The Study Of Celecoxib Effects On TLR4, NF-κB,COX-2 And VEGF Expression In Experimental Choroidal Neovasculariztion

Objective:Choroidal neovascularization (CNV)-related diseases is a major cause of visual disability. Many studies confirmed that inflammation and immune activation was of great importance to the formation and development of CNV.Celecoxib is a kind of ordinary non-steroid anti-inflammatory drug. Inflammation induces the expression of cyclooxygenase-2 (COX-2), resulting to accumulate of PG-like molecules that accelerate inflammation in reverse, while, celecoxib restrains emergent of PG-likemolecules by inhibiting the expression of cyclooxygenase-2 distinctively, so celecoxib acts on anti- inflammatory and analgesia to a large extent.In order to understand the influence of inflammation and immune activation to CNV and explore the mechanism and therapy of CNV, we established the experimental CNV model induced by laser photocoagulation and detected the expression of TLR4,NF-κB,COX-2 and VEGF in CNV membrane and the effect of celecoxib on the experimental CNV model.Methods:1 The study of the expression of TLR4 and NF-κB in experimental CNV model1.1 We randomly divided 10 healthy rats into two groups including the control group and laser group . In laser group ,the eyes were irradiated by a series of Krypon laser (647 nm wave length,100μm spot size, 120mW power,0.05second duration) lesions around the optic nerve head.1.2 Photocoagulation fundus fluorescein angingraohy (FFA) was performed on day 7 in laser group to observed the formation of CNV. 1.3 A rat of the two groups were sacrificed randomly on days 3,7,14,21,30 after laser photocoagulation, the eyes were enucleated and paraffin wax slices were manufactured, then histopathlogic examination processed. Then immunohistochemistry examination were performed for TLR4 and NF-κB. HPIAS-1000 image analysis system software was adapted to analysis test results.2 The study of the influence of celecoxib on the expression of TRL4,NF-κB,COX-2 and VEGF in experimental model of CNV in the BN rats2.1 We randomly divided 15 healthy rats into three groups including the control group, laser group and Celecoxib group. Celecoxib was administrated in celecoxib group by oral gavage (50 mg/kg,twice a day). After a week, the rats received krypon laser in laser group and celecoxib group, and CNV was induced. FFA was performed on days 3 , 7, 14, 21 , 30 after laser photocoagulation.2.2 The rats were sacrificed on 6h after photocoagulation,the eyes were enucleated and paraffin wax slices were maked for the control group and"the thickest CNV membranes"in laser group and celecoxib group. Histopathologic and immunohistochemistry examination were performed to detect the expression of TRL4,NF-κB,COX-2 and VEGF. HPIAS-1000 image analysis system software was adapted to analysis test results.2.3 Total RNA were abstracted from choroidal tissue and the mRNA of TRL4,NF-κB,COX-2 and VEGF gene was detected by RT-PCR.Results:1 The study of the expression of TLR4 and NF-κB in experimental CNV model1.1 7 days after photoagulation,little fluorescence leakage appeared in early stage,and fluorescence leakage expanded evidently in late stage.It shows that CNV were formed.1.2 The results showed that Bruch membrane ruptured.Meanwhile, macrophages infiltrated, RPE cells proliferated and migrated ,and fibroblasts increased. 1.3 Immunohistochemistry examination indicated that the expression of TLR4 and NF-κB was a little weak in the control group, but that ascended on 3-7d and descended later in laser group.2 The study of the influence of celecoxib on the expression of TRL4,NF-κB,COX-2 and VEGF in experimental model of CNV2.1 CNV both increased the highest on day 21 after photocoagulation in laster group and celecoxib group,and the incidence of CNV is clearly lower in celecoxib group than that in laser group(P<0.01).2.2 Immunohistochemistry examination demonstrated that the expression level of TLR4,NF-κB,COX-2,VEGF was positive in CNV membranes in the laser group, the results showed that the expression rising then declined with the time. The expression of TLR4 and NF-κB increasing on days 3 and reaching the peak on days 7.The expression of COX-2 and VEGF increasing on days 7 and reaching the peak on days 21. Immunohistochemistry examined the expression of TLR4,NF-κB,COX-2,VEGF in CNV membranes. The result showed that they significantly declined in celecoxib group on peak days 7-21 after photocoagulation (P<0.01).2.3 RT-PCR showed the mRNA expression of TLR4,NF-κB,COX-2 and VEGF gene. The trend was the same as that of immunohistochemistry examination.Conclusion:1 The signal route of NF-κB may be activated by TLR4,and the expression of blood vessel growth factors be induced through stimulating inflammation in experimental CNV.2 The studies demonstrated that Celecoxib could reduce the incidence of experimental CNV, we inferred that celecoxib may inhibit the expression of NF-κB, which could induced expression of TRL4,COX-2 and VEGF.