Expression Of High Mobility Group Protein 1(HMGB1)and Toll-like Receptor 9(TLR9)in Oxygen-induced Retinopathy Model

BackgroundROP is a kind of immature retinal vascular proliferative blindness which occurs in premature infants.The pathological mechanism has not been clarified.The important pathogenic factors of ROP are low gestational age,hypoxia,inflammation and infection.OIR mouse model is a classic model for ROP study.In recent years,the relationship between inflammation and ROP neovascularization has attracted much attention.High mobility group protein 1(HMGB1)is a highly conserved nuclear DNA binding protein,which is an important late inflammatory mediator.TLR9 is an important member of TLRs family.It can coordinate wound healing and induce angiogenesis by activating the expression of downstream inflammatory cytokines.In this study,OIR model was established.The expression of HMGB1 and TLR9 in retina was detected by Western blot and immunofluorescence staining.To explore the role of both proteins in the pathogenesis of retinal neovascularization,and a new idea for clinical drug treatment was provided.ObjectiveTo explore the expression of high mobility group protein 1(HMGB1)and Toll-like receptor 9(TLR9)in mouse oxygen-induced retinopathy(OIR).MethodsA total of 30 C57BL/6 newborn pups were randomly divided into OIR group and normal control group,15 pups group.The normal control group was fed in room air.7-d-old(p7)mouse pups with nursing mothers are subjected to hyperoxia(75% oxygen)for 5 d,on p12,they are returned to room air for 5 day.Both groups were killed on the P17.Isolectin B4 staining of retinal flat is used to observe the new neovascularization.The expression levels of HMGB1 and TLR9 proteins were detected by Western blot and immunofluorescence staining.Results1.Compared with the normal control group,the structure of the retina in the OIR group is disordered,the branches of blood vessels are significantly reduced,and the blood vessels are tortuous and disordered.There is a vascular occlusion zone in the center of the retina,and a large number of neovascular clusters can be seen at the junction of the occlusion zone and the peripheral vascular zone.2.Immunofluorescence staining: In the control group,the structure of each layer of retina was regular,and HMGB1 protein(red)was mainly expressed in the ganglion cell layer,with a small amount of expression in the inner and outer nuclear layers;in the OIR group,the retinal nerve fiber layer was thickened,and the structure of each layer was disordered,and the expression of HMGB1 protein(red)in the ganglion cell layer,inner nuclear layer,and outer nuclear layer was significantly increased compared with the control group(t=﹣10.38,p<0.001).In the control group,TLR9 protein(red)was mainly expressed in the ganglion cell layer and the nuclear layer;the positive expression of TLR9 protein(red)in the ganglion cell layer and the nuclear layer in OIR group was higher than that in the control group(t=﹣14.37,p<0.001).3.Western blot results showed that HMGB1 and TLR9 protein expression levels were higher than those in the control group(t=9.515,p=0.001 and t=﹣2.993,p=0.040).Conclusion1.HMGB1 protein and TLR9 protein were expressed in the retina of control group and OIR group.2.Compared with normal control group,the expression of HMGB1 and TLR9 proteins was increased in the retina of OIR group,and both of them may be related to the development of retinal lesions and angiogenesis.