Study On The Inhibitory Effect Of Anti-Scg3 On Corneal Neovascularization

Objective:To investigate the effect of secretogranin III（Scg3/Sg III）antibody on corneal neovascularization in rabbit eyes and to make a preliminary exploration of the mechanism of its effect.Methods:Forty-five healthy adult New Zealand white rabbits were selected,and the right eye was uniformly selected for experimental manipulation.The corneal neovascularrization（CNV）model was established by the alkali-burn method.The New Zealand white rabbits were randomly divided into three groups:high concentration group（0.5μg/m L anti-Scg3 group）,low concentration group（0.1μg/m L anti-Scg3 group）and control group（1×phosphate buffered solution（PBS）group）,15 rabbits in each group.Subconjunctival injection of 0.5μg/m L anti-Scg3,0.1μg/m L anti-Scg3 and 1×PBS solution respectively,every two days since the date of burn.The growth of neovascularization was observed under slit lamp microscope and photographed at 1,4,7,10 and 14 days after alkali-burn injury;the CNV and its blood flow signal were observed by corneal confocal microscope;the area and length of CNV were measured by image analysis software;the corneas of three New Zealand white rabbits were randomly selected and removed after euthanasia at each time point,and frozen sections of pathological tissues were made separately.The m RNA of corneal tissue was extracted,and the expression of platelet endothelial cell adhesion factor（CD31）and lymphatic vessel endothelial hyaluronan receptor-1（LYVE-1）in corneal tissue was detected by immunofluorescence,and the expression of vascular endothelial growth factor（VEGF）was detected by Real-time PCR.Results:At1 day after alkali-burn injury,slit lamp examination revealed that vasodilatation of the corneal rim was visible in all three groups.Four days after alkali-burn injury,CNV growth along the corneal edge toward the central cornea was seen in all three groups,and CNV length and area in the high concentration group and low concentration group were lower than those in the control group,and the differences were statistically significant(P _{length-high-4d}<0.01,P_{length-low-4d}<0.01,P_area-high-4d<0.01,P_area-low-4d<0.01),although CNV length in the high concentration group was not significantly different from that of the low-concentration group（P>0.05）,the CNV area of the high-concentration group was significantly lower than that of the low-concentration group（P<0.05）.At 7 days after alkali-burn injury,the CNV length of the control group was close to the edge of the alkali burn area;at 10 days after alkali-burn injury,the CNV length of the control group reached the edge of the alkali burn area;at 14days after alkali-burn injury,the CNV length of the control group crossed the edge of the alkali-burn area,and the CNV growth of the high concentration group was significantly lower than that of the remaining two groups.At 7,10and 14 days after the burn,the CNV length and area of the high and low concentration groups were significantly lower than those of the control group(P_{length-high-7d}<0.01,P_{length-low-7d}<0.01,P_area-high-7d<0.01,P_area-low-7d<0.01,P_{length-high-10d}<0.01,P_{length-low-10d}<0.05,P_{area-high-10d}<0.01,P_area-low-10d<0.01,P _{length-high-14d}<0.01,P_{length-low-14d}<0.01,P_{area-high-14d}<0.01,P_area-low-14d<0.01),and both CNV length and area were also lower in the high concentration group than in the low concentration group(P_length-7d<0.05,P_area-7d<0.05,P_length-10d<0.05,P_area-10d<0.01,P_length-14d<0.01,P_area-14d<0.01),showing a concentration effect relationship.Examination of the CNV with corneal confocal microscopy revealed clear flowing blood cells visible within the canal diameter.Immunofluorescence detection revealed that after corneal alkali-burn injury,CD31 and LYVE-1 fluorescence in the high and low concentration groups were weaker than the fluorescence expression in the control group at the same time point.After corneal alkali-burn injury,VEGF showed a trend of rising and then falling in all three groups,and the expression of VEGF in the control group reached its highest peak at 7 days after burn,which was significantly higher than that at 1 day after alkali burn（P<0.01）,and reached its highest peak at 4days after burn in the low concentration group and high concentration group,which was significantly higher than that at 1 day after alkali burn in the same group(P_high<0.01,P_low<0.01),after which the expression gradually decreased The difference in VEGF expression between the high concentration group,low concentration group and control group at 1 day after alkali-burn injury was not statistically significant（P>0.05）;at 4,7,10 and 14 days after burn,VEGF expression was lower in the high concentration group and low concentration group than in the control group(P _high-4d<0.01,P_low-4d<0.05,P_high-7d<0.01,P_low-7d<0.01,P_high-10d<0.01,P_low-10d<0.05,P _high-14d<0.01,P_low-14d<0.05),and VEGF expression was lower in both the high concentration group than the low concentration group(P_4d<0.05,P_7d<0.01,P_10d<0.05,P_14d<0.05).Conclusion:The ability of anti-Scg3 to effectively inhibit alkali burn-induced corneal neovascularization may be related to the fact that anti-Scg3 regulates the expression of VEGF through some pathway.