Balanced activation of Nrf-2/ARE mediates the protective effect of sulforaphane on keratoconus in the cell mechanical microenvironment

Abstract

Keratoconus (KC) is a progressive degenerative disease that usually occurs bilaterally and is characterized by corneal thinning and apical protrusion of the cornea. Oxidative stress is an indicator of the accumulation of reactive oxygen species (ROS), and KC keratocytes exhibit increased ROS production compared with that of normal keratocytes. Therefore, oxidative stress in KC keratocytes may play a major role in the development and progression of KC. Here, we investigated the protective effect of sulforaphane (SF) antioxidants using a hydrogel-simulated model of the cell mechanical microenvironment of KC. The stiffness of the KC matrix microenvironment in vitro was 16.70 kPa and the stiffness of the normal matrix microenvironment was 34.88 kPa. Human keratocytes (HKs) were cultured for 24 h before observation or drug treatment with H₂O₂ in the presence or absence of SF. The levels of oxidative stress, nuclear factor E2-related factor 2 (Nrf-2) and antioxidant response element (ARE) were detected. The high-stress state of HKs in the mechanical microenvironment of KC cells compensates for the activation of the Nrf-2/ARE signaling pathway. H₂O₂ leads to increased oxidative stress and decreased levels of antioxidant proteins in KC. In summary, SF can reduce endogenous and exogenous oxidative stress and increase the antioxidant capacity of cells.

Figure 1

Stiffness measurement of the PA hydrogel. (a) A picture of the production of the PA hydrogel. (b) An AFM cantilever attached to a silica sphere. (c) Raw data of the representative force–distance curve (approach part). (d) A representative force–indentation curve converted from (c) by subtracting the measured deflection from the known response of the cantilever. (e) The stiffness of the PA hydrogels. The error bars indicate the SDs; n = 3 per group.

Figure 2

Human keratocytes (HKs) identification. (a) Detection of vimentin in HKs (immunofluorescence, × 200). Bar = 100 μm. DAPI, 4'-6-diamidino-2-phenylindole. (b) Percentage of vimentin-positive cells (%) under different culture conditions. Tukey's post hoc test was used to identify significant differences between samples. The data are presented as the means ± SDs; n = 3 per group. n.s., not significant.

Figure 3

Generation of ROS in HKs. (a) The production of ROS in HKs was detected by DHE staining. Bar = 100 μm. (b) ROS levels in HKs. Tukey's post hoc test was used to identify significant differences between samples. The data are presented as the means ± SDs; n = 3 per group. **P < 0.01, ***P < 0.001, n.s., not significant.

Figure 4

Detection of oxidative stress-related indicators under different culture conditions. (a) MDA content. (b) SOD. (c) NO. (d) ·OH. Tukey's post hoc test was used to identify significant differences between samples. The data are presented as the means ± SDs; n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the 34.88 kPa group. ^#P < 0.05, ^##P < 0.01 compared to the 16.70 kPa group. ⁺P < 0.05, ⁺⁺P < 0.01 compared to the 16.70 kPa-SF group. ^&&P < 0.01, ^&&&P < 0.001 compared to the 16.70 kPa group. ^P < 0.05, ^^^P < 0.001 compared to the 34.88 kPa-H₂O₂ group. ^$P < 0.05, ^$$P < 0.01 compared to the 16.70 kPa-H₂O₂ group. ^@P < 0.05, ^@@P < 0.01, and ^@@@P < 0.001 compared to the 16.70 kPa-H₂O₂-SF group.

Figure 5

Generation of ROS in HKs after H₂O₂ treatment. (a) DHE staining was performed to detect the production of ROS. (b) ROS levels in corneal stromal cells. Tukey's post hoc test was used to identify significant differences between samples. The data are presented as the means ± SDs; n = 3 per group. ***P < 0.001, n.s., not significant.

Figure 6

Expression levels of Nox-2, Nox-4, Nrf-2 and HO-1 in HKs under different experimental conditions. (a-d) The upper panels are protein grayscale images of Nox-2, Nox-4, Nrf-2 and HO-1 in Western blot analysis. The lower panels are the corresponding relative expression levels. The original blots are presented in S Figs. 5-9. Tukey's post hoc test was used to identify significant differences between samples. The data are presented as the means ± SDs; n = 3 per group. ***P < 0.001 compared to the 34.88 kPa group. ^###P < 0.001 compared to the 16.70 kPa group; ⁺⁺⁺P < 0.001 compared to the 16.70 kPa-SF group.

Figure 7

Determination of Nox-2, Nox-4, Nrf-2 and HO-1 mRNA levels under different experimental conditions. (a) SF downregulated Nox-2 and Nox-4 and upregulated Nrf-2 and HO-1 in HKs in the KC mechanical microenvironment. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the 34.88 kPa group. ^##P < 0.01, ^###P < 0.001 compared to the 16.70 kPa group. ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 compared to the 16.70 kPa-SF group. (b) SF downregulated Nox-2 and Nox-4 and upregulated Nrf-2 and HO-1 in HKs in the KC mechanical microenvironment after H₂O₂ treatment. **P < 0.01, ***P < 0.001 compared with the 34.88 kPa-H₂O₂ group. ^#P < 0.05, ^###P < 0.001 compared to the 16.70 kPa-H₂O₂ group. ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 compared to the 16.70 kPa-H₂O₂-SF group. The housekeeping gene used for normalization was β-actin. The fold change in gene expression relative to that of β-actin was calculated as 2^−ΔΔct (mean ± SD, n = 3). Tukey's post hoc test was used to identify significant differences between samples. The 34.88 kPa group, normal corneal mechanics microenvironment group. The 16.70 kPa group, KC mechanical microenvironment group.

Figure 8

Evaluation of Nox-2, Nox-4, Nrf-2 and HO-1 expression in HKs by immunofluorescence after H₂O₂ treatment. (a-d) Representative micrographs of HKs obtained from each group stained with an anti-Nox-2 antibody (red, a), an anti-Nox-4 antibody (green, b), an anti-Nrf-2 antibody (red, c), and an anti-HO-1 antibody (green, d) are shown in the upper panels. The middle panels show the corresponding images of DAPI-stained sections (blue). The lower panels show merged images. Scale bar = 100 μm. DAPI, 4'-6-diamidino-2-phenylindole. (e) The mean fluorescence intensity of Nox-2, Nox-4, Nrf-2 and HO-1 in the experimental groups. The data are presented as the means ± SDs; n = 3 per group. **P < 0.01, ***P < 0.001. The 10⁶ kPa group, included conventional cell culture stiffness groups (the matrix stiffness of the rigid plastic dishes). The 34.88 kPa group, normal corneal mechanics microenvironment group. The 16.70 kPa group, KC mechanical microenvironment group.

Figure 9

Expression levels of Nox-2, Nox-4, Nrf-2 and HO-1 in HKs under different experimental conditions after H₂O₂ treatment. (a-d) Representative immunoblotting showing the protein levels of Nox-2, Nox-4, Nrf-2 and HO-1 in HKs (upper panel) and densitometric analysis of the expression of Nox-2, Nox-4, Nrf-2 and HO-1 relative to the loading control (lower panel). The original blots are presented in S Figs. 10-14. Tukey's post hoc test was used to identify significant differences between samples. The data are presented as the means ± SDs, n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001, vs. the 34.88 kPa-H₂O₂ group. ^#P < 0.05, and ^###P < 0.001, vs. the 16.70 kPa-H₂O₂ group. ⁺P < 0.05, ⁺⁺⁺P < 0.001, vs. the 16.70 kPa-H₂O₂-SF group.