| BackgroundFungal keratitis(FK)is a serious infectious disease of the cornea in which the Ctype lectin receptor is involved in recognizing fungal pathogens and mediating the host’s immune response in the presence of fungal infection.However,the mechanism by which the Dendritic cell-associated C-type lectin-2(Dectin-2)receptor mediates the immune response in corneal tissue is not known.ObjectivesTo investigate the expression of Dectin-2 receptors among C-type lectin receptors in fungal keratitis and the mechanism of Dectin-2 receptors in the immune regulation of fungal infectious keratitis,and to understand the role of Dectin-2 receptors in the recognition and defence of corneal tissue against fungi.MethodsFifty clean-grade,C57BL/6 healthy mice,female,6-8 weeks old,weighing between 15-22 g,were selected and no structural or functional lesions were found in the ocular corneal tissue by slit lamp microscopy.The mice were randomly divided into three groups: fungal keratitis group,injury control group and blank control group.Forty of them were used to establish the models of fungal keratitis group and injury control group;10 were used for the blank control group without any treatment.The right eye was used as the Candida albicans experimental group and the left eye was used as the injury control group.The keratitis model of Candida albicans infection in mice was established by the corneal scratch method.In the injury control group,only the corneas of the mice were injured with 29 G needles and equal doses of saline were administered.After 1d,2d,3d and 5d of modeling,the mice were observed under a slit lamp microscope and photographed to record the changes in the corneas of the mice after infection with the fungus.The mice were executed according to the above time and the corneal tissues of each group were taken for the subsequent experiments.One(2 eyes)of the blank control group was used for hematoxylin-eosin staining for pathological observation,and one(2 eyes)was used for immunohistochemical detection of Dectin-2receptor expression in mouse corneal tissues;one(1 eye)of the fungal keratitis group and one(1 eye)of the injury control group were used for HE staining for pathological observation,and one(1 eye)of each group was used for The expression of Dectin-2receptor in mouse corneal tissues was detected by immunohistochemistry,and 4 eyes were taken each time for Real-time PCR and Western blot to detect the mRNA and protein expression of Dectin-2 receptor,IL-1β and CARD9 in corneal tissues respectively.Results1.Morphology and HE staining results of a fungal infection model in miceThe keratitis model of Candida albicans infection could be successfully established in mice by adopting the corneal scratch method.Slit lamp microscopic observation showed that the corneal structure of the blank control group was intact and without edema.The damaged control corneas showed slight edema on the first day,which gradually returned to normal at subsequent times.In the fungal keratitis group,white ulcerated foci,poorly defined borders,reduced transparency in the focal area of the cornea and hairy glass-like cloudiness were observed.The pathological results showed that the corneal structures of the blank control and damaged control groups were intact,without edema and inflammatory cell infiltration,while the fungal keratitis group showed loss of the preelastic layer of the corneal epithelium at the ulcer and neutrophil infiltration in the stroma.2.Real-time PCR resultsDectin-2 receptor and CARD9 mRNA expression were not detected in the blank control group,and the relative expression of IL-1β mRNA was low;Dectin-2 receptor,IL-1β and CARD9 mRNA were expressed in both the injury control and fungal keratitis groups.The relative expression of Dectin-2 receptor,IL-1β and CARD9 mRNA did not change significantly in the injury control group at different time points;in the fungal keratitis group the expression of Dectin-2 receptor mRNA increased significantly,reaching a peak at 3d,with statistical significance compared to the injury control group at all time points(p<0.05).In the fungal keratitis group,the expression of IL-1β and CARD9 mRNA also increased,reaching a peak at 3 d and gradually decreasing over time,with a statistically significant difference compared to the injury control group at each time point(p<0.05).3.Western blotThe expression of Dectin-2 receptor,IL-1β and CARD9 was significantly higher in the fungal keratitis group at different time points compared to the injury control group,and the difference was statistically significant(p<0.05).4.Immunohistochemical resultsDectin-2 receptor was basically not expressed in the blank control group and Dectin-2 receptor was mainly expressed in the corneal epithelium in the fungal keratitis group.ConclusionThe Dectin-2 receptor is a pattern recognition receptor that may be involved in the intrinsic immune response to fungal keratitis in mice.CARD9 was up-regulated after Candida albicans stimulation and may be involved in the inflammatory response of corneal tissue in fungal keratitis. |
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