Role Of Aerobic Glycolysis In The Immune Inflammatory Response Of Microglia Induced By Amyloid-β

Alzheimer’s disease（AD）,a slow-progressive neurodegenerative disease that begins with mild memory loss and ends with severe impairment of cognitive and executive function.The main pathological manifestations of AD areβ-amyloid plaques,neurofibrillary tangles,vascular amyloidosis,and loss of neurons and synapses.Recent studies have found that neuroinflammation plays an important role in the occurrence and development of AD.And the activation of microglia and the generation of inflammation are the main manifestations of AD neuroinflammation.Microglia are immune cells in the brain that become inflamed and further activated in response to external stimulation.When microglia cells enter the over-activated phase,they produce neurotoxic substances,which aggravate the process of central nervous system diseases.In this study,mouse microglial cell line BV2 cells and C57BL/6N mice will be used as research objects to study the influence of Aβon the activation state of microglia cells in the development of AD disease,observe the role of aerobic glycolysis in amyloid-βprotein-induced immune inflammation,and clarify the regulatory role of aerobic glycolysis in Aβ-induced neuroinflammation.Objective:The effects of Aβon the activation of microglia during the development of AD disease were studied using mouse microglia cell line BV2cells and C57BL/6N mice.The time-dependent regulation of aerobic glycolysis of microglia stimulated by Aβon cellular immune inflammatory response was observed,and in acute AD animal model to verify the regulation of aerobic glycolysis on the expression of early inflammatory factors in mouse hippocampus,to clarify the regulation of aerobic glycolysis on neuroinflammation induced by Aβ.Methods:1.MTS experiments examined the effect of Aβ_1-42 on BV2 cell proliferation at different time and dose.2.RT-qPCR method examined the effects of Aβ_1-42 on the IL-1βmRNA expression of BV2 cytokines.3.Using 2DG to inhibit the glycolysis process of BV2 cells,RT-qPCR method examined the effects of BV2 inflammatory factor IL-1βmRNA and i NOS mRNA expression after Aβ_1-42 treatment.Western blot methods examined the time-dependent changes of BV2 inflammatory factor pro-IL-1βprotein expression after Aβ_1-42 treatment.Griess assay was used to detect the NO secretion.4.sh RNA interfering HK2 genes in BV2 cells,knock down BV2 cell HK2 gene inhibits glycolysis process.RT-qPCR method examined the temporal correlation in the IL-1βmRNA and i NOS mRNA expression of BV2cytokines.Western blot methods examined the effects of BV2 cytokines pro-IL-1βproteins.Griess assay was used to detect the NO secretion.5.After intraperitoneal injection of wild-type C57BL/6N mice at the age of 7.5 months for 2DG with 3 days,Aβ_1-42 was stereotaxically injected into bilateral hippocampus to establish an acute model of AD.RT-qPCR assay was used to detect the expression of inflammatory cytokines IL-1βmRNA and TNF-αmRNA in the hippocampus of AD acute model mice after 2DG administration.Results:1.MTS results showed that BV2 cells were treated with 2.5μM and 5μM Aβ_1-42 for 6 h,and the cell proliferation effect was most obvious at 12 h.At 24 h,the proliferation effect of Aβ_1-42 on BV2 cells disappeared.2.RT-qPCR results showed that the inflammatory factors IL-1βmRNA expression of increased significantly and decreased at any time after treatment with different concentrations of BV2 cells.The inflammatory effect of 5μM was the most obvious,so the Aβ_1-42 concentration of 5μM was selected in the follow-up experiment.3.RT-qPCR results showed that 2DG could not inhibit the changes of inflammation in 3 h,but 6 h,12h and 24 h could inhibit the expression of Aβ_1-42 induced BV2 inflammation cytokines.Western blot experiments show consistent results with the above.Griess assay showed that 2DG inhibited the secretion of inflammatory cytokine NO induced by Aβ_1-42 in BV2 cells after24 h.4.RT-qPCR results showed that knockdown HK2 effectively inhibited the expression of Aβ_1-42 induced BV2 cytokines IL-1βmRNA 12 h and 24 h.Western blot experiments show consistent results with the above.Griess assay showed that HK2 knockdown effectively inhibited the expression of inflammatory cytokine NO induced by Aβ_1-42 in BV2 cells at 12 h and 24 h.5.RT-qPCR results showed that the mRNA of inflammatory factors increased significantly on day 1 after the establishment of the acute model of AD at 7.5 months.the Aβ_1-42 stimulated hippocampal inflammatory factors IL-1βmRNA and TNF-αmRNA decreased after 2DG intraperitoneal injection.Conclusions:1.Aβ_1-42 induced the dynamic changes of cell proliferation and inflammatory cytokines expression in BV2 cells.2.Aerobic glycolysis can regulate the expression of inflammatory cytokines induced by Aβ_1-42 in BV2 cells,and the inhibitory effect is more obvious in over-activation phase.3.Aerobic glycolysis can regulate the expression of inflammatory cytokines mRNA in the hippocampus of acute AD model mice.