Protection of sulforaphane against hydrogen peroxide-induced bovine trabecular meshwork cell apoptosis

Background

Evidences indicated that oxidative stress damage is an essential pathological process in primary open angle glaucoma.Sulforaphane (SFN) can play an antioxidative stress role to many tissues and cells by activating Nrf2/ARE single pathway.However, whether SFN has a protective role to oxidative stress induced damage of trabecular meshwork cells is still unclear.

Objective

This study was to investigate the antioxidant effect of SFN against H₂O₂-induced oxidative damage in bovine trabecular meshwork cells.

Methods

Trabecular cells were isolated from fresh black bovine eyeballs and primarily cultured and passaged.The third generation of cells were incubated to 96-well dish at a density of 1×10³/well for 24 hours and divided into 4 groups.The cells were incubated using 100 μl serum-free medium in the blank control group.Oxidative damage models were established by adding 100 μmol/L H₂O₂ (100 μl) in medium in the H₂O₂ group.The cells were cultured with the medium containing 10 μmol/L SFN (100 μl) in the SFN group, and 100 μl H₂O₂at the final concentration of 100 μmol/L was added in the SFN-treated cell medium in the SFN+ H₂O₂ group.The cell vitality in various groups was assayed by using cell counting kit-8 (CCK-8). The apoptosis rate of the cells was detected by Annexin V-FITC/PI double-staining with flow cytometry.

Results

Cultured cells showed a spindle shape with uniform size, abundant cytoplasm, numberous pigmented particles and big nucleolus.The relative cell vitality reduced to (67.00±1.27)% and (80.00±6.25)% in the H₂O₂ group and SFN+ H₂O₂ group in comparison with 100% in the blank control group, and the cell vitality in the SFN+ H₂O₂ group was lower than that in the SFN group but higher than that in the H₂O₂ group (both at P<0.01). The mean apoptosis rate was (11.33±0.77)%, (32.31±1.03)%, (10.44±0.68)% and (17.68±0.21)% in the blank control group, H₂O₂ group, SFN group and SFN+ H₂O₂ group, respectively, showing a significant difference among the groups (F=539.96, P<0.01), and the apoptosis rate in the SFN+ H₂O₂ group was significantly lower than that in the H₂O₂ group but higher than that in the blank control group and SFN group (all at P<0.01).

Conclusions

SFN can improve the antioxidative stress ability of trabecular meshwork cells and alleviate the damage induced by oxidative stress.

Isothiocyanates; Thiocyanates/pharmacology; Cells, cultured; Hydrogen peroxide/toxicity; Oxidative stress/drug effects; Trabecular meshwork cells/drug effects; Apoptosis; Animals, cattle; Sulforaphane