The Effect Of Celecoxib On The Expression Of COX-2,MMP-2 On Corneal Neovascularization In Rat And Mechanism Preliminary Study

BaqgroundCorneal neovascularization (corneal neovascularization, CNV) is a variety of ocular diseases such as infectious corneal diseases, ocular chemical injury, thermal burns, non-infectious corneal diseases, the salient features of corneal degeneration, is one of postoperative corneal transplant rejection risk factors. Often leads to loss of normal corneal transparency is the main reason for blindness. The lack of normal corneal tissue and lymph vessels, the anatomic characteristics of the cornea to some extent prevent the cornea angiogenesis, mainly depends on the presence of anti-angiogenic factors and the formation of anti-vascular stromal penetration barrier. In trauma, inflammation, corneal transplant rejection and induction of other reasons, to maintain the balance of factors avascular cornea is broken, the result in corneal neovascularization. Present study found that inflammation, hypoxia, a variety of angiogenic factors increases, inhibition of angiogenesis factor reduction, corneal edema, corneal nerve injury and many other factor s involved in various processes. The specific mechanism is not fully understood.Current clinical treatment of all causes of corneal neovascularization many ways, such as vascular surgery or laser quiescent consider the active growing season to consider drugs such as corticosteroids, but the result was not ideal, and the long-term use prone to serious side effects, including intraocular pressure, delayed wound healing, cataracts and other systemic side effects. Recent attempts to selective cyclooxygenase-2 (COX-2) inhibitor used in ocular neovascular diseases, has been made in the rat model results. Mainly used in diabetic retinopathy, choroidal neovascularization, corneal neovascularization, and so on. Celecoxib (Celecoxib, selective Cox-2 inhibitors, non-steroidal anti-inflammatory drug NSAID in one) is a highly selective COX-2 inhibitor, on the intensity of COX-2 inhibition than COX-1 strong 375 times. NSAID widely used in clinical practice and treatment of rheumatoid arthritis, familial adenomatous polyp, pain, especially in recent years, the constant depth of the drug, found that addition to the general non-NSAID role, also has anti-tumor, anti-angiogenesis and inhibition of corneal graft rejection after transplantation hungry.COX (cyclooxygenase, COX) is the limiting enzyme in prostaglandin synthesis, are known to have three isomers, namely COX-1, COX-2 and COX-3, in which COX-2 as "inducible expression", it is not detected in most normal tissues, while cytokines, growth factors, oncogenes and tumor promoting agent is upregulated on a variety of stimuli involved in inflammation, cell proliferation and differentiation. MMP (matrix metalloproteinases MMPs) are a family of endogenous Zn-dependent protease family, the degradation of extracellular matrix (extracellular matrix ECM) and basement membrane, and promote neovascularization, tumor invasion and metastasis on the formation of CNV important role. Two protease corneal angiogenesis in the promotion play an important role.Therefore, our experiment by local application of selective cyclooxygenase-2 inhibitor celecoxb, observed on the suture-induced corneal neovascularization, HE staining to observe pathological changes in rat corneal effects. Detection of corneal tissue by COX-2, MMP-2 protein expression changes of corneal angiogenesis in the process of possible mechanism PartⅠInhibitory effect of celecoxib on rat corneal neovascularizationObjectiveTo explore the inhibitory effect and mechanisms of celecoxib on rat corneal neovascularization and observe its effect on the expression of MMP-2 on rat cornealMethod30 healthy SPF SD (Sprague-Dawley) rats were induced corneal neovascularization by stitching cornea.The rats were devided randomly grouped (A, B) a total of two groups, A groups:corneal neovascularization group, B group: celecoxib solution group.And compare with normal rats. The growth of cornea neovascularization(CNV) was observed by slit lamp microscope on each postoperative day and the area of CNV were recorded on 4th,7th,14th days.On the7th, the appropriate corneal was taken for histopathological examination, and the expression of matrix metalloproteinase-2 (MMP-2) was detected by immunohistochemistrySPSS 13.0 statistical package was used for data analysis, The data was recorded by mean±standard deviation. Compare the area of