Aβ_1-42 Induces Aerobic Glycolysis And Promotes Microglial Inflammation Through The EphA2/p38 MAPK Pathway

As the most common neurodegenerative disease,Alzheimer’s disease（AD）is characterized by senile plaques formed by deposition of amyloidβ（Aβ）and neurofibrillary tangles（NFTs）caused by hyperphosphorylation of tau protein.Aβpromotes inflammation in microglia,leading to the release of inflammatory cytokines such as interleukin-1β（IL-1β）and tumor necrosis factor-α（TNF-α）.Studies have shown that glucose metabolism is closely related to inflammation mediated by activation of microglia.Metabolic transition from oxidative phosphorylation（OXPHOS）to aerobic glycolysis has been a marker of early glycolytic reprogramming controls microglial inflammatory activation.In addition,recent studies have found that Aβcan lead to microglial metabolic reprogramming,manifested as increased aerobic glycolysis level.However,the role and mechanism of Aβin the inflammatory response of AD microglia remains unclear.In this study,the mouse microglia BV2 cells and 6-month-old C57BL/6N male mice were used as the cell and animal models to investigate the effects of Aβ_1-42on the expression of inflammatory cytokines and aerobic glycolysis,to detect the changes of Aβ_1-42on microglia gene expression profile after inhibiting aerobic glycolysisby RNA sequencing（RNA-seq）technology,and to study the mechanism of microglial metabolic reprogramming induced by Aβ_1-42in inflammatory response.Part one Dose-effect experiments of Aβ_1-42promoting microglial proliferation and inflammationObjective:The mouse microglia BV2 cells were used as the research models to investigate the effects of different concentrations of Aβ_1-42on the proliferation,activation and expression of inflammatory factors of BV2 cells.Methods:The effect of Aβ_1-42on BV2 cell proliferation in different concentration groups were observed by MTS assay.The changes of counts and morphology of BV2 cell were observed by the microscope under the stimulation of Aβ_1-42.RT-PCR and Western blot was used to detect the effect of different concentration Aβ_1-42on IL-1βand TNF-αm RNA levels.Results:MTS assay showed that Aβ_1-42had dose-effect on the proliferation of microglia,and 2.5μM and 5μM Aβ_1-42had the most significant effect on the proliferation of BV2 cells for 12 h.Under the microscope,the counts of BV2 cells stimulated by Aβ_1-42for 12 h was significantly higher than that of Con group,and the morphology changed from branching to amoeba,accompanied by cell body enlargement.RT-PCR and Western blot results showed that the m RNA and protein expressions of IL-1βand TNF-αin 2.5μM,5μM and 10μM groups were significantly higher than those in Con group,and the m RNA and protein expressions of IL-1βand TNF-αin 5μM group were the most significant.Therefore,in the subsequent experiments,5μM was the concentration of Aβ_1-42.Conclusions:1.Low concentration（5μM）of Aβ_1-42promoted the proliferation of BV2cells.2.Low concentration（5μM）of Aβ_1-42induced BV2 cell morphology from branching to amoeba,accompanied by cell body enlargement.3.The m RNA and protein expression of inflammatory factors IL-1βand TNF-αwere significantly increased when BV2 cells were stimulated with 5μM Aβ_1-42for 12 h.Part two Aerobic glycolysis regulates the microglia-induced inflammatory response of Aβ_1-42Objective:The mouse microglia BV2 cells and SPF healthy 6-month-old C57BL/6N mice were used as the research models to detect whether the microglial inflammation induced by Aβ_1-42was accompanied by aerobic glycolysis.The expression levels of inflammatory factors were measured after microglia aerobic glycolysis was inhibited by 2-deoxy-D-glucose（2-DG）or hexokinase 2（HK2）-sh RNA.Methods:The effect of Aβ_1-42on the content of lactic acid in BV2 cells was detected by lactic acid kit,and the effect of Aβ_1-42on the content of lactic acid in BV2 cells was also detected after pretreatment with 2-DG.The effect of Aβ_1-42on the IL-1βand TNF-αm RNA levels in BV2 cells after pretreatment with 2-DG was detected by RT-PCR.The activation of microglia in the hippocampus of C57BL/6N mice was observed by immunofluorescence staining after stereotactic injection of Aβ_1-42into the brain of mice.The effect of Aβ_1-42on IL-1βand TNF-αm RNA levelsin hippocampus of the C57BL/6N mice after pretreatment with 2-DG was detected by RT-PCR.HK2 was knocked down by sh RNA lentivirus infection in BV2 cells.The knockdown efficiency of HK2 m RNA and protein in BV2 cells infected with HK2-sh RNA lentivirus was detected by RT-PCR and Western blot.The effects of Aβ_1-42on IL-1βand TNF-αm RNA and protein levels in BV2 cells after pretreatment with HK2-sh RNA were detected by RT-PCR and Western blot.Results:The lactate kit showed that Aβ_1-42could increase the content of lactic acid in BV2 cells,but it was inhibited by 2-DG.RT-PCR results showed that pretreatment with 2-DG could significantly inhibit the promotion of Aβ_1-42on the expression of IL-1βand TNF-αm RNA in BV2 cells.Immunofluorescence staining results showed that pretreatment with 2-DG could significantly inhibit the activation of microglia in the hippocampus of C57BL/6N mice induced by Aβ_1-42.RT-PCR results showed that pretreatment with 2-DG could significantly inhibit the promotion of IL-1βand TNF-αm RNA expression in hippocampus of C57BL/6N mice induced by Aβ_1-42.The results of RT-PCR and Western blot showed that HK2 m RNA and protein knockdown efficiency of group 90956 was significantly higher than that of the other two groups.The results of RT-PCR and Western blot showed that HK2-sh