| ObjectiveTo investigate the mechanism of D-galactose-induced intracellular calcium overload which leads to cellular senescence in retinal pigment epithelium(RPE).The content of inflammatory factors released by RPE cells induced by D-galactose induced intracellular calcium overload was investigated.To investigate the effects of the L-type calcium channel blocker verapamil and the anti-aging drug perillal aldehyde on the senescence of RPE cells by regulating calcium overload in RPE cells.Methods1.Establish RPE cell senescence model: RPE cells use 50 m M,100 m M,150 m M,200 m M D-galactose for 24 h,CCK8 is used to detect cell activity,andβ-galactosidase staining is used to evaluate the degree of cell senescence to determine the optimal drug action conditions(100m M D-galactose for 24h)to construct a senescent cell model;Study on the mechanism of intracellular calcium overload and cellular senescence of RPE: Fluo-4 AM calcium ion fluorescent probe was used to detect the trend of calcium ion concentration in RPE cells,and the source and concentration of intracellular calcium ion were clarified.Real-time PCR,western blotting and cellular immunofluorescence were used to observe the change trend of calcium channel in the aging process of RPE cells,and the change trend of apoptosis marker protein Cleaved-Caspase3 and aging marker protein P21 in the aging process of RPE cells was detected by immunoblotting.The L-type calcium channel blocker Verapamil and the anti-aging drug perillal aldehyde(PAE)intervened in the Ca V1.2 channel to observe their effect on intracellular calcium overload during the aging process of RPE cells,and the content of inflammatory factor IL-6 in cell supernatant was detected by enzyme-linked immunosorbent assay(ELISA)to clarify the calcium overload of RPE cells and the secretion of inflammatory factor IL-6.2.Experimental grouping:(1)Control group: no special treatment;(2)D-galactose group: treated with 100 m M of D-galactose for 24 hours;(3)D-galactose + si RNA-Ca V1.2 group:100m M D-galatose+si RNA-Ca V1.2 medium combine treatment for 48 hours;(4)D-galactose + 75μmol/ml verapamil group: medium containing 100 m M D-galactose + 75μmol/ml verapamil was combined for 24 hours;(5)D-galactose + 0.25mg/l perillal group: 100 m M D-galactose + 0.25mg/l perillal aldehyde medium combined treatment for 24 hours.Results1.Comprehensive CCK8 detection and β-galactosidase staining showed that 10 m M of D-galactose had the best effect on RPE cells;the effect of75μmol/ml verapamil and 25mg/l perillal aldehyde for 24 h had a significant inhibitory effect on the aging of RPE cells treated by 100 m M D-galactose.2.Study on the mechanism of D-galactose-induced RPE cell senescence(1)The staining results of β-galactosidase showed that compared with the100 m M D-galactose group,the aging degree of the si RNA-Ca V1.2 group,perillal aldehyde group and verapamil group was significantly reduced(P<0.01).(2)Compared with the 100 m M D-galactose group,the fluorescence expression of calcium ions in the si RNA-Ca V1.2 group,verapamil group and perillal aldehyde group was significantly reduced(P<0.01).(3)The results of western blotting experiments showed that compared with the 100 m M D-galactose group,the expression content of L-calcium-channel protein Ca V1.2 protein was increased(P<0.01)when the si RNA-Ca V1.2 group,verapamil group and perillal aldehyde group were added.(4)The results of q RT-PCR experiments showed that compared with the100 m M D-galactose group,the expression of Ca V1.2 in the si RNA-Ca V1.2group was significantly reduced.(5)The results of cellular immunofluorescence experiments showed that compared with the 100 m M D-galactose group,the fluorescence expression of Ca V1.2 added to the si RNA-Ca V1.2 group was significantly reduced;3.Functional impairment of RPE cells(1)The results of western blotting experiments showed that compared with the 100 m M D-galactose group,the expression of si RNA-Ca V1.2 group,verapamil group and perillal aldehyde group showed that the expression of apoptosis marker protein Cleaved-Caspase3 and aging marker protein P21 were significantly reduced(P<0.01);(2)The results of enzyme-linked immunosorbent experiment showed that compared with the 100 m M D-galactose group,the secretion of inflammatory factor IL-6 in the si RNA-Ca V1.2 group,perillal aldehyde group and verapamil group was significantly reduced(P<0.01).ConclusionsWhen D-galactose acts on RPE cells,it can cause increased influx of extracellular calcium ions through L-type calcium channel Ca V1.2;increase in intracellular calcium ion concentration of RPE cells causes calcium overload,resulting in increased secretion of inflammatory factors IL-6;si RNA-Ca V1.2interference,L-type calcium channel blockers,and perillal aldehydes can significantly inhibit intracellular calcium overload by inhibiting the expression of Ca V1.2 calcium ion channels on cell membranes,thereby inhibiting apoptosis and aging.By inhibiting Ca V1.2 calcium channels,it can also reduce the secretion of inflammatory factors by RPE cells. |
PDF Full Text Request