SUMOylation Inhibitors Exert a Protective Effect on Oxidative
Damage in Retinal Pigment Epithelial Cells Through the
Keap1/Nrf2/ARE Signaling Pathway
Authors: Liang Yilei , Jia Xin , Zheng Fangyuan , Wang Yifan , Fan Yijia , Zhang Haiyu , Dang Ziyao , Wang Lifei 
Published: January 2025

Purpose:
To investigate the effect of the SUMOylation inhibitor TAK981 on
hydrogen peroxide (H2O2)-induced oxidative damage in human retinal pigment
epithelial cells (ARPE-19) and its regulatory mechanism.

Methods:
An oxidative damage model of ARPE-19 cells induced by H2O2 was
established, and 1, 2, and 5 µM TAK981 solutions were administered for intervention
respectively. Normal cells were used as the control group. The viability of the cells in
each group was detected by the methyl thiazolyl tetrazolium (MTT) method. The levels of
superoxide dismutase (SOD) and malondialdehyde (MDA) in each group of cells were
detected by biochemical methods. The levels of IL-1β and TNF-α produced by each
group of cells were detected by enzyme-linked immunosorbent assay (ELISA). The
protein expression levels of Nrf2, HO-1, NQO-1, Keap1, and Sumo1 in each group of
cells were detected by Western blotting. In addition, 2 µM TAK981 and 2 µM TAK981
combined with 10 µM ML385 (an Nrf2 inhibitor) were administered to H2O2-induced
ARPE-19 cells, and the levels of SOD and MDA, IL-1β and TNF-αwere detected again.

Results:
The viability of the ARPE-19 cells decreased with increasing H2O2
concentration (F=19.158, P<0.001). H2O2 treatment at 350 µM was the concentration at
which the cells essentially reached half inhibition (IC50), and the cell oxidative damage
model was successfully established. After intervention with TAK981, cell survival
increased significantly (F=0.098, P<0.001). The differences between the 2 µM and 5 µM
TAK981 groups and the model group were statistically significant (all P<0.01). Compared
with those in the normal group, the MDA content in the model group increased, the SOD
activity decreased, and the release levels of IL-1β and TNF-α increased (all P<0.01).
Compared with those in the model group, the MDA content in the TAK981 group
decreased, the SOD activity increased, and the release levels of IL-1β and TNF-α
decreased. The differences between the 2 µM and 5 µM TAK981 groups were
statistically significant (P<0.05). Compared with those in the normal group, the protein
expression levels of Nrf2, HO-1 and NQO-1 in the model group were greater, whereas
the protein expression levels of Keap1 and Sumo1 were lower (all P<0.05). Compared
with those in the model group, the protein expression levels of Nrf2, HO-1 and NQO-1 in
the TAK981-treated group continued to increase, whereas the protein expression levels
of Keap1 and Sumo1 continued to decrease. The differences in the 5 µM TAK981 group
were statistically significant (P<0.05). In addition, after the combined intervention of
TAK981 and ML385 on H2O2-induced cells, compared with the TAK981-only
intervention on H2O2-induced cells, the cell viability increased, the MDA content
increased, the SOD activity decreased, and the IL-1β and TNF-α release levels
increased. The differences were statistically significant (P<0.05).

Conclusion:
The SUMOylation inhibitor TAK981 activates the Keap1/Nrf2/ARE
signaling pathway, enhances the activity of antioxidant enzymes, and reduces the
production of oxidative stress products and inflammatory factors, thereby exerting a
protective effect on H2O2-induced oxidative damage in ARPE-19 cells. Therefore, it is
suggested that intervention in SUMO regulation can be used as a new therapeutic
target in the AMD disease model, in order to delay the development of AMD by
reducing the oxidative damage of RPE.