LOX-1 And TLR4 Affect Each Other And Regulate The Generation Of ROS In A.Fumigatus Keratitis

Purpose: To explore the relationship between LOX-1 and TLR4 in Aspergillus fumigatus(A.fumigatus)keratitis.To determine LOX-1 and TLR4 can affect each other and regulate inflammation through regulation of the generation of reactive oxygen species(ROS)in A.fumigatus keratitis.Methods: C57BL/6 mice cornea was infected with the active A.fumigatus,and the model of A.fumigatus keratitis was made.The abdominal cavity extracted neutrophils of susceptible C57BL/6 mice were stimulated with inactivated A.fumigatus hyphae.The corneas were pretreated with LOX-1 neutralizing antibody,Polyinosinic acid(Poly(I))(the inhibitor of LOX-1)or CLI-095(the inhibitor of TLR4)separately before the active A.fumigatus infection.LOX-1,TLR4 and IL-1? mRNA and protein expression were detected in normal and infected cornea or neutrophils by real time RT-PCR and Western Blot,while the generation of ROS was detected in the neutrophils by flow cytometry.To observe the changes of ROS in neutrophils after stimulation with inactivated A.fumigatus hyphae.The abdominal cavity extracted neutrophils of susceptible C57BL/6 mice were pretreated with LOX-1 neutralizing antibody,Poly(I)(the inhibitor of LOX-1)or CLI-095(the inhibitor of TLR4)separately before the inactivated A.fumigatus hyphae infection,and then using flow cytometry to detect the changes of ROS generation in these neutrophils of pretreatment.Results: LOX-1,TLR4,IL-1? mRNA and protein levels were up-regulated in C57BL/6 cornea after A.fumigatus infection.LOX-1 neutralizing antibody pretreatment decreased the mRNA expression of TLR4 and IL-1? in C57BL/6 cornea after A.fumigatus infection.To further confirm the result,using Poly(I)(the inhibitor of LOX-1)to block LOX-1,which decreased the mRNA expression of TLR4 and IL-1? in C57BL/6 cornea after A.fumigatus infection.Further detection of protein levels revealed that LOX-1 neutralizing antibody and Poly(I)(the inhibitor of LOX-1)pretreatment decreased the protein expression of TLR4 and IL-1? in C57BL/6 cornea after A.fumigatus infection.CLI-095(the inhibitor of TLR4)pretreatment decreased the mRNA expression of LOX-1 and IL-1? in C57BL/6 cornea after A.fumigatus infection.Further detection of protein levels revealed that CLI-095(the inhibitor of TLR4)pretreatment decreased the protein expression of LOX-1 and IL-1? in C57BL/6 cornea after A.fumigatus infection.Compared with the normal group,LOX-1,TLR4,IL-1? mRNA levels were up-regulated in the abdominal cavity extracted neutrophils of susceptible C57BL/6 mice after inactivated A.fumigatus hyphae stimulation,8 hours after stimulation to peak,followed by decreased expression.The results of flow cytometry showed that ROS generation was increased in C57BL/6 abdominal cavity extracted neutrophils after A.fumigatus hyphae infection,however,ROS generation was decreased in C57BL/6 neutrophils after infection by LOX-1 neutralizing antibody or Poly(I)or CLI-095 pretreatment.Conclusion: LOX-1,TLR4 and IL-1? expression are increased in C57BL/6 cornea after A.fumigatus infection.Inhibition of LOX-1 can reduce TLR4 and IL-1? expression.Inhibition of TLR4 can reduce LOX-1and IL-1? expression.LOX-1 and TLR4 can affect each other and promote inflammation.ROS generation are increased in neutrophils after A.fumigatus hyphae infection.Inhibition of LOX-1 or TLR4 can reduce ROS generation.LOX-1 and TLR4 can affect each other and regulate inflammation through regulation of the generation of ROS in A.fumigatus keratitis.