Effect And Mechanism Of Scg3 On Proliferation Of Human Pterygium Fibroblasts Via SDF-1/CXCR4 Signaling Pathway

Objective:To explore the effects of SecretograninⅢ（Scg3）on the proliferation of human pterygium fibroblasts（HPFs）,and explore the correlation between Scg3 and SDF-1/CXCR4 signaling pathways in HPFs.To explore the molecular mechanism.Methods:Thirty human pterygium tissue specimens were collected from pterygium operations.HPFs were isolated,cultured and identified in vitro.The expression distribution of Scg3 in HPFs was detected by Immunocytochemistry（ICC）.Cell count Kit 8（CCK-8）method was used to detect the proliferative activity of HPFs with different concentrations of Scg3 at 24h and 48h,and the optimal concentration conditions were selected to treat HPFs.Divided into 3groups according to different treatment conditions:Control group was given basal culture medium,Scg3 group was added with basal culture medium containing 60ng/ml Scg3 to treat HPFs,VEGF group was added with 60ng/ml Vascular endothelial growth factor,The basal medium of VEGF was used to treat HPFs,and the HPFs were cultured for 48h.ICC was used to detect the fluorescence expression of stroma cell-derived factor-1（SDF-1）and CXC chemokine receptor 4（CXCR4）in HPFs in each group,and reverse transcription-polymerase chain reaction（Real-Time PCR）,RT-PCR)and Western blotting（WB）were used to detect m RNA and protein expression levels of SDF-1 and CXCR4 in cells.Results:The cultured cells were identified as HPFs.ICC detected Scg3expression in HPFs.CCK-8 assay could promote the proliferation of HPFs with different concentrations of Scg3（P<0.05）,and the optimal concentration was60ng/ml.Control group,60ng/ml Scg3,and 60ng/ml VEGF were cultured with HPFs for 48h,respectively.ICC detection showed that the average fluorescence intensity of SDF-1 in control group,Scg3 group and VEGF group was17.24±0.8063,129.9±5.865 and 128.5±7.388,respectively,and the difference was statistically significant（F=139.9,P<0.05）.The average fluorescence intensity of CXCR4 cells in control group,Scg3 group and VEGF group was17.80±1.776,138.9±2.576 and 127.6±6.234,respectively,and the difference was statistically significant（F=276.1,P<0.05）.The average fluorescence intensity of SDF-1 and CXCR4 cells in Scg3 group and VEGF group was higher than that in control group,the differences were statistically significant(SDF-1:t=19.03,14.97,P_Scg3<0.05,P_VEGF<0.05;CXCR4:t=38.72,16.94,P_Scg3<0.05,P_VEGF<0.05);There was no significant difference in the average fluorescence intensity of SDF-1 and CXCR4 between Scg3 group and VEGF group（SDF-1:t=0.1440,P>0.05;CXCR4:t=1.679,P>0.05）.RT-PCR assay results showed that the relative expression level of SDF-1 m RNA in control group,Scg3 group and VEGF group was 1.00±0.001,1.905±0.07126 and 1.863±0.05040,respectively,and the difference was statistically significant（F=102.7,P<0.05）.The relative expression levels of CXCR4 m RNA in control group,Scg3 group and VEGF group were 1.00±0.002,1.573±0.1009 and 1.558±0.1123,respectively,and the difference was statistically significant（F=14.03,P<0.05）.The relative m RNA expression levels of SDF-1 and CXCR4 in HPFs in Scg3 group and VEGF group were higher than those in control group,and the differences were statistically significant(SDF-1:t=12.70,17.12,P_Scg3<0.05,P_VEGF<0.05;CXCR4:t=5.674,4.971,P_Scg3<0.05,P_VEGF<0.05);There was no significant difference in the relative m RNA expression levels of SDF-1 and CXCR4 in Scg3 group and VEGF group（SDF-1:t=0.4829,P>0.05;CXCR4:t=0.09656;P>0.05）.WB test results showed that the relative expression levels of SDF-1 protein in control group,Scg3 group and VEGF group were 0.9304±0.04537,2.198±0.2497 and2.075±0.06945,respectively,and the differences were statistically significant（F=21.16,P<0.05）.The relative expression levels of CXCR4 protein in control group,Scg3 group and VEGF group were 0.9853±0.009495,2.018±0.1727 and2.331±0.06264,respectively,and the difference was statistically significant（F=43.95,P<0.05）.The relative expression levels of SDF-1 and CXCR4 protein in Scg3 group and VEGF group were higher than those in control group,and the differences were statistically significant(SDF-1:t=4.993,13.79,P_Scg3<0.05,P_VEGF<0.05;CXCR4:t=5.972,21.24,P_Scg3<0.05,P_VEGF<0.05);There was no significant difference in the relative expression levels of SDF-1 and CXCR4protein between Scg3 group and VEGF group（SDF-1:t=0.4752,P>0.05;CXCR4:t=1.701;P>0.05）.Conclusion:Scg3 is expressed in HPFs,and Scg3 can induce proliferation of HPFs.Both Scg3 and VEGF could up-regulate the expression of SDF-1 and CXCR4 in HPFs.Scg3 promotes the proliferation of HPFs,and its mechanism may be related to the regulation of SDF-1/CXCR4 signaling pathway.