Experimental Study On The Regulation Of Inflammation By PAR-2 In Fungal Keratitis In Mice

Purpose:To investigate the expression,function and mechanism of protease-activated receptor-2?PAR-2?in keratitis infected by Aspergillus fumigatus?A.fumigatus?.Methods:1.PAR-2 mRNA expression was tested in normal and infected mice corneas of 12hours,1 day and 2 days by PCR.PAR-2 protein expression was tested in normal and infected mice corneas of 1,2 and 3 days by Western blot.2.Mice corneas were pretreated by 0.25,0.5,1.25mmol/L PAR-2 antagonist?FSLLRY-NH₂?and infected by A.fumigatus.PCR and Western blot were used to test PAR-2 mRNA and protein expression.3.Mice corneas were infected with A.fumigatus with or without pretreatment of FSLLRY-NH₂.Disease severity was documented by clinical score and photographs with a slit lamp.PMN infiltration was assessed by MPO assay and immunofluorescent staining.PCR,Western blot,ELISA tested expression of PAR-2,IL-1?,TNF-?,IFN-?and MIP-2.4.Polymorphonuclear neutrophilic leukocytes?PMNs?were extract by Casein stimulation from the abdominal cavity of mice and then were stimulated of 12,18,24 hours by 75%ethanol-killed A.fumigatus.PAR-2protein expression in PMNs was tested by Western blot.5.PMNs were stimulated with 75%ethanol-killed A.fumigatus with or without pretreatment of FSLLRY-NH₂.PCR,Western blot,ELISA tested expression of PAR-2,IL-1?,TNF-?,IFN-?,MIP-2 and p-ERK1/2.Results:1.PAR-2 m RNA expression was significantly elevated by A.fumigatus at 12hours,1 day,2days,and peaked at 1 day.PAR-2 protein expression was significantly elevated by A.fumigatus at 1,2,3 days,and peaked at 2 days.2.Pretreatment with 0.5 mmol/L and 1.25mmol/L FSLLRY-NH₂ significantly inhibited A.fumigatus induced upregulation of PAR-2m RNA levels.Moreover,1.25 mmol/L FSLLRY-NH₂ inhibition effect is better.However,PAR-2 mRNA had no significant difference after treatment with 0.25 mmol/L FSLLRY-NH₂.1.25 mmol/L FSLLRY-NH₂ inhibition effect of protein level is confirmed.3.FSLLRY-NH₂decreased disease response,PMNs infiltration,and IL-1?,TNF-?,IFN-?,MIP-2 expression compared with infected control.4.In PMNs,PAR-2 protein expression was significantly elevated by 75%ethanol-killed A.fumigatus at 12,18,24 hours and peaked at 18 hours.5.FSLLRY-NH₂ significantly inhibited PAR-2,IL-1?,TNF-?,IFN-?,MIP-2 and p-ERK1/2protein expression compared with infected control.Conclusion:These data provide evidence that A.fumigatus increased PAR-2 expression and elevated disease,PMNs infiltration and proinflammatory cytokines expression through PAR-2,which may be modified by p-ERK1/2.