Dabrafenib Mitigates The Pathology Of Experimental Autoimmune Encephalomyelitis By Inhibiting Ferroptosis-Mediated Neuroinflammation Via Up-regulating Axl

Objective:To investigate the protective effect of receptor tyrosine kinase Axl on experimental autoimmune encephalomyelitis（EAE）mice and its regulatory effect on ferroptosis,as well as the therapeutic effect of Dabrafenib（DA）on neuroinflammation associated with ferroptosis in EAE mice,and to further understand the association between Axl and ferroptosis.It lays a foundation for the research of small-molecule compounds targeted therapy for multiple sclerosis（MS）and even neurodegenerative diseases,which seeks new targets for MS therapy.Methods:1.C57BL/6 mice were used as experimental animals,and EAE mice model was used as animal model of MS disease to verify whether the drug delayed the progression of EAE disease.2.BV2 mouse microglia cells were used as experimental cells,and the cell model was constructed with LPS and Erastin.Firstly,cell viability was detected to determine the optimal intervention concentration of DA.Besides,the cell cycle,ROS and JC-10 levels were detected by flow cytometry in each group.Secondly,immunofluorescence was used to locate the expression of M1/M2 glial cells and Axl and M1 cells.Finally,the detection of expression of neuroinflammation-related proteins and the changes of inflammatory factors in each group by WB,so as to clarify the influence of DA on the inflammatory function of Axl and BV2 cells.The BV2 cell model induced by LPS and Erastin,the cell cycle and ROS levels of each group were detected by flow cytometry,the observation of JC-10 levels of each group by fluorescence microscope,the detection expression of ferroptosis related protein by WB,and the observation ultrastructure of each group by transmission electron microscope.Finally,molecular docking was used to predict the binding pattern of DA and GPX4 to clarify the effect of DA on ferroptosis related neuroinflammation.3.Axl knockout mice were used as experimental animals,and EAE model was used as an animal model of MS.The expressions of Axl and ferroptosis related proteins were detected by WB and immunofluorescence,and the expressions of ACSL4 and GPX4 proteins were identified by immunohistochemistry in different groups.It was further confirmed that DA alleviated inflammatory demyelination of EAE by inhibiting ferroptosis of microglia cells through Axl.Results:1.In EAE mice,H&E staining and Weil results showed inflammatory cell infiltration and myelin sheath loss.Immunohistochemical results showed that ACSL4was positively expressed and GPX4 expression was decreased.The immunofluorescence results showed that both ACSL4 and IBA-1 expression were up-regulated,suggesting that neuroinflammation related to ferroptosis may exist in EAE mice,and ferroptosis may be mediated by microglia cells.DA treatment alleviated symptoms of mice,reducing neurological scores,relieving inflammation,and inhibiting ferroptosis in EAE mice.2.In the LPS-induced inflammatory model,the treatment of DA transformed BV2 cells into M2 cells,blocking S phase,inhibiting ROS response,restoring mitochondrial function,activating the expression of P-JAK2 and P-STAT3 by up-regulating Axl,which can inhibit inflammation in order to playing a therapeutic role in MS.However,ferroptosis was more severe in the LPS and Erastin cell models than in the Erastin group.Treatment with DA can block the S phase of cells and restore mitochondrial function by inhibiting ROS response.In addition,the expressions of Ferritin,GPX4 and System x C-were up-regulated,and the expressions of ACSL4,CD71 and POR were down-regulated,which reduced the degree of intracellular lipid peroxidation and significantly inhibited the ferroptosis of cells.Finally,the binding mode of DA and GPX4 was simulated by molecular docking.It was found that DA and GPX4 are hydrogen bonded,which suggested that DA may inhibit ferroptosis through GPX4.These studies suggest that the neuroprotective effect of DA is related to the inhibition of inflammatory response and ferroptosis,and may be mediated by the receptor tyrosine kinase Axl receptor.3.Compared with the EAE group,the Axl^-/-+EAE group had higher neurological scores,more severe symptoms,and more severe ferroptosis.WB results showed that Ferritin,System x C-and GPX4 decreased significantly,while CD71,ACSL4 and POR increased significantly.Immunohistochemical results showed that ACSL4 was positive,but GPX4 was less expressed.Immunofluorescence results showed up-regulated expression of ACSL4 and IBA-1,suggesting that Axl aggravated the symptoms of EAE mice by promoting ferroptosis in microglia cells.In the DA treatment group,neurological function scores decreased,symptoms were alleviated,Axl expression was up-regulated,and ferroptosis related protein expression levels were reduced,suggesting that DA could regulate the expression of ferroptosis related protein by up-regulating Axl,affecting the level of intracellular iron metabolism and lipid peroxidation,and inhibiting the intracellular ferroptosis.The above studies suggested that DA exerted neuroprotective effects on EAE by acting on Axl receptors to inhibit ferroptosis in microglia cells.Conclusion:1.After Axl gene knockout,the inflammatory response and ferroptosis degree of EAE model were aggravated;2.Axl alleviated inflammatory demyelination of EAE by inhibiting ferroptosis in microglia.3.DA may have a therapeutic effect on ferroptosis related neuroinflammation in EAE mice by up-regulating Axl to inhibit ferroptosis in microglia cells.