Effects Of TRPC1,3,And 6 On High Glucose-Induced Human Retinal Vascular Endothelial Cells And Its Mechanism

ObjectiveMost members of the permeable channel Transient Receptor Potential Canonicals(TRPCs)are widely distributed on various endothelial cell membranes.It has been confirmed that TRPCs are involved in neovascularization after vascular intima injury in many tissues.However,whether TRPCs play a regulatory role in the development of diabetic retinopathy has rarely been reported.In the present study,we selected TRPC1,3,and 6to determine their roles and mechanism in the proliferation,migration,and lumen formation of human retinal vascular endothelial cells.Materials and Methods1.HREC was divided into three groups,respectively,containing 5.5 mM glucose,30 mM glucose and 24.5 mM mannitol + 5.5 mM glucose medium,and the total protein was extracted after 24 and 48 hours of intervention,and high glucose was detected for different subtypes of TRPC.Protein expression effects of TRPC1,TRPC3,TRPC6);2.HREC was divided into 5 groups(30 mM glucose,30 mM glucose + 2mg/LSKF 96365,30 mM glucose + 4 mg/LSKF 96365,30 mM glucose + 8mg/L SKF 96365,30 mM glucose + 16 mg/L SKF 96365,Note: SKF96365 is a TRPC channel blocker)After 24 and 48 hours of intervention,the total protein was extracted and the effect of TRPC channel inhibitor on the expression of VEGF protein in high glucose-induced cells was detected;3.The HRECs were divided into three groups,starved for 24 hours,and then normal medium containing 5.5 mM glucose,30 mM glucose,and 24.5 mM mannitol was added,and the cells were transferred to a 12-well plate in which slides were placed,and 4 complexes were set.The wells were cultured for 24 hours to adhere to the cells,and the slides were taken out for immunohistochemistry of cell slides to observe the quantitative and localized expression of TRPC1,TRPC3 and TRPC6 under the influence of high glucose,and the records were recorded by inverted phase contrast microscope;4.The HRECs were divided into 5 groups,and after 24 hours of starvation treatment,30 mM glucose,30 mM glucose + 2 mg/LSKF 96365,30 mM glucose+ 4 mg/LSKF 96365,30 mM glucose + 8 mg/L SKF 96365,30 mM glucose +16 mg/L SKF 96365 were added,and the cells were added.In the 24-well plate of the slide,4 duplicate wells were set,and the cells were attached to the cells.After intervention with different concentrations of TRPC intervention agent SKF96365 high glucose medium for 24 h,the slides were taken out for immunohistochemistry of cell slides.Quantitative and localized expression of VEGF in the presence of TRPC channel inhibitors,recorded by inverted phase contrast microscopy.Result1.Western blot analysis showed that the expression of TRPC1 wasincreased at 24 °C after 24 hours and 48 hours after high glucose induction(P<0.05).The time-dependent expression was not clear.There was no significant difference in TRPC1 protein expression between hyperosmolar group.There was no significant difference in the expression of TRPC3 and TRPC6 protein levels(P>0.05);2.Western blot analysis showed that high glucose induced human retinal vascular endothelium inhibited VEGF expression in 4,8,and 16 mg/L SKF96365 intervention groups at 24 and 48 hours after treatment with different concentrations of TRPC channel inhibitor SKF96365,in a concentration-and time-dependent manner(P<0.05);3.The results of immunohistochemistry(IHC)showed that the expression of TRPC1 was increased at the protein level after 24 hours of high glucose-induced retinal vascular endothelial cell line(P<0.05).It can be seen that the localization of TRPC1 in the cell is mainly in the cell membrane.With cytoplasm.There was no significant difference in the expression of TRPC3 and TRPC6 protein levels(P>0.05);4.Cellular slide immunohistochemistry(IHC)results showed that high glucose induced human retinal vascular endothelium was treated with different concentrations of TRPC channel inhibitor SKF96365 for 24 hours,2 mg/L,4mg/L,8 mg/L,16 mg.The expression of VEGF in the /L SKF96365 intervention group was significantly down-regulated(P<0.05)in a concentration-dependent manner.It can be seen that the localization of VEGF in cells is mainly in the cell membrane and perinuclear.Conclusion1.High glucose can induce the increase of TRPC1 protein expression in human retinal vascular endothelial cells;2.Blocking the TRPC pathway inhibits high glucose-induced human retinal vascular endothelial cells down-regulation of VEGF expression.