Experimental Study On The Role Of Intracellular Calcium Overload Mediated By Calcium Channel Cav1.3 In The Aging Of RPE Cells

ObjectiveThis study discussed the role of L-type calcium channel Cav1.3 in the aging process of RPE cells.MethodsIn the experiment,human RPE cell line(ARPE-19)was selected to explore the key genes in the aging process of RPE cells through bioinformatics analysis.The cell aging model was established by D-gal acting on RPE cells.The effect of different concentrations of D-galactose on RPE cell viability was detected by CCK8 method β-Galactosidase(SA-β-Gal)staining was used to detect the aging of RPE cells.Cells were transfected with Cav1.3 small interfering RNA(si RNA),and the Cav1.3 gene was silenced.The transfection efficiency of RPE cells was observed under fluorescence microscope,and the silencing efficiency was determined by q PCR.The fragment with the highest Cav1.3 si RNA inhibition rate was screened for subsequent experiments.The intracellular calcium concentration was detected by fluo-4 fluorescence probe,the change of mitochondrial membrane potential expression was detected by JC-1 staining,the expression level of reactive oxygen species(ROS)was detected by DEFH-DA probe method,and the expression level of L-type calcium channel Cav1.3,caspase-9 RNA(m RNA)was detected by real-time real-time real-time fluorescence quantitative PCR(q PCR).ResultsBioinformatics analysis showed that CACNA1D(Cav1.3)was one of the key regulatory factors in the aging process of RPE cells.CCK8 results showed that compared with the control group,D-galactose could inhibit the proliferation and metabolic activity of RPE cells in the aging group in a concentrationdependent manner.SA-β-Gal staining results showed that with the increase of D-gal concentration,cell senescence increased,and the senescence of Cav1.3si RNA group decreased.Cav1.3 si RNA can be successfully transfected into RPE cells(the transfection efficiency is 77.34%).The silencing effect of Cav1.3gene was detected by q PCR,indicating that the transfection and silencing effect are good.Fluo-4 fluorescence probe results showed that the intracellular calcium concentration increased in the aging group and decreased in the si RNA group.The results of JC-1 staining showed that the mitochondrial membrane potential decreased in the aging group and increased in the si RNA group.The experimental results of DEFH-DA probe showed that the ROS in the cells of aging group increased,while the ROS in the cells of si RNA group decreased.The results of q PCR showed that the relative expression of Cav1.3m RNA in the aging group was higher than that in the control group.The relative expression of caspase-9 m RNA decreased.ConclusionsCav1.3 can mediate the calcium overload in RPE cells,and then cause the decrease of mitochondrial membrane potential,the damage of mitochondrial function,the increase of ROS production in cells,and the enhancement of cell aging;After small RNA interference with Cav1.3,the intracellular calcium concentration of RPE cells decreased significantly,the mitochondrial damage weakened,ROS production decreased,and cell senescence weakened.