| Objective: To investigate the expression,function and mechanism of aryl hydrocarbon receptor(Ah R)in the cell and tissue of dry eye mice.Methods: 1.Dry eye models of mice were developed by combining hypodermic injection of scopolamine solution with simulated dry environment.Ah R localization was detected in conjunctival goblet cells by immunohistochemistry.Ah R mRNA and protein expressions were tested in conjunctival tissues and conjunctival goblet cells of normal and dry eye mice by PCR and ELISA.2.Dry eye models of mice were established and mice conjunctivas were treated by Ah R antagonist 6-formylindole [3,2-b] carbazole(FICZ)for 1,3,5,7 days to measure the tear volume by using phenolic cotton line method,observe the corneal injury stained with sodium fluorescein under a slit-lamp,and record dry eye clinical scores;The quantity of goblet cells in normal,dry eye and FICZ-treated conjunctivas was assessed by HE staining,PAS staining and immunohistochemistry.PCR and ELISA were used to test Ah R,MUC5 AC,inflammatory cytokines TNF-α,IL-1β,IL-6 and IL-8 mRNA and Ah R and MUC5 AC protein expressions in conjunctivas of normal group,dry eye control group and FICZ-treated group;Conjunctival goblet cells were extracted from conjunctivas of dry eye mice by tissue cultivation.Ah R and MUC5 AC mRNA and protein expressions in conjunctival goblet cells of mice were tested by PCR and ELISA.TNF-α,IL-1β,IL-6 and IL-8 mRNA expressions were tested by PCR.3.Dry eye models of mice were established and mice conjunctivas were treated by Ah R inhibitor 2-methyl-2H-pyrazole-3-carboxylic acid(2-methyl-4-o-tolylazo-phenyl)-amide(CH223191)for 1,3,5,7 days.PCR and ELISA were used to test Ah R,MUC5 AC,TNF-α,IL-1β,IL-6 and IL-8 mRNA and Ah R and MUC5 AC protein expressions in conjunctivas of normal group,dry eye control group and CH223191-treated group; Conjunctival goblet cells were extracted from conjunctivas of dry eye mice by tissue cultivation.Ah R and MUC5 AC mRNA and protein expressions in conjunctival goblet cells of mice were tested by PCR and ELISA.TNF-α,IL-1β,IL-6 and IL-8 mRNA expressions were tested by PCR.4.Conjunctival goblet cells of dry eye mice were treated with FICZ and p-ERK1/2 inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene(U0126)after FICZ pretreatment.PCR and ELISA tested mRNA and protein expressions of Ah R and MUC5 AC.Western blot tested protein expressions of p-ERK1/2 and ERK1/2.Results: 1.Ah R expressions were detected in conjunctival tissues and conjunctival goblet cells of dry eye mice and the expressions of Ah R mRNA and protein were lower than in normal conjunctival tissues and conjunctival goblet cells.2.FICZ increased the tear volume,reduced corneal injury and significantly decreased dry eye clinical scores compared with dry eye control group;The number of conjunctival goblet cells was increased by FICZ.Ah R and MUC5 AC mRNA and protein levels in conjunctivas of dry eye mice were significantly elevated by FICZ compared with dry eye control group,Ah R and MUC5 AC mRNA levels peaked at 5 days,Ah R and MUC5 AC protein levels peaked at 7 days,inflammatory cytokines TNF-α,IL-1β,IL-6 and IL-8 mRNA expressions were reduced by FICZ;Ah R and MUC5 AC mRNA and protein expressions in conjunctival goblet cells of dry eye mice were significantly increased by FICZ,TNF-α,IL-1β,IL-6 and IL-8 mRNA expressions were reduced by FICZ.3.Ah R and MUC5 AC mRNA and protein levels in conjunctivas of dry eye mice were significantly reduced by CH223191 compared with dry eye control group,TNF-α,IL-1β,IL-6 and IL-8 mRNA expressions were increased by CH223191;Ah R and MUC5 AC mRNA and protein expressions in conjunctival goblet cells of dry eye mice were significantly induced by CH223191,TNF-α,IL-1β,IL-6 and IL-8 mRNA expressions were elevated by CH223191.4.Ah R and MUC5 AC mRNA expressions and Ah R,MUC5 AC,p-ERK1/2 and ERK1/2 protein expressions in conjunctival goblet cells of dry eye mice were significantly reduced by U0126 after FICZ pretreatment compared with FICZ-treated group.Conclusion: Ah R expressions decreased in conjunctival tissues and conjunctival globet cells of C57BL/6 dry eye mice,which suggested that Ah R affected dry eye.The activated Ah R promoted MUC5 AC secretion,decreased TNF-α,IL-1β,IL-6 and IL-8 expressions and relieved the inflammation of dry eye by p-ERK1/2 pathway. |