neovascularization at each time point in two groups with repeated measure analysis of variance; Statistical tests were considered significant when P values were less than 0.05.ResultOn 4th,7th,14th days, the neovascularization area of CNV control group were(0.138±0.154)mm2,(0.663±0.139)mm2,(0.730±0.143)mm2 respectively; Celecoxib subconjunctival injection group were (0.063±0.154) mm2,(0.425±0.129) mm2,(0.529±0.139) mm2; in normal control group no neovascularization was founded. Comparison between groups was statistically significant difference, P<0.05. The neovascularization area of in Celecoxib subconjunctival injection group was significantly lower than CNV control group.and at different observing times the neovascularization area of in two groups were significant difference, P<0.05.On the 7th postoperrative day,by light microscope,there is a large amout of neovascularization in cornea of group A and many lymphocytes and fibroblasts infilitrat the cornea.In contrast, the number of neovascularization in cornea of group B is less than of group A and with light inflammatory reaction.In normal corneal tissue, MMP-2 was not expressed or weak expression in the epithelial basement membrane; On the 7th in control group MMP-2 expression was significantly increased, the positive reaction to brown or brown intracytoplasmic granules, and its expression mainly in parts of the formation of new blood vessel area; The neovascularization area of Celecoxib subconjunctival injection group were markedly reduced compared with CNV control group, and the expression of MMP-2 reduced corresponding in the corneal stroma neovascularization region.ConclusionCelecoxib by the local application is effective in inhibition of corneal neovascularization induced by sutures,which may be associated with supppression the expression of MMP-2, and its inhibitory effect on MMP-2 may be one of the mechanisms Celecoxib inhibited CNV.PartⅡEffect of celecoxib on the expression of COX-2 and MMP-2 in rat corneal neovascularization induced by suturingObjectiveTo detect the effcts of topical application of celecoxib on the expression of COX-2 and MMP-2 on rat corneal neovascularization.And to explore the possible mechanisms of celecoxib to inhibit corneal neovascularization.Method33 healthy SPF SD (Sprague-Dawley) rats were induced corneal neovascularization by stitching cornea.Animals were randomized into celecoxib solution group(15 rats,at each time point 3,6eyes), saline control group(15 rats,at each time point 3,6eyes) and blank control group(3rats,6eyes). Each group full cornea corneoscleral were removed On1th,4th,7th,14th,21th days for conventional paraffin blocks, the appropriate corneal was taken for histopathological examination under light microscope morphology COX-2, MMP-2 distribution and expression were detected by with immunohistochemistry and RT-PCR,. SPSS 13.0 statistical package was used for data analysis, The data was recorded by mean±standard deviation., COX-2mRNA, MMP-2mRNA expression of groups used independent sample t test; two genes at each time point was used to compare the variance of factorial design information analysis; Pearson correlation analysised correlation test. With P<0.05 indicated statistically significant difference.ResultTime points after suturing, the expression of COX-2mRNA in CNV control group were 15.381±2.367mm2,67.176±8.845mm2,103.551±4.153mm2 49.286±5.319mm2,9.829±2.722 mm2,6.465±5.540mm2; the expression of MMP-2 mRNA were0.783±0.199 mm2,2.504±0.361mm2,7.433±0.053mm2 2.286±0.111mm2,0.987±0.196mm2; Celecoxib treatment group, the expression of COX-2 mRNA were 3.616±0.556 mm2,25.888±3.650 mm2,44.313±6.125mm2,20.114±8.017 mm2,5.186±0.806 mm2; the expression of MMP-2 mRNA were 0.269±0.072mm2,1.011±0.097 mm2,2.899±0.300 mm2,0.739±0.065 mm2,0.079±0.005 mm2; There was a significant difference in the expression of COX-2 mRNA, MMP-2 mRNA in two groups, (P<0.05); There is defference in cornea COX-2mRNA on different observation days(F=91.909, P=0.000),There is interactive reaction between groups and different observation days (F=14.728,P=0.000)。There is defference in cornea MMP-2mRNA on different observation days (F=658.717, P=0.000), There is interactive reaction between groups and different observation days (F=113.822, P=0.000); correlation analysis show that there is correlation between the two genes.(r= 0.921, P= 0.000).ConclusionThe topical inhibitive effect of Celexicob in corneal neovascularization may be related to inhibit the expression of COX-2 and activation of MMP-2.