LOX-1 Regulates Neutrophil Apoptosis In A.fumigatus Keratitis

Purpose: To determine whether lectin-like ox-LDL receptor(LOX-1)expresses and regulates neutrophil apoptosis in A.fumigatus keratitis.Methods: The mRNA levels of Fas,Fas ligand(FasL),caspase3(CASP3),caspase8(CASP8),caspase9(CASP9),and BCL2 were detected in normal and fungal keratitis corneas of C57BL/6 mice.Pretreated with LOX-1 neutralizing antibody or LOX-1inhibitor Ploy I,RT-PCR,TUNEL staining was used to test mRNA expression and apoptosis.Immunofluorescence assays were used to test neutrophils infiltration and cleaved caspase3 protein expression of C57BL/6 mice corneas.Neutrophils were extracted from C57BL/6 mice,and pretreated with LOX-1 neutralizing antibody or IgG;LOX-1 inhibitor Ploy I or PBS,then stimulated with A.fumigatus.The mRNA levels of Fas,FasL,CASP3,CASP8,CASP9,and BCL2 were evaluated by PCR and expression of cleaved caspase3 protein was tested by western blotting.Neutrophil apoptosis were also assessed by flow cytometry.Results: Fas,FasL,CASP3,CASP8,and CASP9 m RNA were higher in C57BL/6mice infected corneas than normal corneas,on the contrary,BCL2 mRNA lower.After treatment with LOX-1 neutralizing antibody or LOX-1 inhibitor Ploy I,the mRNA levels of Fas,FasL,CASP3,CASP8,andCASP9,cleaved caspase3 protein and TUNEL-positive cells in C57BL/6 corneas were significantly decreased compared with treatment with control IgG or control PBS,instead BCL2 mRNA increased.Neutrophils infiltration was significantly decreased after treatment with LOX-1 neutralizing antibody or LOX-1inhibitor Ploy I compared with IgG or PBS control.Fas,FasL,CASP3,CASP8,and CASP9,and cleaved caspase3 protein were significantly decreased after treatment with LOX-1 neutralizing antibody or LOX-1 inhibitor PloyI compared with IgG or PBS control in neutrophils,but BCL2 mRNA increased,correlated with significantly less neutrophils apoptosis.Conclusion: These data provided evidence that LOX-1 inhibition can reduce the expression of pro-apoptotic gene,Fas,FasL and anti-apoptotic gene,neutrophils infiltration,caspase-3 activation and corneal cells apoptosis in fungal infected corneas of C57BL/6 in vivo.Meanwhile,LOX-1 inhibition can also reduce the expression of Fas,FasL,CASP3,CASP8,CASP9 mRNA,and increase BCL2 mRNA,decrease caspase-3activation and the apoptotic rates of neutrophils in vitro.