Mechanism Of Celecoxib Inhibiting The Expression Of Retinal VEGF In Diabetic Retinopathy Rats Via JAML

Background Diabetic retinopathy(DR)is a severe complication of diabetes(DM).The main symptom of DR is permanent loss of sight,which ultimately leads to blindness.As diabetes becomes more prevalent,the number of people with diabetic retinopathy will increase,and tend to be younger.Therefore,clarifying the pathogenesis of DR is an urgent problem.JAML is a new type of adhesion molecule that has been discovered to treat acute promyelocytic leucopenia.It plays an active role in immunity,inflammation and cell proliferation.Recently,low expression of JAML was observed in epithelial and endothelia cells.Our results showed that JAML was expressed in vascular endothelial cells.Celecoxib is commonly used in the treatment of inflammatory-related disorders.Recently,it has been demonstrated that celecoxib may inhibit the migration of vascular endothelial cells.On the basis of previous experiments,the author intends to investigate the role of celecoxib in treating diabetic retinopathy by means of JAML-mediated signaling.Objective: To study the effect and mechanism of celecoxib on retinal vascular endothelial growth factor in rats with diabetic retinopathy.Methods: 45 SD rats with a body weight of 200 grams were randomly divided into3 groups: normal control group(NC),diabetic retinopathy group(DR)and celecoxib intervention diabetic retinopathy group(DR+C).Model of DR was established by intraperitoneal injection of STZ and high-glucose HUVEC cells.1.The protective effect of celecoxib on the retina of diabetic retinopathy rats.One month after the successful establishment of the DR model,the DR+C rats were administered intragastrically with celecoxib,and the other two groups were given with sodium citrate.Two months later,the rats were killed,and their eyes and serum were collected for further experiments.After modeling,the amount of diet and drinking water was observed once a week;weight and blood glucose were recorded once a week.HE staining was used to examine the pathological changes of retina.IHC was used to detect the distribution of TNF-α,IL-10,Vcam-1.The expression of HIF1-α,VEGF was detected by western blot.Serum insulin and total cholesterol(TC)were measured.2.Mechanism of the reversal of diabetic retinopathy by celecoxib.Cells were classified into three groups: NG,HG,HG+C.HUVEC cells were cultured with 30 m M glucose,and HC+C was treated with 10μM celecoxib.The toxicity of cell proliferation was measured by CCK-8 kit,and the production of ROS was detected by ROS.Expressions of JAML,PI3 K,P-PI3 K,HIF1-α and VEGF in cells were detected by western blot.Western blot was used to detect the level of JAML,PI3 K and P-PI3 K in retinal tissues too.Result:1.Changes in body weight,blood glucose,diet and drinking water: The body weight of rats in DR+C group and DR group was significantly lower than that in NC group(both P< 0.01).FPG in both DR+C and DR group was significantly higher than that in NC group(both P<0.01).There was no statisic difference in food intake between the three group.Compared with NC group,the water intake of DR+C group and DR group increased significantly(both P<0.01).2.Retinal histopathological changes: Compared with the NC group,the retinal thickness of DR groups increased(P<0.05),while the retinal thickness of the DR+C became thinner compared with the DR group(P<0.05).The retina of DR+C and DR group is edema and each layer structure is not clearly arranged.In the DR group,the retinal ganglion cell layer of the rats has dilated neovascular expansion,and vacuolar degeneration is visible in the deep retinal tissues.3.The expressions of JAML,PI3 K,P-PI3 K,HIF1-α,VEGF in retinal tissues:The protein levels of DR+C were lower than those in DR group.The level of DR was obviously higher than that in NC group.4.Distributions of TNF-α,IL-10 and Vcam-1 in retinal tissues: The above cytokines in DR+C group were lower than those in DR group(each P<0.01);DR group were higher than NC group(each P<0.01).5.Serum TC and Insulin test results: The serum TC content of DR+C and DR group was higher than that in NC group(P<0.01),and there was no significant difference in TC content between DR+C and DR group(P > 0.05).The serum Insulin content of rats in DR+C and DR group was lower than NC group(both P<0.001),while there was no statistical significance between DR+C and DR group(P>0.05).6.CCK-8 assay indicated that the proliferation rate of celecoxib 10 μM increased significantly(P<0.01).The proliferation rate of 30 μM was lower than that in other groups(each P<0.05).Compared with other groups,the inhibition rate of celecoxib 10μM was significantly lower(each P<0.01),and the percentage of inhibition in 30μm group was obviously increased(each P<0.05).7.Expressions of JAML,PI3 K,P-PI3 K,HIF1-α and VEGF in cells:Compared to HG,the level of JAML,P-PI3 K,HIF1-α and VEGF decreased significantly in HG +C.And the levels of JAML,P-PI3 K,HIF1-α and VEGF in HG group were higher than those in NG group.The expression of PI3 K was not different in all groups.8.Detection of intracellular reactive oxygen species: The level of ROS was higher in HG than in NG Groop(P<0.001).Comparing HG+C cells to HG Groop,ROS in cells reduced significantly(P<0.01).Conclusion: Through PI3K/HIF1-α signaling pathway mediated by JAML,celecoxib down-regulates VEGF expression in diabetic retinopathy,inhibits the release of inflammatory factors and improves DR retinal microangiopathy.