| Objectives: To explore the relative expression level of miR-19 b and JAK/STAT3 signaling pathways in the IgE mediated allergic conjunctivitis model.To investigate the changes of ocular surface inflammation in mice with allergic conjunctivitis after blockade JAK/STAT3 signaling pathway.To test the effect of miR-19 b on TSLP-mediated allergic inflammation and JAK/STAT3 signaling pathway.Method: 1.Thirty BalB/c female mice were randomly divided into control group(PBS),allergic conjunctivitis group(AC),CYT387(JAK/STAT3 pathway blocker)interfering allergic conjunctivitis group(AC+ CYT387),miR-19 b agomir interfering allergic conjunctivitis group(AC+ miR-19b),and agomir negative control allergic conjunctivitis group(AC+control miR),with six mice in each group.2.Use clinical scorings to assess the severity of the mice conjunctivitis.HE staining detected the inflammatory cells and eosinophils infiltrating in ocular surface.ELISA was used to detecting serum IgE levels.Double immunofluorescence staining detected both TSLP and p-STAT3 expression in dendritic cells.RT-qPCR was used to observe mRNA levels of miR-19 b,TSLP and pro-inflammatory factors in cornea,conjunctiva and lymph node tissues of mice.Result: 1.Clinical scores showed that the degree of ocular surface inflammation in the AC group was significantly higher than that in the normal control group.JAK/STAT inhibitor or miR-19 b agomir could also reduce the AC symptoms and inflammatory scores,whereas control miR injection showed similar results with AC.2.HE staining showed that the infiltration levels of inflammatory cells and eosinophils on the ocular surface of the AC group were significantly higher than PBS group(P<0.0001),while those of AC+ miR-19 b group and AC+CYT387 group were significantly lower than AC group(P<0.001).3.ELISA showed that the level of serum IgE in AC group was significantly higher than control group(P<0.01),and the level of serum IgE in AC+CYT387 group and AC+ miR-19 b group was significantly lower than AC group(P<0.05).4.Double immunofluorescence staining showed that after the injection of miR-19 b agomir,the expression of TSLP and p-STAT3 in dendritic cells were significantly reduced compared with those in the AC group.Expression of TSLP in AC+CYT387 group was significantly decreased comparing with AC group.5.RT-qPCR showed that the expression of miR-19 b was significantly decreased in AC group comparing with PBS group,and the level of miR-19 b was significantly increased respectively in conjunctiva,cornea and lymph node tissues in mice after the injection of mir-19 b agomir(P<0.001).The levels of pro-inflammatory cytokines,such as TSLP,IL-1,IL-5 and IL-13,were obviously decreased after mir-19 b overexpression or blockade JAK/STAT pathway(P<0.5).Conclusions: 1.In allergic conjunctivitis model,the expression of mir-19 b was significantly reduced,the expression of targeted regulatory TSLP was significantly increased,and the JAK/STAT3 pathway was activated in dendritic cells.Inhibition of the JAK/STAT signaling pathway or overexpression of mir-19 b can lead to phenotypic inhibition of allergic conjunctivitis and reduced inflammatory response.2.The expression of TSLP in conjunctival,corneal and cervical lymph node tissues induced by allergic conjunctivitis can be significantly blocked by the overexpression of mir-19 b.While TSLP is blocked,phosphorylation of the downstream JAK/STAT signaling pathway is blocked.3.Inhibition of the JAK/STAT pathway or overexpression of mir-19 b can reduce the expression of dendritic cell maturation and Th2 inflammatory responses,thereby affecting the occurrence and development of allergic conjunctivitis. |
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