Study On The Role Of Hv1-ko Regulates Autophagy Of Microglia In Demyelination Of Central Nervous

[Objective]Demyelination disease of central nervous system is a group of diseases which cause the absence of myelin sheath in brain and spinal cord as the main pathological feature.Microglia is the immune cell implanted in the central nervous system and play an important role in the pathological process of central nervous demyelination disease.Hv1 is a voltage-gated proton channel expressed on microglia in the central nervous system^[1].This study intends to take proton channel Hv1 as the target to investigate the changes of autophagy in microglia after the knockout of Hv1 gene,and study its role in demyelination of central nervous system.[Method]In this experiment,we injected lysophosphatidylcholine?LPC?on one side of the corpus callosum?CC?in the Hv1 knockout(Hv1^-/-)and wild type?WT?mice,set up animal model of demyelination in central nervous system.We selected Sham,5d,10d,28d after the operation as group of experiment,collected mouse's brain,through the Luxol fast bule?LFB?staining,Western Blot and immunofluorescence staining to observe demyelination and autophagy level's change,through electron microscopy to compare the Myelin loss and autophagosome formation in CC.The above experimental results were statistically analyzed to compare the difference in demyelination severity and autophagy level between the Hv1^-/-and WT mouse.[Result]LFB staining results of the mouse's CC showed that demyelination degree in Hv1^-/-group was significantly reduced compared with WT group at the time points of 5d,10d and 28d after the demyelination model was established.Western blot results indicated that compared with WT group,LC3II/LC3I ratio in Hv1^-/-group were down-regulated,at the time point of 5d,the difference was most significant.Immunofluorescence staining showed that in the CC of Hv1^-/-group at 5d after operation,LC3-positive microglia were significantly reduced compared with WT group,which means autophagy level of microglia in Hv1^-/-group was decreased compared with WT group.The changes of autophagosomes in two groups of mouse were observed by electron microscopy,indicated that the autophagosomes in the microglia of the Hv1^-/-group were significantly decreased compared to those in WT group,which means the autophagy level of the microglias in the CC of the Hv1^-/-group was significantly lower than that in the WT group.The demyelination in the two groups were observed by electron microscopy,too.G-ratio of Hv1^-/-group was lower than that of WT group,means that the myelin of Hv1^-/-group was thicker than that of WT group,and the less demyelination of nerve fibers in Hv1^-/-group.[Conclusion]In the LPC-induced mouse model of demyelination in central nervous system,knockout of Hv1 gene may through the pathway of inhibiting microglia autophagy to alleviate demyelination,suggesting that Hv1 gene is expected to become a new target for the treatment of demyelination disease of central nervous system.