RNA pretreatment significantly inhibited the promoting effect of the m RNA and protein expression of inflammatory factors in BV2 cells,such as IL-1βand TNF-α.Conclusions:1.Aβ_1-42can enhance the aerobic glycolysis process in BV2 cells.2.Inhibition of aerobic glycolysis by 2-DG or HK2-sh RNA significantly down-regulated the promoting effect of Aβ_1-42on the expression of inflammatory factors,such as IL-1βand TNF-α.3.When 2-DG was given to inhibit the glycolysis process in the hippocampus of C57BL/6N mice,the activation effect of Aβ_1-42on microglia was significantly down-regulated.Part three Transcriptome sequencing analysis of HK2 knockdown BV2 cells stimulated by Aβ_1-42Objective:To further study the mechanism of aerobic glycolysis of microglia induced by Aβ_1-42on its immune inflammatory response,we selected the mouse microglia BV2 cells were used as the research models.Then,the differentially expressed genes（DEGs）were screened by RNA-seq after pretreatment with HK2-sh RNA in BV2 cells,and some DEGs were selected for validation to investigate the regulation of aerobic glycolysis on the inflammatory response of BV2 cells induced by Aβ_1-42.Methods:BV2 cells after pretreatment with HK2-sh RNA were stimulated with Aβ_1-42,and then m RNA was extracted 12 h later for RNA-seq and bioinformatics analysis and verification.After constructing the library,the whole transcriptome gene sequence was obtained by high-throughput sequencing technology,and the quality of the sequencing data was evaluated.DEGs results were obtained by analysis software,and the heat map and volcano map of m RNA cluster analysis were drawn.Meanwhile,the gene ontology（GO）of DEGs and enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes（KEGG）was analyzed.Partial DEGs were verified by RT-PCR.Results:The results of sequencing quality showed that Q30 of all samples could reach more than 90%,which could be used for follow-up studies.The gene expression level was high,and the overall distribution of FPKM values among groups was consistent.Cluster analysis results showed that compared with the control group,543 DEGs were identified in HK2-sh RNA pretreated samples,including 346（63.72%）up-regulated genes and 197（36.28%）down-regulated genes.KEGG enrichment analysis results revealed 322 signaling pathways,including 214 up-regulated pathways and108 down-regulated pathways.Then,we select four DEGs,chemokine ligand2（Cxcl2）,dual specific phosphatase 10（Dusp10）,erythropoietin-Producing Hepatocellular receptor A2（Eph A2）and matrix metallopeptidase 9（Mmp9）to verify,and RT-PCR results showed that Cxcl2 and Eph A2 m RNA expression was significantly down-regulated,which was consistent with RNA-seq results.Conclusions:1.GO analysis showed that 346 DEGs were significantly up-regulated and 197 DEGs were significantly down-regulated.2.KEGG analysis showed enrichment of 214 up-regulated signaling pathwaysand108down-regulatedsignalingpathways.Glycolysis/gluconeogenesis,MAPK signaling pathway,IL-17 signaling pathway,TNF signaling pathway and other signaling pathways related to glycolysis and inflammation were significantly down-regulated.3.RT-PCR results showed that Cxcl2 and Eph A2 m RNA expression were significantly down-regulated after HK2-sh RNA pretreatment.Part four Aerobic glycolysis induces Aβ_1-42to cause inflammatory responses in BV2 cells by Eph A2/p38 MAPK pathwayObjective:The mouse microglia BV2 cells were used as the research models to analyze the effects of the phosphorylated proteins and total proteins levels of JNK mitogen-activated protein kinase（JNK MAPK）,extracellular-signalregulated protein kinase mitogen-activated protein kinase（Erk MAPK）,p38 mitogen-activated protein kinase（p38 MAPK）and the protein expression levels of Eph A2 after pretreatment with HK2-sh RNA and then analyze the effects of the phosphorylated proteins and total proteins levels of p38 MAPK and the protein levels of Eph A2,IL-1βand TNF-αafter pretreatment with Eph A2-si RNA.Methods:The effects of Aβ_1-42on the expression of Eph A2 protein and the phosphorylated protein and total protein of JNK MAPK,Erk MAPK,p38MAPK in BV2 cells after pretreatment with HK2-sh RNA were detected by Western blot.The infection efficiency of Eph A2 in BV2 cells after pretreatment with Eph A2-si RNA was analyzed by Western blot.The expressions of Eph A2 protein and p38 MAPK phosphorylated proteins and total proteins as well as inflammatory factors IL-1βand TNF-αwere detected by Western blot after pretreatment with Eph A2-si RNA.Results:Western blot results showed that compared with the Con group,the expression of Eph A2 protein and phosphorylated p38 MAPK protein in pretreated with HK2-sh RNA BV2 cells were significantly decreased,but the phosphorylation and total protein levels of Erk1/2 and JNK were not significantly changed.Western blot results showed that Eph A2 protein levels were significantly decreased in Eph A2-si RNA pretreated BV2 cells,and the expressions of p38 MAPK phosphorylated protein and inflammatory factors IL-1βand TNF-αwere also significantly decreased.Conclusions:1.Inhibition of aerobic glycolysis in BV2 cells significantly down-regulated the promotion effect of Aβ_1-42on the expression of Eph A2protein and p38 MAPK phosphorylated protein.2.Eph A2 knockdown significantly down-regulated thepromotion effect of the expression of Eph A2 protein,p38 MAPK phosphorylated protein and inflammatory factor in BV2 cells by Aβ_1-42.3.Aerobic glycolysis induced by Aβ_1-42regulates the expression of inflammatory factors in BV2 cells through Eph A2/p38 MAPK signaling pathway.