| Objectives:To confirm the protective effects of scutellarin in inhibiting pro-inflammatory mediators in LPS-activated BV-2 microglia and hypoxia-ischemia brain damage-induced microglia.Then to compare the changes of miRNAs that involved accelerating or alleviating neuroinflammation in LPS-induced and scutellarin pretreatment BV-2 microglia by miRNA chips of high flux.Furthermore,to screen and identify the target gene that purpose microRNA regulated.The mechanism and signaling pathway of certain miRNA that regulated in neuroinflammation of activated microglia in developing brain by scutellarin should be verified in vitro and in vivo.Methods:Part 1.In vitro,to observe the expressions of the pro-inflammatory mediators(TNF-α,IL-1β and iNOS)in activated BV-2 microglia inducing by LPS were detected by the ELISA,Western blot and double-label immunofluorescence assays.In the model of HIBD 7-day-old KM mice induced by Rice-Vannucci method,expressions of the pro-inflammatory mediators(TNF-α,IL-1β and iNOS)in activated microglia of the brain were detected by the ELISA,Western blot and double-label immunofluorescence assays in HIBD-ld,3d and 7d.Neurologic function of neonatal mice were observed by neurological scores at all time points.Part 2.The aim of this part was to silt out miRNAs that expressed differentially and explore the probable mechanism of certain miRNAs in participating neuroinflammation in LPS-induced and scutellarin pretreatment BV-2 microglia.miRNA microarrays were touched on detecting the certain miRNAs that differentially expressed in the three groups of activated BV-2 microglia model.Certain miRNA target protein was matched by Targetscan and GENE bank.In vivo,microglia were treated by miRNA-7036a agomir or miRNA-7036a antagomir before LPS and scutellarin used.Pro-inflammatory mediators(TNF-α,IL-1β and iNOS)and target protein(MAPT)were detected by qPCR,Western blot,ELISA and double-labeling immunofluorescence.The results were then confirmed by rescue experiment and gene interference.The relationship between miRNA-7036a and MAPT were validated by dual-luciferase reporter assay and RNA fluorescence in situ hybridization(RNA-FISH).To design mir-7036a-5p over-expression vector and MAPT dual luciferase reporter gene vector through databases,and detect the relationship between them.The co-localization between mir-7036a-5p and MAPT was verified by RNA-FISH experiment.Part 3.To explore the signaling pathway which correlated with MAPT by String database and KEGG database and the mechanism of scutellarin alleviate neuroinflammation of activated microglia by modulating miRNA-7036a/MAPT/PRKCG/ERK Signaling Pathway in vitro and in vivo.The expressions of PRKCG and ERK were then validated by qPCR and Western blot after over-expressing or suppressing in vitro.Meanwhile,all of them were detected by qPCR,Western blot and double-labeling immunofluorescence in hypoxia-ischemia brain damage model.Results:① In vitro,the results showed that the level of pro-inflammatory mediators(TNF-α,IL-1β and iNOS)in LPS-activated BV-2 microglia group were significantly increased(p<0.05).While all of them were reduced in scutellarin pretreatment group(p<0.05).In vivo,compared to the sham group,7-day-old KM mice performed lower neurological scores at all HIBD groups(p<0.05).Scutellarin improved the Grip test scores of neonatal mice in the HIBD-3d group and the tail suspension test scores in the HIBD-3d and 7d groups conspicuously(p<0.05).The expressions of pro-inflammatory mediators(TNF-α,IL-1β and iNOS)in HIBD group were obviously increased.While all of them were inhibited effectively by the treatment of scutellarin.②The data of miRNA microarrays showed that the expression levels of miRNA-7036a,miRNA-6912 and miRNA-181a were changed in LPS-activated BV-2 microglia group and scutellarin pretreatment group.LPS may induced neuroinflammation by up-regulation expression of miRNA-7036a and down-regulation expressions of miRNA-6912 and miRNA-181a.On the contrary,scutellarin reversed the expression levels of these three miRNAs.Consistency of these results and miRNAs microarrays were then evaluated by qPCR.Matching with Targetscan and GENE bank,the result suggested that scutellarin may inhibit the neuroinflammation in activated microglia via miRNA-7036a and its target gene MAPT.In vitro,the result showed that the level of MAPT mRNA significantly down-regulated,while the expressions of p-tau and pro-inflammatory mediators(TNF-α,IL-1β and iNOS)in LPS-activated BV-2 microglia group were significantly increased under circumstance of using mir-7036a-5p agomir(p<0.05).The protective effects of scutellarin in inhibiting neuro-inflammation in LPS-activated BV-2 microglia were weakened by mir-7036a-5p agomir.Though mir-7036a-5p antagomir reversed the result,sh-MAPT were totally rescued its phenotype.The relationship between miRNA-7036a and MAPT were validated by dual-luciferase reporter assay,and RNA-FISH showed that the co-localization of mir-7036-5p and MAPT of BV-2 microglia was in the cytoplasm.③ Gene set enrichment analysis showed that MAPT/PRKCG/MAPK signaling pathway may participate in the procedure of scutellarin alleviating neuroinflammation though modulating miRNA-7036a.In vitro,the results showed that the expression of MAPT/PRKCG/ERK signaling pathway in LPS-activated BV-2 microglia group were increased after over-express mir-3036-5p.While all of them were reduced in scutellarin pretreatment group or down-regulate mir-3036-5p group(p<0.05).The protective phenotype of mir-7036a-5p were intervened by sh-MAPT,and the expression of MAPT/PRKCG/ERK signaling pathway went back up(p<0.05).In vivo,compared to the sham group,qPCR,Western blot and double-label immunofluorescence assays results showed that the level of MAPT/PRKCG/ERK signaling pathway in HIBD group were obviously increased at all time points.While both of them were reduced in Scutellarin treatment group.Conclusions:The results showed that scutellarin played protective roles on alleviating microglia-mediated neuroinflammation though reducing pro-inflammatory mediators(TNF-α,IL-1β and iNOS)in LPS-activated microglia and HIBD-induced P7 neonatal mice.miRNA-7036a may involve in the regulation of the procedure of anti-inflammation mechanism in scutellarin by targeting miRNA-7036a/MAPT pathway.Scutellarin exerted the anti-neuroinflammation effect in LPS-stimulated BV-2 microglia and HIBD-induced neonatal mice by binding to miRNA-7036a on MAPT/PRKCG/ERK signaling pathway. |
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