Expression And Role Of CGRP In Mouse Aspergillus Fumigatus Keratitis

Purpose:To investigate the expression and role of calcitonin gene-related peptide?CGRP?in the mouse models induced by Aspergillus fumigatus?A.fumigatus?.Methods:1.Specific pathogen-free C57BL/6 mice was randomized into a control group and A.fumigatus keratitis group.Mouse cornea after infection of A.fumigatus on 1d,3d and 5d was established by intratumoral injection which 2?L A.fumigatus conidia suspension?2.5×10⁴CFU/?L?was injected.The cornea photography was assessed under the slit lamp and the clinical score was recorded after infection.Western blot,real-time PCR and immunohistofluorescence analysis was applied to detect CGRP expression in cornea of both groups.2.In vitro,tests were conducted with C57BL/6 mice macrophages to investigate CGRP expression after interaction with A.fumigatus.The mRNA and protein expressions of CGRP were detected by real-time PCR and Western Bolt.The localization of CGRP in the normal or infected macrophages?8h?of mice was detected by immunocytofluorescence staining.3.Macrophages were pretreated with exogenous CGRP or CGRP8-37 30min,then stimulated with A.fumigatus hyphae.The mRNA expression of IL-1?,TNF-?,IL-6,MIP-2,IL-10 and TGF-?in macrophages was tested by real-time PCR.We also used enzyme linked immunosorbent assay?ELISA?to examine IL-1?and TNF-?protein levels in macrophages supernatant after stimulation with A.fumigatus hyphae.Results:1.Compared with the control group,CGRP mRNA and protein level was significantly elevated at 1d,3d and 5d after A.fumigatus infection of mice cornea,and peaked at 3days.And clinical scores indicated statistically significant differences at 1,3and 5day between control group and A.fumigatus-infected group 2.CGRP mRNA and protein level were significantly increased by A.fumigatus at 4h,8h,12h in A.fumigatus-infected macrophages than in control group,and peaked at 8h.Immunocytofluorescence staining?ICF?images showed CGRP protein in primary macrophages without or with exposure to A.fumigatus.3.The macrophages of mice were pretreated with exogenous CGRP or CGRP_8-37-37 before A.fumigatus infection.CGRP treatment reduced IL-1?and TNF-?mRNA and protein expression,and the mRNA level of IL-6.While,exogenous CGRP increased the expression of IL-10 compared with the A.fumigatus infection group.No significant difference was detected on mRNA level of MIP-2 and TGF-?.Pretreatment with CGRP_8-37-37 before infection of A.fumigatus increased IL-1?and TNF-?production at mRNA and protein levels compared with the A.fumigatus infection group,and elevated the IL-6 mRNA level.No significant difference was detected on mRNA level of IL-10,MIP-2 and TGF-?.Conclusions:CGRP expression was elevated in mice cornea with A.fumigatus keratitis.It is suggested that CGRP was involved in the inflammatory process of A.fumigatus keratitis.The exogenous CGRP reduced the expression level of pro-inflammatory factors and increased the expression of anti-inflammatory factors.While pretreatment with CGRP8-37 can increase the expression of pro-inflammatory factors.It suggested that CGRP might play a protective role against inflammation in A.fumigatus keratitis.