Acetylcholinesterase function in apoptotic retina pigment epithelial cells induced by H2O2

Abstract

Aim: To investigate the acetylcholinesterase (AChE) expression involved in retina pigment epithelial (RPE) apoptosis induced by higher concentrations H2O2.

Methods: The human retinal pigment epithelium cell line ARPE-19 was from ATCC (Rockville, MD). Cultured ARPE-19 cells were treated with H2O2 at 0, 250, 500, 1 000, 2 000µmol/L and cell viability was measured with MTT assay. AChE expression and DNA fragments were analyzed by immunocytochemistry, TUNEL and PARP-1 Western blotting.

Results: Immunofluorescence detected AChE exist in the normal human retinal tissue. When H2O2 >500µmol/L, AChE expression showed an increase after 2h, and this concentration was selected for the present study. RPE cell was induced with 1 000µmol/L H2O2 for 2h, compared to the control group, cell activity decline detected by MTT, AChE and PARP-1 protein expression was significantly increased detected by Western blotting. AChE immunofluorescence staining was positive in RPE cell after H2O2 incubate 2h. In addition, pretreatment with 100µmol/L epigallocatechin gallate (EGCG), cell viability increased from 31.20%±3.90% to 70.23%±12.96%.

Conclusion: AChE is weakly expressed in normal human RPE cells. Stimulation with H2O2 caused the stable increase of AChE expression in RPE cells, which may indicate that AChE may be an important role in AMD.

Keywords: acetylcholinesterase; age-related macular degeneration; oxidative stress; retina pigment epithelial cells.

Figure 1. HE and AChE immunofluorescence staining in normal human retina tissue

HE staining of human retina (A), AChE immunofluorescence is green, DAPI specificity in the nucleus (blue) is merged (400×).

Figure 2. The time and concentration dose of RPE cell treated by H₂O₂

Detect the RPE cells viability via MTT assay and the result shows that: treated concentration are shown at A (0, 500, 1 000, 2 000µmol/L), with the increase concentration of H₂O₂, the cell survive rate decrease, as well as treated time at B (0, 60, 120min) when the H₂O₂ concentration is 1 000µmol/L, 120min has the high rate about 50%, ^aP<0.05.

Figure 3. Induction apoptosis of RPE cells treated with H₂O₂ by morphology and TUNEL

Control group (a,d), after 2h following H₂O₂ (1 000µmol/L), these is cell fragmentation, floating and death (b,e), 2h following H₂O₂ (1 000µmol/L) and 100µmol/L EGCG (c, f) pre-treatment, cell number is more (A). The HoChest 33258 staining is positive, AChE immunofluorescence and TUNEL staining is negative in control group. HoChest 33258, AChE immunofluorescence and TUNEL all positive staining in H₂O₂ (1 000µmol/L) group 400×(C).

Figure 4. After different concentrations of H₂O₂ incubated for 2h, the AChE protein is detected by western blot (A).

With the higher concentration of H₂O₂, cleaved-PARP, AChE and beta-actin Western blot, cleaved-PARP, AChE protein show an increase trend. (B) Western blot gray value quantitative is analyzed (ImageJ software), ^aP<0.05, statistically significant.