The Effect Of MI-3 On Microglial Neuroinflammatory Factors And Its Mechanism

Object: Microglia are important immune cells in the central nervous system.When stress or injury occurs,they can secrete pro-inflammatory factors,mediate inflammatory responses,and produce cytotoxic effects.Menin,the protein product of MEN1 gene,interacts with mixed lineage leukemia protein（MLL）as an oncogenic accessory protein,and is involved in the proliferation and differentiation of hematopoietic stem cells.Menin protein is also expressed in the central nervous system and is involved in the occurrence and development of many nervous system diseases.MI-3,as a specific inhibitor of menin-MLL interaction,has been shown to be a potential target for the treatment of acute leukemia with MLL rearrangement.Currently,the role of menin-MLL interaction in models of microglial neuroinflammation has not been investigated and remains unclear.This study mainly explored the effect and underlying mechanism of MI-3,a specific inhibitor of menin-MLL interaction,on the development of microglial neuroinflammation model.Methods: First,the BV2 microglia were stimulated with LPS to construct a sepsis model,and the changes of menin and MLL proteins under LPS stimulation were observed.MI-3 was used for intervention to observe the expression of inflammatory factors（i NOS 、 TNF-α 、 IL-1β）,and to explore whether MI-3 could alleviate LPS-induced microglial neuroinflammation.Continue to use the above model to observe the changes of PI3 K phosphorylation,NFκ B phosphorylation and nuclear translocation,and explore whether MI-3 can alleviate LPS-induced activation of PI3K/NFκ B axis in microglia.On the basis of confirming that MI-3 can alleviate the neuroinflammation of microglia caused by LPS,the method of indirect co-culture of BV2 microglia and N2 a cells was used to investigate whether MI-3 could further protect N2 a cells from the neurotoxic effects of LPS-stimulated BV2 cells after alleviating LPS-induced microglial inflammation.Results:（1）After 50 ng/m L LPS treatment for 8/12 hours,the expression of menin protein was significantly increased compared with the baseline level（P8h=0.0406,P12h=0.0018）.After 50 ng/m L LPS treatment for 8/12 hours,the expression of MLL protein was significantly increased compared with the baseline level（P8h=0.0125,P12h=0.0021）.Menin protein and MLL protein were treated with different concentrations of LPS（10 ng/m L,50 ng/m L）for 8 hours,and the expressions of menin and MLL proteins increased after 50 ng/m L LPS treatment compared with the baseline levels（Pmenin=0.0096,PMLL=0.0219）（2）MI-3,an inhibitor of menin-MLL interaction,can down-regulate the expressions of i NOS（p=0.0039）,IL-1β（p=0.0008）and TNF-α（p=0.0011）in microglia after LPS stimulation.（3）MI-3 could down-regulate the expression of PI3 K phosphorylation in microglia after LPS stimulation（p=0.0028）.After adding the PI3 K inhibitor LY294002 at the same time,MI-3 failed to further down-regulate the expression level of pro-inflammatory mediator IL-1β in microglia stimulated by LPS（p=0.9997）.（4）MI-3 could inhibit LPS-induced phosphorylation of NFκ B-p65 in microglia（p=0.0133）and nuclear translocation of NFκ B-p65（p=0.0331）.（5）MI-3 protects N2 a cells from LPS-induced neurotoxicity in microglia.That is,MI-3and LPS treatment of BV2 cells can reduce the expression of caspase3 in N2a（p=0.0028）,and can improve the survival rate of N2 a cells（p=0.0293）.Conclusion: Menin-MLL interaction is involved in the regulation of microglial inflammatory response.MI-3,a specific inhibitor of menin-MLL interaction,can attenuate microglial inflammatory response and neurotoxicity through PI3K/NFκ B signaling pathway.