Cannabinol-activated FOXO3a Down-regulates The Expression Of VEGF In Retinal Microvascular Endothelial Cells Under High Glucose Condition

Objective:To observe the effect of cannabinol(CBD)on the expression of vascular endothelial growth factor(VEGF)in human retinal microvascular endothelial cells(HRCECs)under high glucose and to explore its mechanism.Methods:HRCECs were cultured in vitro and divided into 6 groups: P0 group(blank control): only equal volume of medium was added;P1 group(solvent control group): 0.5% ethanol was added as solvent control;P2group(low-sugar control group)+0.5% ethanol: 5.5 mol/l of glucose +0.5% ethanol;P3 group(high-sugar control group): 22 mmol/l of glucose+ 0.5% ethanol;P4 group: P3+1 ?mol/L CBD;P5 group: P3+10?mol/L CBD.After CBD treatment,MTT was used to detect cell proliferation,real-time quantitative PCR and Western blot were used to detect the expression of VEGF mRNA and protein respectively.At the same time,the phosphorylation of nuclear transcription factor FOXO3 a and the expression of FOXM1 protein were detected by Western blot,and thenuclear translocation of FOXO3 a was detected by immunofluorescence.Finally,the binding of FOXO3 a and FOXM1 to the promoter region of vascular endothelial growth factor was detected by chromatin coprecipitation technique.Results:1.MTT results showed that at the same time point,the cell proliferation rate of the high-sugar group was significantly higher than that of the low-sugar group and the control group.After adding 1 ?mol/L and 10?mol/L CBD respectively,the cell activity decreased significantly compared with the high-sugar group.The difference was statistically significant.2.There was no significant change in the expression of vascular endothelial growth factor(VEGF)in P1 solvent control group and P2 hypoglycemia group,but the expression of vascular endothelial growth factor(VEGF)in P3 hyperglycemia group was significantly increased.After CBD treatment,the expression of VEGF in P4 and P5 groups decreased significantly.In addition,Western blot results also showed that the expression level of VEGF protein was similar to that of RNA.3.Western blot results showed that the phosphorylation level of FOXO3a253 serine(Ser253)was lower in P0 blank group and P1 solvent control group,and increased in P2 low-sugar group and P3 high-sugar group in varying degrees.The phosphorylation level of Ser253 decreased with theaddition of 1 and 10 ?mol/L CBD.Immunofluorescence results also showed that FOXO3 a was mainly located in the nucleus of P0 group.Under high and low sugar conditions,FOXO3 a content in the nucleus decreased,while FOXO3 a content in the cytoplasm increased significantly.After CBD treatment,FOXO3 a translocation was further promoted to the nucleus.In addition,the expression of FOXM1 protein was significantly decreased after CBD treatment.4.Chromatin coprecipitation results showed that the binding level of FOXO3 a and VEGF was higher in P0 group,and the binding level of FOXO3 a and VEGF was lower in high sugar group than in P0 group,while the level of FOXO3a-DNA complex increased significantly after CBD treatment at different concentrations.Contrary to FOXO3 a,the level of FOXM1-DNA complex after CBD treatment was significantly lower than that of high-sugar treatment.Conclusions:1.Cannabinol can inhibit the proliferation of retinal microvascular endothelial cells under high glucose.2.Cannabinol affects the binding of nuclear transcription factors FOXO3 a and FOXM1 to the promoter of vascular endothelial growth factor,and ultimately inhibits the expression of vascular endothelial growth factor.