Effects of O6-methylguanine-DNA methyltransferase on DNA damage and ap-optosis of lens epithelial cells in hydrogen peroxide-induced oxidative dam-age models
10.13389/j.cnki.rao.2024.0180
- VernacularTitle:MGMT在双氧水诱导的氧化损伤模型中对晶状体上皮细胞DNA损伤和凋亡的影响
- Author:
Weihua CAI
1
;
Renjian GUO
Author Information
1. 215028 江苏省苏州市,上海交通大学医学院苏州九龙医院
- Keywords:
age-related cataract;
oxidative damage;
O6-methylguanine-DNA methyltransferase gene
- From:
Recent Advances in Ophthalmology
2024;44(12):955-960
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the ability of the DNA-repair gene O6-methylguanine-DNA methyltransferase(MG-MT)to repair DNA damage and control apoptosis of lens epithelial cells(LECs)in an oxidative damage environment.Methods The cultured LECs were treated with different concentrations of hydrogen peroxide(H2O2)(0 μmol·L-1,200μmol·L-1,400 μmol·L-1,and 600 μmol·L-1)to confirm the effects of different concentrations of H2O2 on LECs.The LECs were divided into Control group(without treatment),H2O2 group(adding 400 μmol·L-1 H2O2),H2O2+HA group[adding 400 μmnol·L-1 H2O2 and hemagglutinin(HA)]and H2O2+OE-MGMT group[adding 400 μmol·L-1 H2O2 and 5 μL pcDNA3.1-MGMT(overexpression plasmid)and 12 μL Lipofectamine8000(liposome transfection reagent)].Cell viability was detected by Cell Counting Kit-8(CCK-8).Western blot was used to detect the expression levels of proteins(DNA dam-age marker protein γH2A and pro-apoptotic protein Bax/Bcl2)in anterior capsular tissue of lens of age-related cataract(ARC)patients(ARC group)and other groups.DNA damage repair ability(DNA damage marker 15A3)of LECs in each group was detected by immunofluorescence,and apoptosis in each group was detected by TUNEL staining.Results Western blot results showed that the relative protein expression level of MGMT in LECs of the Control group and the ARC group was 1.000±0.002 and 0.597±0.001,and the difference was statistically significant(P＜0.0001).The relative pro-tein expression level of MGMT in LECs decreased with the increase of H2O2 concentration.The relative expression level ofγH2A in the Control group,H2O2 group,H2O2+HA group and H2O2+OE-MGMT group was 1.000±0.005,1.424±0.011,1.359±0.009,and 0.586±0.004,respectively,and the expression level of Bax/Bcl2 was 1.000±0.003,2.132±0.017,1.491±0.006,and 0.687±0.008,respectively,with those expression levels in the H2O2+OE-MGMT group being signifi-cantly lower than the H2O2+HA group(all P＜0.001).Immunofluorescence detection results showed that the relative ex-pression level of 15A3 in the Control group,H2O2 group,H2O2+HA group and H2O2+OE-MGMT group was 1.000±0.128,1.842±0.207,2.104±0.267,and 0.723±0.073,respectively,with the fluorescence intensity of 15A3 staining in the H2O2+OE-MGMT group being significantly lower than the H2O2 group(P＜0.001).TUNEL staining results showed that the fluorescence intensity of apoptosis in the Control group,H2O2 group,H2O2+HA group and H2O2+OE-MGMT group was 1.800±0.002,13.660±0.002,18.000±0.011,and 2.150±0.003,respectively,with decreased fluorescence intensity of apoptosis in the H2O2+OE-MGMT group compared with the H2O2 group(P＜0.000 1).The results of CCK-8 showed that the cell viability of the Control group,H2O2 group,H2O2+HA group and H2O2+OE-MG-MT group were 100.09±1.54,57.09±2.09,60.39±3.87,and 74.16±1.96,respectively,with increased cell viability in the H2O2+OE-MGMT group compared with the H2O2 group(P＜0.05).Conclusion MGMT expression is reduced in the anterior capsular tissue of ARC patients and the H2O2-induced LEC oxidative damage models.MGMT overexpression is involved in the double-strand break repair pathway and base excision repair pathway to promote the repair of DNA damage in LECs,thereby increasing cell vitality,reducing apoptosis,and regulating the occurrence and development of ARC.
