Therapeutic Effect And Mechanism Of P2X7 Receptor Inhibitor BBG In EAE Mice

Objective:To investigate the effect of purinergic receptor P2X ligand-gated ion channel 7（P2X7R）inhibitor Brilliant Blue G（BBG）on the inflammation and demyelination of spinal cord,and to explore the potential mechanism with focus on P2X7R/NOD-like receptor thermal protein domain associated protein 3（NLRP3）signaling pathway in experimental autoimmune encephalomyelitis（EAE）mice.Methods:In this study,EAE mice were induced by Myelin Oligodendrocyte Glycoprotein_35-55(MOG_35-55),followed by BBG injected intraperitoneally every other day since 24 hours post-induction in EAE+BBG group.The neurological deficits of mice were evaluated by Kono’s scores daily.Luxol Fast Blule（LFB）and hematoxylin and eosin staining（H&E）staining were used to observe the demyelination and inflammation of the spinal cord,while immunohistochemistry（IHC）staining was employed to evaluate the activation of microglia in the spinal cord.Proteins related to P2X7R/NLRP3 signaling pathway were detected by IHC staining and western blot.Results:Around 14 days post-immunization（DPI）,EAE mice started to display neurological deficits and reach peak at about 21 DPI.Compared to EAE+PBS mice,the symptom onset of EAE+BBG group was delayed by about 2 days（13.92±0.358 vs15.75±0.494,P<0.05）,and the maximal score of neurological deficits was significantly decreased（3.108±0.112 vs 1.375±0.125,P<0.05）.Compared to EAE+PBS mice,the demyelination areas of white matter were significantly reduced（30.530±1.956 vs7.348±1.231,P<0.05）by LFB staining,as well as the inflammatory infiltrated areas（3.653±0.086 vs 2.125±0.136,P<0.05）by H&E staining in EAE+BBG group.Further,the expression of Iba-1⁺microglia in the spinal cord of EAE+BBG group was significantly lower than that in EAE+PBS group（P<0.05）revealed by IHC staining.The Moreover,the expression of NLRP3 inflammasome related proteins in the spinal cord of EAE mice decreased after BBG treatment,followed by downregulated expression of IL-1βand IL-18,which were evaluated by IHC staining and Western Blot.Conclusion:P2X7R inhibitor,BBG,effectively reduced the inflammatory cell infiltration and demyelination in the EAE spinal cord,thus delayed the symptom onset of EAE with reduced severity.BBG inhibited the activation of NLRP3 inflammasome and antagonized the downstream inflammatory factors,thereby playing a neuroprotective role in EAE.