| Chapter OneObjectiveAn experimental choroidal neovascularization(CNV)animal model was established by using a 532nm frequency-doubled laser,which lay the experimental foundation for CNV-related research.Methods1.A 532nm laser was used to establish a CNN model in BN rats.Fundus Fluorescein Angiograph(FFA)and Indocyanine green Angiography(ICGA)were performed at Id,3d,5d,7d,14d,and 21d after photocoagulation respectively to observe the changes in CNV.2.Ocular histopathological specimens were made,and the central thickness of CNV at different time points after photocoagulation were observed by HE staining.3.Ocular histopathological specimens were made,and the expression of CD 105 at different time points after photocoagulation were observed by immunohistochemistry.Results1.There was no leakage of fluorescein after 1d of photocoagulation;there was only a small amount of fluorescein after 3d and 5d of photocoagulation.After 7 days of photocoagulation,the CNV generation rate was 70.18%,and the mean density value was 128.30 ± 11.21.After 14 days of photocoagulation,the CNV generation rate was 78.18%,and the mean density value was 182.12 ± 6.59,the difference was significant(P<0.05)when compared with the group of 7 days after photocoagulation.After 21 days of photocoagulation,the CNV generation rate and the mean density of fluorescence leakage were not significantly different from those 14 days after photocoagulation.2.The HE staining results showed that the central thickness of CNV increased gradually with the time of photocoagulation(P<0.05).HE staining results showed that:with the increase of time after photocoagulation,the central thickness of CNV gradually increased,and the thickness of CNV was 48.92±2.81?m after 7 days of photocoagulation,which was significantly increased compared with 1d after photocoagulation(P<0.05);after photocoagulation The thickness of CNV at 14d was 61.98±5.06?m,which was significantly higher than that at 7d after photocoagulation(P<0.05);the thickness of CNV at 21d after photocoagulation was 61.78±4.03?m,which was not statistically different from the 14d group.3.The expression of CD 105 in the retinal tissue of the normal group was negative,and a small amount of brown-yellow reactants were observed at the model group after 1d,3d,and 5d.The brown-yellow reactants were gradually increased at 7d model group(P<0.05),14d model group were increased when compared with 7d(P<0.05),and there is no significant different between 14d model group and 21d model group.Conclusions1.Application of 532nm laser photocoagulation can successfully establish CNV model in BN rats.2.CNV grows rapidly from 7d to 14d after photocoagulation and gradually stabilizes in 21d.This model has the advantages of fast molding speed,high molding rate,good repeatability and low cost,which can be used as an animal model for CNV basic research.3.Fundus photography,FFA,ICGA,HE staining and immunohistochemistry can dynamically observe changes in CNV.Chapter TwoObjectiveTo observe the effect of curcumin on the CNV of Brown Norway(BN)rats and the effect on the activity of AKT/p-AKT/HIF-1?/VEGF signaling pathway,which can provide experimental basis in CNV treatment.Methods1.A 532nm laser was used to establish a CNV model in BN rats.Rats after modeling were randomly divided into a model group,a ranibizumab group,a curcumin low,medium,and high dose group.FFA and ICGA examination were performed after photocoagulation 14 days.2.Ocular histopathological specimens were made,and the changes in central thickness of CNV were observed by HE staining.3.Ocular histopathological specimens were made,and the expression of AKT/p-AKT/HIF-1?/VEGF signaling pathway-related proteins were observed by immunohistochemistry.4.Detect the mRNA expression of AKT/HIF-1?/VEGF factor in CNV tissues by RT-qPCR.5.Detect the protein expression of AKT/p-AKT/HIF-1?/VEGF factor in CNV tissues by Western-blot.Results1.CNV generation rates in the model group,the ranibizumab group,the low,medium and high dose curcumin were 78.18%,73.21%,77.19%,75.86%,74.55%,and there was no statistical difference between the groups.The average optical density values were 182.12±6.59,119.22±8.03,166.45±8.33,164.34±5.69,149.22±6.45,the ranibizumab group was significantly lower than the model group(P<0.05),the low-dose,middle-dose and high-dose groups were significantly higher than the ranibizumab group(P<0.05),and the curcumin high-dose group was significantly lower than the model group(P<0.05).2.The retinal tissue structure of BN rats in normal group was clear and neatly arranged.The central thickness of CNV in the ranibizumab group was significantly reduced compared with the model group(P<0.05);the curcumin low/middle-dose group had no significant difference when compared with the model group.While the curcumin high-dose group was significantly reduced than the model group(P<0.05),but increased when compared with ranibizumab group(P<0.05).3.Immunohistochemistry results showed that AKT,p-AKT,HIF-1?,and VEGF factors were negative in the retinal tissue structure of BN rats in the normal group,and no brown-yellow reactants were found.The expression of AKT,p-AKT,HIF-1?,and VEGF factors in the model group were higher than that in the normal group(P<0.05)after 14 days photocoagulation;the ranibizumab group