AIM: To study the effect of leech extract on the expression of vascular endothelial growth factor(VEGF)in retinoblastoma(WERI-RB-1 cells)and the related molecular mechanism.

METHODS: WERI-RB-1 cells cultured in vitro were divided into control group, 0.04U/mL leech extract group and 0.08U/mL leech extract group. The control group was cultured with complete medium for 48h, and the leech extract group was cultured with 0.04 and 0.08U/mL drug-containing medium for 48h, respectively. The expression level of VEGF in conditioned medium of cell culture was detected by ELISA. The mRNA levels of hypoxia-inducible factor-1a(HIF-1a)and matrix metalloproteinase-9(MMP-9)were detected by RT-PCR. The expression levels of HIF-1a, MMP-9, phosphatidylinositol 3-kinase(PI3K)and human phosphorylated AKT(p-AKT)were detected by Western Blot.

RESULTS: Compared with the control group, the expression of VEGF in the conditioned medium with the concentration of 0.04U/ mL and 0.08U/mL of leech extract was decreased(P<0.05), and the inhibition rate was 32.43% and 38.92%, respectively. The expression levels of HIF-1a and MMP-9 mRNA were significantly decreased(P<0.05). The inhibition rates of HIF-1a mRNA expression of the two leech extracts were 27.64% and 24.75%, respectively. The inhibition rate of MMP-9 mRNA expression was 43.97% and 51.48%, respectively. The protein expression levels of HIF-1a, MMP-9, PI3K and p-AKT were significantly decreased compared with the control group(P<0.05). The inhibition rates of 0.04 and 0.08U/mL leech extracts on the protein expression of HIF-1a, MMP-9, PI3K and p-AKT were 55.81% and 43.85%, 39.49% and 47.23%, 33.27% and 29.83%, 52.07% and 30.21%, respectively.

CONCLUSION:Leech extract may inhibit the expression of VEGF in WERI-RB-1 cells via the VEGF/PI3K/AKT pathway or HIF-1a and MMP-9 factors.