| BackgroundFungal keratitis is a kind of inflammatory disease caused by fungal infection in the cornea tissue,which was firstly reported by Leber in 1878 and is a common,more stubborn fungal infection.Because of the difficulty of early diagnosis and treatment of this disease,it often causes corneal ulcer or perforation,progresses to endophthalmitis and results in visual impairment or blindness.In recent years,due to the extensive use of antibiotics,cortical hormones,immunosuppressants and other drugs in clinical,the incidence of fungal keratitis in China is increasing,especially in male and rural residents.Fungal keratitis has been the second primary blinding eye disease following cataract,putting enormous economic burdens to people’s families and social development.Fusarium and Aspergillus are the main strains causing ocular fungal infection,and the ocular infection caused by aspergillosis is mainly caused by Aspergillus and flavus.Corneal infections have become major pathogens in some areas.At present,there are many patients with fungal infection,but few effective drugs can be used,makes treatment is more difficult.Therefore,it is very important to study the etiology,pathology,immunology,diagnosis and epidemiology of this disease in early diagnosis and prevention.The cornea is a spherical crown,with thin center and thick edge,is composed of 5layers of corneal cells and connective tissue,its mechanical barrier can resist to fungus infection.Corneal epithelial cells and stromal cells can express some pattern recognition receptors(PRRs)to activate the innate immune system by pathogenic molecular model(PAMPs),inducing specific adaptive immune response to remove pathogens and repair the damage of pathogens to human body.Ariizumi et al.in 2000 firstly discovered dendritic cells related to C-type lectin-1(dectin-1),which is a receptor of the surface-specific expression of dendritic cells,and is widely distributed in dendritic cells,monocytes,B lymphocytes and some T-lymphocyte subsets,as well as in mouse microglia.Recent studies have shown that dectin-l can recongnizeβ-1,3/β-1,6-glucan and carbohydrates on the surface of fungi and bacteria.During fungal infection,Dectin-1 participates in the proliferation of Th17cells,which produce IL-17a,IL-17f,IL-6,and TNF-ɑto mobilize,raise and activate neutrophils for counteracting the immune response to extracellular fungal and bacterial infections.The IL-17 produced by Th17 cells can effectively mediate the excitatory process of neutrophil mobilization,thus effectively induce the inflammatory response of tissues.Because of the Tyr238x mutation,Dectin-1 cannot be expressed on the cell membrane,resulting in reduced the production of IL-17 and increased susceptibility to fungal infections.IL-12 and TNF-αfactors also play an important role in the antifungal response.Dectin-1 receptor has transmembrane structure,belonging to the C-type lectin receptor family,which can identify exogenous pathogens,such as fungi and mycobacteria,and also can recognize the endogenous ligand of T lymphocytes.Because the receptor recognizes fungi,it can be found in patients with fungal keratitis.And in the clinical diagnosis and treatment of patients,we can achieve good results by interfering with the role of Dectin-1 receptor.We note that there are some studies on the Dectin-1 receptor in the clinical diagnosis and treatment of fungal keratitis in China,but it needs to study it in detail.According to this,we carry out this research.ObjectiveThe pathological changes of rat cornea before and after the construction of the animal model of fungal keratitis,the expression of Dectin-1 receptor and the relationship between Dectin-1 receptor with IL-17,IL-12 and TNF-αwere discussed in this study.These results can reveal the role and mechanism of Dectin-1 receptor in antifungal keratitis,providing certain data for clinical diagnosis and new therapeutic target sites.Materials and Methods1.Establishment of animal model(1)Animals and groupsThe 60 Wistar-clean and healthy rats,weight between 200g-250g,were selected by the school Laboratory Animal Center.No structural and functional lesions were observed in the cornea tissue of the eyes after the slit lamp microscope.The rats were divided into three groups:experimental group 1,experimental Group 2 and control group,the number of mice were 25,25 and 10.(2)Establishment of animal model1)The bacterium Aspergillus strain(number 3.0772),which is provided by the storage and management center of common microbial strains in China,is prepared into a bacterial suspension with a concentration of 1x10~8 mycelium/ml.2)The corneal curvature radius of the experimental rat was measured and the mean value was calculated as the final.The corneal contact lens prepared by sealing film for corresponding radius of curvature,were soaked and disinfected with the ethanol with concentration of 75%.3)The 25 rats in the experimental group 1 were treated with laminaria(dectin-1specific inhibitor),and the 25 rats in the experimental group 2 were treated with saline.4)The 50 rats in the experimental group 1 and 2 were drilled around 2mm circular prints in the middle of the cornea.5)The bacterial suspension was dropped on the cornea of the rat,and the corneal contact lens was used to cover the eyelid,to ensure the target strain infect rats and to establish the rat model of fungal keratitis.2.Experimental methodAfter the establishment of the animal model of fungal keratitis,on the 1th Day,2nd day,3rd day and 5th day,the rat corneal epithelium of the control group,experimental Group 1,experimental Group 2 were taken respectively.(1)Pathological observation;(2)The expression of Dectin-1 in corneal tissue of rats was observed by immunohistochemistry;(3)The mRNA expression levels of Dectin-1,TNF-α,IL-17 and IL-12 in rats were measured and compared by QRT-PCR technique.Result1.Pathological ResultsThe cornea of the rats in the control group were intact,without edema and inflammatory cell infiltration.The cornea of the rats in experimental group 1 and 2,were thickening of edema,absence of anterior elastic layer of ulcer,neutrophil infiltration in anterior chamber and matrix.2.Immunohistochemical ResultsThe expression of dectin-1 receptor in the corneal tissue of rats in the control group and the experimental group 1 was weak positive,and the expression of dectin-1receptor was strong positive in the Rats in the experimental group 2 after 1d.3.QRT-PCR Detection Results(1)The Dectin-1,TNF-αand IL-12 were expressed in the cornea tissue of the rats in the control group,experimental Group 1 and experimental Group 2.(2)There was no IL-17 expression in the corneal tissue of the rats in the control group and the experimental group 1.(3)After 1d,the expression of Dectin-1 in the corneal tissue cells of the rats in the experimental Group 2 was significantly higher than that in the control group and the experimental Group 1(P<0.05),the expression of Dectin-1 in the corneal tissue of the rats in the experimental Group 1 was slightly lower than that in the control group,but there was no statistical difference(P>0.05).(4)The expression of IL-12 and TNF-αin the corneal tissue of the rats in the experimental Group 1 was significantly higher than that in the control group(P<0.05).(5)In the experimental group 2,the expression of IL-12 in the cornea tissue of the rats increased in 1d~3d,and the expression of TNF-αincreased in 1d~5d.Conclusion1.After fungal infection,the expression of Dectin-1 in the cornea of the rat was increased,which may be closely related to the response degree of inflammatory reaction.2.The synthesis of IL-17 may depend only on the intracellular signaling pathway mediated by Dectin-l receptor.3.IL-12 plays the role in the early stage of fungal infection.Dectin-l receptor is involved in expression of IL-12,and IL-12 is also induced by other types of cell receptors.4.TNF-αfactor is involved in the corneal inflammatory response of fungal keratitis,and the expression level of the factor is not significantly related to the Dectin-1 receptor,which may be regulated by other receptor-induced signaling pathways in the cell. |
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