was lower than the model group(P<0.05).The expression of AKT,p-AKT,HIF-1?,and VEGF factors in the low-dose and middle-dose group were not significantly different from the model group;while the expression of the curcumin high-dose group was decreased than the model group(P<0.05);but increased when compared with ranibizumab group(P<0.05).4.The mRNA results showed that 14 days after photocoagulation,the relative expression levels of AKT,HIF-la and VEGF mRNA in the model group were higher than the normal group(P<0.05);the ranibizumab group was lower than the model group(P<0.05).There was no significant difference between curcumin low/medium-dose group and the model group(P<0.05);while curcumin high-dose groups were decreased from the model group(P<0.05),but increased when compared with ranibizumab group(P<0.05).5.Westernblot results showed that there was no significant difference in the relative expression of AKT protein between every group at 14 days after photocoagulation.The relative expression of p-AKT protein in the model group was higher than that in the normal group(P<0.05),and the ranibizumab group was lower than in the model group(P<0.05),the curcumin low/medium-dose groups were not significantly different from the model group(P<0.05),and the curcumin high-dose group was decreased than the model group(P<0.05).The relative expression levels of HIF-la protein was higher in the model group than in the normal group(P<0.05),and the ranibizumab group were lower than in the model group(P<0.05).There was no significant difference between the model group and the low/medium-dose group.The relative expression levels of HIF-1? protein was lower in the curcumin high-dose group than in the model group(P<0.05),higher than ranibizumab group(P<0.05).The relative expression levels of VEGF protein was higher in the model group than in the normal group(P<0.05),and the ranibizumab group were lower than in the model group(P<0.05).There was no significant difference between the model group and the low-dose group.The relative expression levels of HIF-1? protein was lower in the curcumin medium/high-dose group than in the model group(P<0.05).Conclusions1.Curcumin(400mg/Kg/d)has an inhibitory effect on CNV model in BN rats.2.The mechanism may be related to inhibiting the activation of AKT/p-AKT/HIF-la/VEGF signaling pathway and reducing the expression of related factors.Chapter ThreeObjectiveTo observe the effects of curcumin on AKT,HIF-1? and VEGF factors in hypochloric model of ARPE-19 cells induced by cobalt chloride(CoCl2).Methods1.ARPE-19 cells chemical hypoxia model was established by CoCl2.The CCK-8 method was used to detect the concentration of the modeling reagent CoCl2,the experimental drug curcumin and the positive control drug ranibizumab,and to detect the effect of curcumin on the activity of ARC1-19 cells induced by CoCl2.2.ARPE-19 cells was divided into normal group,hypoxia model group,ranibizumab group,low-dose,medium-dose and high-dose curcumin group.Effects of curcumin on ARPE-19 cell hypoxia model in the the expression of AKT,HIF-1? and VEGF mRNA were detected by RT-qPCR.3.ARPE-19 cells was divided into normal group,hypoxia model group,ranibizumab group,low-dose,medium-dose and high-dose curcumin group.Effects of curcumin on ARPE-19 cell hypoxia model in the the expression of AKT,p-AKT,HIF-1? and VEGF protein were detected by Westernblot.Results1.CCK-8 cell viability assay:ARPE-19 cells showed growth first and then suppressed with the concentration of CoCl2 increased.When the final concentration of CoCl2 was ? 200?M,cell viability was significantly reduced(P<0.05).When the final curcumin concentration was 0-100?M,there was no effect on the activity of ARPE-19 cells.The cell activity was significantly reduced(P<0.05)when the the final concentration of CoCl2 was 200?M.The activity of ARPE-19 cells was lower than CoCl2 group(P<0.05)when the final concentration of ranibizumab was 20 ?g/mL,and there was no difference compared with the normal group;the cell viability was lower than that in the CoCl2 group and the normal group(P<0.05)when the concentration was 40?g/mL and 80?g/mL.Therefore,we chose 100?M of CoCl2 as the model concentration;6.25?M,25?M,and 100?M of curcumin as the low-dose,medium-dose,and high-dose groups respectively;20?g/mL of ranibizumab as the positive control drug concentration.2.The mRNA results showed that the relative expression of AKT,HIF-1? and VEGF mRNA in the model group were higher than the normal group(P<0.05);the ranibizumab group were lower than the model group(P<0.05).The relative expression of AKT,HIF-1? and VEGF mRNA in the low-dose and medium-dose were no significant difference compared with the model group.The relative expression of AKT,HIF-1? and VEGF mRNA in curcumin high-dose group were significantly lower than the model group(P<0.05);there was no significant difference with the ranibizumab group.3.Westernblot results showed that the expression of AKT protein was no difference between each group.The relative expression of p-AKT and HIF-1? protein in the model group were higher than those in the normal group(P<0.05),and those in the ranibizumab group were lower than in the model group(P<0.05).The relative expression of p-AKT and HIF-1? protein in curcumin low-dose and medium-dose were no difference