The Effect And Mechanism Of TRPC On Human Retinal Vascular Endothelia Cells Induced By High Glucose

ObjectiveTo investigate the effect of TRPC on proliferation migration tube formation and VEGF expression of human retinal vascular endothelial cells induced by high glucoseMaterials and methods1.HREC were divided into three groups respectively,containing 5.5,30mM glucose and mannitol 30mM culture medium,after 24 and 48 hours of intervention,RNA was extracted from the cells,and the effect of high glucose on the expression of TRPC isoforms(TRPC1,TRPC3,TRPC6)was detected2.HRECweredividedintosixgroups(5.5mM,30mM,30mM+2mg/LSKF96365,30mM+4mg/L SKF96365,30uM+8mg/L SKF96365,30uM+16mg/L SKF96365)and then were added to 96-well plates with about4000 cells per well.After 24 h of starvation,the holes were treated with different concentrations of sugar and SKF96365 for 24 h.Add CCK-8 10?l,37?incubator for 2 to 4 h,enzyme analyzer to detect the 450nm wavelength per hole absorbance(OD),recorded and analyzed.3.HREC was divided into the above six groups,and the cells were uniformly added to a 6-well plate,after the cells were covered with the bottom of the plate,the sterile yellow spear was scratched by the vertical plate bottom.After removing the floating cells,add serum-free medium containing different concentrations of D-glucose and SKF96365 in a 37?incubator containing 5%in the training,Photoshop CS2 software to calculate the migration distance at the time of 0 h,12 h,24 h.4.The cells were treated with the above-mentioned six groups for 24 hours,and about 200?l of cell suspension was inoculated into the upper chamber of the Transwell chamber.The cells were supplemented with 800?l of DMEM medium containing 10%FBS with different components,Each group has 3holes,placed in the incubator for 16 hours fixed,stained:remove the chamber,with a cotton swab head carefully scraped off the room surface of the cells,with4%paraformaldehyde to the next room cells for 30 min,and then 0.1%purple for 20min,washed with PBS,dried at room temperature for 5 minutes and then observed under the microscope and counted the number of cells migrating in each group.5.1.5×10~4 cells and sugar and different concentrations of drug SKF96365were added to 96-well plates containing Matrigel matrix glue according to the above cell group.After incubation for 8 hours in the incubator,the number of lumens was counted and analyzed.6.The cells were divided into six groups according to the above method.After 24 hours of treatment with different concentrations of sugar and drug SKF96365,RNA was extracted and RT-PCR was used to detect the expression of VEGF in mRNA.ResultPCR results show the expression of TRPC1 and TRPC6 in human retinal vascular endothelial cells induced by high glucose was increased in a time-dependent manner(P<0.05).The expression of VEGF mRNA in HREC was decreased in 8,16mg/LSKF96365 group,(P<0.05).CCK-8 results showed that 4,8,16mg/L SKF96365 intervention group inhibited cell proliferation(P<0.05).The results of cell scratches showed that HREC migration distance was significantly decreased at 4,8,16 mg/L SKF96365 intervention group at 12 and24 h(P<0.05).Transwell results showed that the number of migrating cells in 8 mg/L and16 mg/L SKF96365 intervention group was significantly lower than that in the normal group(P<0.05).The results of tube formation in 8,16mg/LSKF96365 group were decreased(P<0.05).Conclusion1.High glucose could induce the expression of TRPC1 and TRPC6 mRNA in HREC.2.Blocking TRPC pathway inhibits high glucose-induced human retinal vascular endothelial cell proliferation,migration,lumen formation,and down-regulates VEGF mRNA expression.