MicroRNA-146a Participates In Allergic Conjunctivitis In Mice By Regulating CD4~+ CD25~- Tcon Cells

Objective:To study the expression and significance of miRNA-146a in mouse model of allergic conjunctivitis,to explore the cellular immunological regulation mechanism of miRNA-146a on allergic conjunctivitis,and to provide a new strategy for the diagnosis and treatment of allergic conjunctivitis.Methods:The hypersensitive conjunctivitis model was established by stimulating BALB/c mice with ragweed pollen(RW).The expression of miRNA-146a was detected by qRT-PCR,and the conjunctiva tissue was stained by Jisam.The number of eosinophilic cells was determined by Elisa and the levels of IL-5 and IL-13 were detected by Elisa,and the numbers of CD4~+CD25~-Tcon cells and CD4~+CD25~+Treg cells were detected by flow cytometry.Construction of miRNA-146a overexpression Lentivirus Vector To observe whether the overexpression of mi RNA-146a can improve the clinical symptoms of allergic conjunctivitis and various immune inflammatory indexes.Tcon cells and Treg cells were isolated and purified to induce hypersensitivity and mimic hypersensitive conjunctivitis at the cell level,and miRNA-146a mimics were constructed as inhibitor of mi RNA-146a and miRNA-146a negative control nucleic acid,and transfected into cells.To observe the effects of different levels of miRNA-146a expression on cell proliferation,IL-5 and IL-13 levels and NF-?B inflammatory signaling pathway.Results:1.After sensitized by RW,the mice showed obvious symptoms of allergic conjunctivitis,the level of RW specific IgE increased in serum,a large number of eosinophilic granulocytes infiltrated in conjunctiva,the expression of IL-5 and IL-13in spleen increased,and Tcon cells proliferated excessively.The proportion of Treg cells increased significantly,while the proportion of Treg cells decreased significantly.In addition,the expression of miRNA-146a in spleen of allergic conjunctivitis mice was significantly down-regulated.After injecting miRNA-146a lentivirus into tail vein of mice,the expression of miRNA-146a in spleen was upregulated significantly.The high expression of miRNA-146a could effectively improve the symptoms of allergic conjunctivitis,decrease the specific IgE in serum,decrease the infiltration of eosinophils in conjunctiva and decrease the infiltration of eosinophils in the conjunctiva.The level of IL-5 and IL-13 in spleen decreased,while the proportion of Tcon cells decreased and the proportion of Treg cells increased.2.After 72 hours of RW stimulation,the proliferation of spleen cells accelerated markedly and the levels of IL-5 and IL-13 increased significantly.The expression of miRNA-146a in spleen cells and purified Tcon cells and Treg cells were down-regulated.But spleen cells transfected with miRNA-146a mimics could counteract these changes.Co-culture of wild type Treg cells and Tcon cells could significantly inhibit the inhibitory effect of Tcon cell colonization.Tcon cells transfected with miRNA-146a inhibitor and then co-cultured with wild type Treg cells on Tcon cells.However,the inhibitory effect of Tcon cells transfected with miRNA-146a mimics and co-cultured with wild-type Treg cells was significantly enhanced.Interestingly,the changes in miRNA-146a in Treg cells The level of expression had no effect on its inhibitory effect.It was further found that miRNA-146a upregulated in Tcon cells could inhibit the epidermis of TRAF6,thus inhibit the activity of NF-?B and decrease the level of IL-5 and IL-13.However,after downregulation of miRNA-146a in Tcon cells,the expression of TRAF6 was increased,and the production of IL-5/IL-13 was increased after activation of NF-?B,which could be cancelled by NF-?B specific blocker Bay.Conclusions:1.The expression of miRNA-146a in spleen of allergic conjunctivitis model mice was significantly down regulated.Up regulation of miRNA-146a expression can effectively improve the symptoms of allergic conjunctivitis in mice.2.The inhibitory effect of CD4~+CD25~+Treg cells on CD4~+CD25~-Tcon cells is affected by the level of miRNA-146a in Tcon cells,and there is a positive correlation between them.3.The expression of miRNA-146a in allergic conjunctivitis Tcon cells down-regulated the activation of NF-?B signaling pathway and increased the levels of IL-5and IL-13 in Tcon cells.Upregulation of miRNA-146a expression in Tcon cells inhibited the activation of NF-?B and reduced cytokine production.