Lentinan Regulates Microglia Through Dectin-1 Receptor To Inhibit Neuroinflammation-mediated Remyelination

Background: Inflammatory demyelinating disease of the nervous system is a disease that involves multiple parts of the brain,spinal cord,and peripheral nerves.Current,there is no drug to cure this disease completely.The development of drugs to treat demyelinating diseases by "promoting myelin regeneration" will bring hope for the complete cure of such diseases.Previous studies have found that Dectin-1 is closely related to the process of remyelination,and β-glucan can promote regeneration and repair after optic nerve system injury through Dectin-1 receptors.Lentinan（LNT）is a kind ofβ-glucan.Studies have shown that it has strong anti-inflammatory and immunomodulatory functions.However,it is not clear whether LNT regulates microglia through Dectin-1 receptors to inhibit inflammation-mediated remyelination.In this study,we systematically investigated the effect of LNT on the inhibition of inflammationmediated remyelination by microglia through Dectin-1 receptors and its possible mechanisms at the cellular and animal levels.This study will provide a potential target and candidate compound for the clinical treatment of demyelinating diseases.Methods: In vitro experiments: Lipopolysaccharide（LPS）induction of BV2 microglia was used to construct the neuroninflammatory model;CCK-8 assay,cellular IF and WB assay were used to evaluate whether LNT on LPS with Laminarin（Lam）blocking Dectin-1 receptor.Lipopolysaccharide（LPS）was selected to induce BV2 microglia to induce inflammatory response;CCK-8 method,cellular immunofluorescence staining and Western blotting assay were used to evaluate the polarization effect of LNT on LPS-induced BV2 microglia and the expression of inflammatory cytokines under the condition of blocking Dectin-1 receptor with Laminarin（Lam）.The detection indexes included Iba1,i NOS,Arg-1,TNF-α,IL-1β,IL-10,BDNF,and Dectin-1.In vivo experiments: C57 mice induced by Cuprizone（CPZ）were used as the animal model of demyelinating disease.The improving effects of LNT on the model mice of demyelination was observed by weight measurement,rotarod test,LFB staining and immunofluorescence staining.After blocking Dectin-1 with Lam,the motor function of mice was evaluated using the rotarod test,the expression changes of myelin in the corpus callosum region were evaluated using LFB staining and MBP staining,and the expression changes of microglia,astrocytes,oligodendrocyte precursor cells were evaluated using Iba1,GFAP,Olig2,Dectin-1 immunofluorescence staining,respectively,as well as Dectin-1 receptor-activated cells in the corpus callosum region.WB was used to detect the expression of i NOS,Arg-1,TNF-α,IL-1β,and BDNF in the corpus callosum region.Results: In vitro experiments: CCK-8 assay showed that LNT inhibited the proliferation of LPS-induced BV2 microglia in a concentration-dependent manner.immunofluorescence staining and Western blotting results showed that LNT significantly inhibited LPS-induced BV2 microglia Iba1 expression,and had a significant inhibitory effect on the highly expressed pro-inflammatory factors（i NOS,IL-1β and TNF-α）,and had significant increase effects on low-expressed anti-inflammatory factors Arg-1 and BDNF.After applying Lam to block Dectin-1,the regulatory effects of LNT on proinflammatory and anti-inflammatory factors were inhibited.In vivo experiments: After the model mice were given LNT continuously for 7 days,monitoring body weight and using the rotarod test experiment revealed that LNT could alleviate the weight loss and motor dysfunction.The results of LFB staining and MBP staining showed that LNT could significantly inhibit demyelination in the model mice.After blocking Dectin-1 with Lam,the results of the rotational test,LFB staining and MBP staining showed that LNT could neither alleviate the motor dysfunction nor effectively inhibit the demyelination of the model mice.The results of immunofluorescence staining showed that the activation of Iba1+,GFAP+ and Olig2+ cells could not be significantly inhibited by LNT in the corpus callosum region.The results of western blotting showed that LNT could not significantly inhibit the expression of i NOS,IL-1β and TNF-α and promote the expression of Arg-1,BDNF and Dectin-1 in the corpus callosum.Conclusion: In this study,we found that LNT can regulate microglial cell polarization,inhibit neuroinflammation,improve motor function and promote remyelination through Dectin-1 receptor,and significantly improve demyelinating disease,which provides a new target and potential candidate compound for clinical treatment of demyelinating disease.