volume and improve the symptoms of neurological deficits.This effect is related to the induction of microglial polarization to M2 type.(5)Netrin-1 can promote PPARγtransfer from nucleus to cytoplasm... |
