Effects of U73122 and SQ22536 on proliferation and TGF-β_2 secretion of RPE cells in vitro

BackgroundPrevious studies highlight the importance of TGF-β2in the development of myopia.It was known that RPE cells is associated with secretion of the TGF-β2.However,how the RPE cells to regulate the secretion of TGF-β2 is unclear.ObjectiveThis study was to evaluate the roles of phospholipase C inhibitor,U73122,and adenylate cyclase inhibitor,SQ22536,on the morphology,proliferation and function of secreting transfer growth factor-β2(TGF-β2)in cultured retinal pigment epithelial cells(RPE).MethodsRetinal pigment epithelial cells were isolated from the eyes of guinea pig and digested and cultured in the DMEM/F12 medium containing 20% fetal bovine serum.The second generation of RPE cells were identified by keratin and plated into 96-well and 24-well dishes at a density of 1×105/well.The medium was shifted into DMEM/F12 medium without fetal bovine serum after cells nearly grew platefully.Different concentrations of U73122 or SQ22536 were added in medium respectively in 24 hours after the first medium change.The same dose of DMSO or ddH2O was used in control group.After another 24 hours,the proliferation of RPE cells was evaluated by using MTT assay.The TGF-β2 secreted by RPE cells was detected using ELISA method at 2,4,6,8,16 hours after the addition of 1×10-5 M U73122 or SQ22536.ResultsCultured cells showed the positive response for keratin.MTT result revealed that the OD value in 5×10-6 mol/L U73122 24 hours group was 0.277±0.020 and that in control group was 0.271±0.005,showing an insignificant difference between them(t=0.771,P0.05).In 24 hours after action of 1×10-5,1.5×10-5,2×10-5 mol/L U73122,the OD values were 0.214±0.011,0.181±0.012 and 0.136±0.009 respectively,showing statistically significant differences in comparison with control group(t=9.008,t=16.050,t=25.483,P0.01).In 5×10-6,1×10-5,2×10-5,5×10-5 mol/L SQ22536 groups,the OD values were 0.133±0.011,0.139±0.009,0.134±0.009 and 0.148±0.013 respectively,and no significant differences were seen among various group(F=1.316,P0.05).The result of the ELISA test showed that the secretion of TGF-β2 was increased after action of U73122 in 4,6,8,16 hours in comparison with control group(P0.01,P0.05),and the levels of TGF-β2in above time points were significantly higher than that of 2 hours(P0.05).In the SQ22536 group,there was considerably difference in the secretion of TGF-β2 between control group and SQ22536 group in 8 hours(t=-3.648,P0.05),but no significant difference was found among various time points(P0.05).ConclusionThe U73122,a phospholipase C inhibitor,can inhibit the proliferation of RPE cells and elevate the secretion of TGF-β2.SQ22536,the adenylate cyclase inhibitor,has no obvious influence on the growth of RPE cells and the secretion of TGF-β2.