AnCoA4 Inhibits The Activation Induced By LPS In Primary Microglia And BV-2 Cells

In recent years,a large number of studies have shown that inflammation in the brain is closely related to the occurrence and development of various acute or chronic neurodegenerative disease,such as Alzheimer's disease?AD?,Parkinson's disease?PD?and Multiple Sclerosis?MS?,etc.Microglia cells play a key role in nerve inflammation,which protects neurons and secretes neurotoxic substances to maintain the microenvironment of neurons.Microglial cells,which are immune active cells of the central nervous system,present a resting state in normal conditions.When the brain is damaged or infected,microglial cells can quickly be activated to play a defensive role,such as activation,devouring,migration and release large amounts of inflammatory mediators and cytokines including tumor necrosis factor-??TNF-??,nitric oxide?NO?and interleukin 6?IL-6?,etc.This inflammatory response not only targets pathogens and tissue fragments in the lesion area,but also damages peripheral neurons and causes secondary brain damage.Therefore,it is important to restrain the excessive activation of microglia to repair brain damage.Ca²⁺is the second messenger in cells,which regulates many life activities,such as cell proliferation,gene transcription,apoptosis,immune response,and neurotransmitter delivery.In the immune system,calcium signaling plays an important role in the activation of lymphocyte immune function.Store operated calcium entry?SOCE?is the main way for extracellular Ca²⁺entering the cells.At the same time,it plays an important role in the process of the generation of cAMP and activation of T lymphocyte.In lymphocytes,SOCE is the main pathway to increase intracellular Ca²⁺,which is used to activate the expression of cytokines and activation of T cells and B cells.The activation of SOCE requires the interaction of endoplasm retinal protein STIM1?matrix molecular interaction between 1?and plasma membrane Ca²⁺release activated calcium channel Orais?its homologue Orai1,Orai2 and Orai3?or transient receptor potential channel?TRPC?interaction.It has been reported that SOCE plays an important role in regulating UDP-induced phagocytosis and LPS?bacterial lipopolysaccharide?-incuced release of inflammatory cytokines.Pharmacology research points out that AnCoA4,the Orai1 specific inhibitors,can interact with the C-terminal of Orai1 protein and directly inhibit calcium influx,and reduce the combination of Orai1 and STIM1,indirectly inhibit SOCE.Although the role of SOCE in microglial cells has been confirmed,the cause of SOCE is complex,and the molecular mechanism of SOCE in the activation of microglia is not yet clear.To explore whether Orai1 protein is involved in SOCE in the process of regulating the microglia activation,if relevant,what role Orai1 actually plays in this process,the mouse microglial cell line BV-2 and the primary microglia of rats were used.We used enzyme-linked immunosorbent assay?ELISA?,calcium imaging technology and FCM to explore Orai1's effects on microglia activation.The results showed that AnCoA4 can significantly inhibit the inflammatory response induced by LPS in primary microglia and BV-2 cells.It can inhibit not only the activation on the form but also the effect on the function,reducing inflammation reaction and production of cytokines,such as TNF-?,IL-1?and NO.In addition,AnCoA4 has a certain inhibitory effect on the proliferation of BV-2 cells and the apoptosis of primary microglia induced by LPS.Calcium imaging results showed that,Tg-triggered SOCE existed in microglia and Orai1 involved in the process of SOCE,while,there may be other calcium channel and related proteins involved except Orai1,such as the TRPC channel and Orai2 and Orai3.According to the results above,this study concluded that inhibiting Orai1 can reduce the excessive activation of microglia and inhibit its proliferation and apoptosis induced by LPS.It provided new clues to explore Orai1 and STIM1 mediating SOCE in the microglia further,and a new basis for reaserching nervous system degenerative disease caused by excessive activation of microglia.