Effects Of SMP30 Expression On Cell Growth And Oxidative Activity Of Human Lens Epithelial Cells Under Acute Oxidative Condition

Purpose:This study aims to observe the effect of changes in the content of intracellular Senescence Marker Protein 30(SMP30)on the physiological function of human Lens Epithelial Cells(HLEC)SRA01/04 in the early stage of acute oxidative stress,and to provide further research on the etiology and development of cataracts.Methods:?Selection of the most appropriate H2O2 concentration:SRA01/04 in the logarithmic growth phase was implanted into a 96-well plate with different concentrations of H2O2(150 ? MOL·L-1?200 ? MOL·L-1?250 ? MOL·L-1?300 ? MOL·L-1?350 ? MOL·L-1?400 ? MOL·L-11?450 ? MOL1 L-1)for 2 h,and the optimal H2O2 concentration is selected by CCK8 method to establish the cell model of early stage of acute oxidative stress.?Establishment of the cell model:According to our previous research study,the cell model was established by transfecting SRA01/04 with lentivirus:including OE(SMP30 over expression)group?NCOE(Negative Control Over Expression)group?KD(Knock Down SMP30)group?NCKD(Negative Control Knock Down)group.After the successful transfection,q-PCR(Real-time Quantitative Polymerase Chain Reaction)was used to determine whether the model was successfully established,and then the model was cultured in a medium with the optimal concentration of H2O2.?Detection of cell function:Cell viability was measured by Kit-8(Cell Counting Kit-8,CCK-8),and the proliferation activity of each group was measured by 5-bromodeoxyuridine(BrdU).Cell cycle analysis and cell apoptosis were performed by flow cytometry;the antioxidant capacity of the cells was measured by an SOD assay kit(WST-1 method).RESULTS:?By means of the pre-experiment,we determined that the most appropriate H2O2 concentration in the cell model of acute oxidative stress was 300 ? MOL·L-1.?q-PCR was used to determine the transfection efficiency of four groups.Compared with the NCOE group,the expression abundance of the OE group was 18.378 times;compared with the NCKD group.The silencing efficiency of the KD group cells was about 94%.? Compared with the NCOE group and the CON group,the cell viability of the OE group was increased(p<0.05);and compared with the NCKD group and the CON group,the cell viability of the KD group was decreased(p<0.05).The Brdu proliferation assay showed that compare with CON group(5.618±0.295),the cell proliferation of OE group(7.681±0.490)was increased(p<0.01),Compare with NCOE group(5.886±0.387),the cell proliferation of OE group(7.681±0.490)increased(p<0.01);Compare with CON group(5.618±0.295),the cell proliferation of KD group(4.777±0.353)was decreased(p<0.05),Compare with NCKD group(5.494±0.067),the cell proliferation of KD group(4.777±0.353)was decreased(p<0.05).Flow cytometry analysis indicated that the cells of OE group in the late stage of DNA synthesis and the stage of division was significantly increased under acute oxidative stress.While in the early stage of DNA synthesis,the number of cells was decreased.The cells in the KD group were significantly reduced in the cell division stage,and the number of cells in the pre-DNA synthesis stage and DNA synthesis stage was not significant changed.The apoptosis rate of the OE group(1.94±0.066)was lower than that of the NCOE group(2.28±0.059)(p<0.01);meanwhile,compared with the NCKD group(1.86±0.107),the apoptosis rate of KD group(6.28±0.176)was significantly increased(p<0.01).The activity of SOD of each group was detected by SOD assay kit(WST-1 method).Compared with the NCOE group(10.734±0.651),the activity of SOD in the OE group(17.523±0.975)was increased(p<0.01),compared with the CON group(11.985±0.364),the activity of SOD in the OE group(17.523±0.975)was increased(p<0.01);compared with the NCKD group(10.781±0.060),the activity of SOD in the KD group(8.150±0.506)was decreased(p<0.01),compared with the CON group(11.985±0.364),the activity of SOD in the KD group(8.150±0.506)was decreased(p<0.01).Conclusion:The most appropriate H2O2 concentration and time for establishing SRA01/04 cell model in the early stage of acute oxidative stress is 300 ?MOL·L-1 and 2 hours respectively.When SRA01/04 is in the early stage of acute oxidative stress,the increase of SMP30 content could effectively promote the cell growth activity,proliferation ability,antioxidant capacity,and inhibit cell apoptosis.Increasing the amount of SMP30 in the cell may inhibit or delay the cataracts caused by oxidative stress in HLEC SRA01/04.