The Protective Effect Of Slit2-n On VEGF165-induced Proliferation Of HUVEC And HMVEC And Its Mechanism

Objective: To explore the effect and mechanism of Slit2-N on the VEGF165-induced proliferation of HUVEC and HMVEC cells aiming to lay a foundation of finding a new target to treating CNV.Methods:1.HUVEC and HMVEC cell lines were cultivated in vitro.2.The appropriate working concentrations of Slit2-N and VEGF165 treated to HUVEC and HMVEC cells were measured by CCK-8 assay.3.The effect of Slit2-N on the VEGF165-induced proliferation of HUVEC and HMVEC was measured by CCK-8 assay.4.The proliferation index of each group was measured by CFSE staining flow cytometry assay.5.The apoptosis rates of Control group,Slit2-N group,VEGF165 group and Slit2-N and VEGF group were measured by flow cytometry.The expressions of p-AKT,AKT,p-ERK1/2,ERK1/2 in each group of HUVEC and HMVEC were measured by Western Blot.Results:1.The minimum concentrations of VEGF165 that could induce the proliferation of HUVEC and HMVEC cells were 25ng/ml and 500 pM.The two concentrations were selected as working concentrations in HUVEC and HMVEC cells.The maximum concentrations of Slit2-N that couldn't affect the vitality of HUVEC and HMVEC were both 16 nM and it was selected as working concentration,too.2.After 48 hours of treatment,the results of CCK-8 assay showed that the number of cells in Slit2-N groups and control groups were not significantly different in both HUVEC and HMVEC(p?0.05).The cell numbers of VEGF165 groups were significantly higher than control groups(p ? 0.01 in HUVEC,p ? 0.001 in HMVE)and they were significantly lower in Slit2-N + VEGF165 groups than those in VEGF165 groups(p?0.05 in HUVEC,p?0.001 in HMVEC).3.The results of Annexin-V FITC and PI double staining flow cytometry showed that the apoptosis rates of Slit2-N+VEGF165 groups and VEGF165 groups in HUVEC and HMVEC were not significantly different at the time points of 24 h,48h and 72 h.4.The CFSE flow cytometry assay results showed that the proliferation indexes in each group of HUVEC were(31.63�0.28),(31.88�0.92),(42.12�1.68),(32.71�0.32)and those of HMVEC were(12.54�1.08),(13.39�0.15),(15.74�0.51),(12.89�0.90).The proliferation indexes of the cells in Slit2-N groups and control groups were notsignificantly different(p ? 0.05 in both HUVEC and HMVEC).The proliferation indexes of the cells in VEGF165 groups were significantly higher than those in control groups(p ? 0.001 in HUVEC,p ? 0.01 in HMVEC)and they were significantly lower in Slit2-N+VEGF165 groups than in VEGF165 groups(p?0.001 in HUVEC,p?0.01 in HMVEC).5.The Western Blot results showed that the relative expression of p-AKT in Slit2-N group and control group were not significantly different(p ? 0.05 in both HUVEC and HMVEC).It was significantly higher in VEGF165 group than that of control group(p<0.001 in HUVEC,p<0.01 in HMVEC).The relative expression of p-AKT was significantly lower in Slit2-N+VEGF165 group than that of VEGF165(p<0.001 in HUVEC,p< 0.01 in HMVEC).The relative expressions of p-ERK 1/2 were not significantly different(p?0.05 in HUVEC and HMVEC).It was higher in VEGF165 group than that of control group(p<0.001 in both HUVEC and HMVEC).However,the relative expressions of p-ERK1/2 were not significantly different between Slit2-N +VEGF165 group and VEGF165group(p?0.05 in HUVEC and HMVEC).Conclusion: Slit2-N can inhibit the VEGF165-induced proliferation of HUVEC and HMVEC through AKT pathway,may not through ERK1/2pathway.It lays a foundation of finding a new treating target towards CNV.