Boxb Mediate BALB/c Mice Corneal Inflammation Through A TLR4/MyD88-dependent Signaling Pathway In Aspergillus Fumigatus Keratitis

Purpose: To investigate whether high-mobility group box 1(HMGB1)Boxb exacerbates BALB/c mice corneal immune responses and inflammatory through the TLR4/My D88-dependent signaling pathway in Aspergillus fumigatus keratitis.Methods: The effects of Boxb on the innate immunity and inflammatory response of Aspergillus fumigatus keratitis in mice were studied from the BALB/c mice model of Aspergillus fumigatus keratitis and the extracorporeal extraction of macrophages from the peritoneal cavity of BALB/c mice with inactivated Aspergillus hypha.BALB/c mice were randomly divided into four groups: PBS preconditioning group,Boxb preconditioning group,PBS pretreated Aspergillus fumigatus keratitis group,Boxb pretreated Aspergillus fumigatus keratitis group.The cornea of BALB/c mice was pretreated with PBS and Boxb subconjunctival injection for 1 days.The corneal morphology of mice was observed.A mouse model of Aspergillus fumigatus keratitis was made with the infection of Aspergillus fumigatus mycelium.After 1 days,the infection of the cornea tissue was observed and the mice were killed to collect corneal tissue.BALB/c mice were pretreated intraperitoneally with sodium thioglycolic acid.After 1 weeks,mouse peritoneal macrophages were extracted.PBS,Boxb,Boxb+CLI-095(TLR4 inhibitor)and Boxb+DMSO(TLR4 inhibitor solvent)were pretreated for 2 hours after adding inactivated Aspergillus fumigatus mycelium.The cells were collected for 12 hours for m RNA detection,24 hours for protein detection.Real-time RT-PCR technique was used to detect the expression of HMGB1,pattern recognition receptor TLR4,pro-inflammatory factor TNF-alpha and IL-1 beta m RNA in mouse cornea tissue and mouse peritoneal macrophages.The expression of pattern recognition receptor TLR4,pathway protein My D88 and proinflammatory factor TNF-alpha in mouse corneal tissues were detected by Western blot.Western blot was used to detect the expression of pattern recognition receptor TLR4 and pathway protein My D88 in peritoneal macrophages of mice.Results: The expression of HMGB1,pattern recognition receptor TLR4,pathway protein My D88,proinflammatory factor TNF-alpha and IL-1 beta in BALB/c mouse corneas pretreated with PBS and Boxb were almost the same,but in the BALB/c Aspergillus fumigatus mouse keratitis model,Boxb preconditioning group HMGB1,pattern recognition receptor TLR4,pathway proteins My D88 and proinflammatory cytokines TNF-alpha and IL-1 beta increased significantly compared with the PBS pretreatment group.In the BABL/c mouse peritoneal macrophages,compared with Boxb+DMSO preconditioning groups,the Boxb preconditioning group HMGB1,pattern recognition receptor TLR4,pathway protein My D88 and proinflammatory factor IL-1 beta,and TNF-alpha expression increased.The Boxb plus CLI-095 pretreatment group HMGB1,pattern recognition receptor,the pro-inflammatory factor IL-1beta,TNF-alpha elevation was suppressed.Conclusion: In the BALB/c mice model of Aspergillus fumigatus keratitis,HMGB1,pattern recognition receptor TLR4,pathway protein My D88,and proinflammatory factor IL-1 beta,TNF-alpha in the corneal tissue of Boxb pretreated mice were higher than PBS preconditioning group,indicating that Boxb can promote the inflammatory response of Aspergillus fumigatus keratitis in BALB/c mice through TLR4/My D88 pathway.The addition of Boxb to the macrophages of Aspergillus fumigatus inactivated mycelium infection can promote HMGB1,pattern recognition receptor TLR4,pathway protein My D88 and proinflammatory factor IL-1 beta,TNF-alpha,while TLR4 inhibitor CLI-095 is added to HMGB1,pattern recognition receptor TLR4,pathway protein My D88 and proinflammatory factor IL-1 beta and TNF-alpha was decreased.It is suggested that Boxb can promote the inflammatory response of Aspergillus fumigatus keratitis in BALB/c mice through TLR4/My D88 signaling pathway.