Selenoprotein P controls oxidative stress in cornea

Abstract

The ocular surface is always attacked by oxidative stress, and cornea epithelial cells are supposed to have their own recovery system against oxidative stress. Therefore we hypothesized that tears supply key molecules for preventing oxidative stress in cornea. The potential target key molecule we focused is selenoprotein P (SeP). SeP is a carrier of selenium, which is an essential trace element for many animals, for oxidative stress metabolism in the organism, and was extremely expressed in lacrimal gland. An experiment was performed with SeP eye drops in a rat dry eye model, prepared by removing the lacrimal glands. The anticipated improvement in corneal dry eye index and the suppression of oxidative stress markers were observed in SeP eye drop group. Furthermore, the concentration of SeP was significantly higher in dry eye patients compared with normal volunteers. Collectively, we concluded that tear SeP is a key molecule to protect the ocular surface cells against environmental oxidative stress.

Figure 1. Preparation of dry eye rat model.

a: Measurement of tear volume secreted from normal eye or lacrimal gland-removed eye of rats. The vertical axis shows tear volume measured by cotton thread. Results are expressed as mean ± S.D. Dunnett's test was used to determine the significance of differences. * and **indicates a significant difference from the result in normal side, P<0.05 and <0.01, respectively. b: Corneal fluorescein staining in normal rats and rats with dry eye. The vertical axis shows score of the fluorescein-stained area. Dunnett's test was used to determine the significance of differences. ** indicates a significant difference from the result at 0 week, P<0.01 (n = 10). c: Photo of cornea stained by fluorescein after 4 weeks treatment. d: Increase of marker during oxidative stress. Ratio of 8-OHdG content in the cornea between the dry eye and normal eye. Results are expressed as mean ± S.D. Dunnett's test was used to determine the significance of differences. * and *** indicates a significant difference from the result in 1-week removal, P<0.05 and <0.005, respectively. e, f: Oxidative stress markers in cornea of normal rats and rats with dry eye. Contents of 8-OHdG (e) and HEL (f) in a dry eye and normal corneas. t-test was used to determine the significance of differences. * indicates a significant difference from the result in normal side, P<0.05. Results are expressed as the mean ± S.D. (n = 10).

Figure 2. Effects of SeP eye drops on the cornea of a dry eye rat model.

a: Photo of cornea stained by fluorescein after 3 weeks of SeP eye drop administration. b: Fluorescein score of cornea in normal rats (Normal), in dry eye rats treated with PBS (PBS, closed bar). Results are expressed as mean ± S.D. Dunnett's test was used to determine the significance of differences. * and *** indicates a significant difference from the result in PBS treatment, P<0.05 and <0.005, respectively. c: Fluorescein score of cornea in dry eye rat treated with PBS or SeP. d: Ratio of 8-OHdG content in cornea of dry eye between SeP and PBS treatment. Results are expressed as mean ± S.D. T-test was used to determine the significance of differences. *, **, and *** indicates a significant difference from the result in PBS treatment, P<0.05, <0.01, and <0.005, respectively (n = 10).

Figure 3. Effect of selenium deficiency on CEPI cells.

Selenium deficiency-induced GPx activity and HEL production in CEPI cells. Open triangle shows the ratio of GPx activity and closed cycle shows ratio of HEL content. Data are mean ± S.D. (n = 6).

Figure 4. SeP addition suppresses oxidative stress in selenium-deficient CEPI cells.

a: Recovery of GPx activity by addition of SeP. Fifty µg/mL SeP was added to culture medium and incubated for 0, 1, 2, or 3 days. ** indicates a significant difference from the result in Se-, P<0.01 (n = 8). b: Recovery of GPx activity by addition of SeP (5 or 50 µg /mL) and incubated for 3 days. **P<0.01 and ***P<0.005, respectively (n = 6). c, d: Suppression of production of 8-OHdG. (c, n = 8) and HEL (d, n = 5) in CEPI cells. SeP (0.5, 5 or 50 µg /mL) was added to medium and cells were incubated for 3 days. *P<0.05. All results are expressed as mean ± S.D. Dunnett's test was used to determine the significance of differences. Se+: GPx activity of CEPI cells cultivated in normal medium. Se-: GPx activity of CEPI cells cultivated in selenium-depleted medium.

Figure 5. SeP suppresses lipid peroxidation in CEPI cells.

The vertical axis shows peroxidation rate calculated from fluorescence intensity. Results are expressed as the mean ± S.D. (n = 4). Dunnett's test was used to determine the significance of differences. ** and *** indicate a significant difference from the result of 0 µg/mL SeP addition, P<0.01 and <0.005, respectively.

Figure 6. SeP in tears.

a, b: Concentration of SeP in human tears. Fluorescein (a) and Rose Bengal (b) Scores are indices of dry eye diagnosis. Dry eye is indicated with fluorescein scores of ≥1, or when the Rose Bengal Score is ≥3. c: SeP expression in rat lacrimal glands. The vertical axis shows expression level of SeP in each tissue compared with liver. The expression level in the liver defined 1. All results are expressed as mean ± S.D.