Curcumin Through Notch Signal Pathway Inhibit The Corneal Neovascularization In Vivo And In Vitro

Objective1.Establish umbilical vein endothelial cells(HUVECs)in vitro model and discuss the influences of curcumin on the proliferation,migration and inhibition mechanism and targets.2.Establish the rabbit model of new blood vessels after corneal alkali burn and observe the inhibitory effect of curcumin on corneal neovascularization(corneal neovascularization,CNV),preliminary study the mechanism of curcumin inhibition and targets.Methods1.Using the enzyme digestion method to obtain and develop the original generation of umbilical vein endothelial cells,then obserbe and identify the cells.(1)with different concentrations of curcumin medium(1,5,7.5,10,20 umol/L)and different time culture endothelial cells 12 h,24h,48 h,CCK8 method for determining the effects of curcumin on HUVECs proliferation;(2)with different concentrations of curcumin medium(5,10 umol/L)training for 24 h,scratch method to explore the influence of curcumin on HUVECs migration;(3)the cell is divided into 5 groups with different culture medium,the control group(containing 0.1% DMSO),group A(including 5 umol/L curcumin),group B(including 10 umol/L curcumin),group C(including 10 ng/ml VEGF),group D(including 10 ng/ml VEGF + 10 umol/L curcumin),training after 48 h,using reverse transcription polymerase chain reaction(RT-PCR)to detect the cell of relative m RNA expression of Notch1,Dll4 and Bcl-2.2.Take 24 rabbits after alkali burn,randomly divided into control group and curcumin group,twelve rabbits(12 eyes)were in each group.Control group given 0.1% DMSO saline subconjunctival injection,curcumin group was given subconjunctival injection of 40umol/L curcumin solution,0.5ml/time,1time/2day.Every day observe the corneal neovascularization,and after alkali burn 1d,7d and 14 d use slit lamp camera take pictures,record length of CNV and calculated CNV area;After alkali burn 7d,10 d,14d randomly selected three rabbits from two groups,take the cornea away after using enzyme-linked immunosorbent experiment(enzyme linked immuno sorbent assay ELISA)to determin the Notch1,Dll4 and Bcl-2 protein content.Results1.(1)The endothelial cells spread on the bottom of the dishes in 2h,then coalesced and grew to from confluent monolayers of polygonal cells within 24 h.The cultured cells were identified as HUVECs.the effect of curcumin on proliferation and migration of human umbilical vein endothelial cell(HUVECs)is in a time-dependent manner and a dose-dependent manner;(2)The RT-PCR results show that the group A and B compared with control group,the Notch1,Dll4 and Bcl-2 in the expression of m RNA level difference was statistically significant(p<0.05),and increased with the concentration of curcumin,the expression of m RNA reduced in turn.Notch1 m RNA expression in C group compared with control group not significant(p > 0.05),but the m RNA expression of Dll4,Bcl-2 was obviously higher than that of control group,the difference was statistically significant(p<0.05).Notch1 m RNA expression in group D significantly lower compared with group C,the difference was statistically significant(p<0.05),and there was no statistically significant difference compared with group B(p>0.05),group D the m RNA expression of Dll4,Bcl-2 in a significant decrease in the C group,the difference was statistically significant(p<0.05).2.(1)After alkali burn1~5d,two groups of corneal neovascularization area is not significant;after alkali burn 7d,corneal neovascularization area of curcumin group was(20.56 ± 2.09)mm2,the control group was(27.92 ± 1.28)mm2,comparing the two groups was statistically significant(t=5.213,p< 0.05);after alkali burn 14 d,corneal neovascularization area of curcumin group was(11.86±1.23)mm2,the control group was(31.98±2.48)mm2,comparing the two groups was statistically significant(t=12.585,p<0.05).(2)ELISA results showed that 7d after alkali burn Notch1,Dll4 and Bcl-2 protein content in the control group were relatively high content of curcumin group,the difference was statistically significant(p<0.05);10d three kinds of protein content in the control group continued to increase,lower protein content of curcumin group is not obvious,the expression of stability,are similar between the two groups was statistically significant(p<0.05);14d in the control group and the protein content of curcumin group were lower,the Bcl-2 protein is similar between the two groups was statistically significant(p<0.05).Line of Spearman rank correlation analysis found that corneal tissue Dll4 and Bcl-2 protein expression were positively correlated,the correlation coefficient was 0.750 in the control group,the curcumin group was 0.667(at the 0.05 level,correlation significantly).Conclusions1.collagen enzyme digestion method can be used to obtain HUVECs,Ⅰbuilding a successful model of the vascular endothelial cells in vitro.Curcumin in 5 umol/L ~ 20 umol/L range can inhibit HUVECs proliferation and migration.2.In the early time,the new angiogenesis in rabbit corneal alkali burns,curcumin can reduce bulky angiogenesis in the cornea.3.Curcumin can significantly reduce the Notch signaling pathway in the expression of related genes and proteins,which may be one of the mechanism of inhibition of corneal neovascularization.