Effects Of Naringenin Complexed With Copper (Ⅱ)/β-cyclodextrin With Free Ligand On Laser-induced Choroidal Neovascularization In Rats

Objective:To compare the effects of naringenin complexed with copper (Ⅱ)/β-cyclodextrin with free ligand on laser-induced choroidal neovascularization (CNV) in rats,and research the mechanism.Methods:Selecting50Male Brown Norway rats(100eyes), including40rats were anesthetized to receive krypton laser to induce CNV and only10rats(20eyes) were owe to normal group.40model rats were divided randomly into control,naringenin and its complexes of Cu/β-cyclodextrin groups,10rats each group. After making models,Naringenin and its complexes (20mg/kg/d)were given once-daily through intraperitoneal (i.p.) injection after laser treatment for30days,and normal group did not give medicine. Each group after modeling30d, routine to be put animals to death, take the eyeball, and obtain the retina-choroid-sclera complex specimens from each group.The sizes of CNV lesions were measured by choroidal flatmounts with fluorescein isothiocyanate dextran. Use the method of immunohistochemical examination to test retina and choroid neovascularization of VEGF and COX-2expression.The protein and mRNA of COX-2. VEGF, PI3K、MAPK、MMP-2and MMP-9were examined by Western blotting and RT-PCR.Proteomic analyses of five groups were made using mass spectrography and bioinformatics to detect the protein expression level.Results:1.naringenin complexed with copper ion was easy to dissolve in DMSO, complexes of β-cyclodextrin can improve the solubility of naringenin11.8times in water,and they improved the bioavailability of naringenin.2.Observe CNV of the model rats and calculate the area (×103μm2):model control group was34.56±1.76, narigenin group was20.90±1.47, complexes of Cu group was13±1.43,complexes of β-cyclodextrin group was13.24±1.33.The animals treated with naringenin and its complexes showed significant decrease in their average CNV sizes, compared with the vehicle-treated animals(P<0.05,0.01).Especially the sizes of naringenin’s complexes of Cu/β-cyclodextrin groups were less than naringenin group(P<0.05).But there was no difference between naringenin’s complexes of Cu/β-cyclodextrin groups(P>0.05).3.The immunohistochemical examination showed:tissue photocoagulation area have scarring and tan positive granular cell expression, model control group had a large amount of brown granules, and high expression of COX-2/VEGF.Naringenin and its complexes groups had loss of brown granules in the areas of photocoagulation than model group, and COX-2/VEGF weakly expressed.4.mRNA expression:Compared with the vehicle-treated animals, the mRNA expressions of VEGF、COX-2and so on were significantly higher in the model control group (P<0.05,0.01). The mRNA expressions of VEGF、COX-2and so on were weaker in the Naringenin and its complexes groups than the model control group except,especially complexes of Cu/β-cyclodextrin group.Besides P38MAPK,differences were statistically significant (P<0.05). But there was also no difference between naringenin’s complexes of Cu/β-cyclodextrin groups(P>0.05).5.Western blotting:The protein expression existed certain differences between each group, the differences were statistically significant (P<0.05,0.01).Protein expression level of model group was higher than in normal group, the difference had statistical significance (P<0.05,0.01).naringenin’s complexes groups’ protein expression were lower than the naringenin group, expect P38MAPK. PI3K、MMP-2,differences were statistically significant (P<0.05), but there was no statistically significant between the two groups (P>0.05).6.Proteomic analyses:each group has a larger difference, apart from narigenin complexed of Cu/β-cyclodextrin group, the differences between other groups were statistically significant (P<0.05).Conclusions:Compared with narigenin,complexes with copper ion have better biological activity, complexes of β-cyclodextrin significantly enhanced the drugs’ water solubility,the effect of they restraining CNV strengthen.We observed the effect of narigenin complexed with Cu/β-cyclodextrin on related signal channel and cytokines around cox-2.To a certain extent, COX-2can adjust the expression of VEGF、MMP-2、MMP-9,and influence the activation of PI3K、MAPK signal pathway.From macro to micro level,illustrate narigenin complexed of Cu/β-cyclodextrin to inhibit the formation and progress of CNV and the related mechanism.