The Role And Mechanism Of TLR4 Signal Inhibition In Alkali-burn Induced Corneal Neovascularization

Objective:To investigate the role and molecular mechanism of Toll-like receptor 4（TLR4）signal in alkali burn（AB）-induced corneal neovascularization（CNV）.Methods:The corneal AB model was built by using C57BL/10Sc N background and 8 weeks male wild-type（WT）mice,and the same background TLR4 knockout(TLR4^-/-)mice.The study was divided into three sections.Section 1 was NC group and AB group,with15 WT mice in each group.Section two was WT-NC group,WT-AB group,TLR4^-/--NC group and TLR4^-/--AB group,with 15 mice in each group.Section 3 was NC group,AB group and Tak242-AB group,with 15 WT mice in each group.Intraperitoneal injecting the 1.25%tribromoethanol（0.2 m L/10 g）for anesthetizing mice.A round piece of 2 mm diameter filter paper was dipped in a concentration of 1M Na OH and attached to the left eye for 40 s.Subsequently,the left eye was immediately rinsed with20 ml of normal saline solution to remove the residual Na OH.Routine postoperative feeding was performed.Corneal neovascularization was observed by slit lamp microscope at D0,3,7 and 14 after alkali-burn,and the percentage of containing CNV area was quantified by Robert formula.Mice were sacrificed after anesthesia with tribromoethanol on D14,and the left eye was removed.H&E staining was used to evaluate the changes of corneal thickness.Western blot,immunohistochemistry and RT-PCR were used to detect the expression of VEGF-A,My D88 and NF-κB.The expressions of IL-1β,IL-6 and TNF-αwere detected by RT-PCR.In addition,a specific inhibitor of TLR4,Resatorvid（Tak242）,was used to treat the alkali-burn cornea of WT mice.Corneal neovascularization was observed by slit lamp microscope,western blot,immunohistochemistry and RT-PCR were used to detect the expression of VEGF-A,My D88 and NF-κB.The expressions of IL-1β,IL-6 and TNF-αwere detected by RT-PCR.Result:1.After corneal alkali burn,CNV grew from the edge of the cornea and gradually invaded the center of the cornea.The area of neovascularization gradually increased with time on D3,7 and 14.The results of H&E staining showed that alkali-burn significantly increased the thickness of the corneal stroma.RT-PCR and Western blot results showed that the expression of TLR4 in the cornea obviously increased after alkali-burn.2.TLR4 KO had notably reduced CNV area and corneal stroma thickness.The m RNA expression levels of TLR4 down-stream signal My D88 and NF-κB were down regulated;VEGF-A,inflammatory factors IL-1β,IL-6 and TNF-αwere also decreased in TLR4^-/-mice cornea.The results of Western blot showed that the expression of My D88,NF-κB and VEGF-A protein was decreased in TLR4^-/-mice after corneal alkali-burn.Immunohistochemistry showed that TLR4 KO reduced the positive immunoactivity of VEGF-A.However,the m RNA expression levels of TRIF-dependent pathway TRIF and IRF3 was not different from that of WT mice after alkali-burn.3.Compared with AB group,Tak242 reduced the area of neovascularization and the corneal thickness stroma.In addition,the m RNA expression levels of My D88,NF-κB,VEGF-A and inflammatory factors IL-1β,IL-6 and TNF-αin the cornea of Tak242treatment mice were also decreased.Similar results were also replicated in Western blot and immunohistochemistry experiments.However,there was no difference in the m RNA expression levels of TLR4,TRIF and IRF3 in the cornea of Tak242 treated mice compared with AB group.Conclusion:TLR4 signaling and related inflammatory factors were activated in the alkali-burn cornea.TLR4 inhibition attenuated AB-induced CNV and down regulated the expression of My D88,NF-κB,VEGF-A,and inflammatory factors.Therefore,the results of TLR4 mediating alkali-burn induced corneal vasculature in My D88dependent way may provide a theoretical basis for the clinical treatment of CNV and angiogenic diseases.