The Effect Of HOGG1 In Retinal Pigment Epithelium Cells On Oxidative Stress And Apoptosis Induced By TNF-α

Retinal pigment epithelium(RPE) is located in the outermost layer of the retina, it plays an importmant role in maintaining normal function of retina by absorpting scattering light, making regeneration and synthesis of retina visual pigment, keeping renewal and metabolism of the photoreceptors, and forming the blood-retinal barrier. RPE cells’ oxidative damage and apoptosis play an important role in the pathogenesis of a wide variety of retinal diseases. Tumor necrosis factor alpha mediates inflammatory response in the pathogenesis of these diseases.TNF alpha could induce cells to produce more reactive oxygen species (ROS). ROS could attack DNA, results in oxidative damage. In oxidative damage of DNA, the oxidation of the eight carbon atoms of guanine is the most common form, which is called 8-oxoguanine(8-oxodG), and is an internationally recognized sensitive index and biomarker for DNA oxidative damage evaluation. In mammalian,8-hydroxy guanine DNA glycosylase enzyme(OGGl) could specific repair 8-oxodG.OGG1’s human homologue is known as hOGGl,which is the key enzyme of base excision repair pathways (BER). RPE cells are vulnerable to be attacked by various factors, resulting in oxidative DNA damage. If these DNA damage were not promptly repaired, apoptosis of RPE cells may be induced. Recent research showed that with lower OGG1 expression in mice, TNF alpha could not process inflammatory response. In these studies, we investigated hoggl gene’s role on the TNF alpha-induced oxidative damage and apoptosis in RPE cells.In the first part, we established an retinal pigment epithelial cells(ARPE-19) model with high express of hOGGl-la. Firstly hOGG1-la recombinant adenovirus expression vector pAd/CMV/V5-DEST-hoggl and negative control pAd/CMV/V5-GW/lacZ were identified. Then the two vectors were packaged in human embryonic kidney cells (293A). To obtian high titer virus solution, the virus was repeatedly amplified in 293A cells, and concentration of virus was measured by plaque formation assay. pAd/CMV/V5-DEST-hogg/, pAd/CMV/V5-GW/lacZ were transfected into ARPE-19 cells respectively by a MOI of 30:1. Then the expression of hOGGl-la protein in these three cell lines (ARPE-19 cells transfected pAd/CMV/V5-DEST-hoggl (RPE-Ad-hoggl), ARPE-19 cells transfected pAd/CMV/V5-GW/lacZ(RPE-Ad-lacZ) and non-transfected ARPE-19 cells) were detected by western blot assay. The results showed that the hOGGl-la protein expression in RPE-Ad-hoggl cell line was significantly higher than in the other two RPE cell lines, and the expression of hOGG1-la protein was not significantly different between RPE-Ad-lacZ cell line and non-transfected ARPE-19 cell line.In the second part the expression of 8-oxodG and ROS production in these three cell lines(ARPE-19 cells transfected pAd/CMV/V5-DEST-hoggl(RPE-Ad-hogg1), ARPE-19 cells transfected pAd/CMV/V5-GW/lacZ(RPE-Ad-lacZ) and non-transfected ARPE-19 cells) responsed to hydrogen peroxide (H2O2) were observed. 100μmol/L H2O2 was treated on these three ARPE-19 cell lines for 2h respectively. Then the intracellular ROS production and the formation of 8-oxodG were detected by FCM and immunohistochemistry respectively. It was found that, compared to non-transfected ARPE-19 cells and RPE-Ad-laeZ cell line, RPE-Ad-hoggl cell line producted less ROS and had less 8-oxodG formation (P＜0.001). We made an conclusion that ARPE-19 cells with a high expression of hOGG1 could relieve the oxidant to reduce H2O2-caused oxidative damage.In the third part, TNF alpha-induced oxidative stress and apoptosis in different hOGGl-la expression ARPE-19 cell lines were observed. After RPE-Ad-hogg1, RPE-Ad-lacZ and non-transfected ARPE-19 cell lines were treated with TNF alpha of different concentrations for different time respectively, the survival rates of the three cell lines were determined by MTT. The results showed that within 24h, with the increasing of TNF alpha concentration and prolong of time, the survival rates of all these three cell lines were decreased (P<0.05). Compared 24h and 36h and 48h after treated with TNF alpha under same concentration, the survival rates had no significant difference among all three cell lines (P>0.05). RPE-Ad-hoggl cells presented a higher survival rates (P＜0.05). Then the intracellular ROS production and 8-oxodG formation of three cell lines treated with 50 ng/mL TNF alpha for 24h were detected by FCM and Western blot seperately. The results showed that, compared to RPE-Ad-lacZ and non-transfected ARPE-19 cell lines, the production of ROS was less and 8-oxodG formation was significantly lower (P<0.0001) in RPE-Ad-hoggl cell line.After RPE-Ad-hoggl, RPE-Ad-lacZ, non-transfected ARPE-19 cell lines were treated with 50 ng/mL TNF alpha for 24h, apoptosis ratio was tested by flow cytometry(FCM). The results showed that TNF alpha could increase apoptosis ratio of all three cell lines. Compared with RPE-Ad-lacZ cell line, apoptosis rate of RPE-Ad-hoggl cell line was significantly lower(P<0.05). Then the transcript change of Fas/FasL apoptosis pathway related gene, FasL, caspase3 and caspase8 were detected by fluorescence quantitative PCR method. The results showed that the expression of mRNA levels of each above gene were raised. But compared with the RPE-Ad-lacZ cell line, the mRNA levels of FasL, caspase3, caspase8 gene in RPE-Ad-hoggl cell line were all significantly lower. We conclude that as a DNA repair enzyme, hOGG1 played an important role in TNF alpha-induced oxidative damage in ARPE-19 cells. In addition, hOGGl could block the Fas/FasL apoptosis pathway and inhibit TNF alpha-induced apoptosis.