with model group(P<0.05).The relative expression of p-AKT and HIF-1? protein in curcumin high-dose group was significantly lower than the model group(P<0.05),and there was no significant difference with the ranibizumab group.The relative expression of VEGF protein in the model group was higher than the normal group(P<0.05),and the ranibizumab group was lower than the model group(P<0.05).The relative expression of VEGF protein in curcumin low-dose group was no significant difference from the model group(P<0.05).The relative expression of VEGF protein in curcumin medium-dose and high-dose groups were significantly lower than model group(P<0.05),and there was no significant difference from the ranibizumab group.Conclusions1.Chemical hypoxia model of APRE-19 cells can successfully establish by CoCl2.2.It can promote the the expression of AKT,HIF-1?,and VEGF mRNA and the p-AKT,HIF-1?,and VEGF proteins in ARPE-19 cells when CoCl2 at 100?M.3.Curcumin at a final concentration of 100?M can reduce the expression of AKT,HIF-1? and VEGF mRNA and p-AKT,HIF-1? and VEGF proteins in ARPE-19 hypoxia model.4.Curcumin protects ARPE-19 cells from hypoxia induced by CoCl2 when Curcumin at 100?M.Chapter FourObjectiveTo observe the effects of conditioned-medium of ARPE-19 cells on proliferation,migration,invasion,and tube formation of HUVEC cell.Methods1.ARPE-19 cell conditioned medium were divided into normal group,model group,ranibizumab group,curcumin low-dose,medium-dose and high-dose group.The effect of ARPE-19 cells conditioned-medium on HUVEC cell activity were observed by CCK-8 method.2.Cell scratch test were used to observe the ARPE-19 cells conditioned-medium on HUVEC cell horizontal migration.3.Transwell chamber migration experiment were used to observe the condition of ARPE-19 cells conditioned-medium on HUVEC cell vertical migration.4.Transwell chamber invasion experiment were used to observe the condition of ARPE-19 cell conditioned-medium on HUVEC cell invasion.5.Matrigel lumen formation experiment were used to observe the condition of ARPE-19 cell conditioned-medium on HUVEC cell lumen formation.Results1.There was no significant difference between the different condition-medium groups on HUVEC cell activity.2.HUVEC cell horizontal migration results:when compared with the normal group,the horizontal migration of HUVEC cells in the model group was significantly increased(P<0.05).Compared with the model group,the horizontal migration of the ranibizumab group was significantly reduced(P<0.05).The horizontal migration of the low-dose,medium-dose,and high-dose groups were significantly reduced compared with the model group(P<0.05).There was no statistically significant difference between the high-dose group and the ranibizumab group(P<0.05).3.HUVEC cell vertical migration results:when compared with the normal group,the vertical migration of HUVEC cells in the model group was significantly increased(P<0.05);compared with the model group,the vertical migration of the ranibizumab group was significantly reduced(P<0.05).There was no significant change in vertical migration in the low-dose and medium-dose curcumin-conditioned medium group when compared with the model group.The vertical migration of the high-dose curcumin conditioned-medium group was significantly reduced when compared with the model group(P<0.05),and there was no significant difference between the high-dose group and the ranibizumab group(P<0.05).4.HUVEC cell invasion results:when compared with the normal group,HUVEC cell invasion in the model group was significantly increased(P<0.05),the invasion of ranibizumab group was significantly reduced(P<0.05)when compared with the model group.There was no significant change in cell invasion in the low-dose and medium-dose curcumin conditioned-medium group when compared with the model group.The invasion in high-dose curcumin conditioned-medium group was significantly reduced compared with the model group(P<0.05),and there was no significant difference between the high-dose group and the ranibizumab group(P<0.05)5.HUVEC cell lumen formation rusults:when compared with the normal group,the lumen formation of HUVEC cells in the model group was increased significantly(P<0.05).The lumen formation of the ranibizumab group was significantly reduced(P<0.05)when compared with the model group.Compared with the model group,there was no significant change in the lumen formation of low-dose curcumin conditioned-medium group.The lumen formation in the medium-dose and high-dose groups were significantly reduced(P<0.05)when compared with the model group,and there was no difference difference between the high-dose group and the ranibizumab group(P<0.05)Conclusions1.Low-dose(6.25?M),medium-dose(25?M)and high-dose(100?M)curcumin conditioned-medium can inhibite horizontal migration of HUVEC cells significantly.High-dose(100?M)curcumin conditioned-medium can inhibite vertical migration of HUVEC cells significantly.2.High-dose(100?M)curcumin conditioned-medium group can inhibit invasion of HUVEC cells significantly.3.High-dose(100?M)curcumin conditioned-medium group can inhibit lumen formation of HUVEC cells significantly.4.Curcumin can inhibit the formation of blood vessels at the cellular level. 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