The Anti-inflammatory Effect Of Tristetraprolin In Fungal Keratitis

Objective:To study the effect and mechanism of Tristetraprolin(TTP)on fungal keratitis(FK).Methods:1.The corneas of C57BL/6 mice were infected by Aspergillus fumigatus(A.fumigatus)to establish animal models of fungal keratitis.Reverse transcription-polymerase chain reaction(RT-PCR)and Western blot were used to detect the expression of TTP m RNA and protein in the corneas of mice infected for 4h,6h,9h,12 h,16h,18 h and 24 h and mice in the control group.Immunofluorescence(IF)was used to detect the expression and localization of TTP in the corneas of mice in the control group and mice infected by A.fumigatus for 24 h.2.Mice were pretreated with TTP siRNA and Arctigenin(ATG),an agonist of Protein phosphatase 2(PP2A)which can dephosphorylate TTP and transform TTP into active state after being activated,to establish animal models of A.fumigatus keratitis.The corneal inflammatory reaction of mice corneas was observed with slit-lamp biomicroscope and the clinical scores were recorded.Western Blot was used to detect the protein expression level of TTP in mice corneas after ATG pretreatment,and RT-PCR was used to detect the effect of TTP siRNA pretreatment on TTP expression in mice corneas.3.Mice were pretreated by subconjunctival injection of ATG and TTP siRNA to establish animal models of A.fumigatus keratitis.Western blot was used to detect the protein expression level of TTP in corneas after ATG pretreatment.The m RNA expression levels of TTP,inflammatory factors such as IL-1β,TNF-α,IL-6 and chemokine CCL-2 in mice corneas were detected by RT-PCR and the expression of protein were detected by ELISA.4.In vitro,we used inactivated mycelium of A.fumigatus to stimulate human corneal epithelial cells(HCECs).RT-PCR and Western blot were used to detect the expression of TTP m RNA and protein in HCECs after 6h,9h,12 h,16h and 24 h infection,respectively.The expression and localization of TTP in HCECs were verified by immunofluorescence staining.After pretreatment HCECs with TTP siRNA or ATG,inactivate hyphae of A.fumigatus was added to stimulate cells.Western Blot was used to detect the protein expression level of TTP in HCECs after ATG pretreatment,and the expression of TTP,IL-1β,TNF-α,IL-6 in HCECs were detected by RT-PCR.5.After pretreatment HCECs with Zn PP(HO-1 inhibitor)for 2h,we used inactivated hyphae of A.fumigatus to stimulate for 12 h and 24 h,respectively.The expression of HO-1 and TTP in HCECs were detected by RT-PCR,and the protein expression of TTP in HCECs was detected by Western blot.Results:1.The expression of TTP m RNA and protein in corneas of mice infected by A.fumigatus were significantly higher than that of normal control group.Immunofluorescence staining showed that the expression of TTP protein in corneal tissues of mice infected by fungi was significantly increased compared with that of normal corneas,and it was mainly located in corneal epithelium.2.After ATG pretreatment,the corneal inflammatory reaction and clinical scores of mice were decreased.TTP siRNA down-regulates the expression of TTP in mice corneas.At the same time,the corneal inflammatory reaction was aggravated,and the clinical scores were increased.3.Compared with A.fumigatus infection group,the expression levels of m RNA and protein of IL-1β,TNF-α,IL-6 and CCL-2 in the corneas of mice injected with TTP siRNA under the conjunctiva increased,while the expression levels of the above inflammatory factors and chemokines in the corneas of mice injected with ATG under the conjunctiva decreased.4.In vitro,compared with the control group,the expression level of TTP m RNA and protein in HCECs increased significantly after stimulated by inactivated mycelium of A.fumigatus.Immunofluorescence results showed that the expression of TTP in infected HCECs increased,and the fluorescence signal was enhanced,mainly located in the nucleus.When the expression level of TTP was decreased after pretreatment with TTP siRNA in HCECs,the expression of IL-1β,TNF-α and IL-6 were increased compared with A.fumigatus infection group.HCECs were stimulated by inactivated mycelium of A.fumigatus after pretreatment with ATG,and the TTP protein in HCECs was decreased,while the expression of inflammatory factors IL-1β、TNF-α and IL-6 decreased significantly.5.In HCECs,the expression of HO-1 and TTP were increased after stimulated by A.fumigatus.Pretreatment HCECs with Zn PP down-regulated the expression of HO-1 in HCECs.Compared with A.fumigatus infection group,the expression level of TTP was decreased.Conclusion:TTP plays an anti-inflammatory role in fungal keratitis.HO-1 mediates the increase of TTP expression and reduces the inflammatory response of fungal keratitis caused by A.fumigatus by inhibiting the expression of downstream inflammatory factors and chemokines mediated by A.fumigatus.