Preliminary Study On The Role Of ERRα In Hypoxia Injury Of Microglia

Spinal cord injury（SCI）is a neurological disorder that results from the destruction of the spinal cord’s structure and function,resulting in neurological disorders and leading to the weakening or loss of skeletal muscle voluntary movements.Hypoxia is a common pathological state after SCI,neurons in the central nervous system are extremely sensitive to changes in the internal environment.Thus,hypoxia not only causes neuronal degeneration and necrosis,but also aggravates the inflammatory responses in the spinal cord.As resident immune cells of the central nervous system,microglia cells can be activated in response hypoxia signal stimulation,and they induce multiple signaling pathways of microglial cells are activated to regulate the pathophysiological process after spinal cord injury.Microglia activation is a highly energy-dependent process,and mitochondria,as the main site of intracellular energy synthesis,play a non-negligible role in this process.Estrogen-related receptorα（ERRα）is a key metabolic regulator in energy metabolism and mitochondrial biogenesis.Its deletion leads to an imbalanced antioxidative stress capacity in the body.Currently,the role of ERRαin hypoxic spinal microglia has not been fully elucidated.Therefore,in this study,we used BV2 microglial cells to establish an in vitro hypoxic model,which we used to clarify the role of ERRαin spinal microglial hypoxic injury.In this study,CoCl₂ was used to stimulate BV2 microglia to construct a cell hypoxia model.Different concentrations of CoCl₂ were used to stimulate BV2 microglia cells,and the concentration and time of CoCl₂ treatment were screened through laser confocal technology combined with CCK-8.The results showed that when the concentration of CoCl₂ was 100μmol/L,the viability of BV2 microglia was not significantly affected,and when the concentration was treated for 8 hours,the expression of HIF-1αin the cell in response to hypoxia was the highest and it was transferred from the cytoplasm to the nucleus,indicating that the BV2 microglia has been hypoxic at this time,so it can be judged that the BV2 microglia hypoxia model has been successfully established.To investigate the role of ERRαin autophagy of hypoxic BV2 microglia,the expression levels of autophagy-related proteins Beclin1 and LC3,as well as the m RNA levels of autophagy gene p62 were detected.The results showed that ERRαsignificantly down-regulated the expression of autophagy-related proteins Beclin1 and LC3,as well as the expression of autophagy-related gene p62 m RNA,suggesting that ERRαinhibited autophagy in hypoxic BV2 microglia.To investigate the changes of ERRαin the NF-κB inflammatory signaling pathway in hypoxic BV2 microglia,the protein expression levels of p65,p38,and IκB-αin the NF-κB inflammatory signaling pathway,and the m RNA expression levels of inflammatory factors IL-1,IL-6,TNF-α,p65,IL-4,and IL-10 were detected in this study.The results showed that ERRαsignificantly down-regulated the phosphorylation level of p38,suggesting that ERRαmay inhibit the activation of p38MAPK signaling pathway.The m RNA levels of pro-inflammatory cytokines IL-1β,IL-6,TNF-α,p65 and IL-10 were significantly down-regulated,and the m RNA levels of anti-inflammatory cytokines IL-4 were significantly up-regulated,suggesting that ERRαmay affect the process of secondary inflammatory response after spinal cord hypoxia injury by regulating the expression of anti-inflammatory cytokines,and prevent the inflammatory pathway from over-activation of injured nerve cells.In order to investigate the effect of ERRαon the proliferation and migration of hypoxic microglia,the protein expression levels of FNDC5,BDNF and ERK 1/2 were detected in this study,and the change of wound healing rate was detected by cell scratch test.The results showed that ERRαincreased the wound healing rate of BV2 microglia,and ERRαsignificantly up-regulated the level of FNDC5 protein and down-regulated the level of BDNF protein,suggesting that ERRαmay promote the proliferation of BV2 microglia by regulating the expression of FNDC5,and thus play a positive role in injury repair.