| Ischemic stroke（cerebral ischemia）is the number one killer of human health,which is characterized by high disability rate,high mortality rate and heavy economic burden.Within a few minutes after cerebral ischemia,neuroinflammatory responses rapidly initiate and further aggravate secondary nerve injury through a series of cellular and molecular cascade reactions.As the first immune responder of ischemic injury,microglia activate rapidly and participate in the regulation of neuroinflammatory response,playing a corresponding nerve damage or neuroprotection function through the M1 or M2 phenotype through polarization.Therefore,it is of great significance to actively explore the function of microglia and the molecular mechanism of polarization in the early stage of cerebral ischemia,and to find new therapeutic targets for regulating neuroinflammatory response,so as to improve the prognosis of patients.After the occurrence of ischemic stroke,the content of lactate in the brain increased rapidly.Previous studies have shown that lactate,a bioactive substance,can shuttle between astrocytes and neurons to play a corresponding regulatory role.At the same time,it has been reported that lactate can regulate the polarization of macrophages,however,as a signaling molecule,the regulation of lactate to the biological function of microglia has rarely been reported.DAB2 is an endocytosis adaptor protein that plays an important role in neuroinflammation and macrophage polarization,but whether its expression is regulated by lactate and thus modulates microglia biological functions have not been reported.The purpose of this study is to explore the mechanism of lactate in regulating microglia polarization through DAB2 under the condition of oxygen glucose deprivation（OGD）,so as to provide new therapeutic targets and ideas for promoting M2-type polarization of microglia as much as possible,to alleviate inflammatory response and improve the prognosis of patients.In this study,we used the mouse middle cerebral artery occlusion（MCAO）animal model to observe the activation and morphology of microglia after cerebral ischemia from the histological dimension.RT-qPCR and Western blot were used to detect the differentially expressed genes and their protein expression.The high-throughput sequencing of mRNA and bioinformatics analysis were used to screen the differentially expressed genesand functional enrichment analysis was performed to explore the mechanism of lactate in regulating microglia polarization through DAB2.The results are as follows:（1）The tissue sections of MCAO mice were stained by HE and immunohistochemical staining.The results showed that there was a clear penumbral zone on the ipsilateral side,the boundary between the injured tissue and the normal tissue,was obvious.The tissue structure was loose,and there were obvious cave-like structures in the ischemic focus.The expression of IBA1,which is the activation marker of microglia,was significantly increased in the injured side.The cell volume increased,and the protrusion became coarse-like amoeba.（2）In vitro OGD 8h cell model was used to simulate the pathological state of brain tissue in the early stage of cerebral ischemia.RT-qPCR and Western blot results showed that iNOS was significantly down-regulated and ARG1 was significantly up-regulated after OGD treatment.High-throughput sequencing were conducted among OGD 8h microglia and classical M1/M2 cell models（induced by LPS and IL4）,and the results of showed that the gene expression profile of microglia after OGD treatment was more favorable to M2 microglia.（3）Exogenous lactate was added to explore the regulation of lactate on microglia polarization under the condition of 8h OGD.RT-qPCR and Western blot results showed that iNOS was significantly down-regulated,and ARG1 was significantly up-regulated.High-throughput sequencing and bioinformatics analysis of mRNA were performed on OGD 8h treated cells and OGD 8h treated cells with exogenous lactate.The results showed that metabolic signaling pathways were significantly up-regulated after lactate added,while signaling pathways related to inflammation,such as Toll-like receptor,MAPK and TNF,were significantly down-regulated.Dab2 and other genes involved in neuroprotection and neuroinflammation were included in the significantly upregulated genes.（4）In order to further explore the regulation and mechanism of lactate on microglia polarization mediated by DAB2,western blot was performed on microglia treated with OGD combined with lactate treatment.The results showed that:compared with the control group,lactate treatment significantly increased DAB2 expression,however,significantly decreased the protein expression of TLR4 and P-p38 / p38.Under the same conditions,interference Dab2 expression significantly upregulated TLR4 and P-p38 / p38 protein expression.Finally,the experiment confirmed that SB203580 treatment could reverse the increasing trend of P-p38 / p38 induced by Dab2 interference..Conclusion:（1）Under the condition of OGD for 8h,microglia were polarized to M2 microglia.（2）Lactate induced a further M2 polarization of microglia under OGD conditions.（3）Under the condition of OGD,lactatemay promote M2-type polarization of microglia through DAB2/TLR4/ p38 MAPK signal axis,inhibiting inflammation and play an important neuroprotective role. |
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