The role of HMOX1-mediated ferroptosis in blue light-induced damage to retinal pigment epithelium

Abstract

Currently, blue light irradiation is frequently encountered in daily life and is widely considered a high-risk factor for retinal damage. In particular, blue light-induced dysfunction and death of the retinal pigment epithelium (RPE) may ultimately contribute to irreversible vision impairment and even blindness. However, the underlying pathogenic mechanism and pathogenically targeted protection against blue light-induced RPE degeneration remain unclear. In this study, through sophisticated biochemical evaluation and high-throughput sequencing, the predominant pathological process during blue light-induced RPE degeneration was confirmed to be HMOX1-mediated RPE ferroptosis, which may be involved in the Nrf2-SLC7A11-HMOX1 hierarchy. Upon further knockdown of HMOX1 with si-HMOX1 or the HMOX1 inhibitor zinc protoporphyrin (ZnPP), specific inhibition of HMOX1 overexpression significantly suppressed RPE ferroptosis. In mice, treatment with ZnPP effectively rescued RPE degeneration and visual function. These results highlighted that HMOX1-mediated ferroptosis might be a potential target for protection against blue light-induced damage to RPE cells.

Keywords: Blue light; Ferroptosis; HMOX1; Retinal pigment epithelium.

Fig. 1

Ferroptosis played an important role in blue light-induced damage of ARPE-19 cells. Blue light (BL) or ferroptosis inducer Erastin-treated ARPE-19 cells with or without ferroptosis inhibitor DFO exposure for 24 h, (A) cell survival and death were detected through live/dead staining. PI for dead cells and Calcein AM for live cells. Scale bar: 100 μm. DMSO treatment alone was used as a control. (B) Quantitative results of the cell viability for the statistical analysis by counting the live and dead cells using ImageJ software. n = 3, one-way ANOVA with Tukey’s post hoc test, **p < 0.01, data presented as mean ± SD. (C) CCK-8 analysis of cell viability by measuring the optical density of each well at 450 nm (O.D./450 nm) in each group. n = 3, one-way ANOVA with Tukey’s post hoc test, **p < 0.01, data presented as mean ± SD. (D) Protein expression level of the ferroptosis markers, GPX4 and ACSL4, and (E) their relative protein quantitation. n = 3, one-way ANOVA with Tukey’s post hoc test, **p < 0.01, data presented as mean ± SD. Original blots are presented in Supplementary Fig. 5. (F) KEGG enrichment analysis^– identified the most significantly influenced pathways in response to blue light treatment. Copyright permission of KEGG pathway maps has been obtained.

Fig. 2

Ferrous ion-catalysed oxidation reaction was involved in blue light-induced ferroptosis in ARPE-19 cells. (A) FerroOrange staining and (B) the relative fluorescence intensity of ferrous ions. Scale bar: 50 μm. n = 3, one-way ANOVA with Tukey’s post hoc test, **p < 0.01, data presented as mean ± SD. (C) CM-H2DCFDA staining and (D) the relative quantitation of intracellular ROS levels in ARPE-19 cells. Scale bar: 50 μm. n = 3, one-way ANOVA with Tukey’s post hoc test, **p < 0.01, data presented as mean ± SD. (E) BODIPY staining of LOS generation in ARPE-19 cells and (F) ratio of oxidative BODIPY. Scale bar: 50 μm. n = 3, one-way ANOVA with Tukey’s post hoc test, **p < 0.01, data presented as mean ± SD.

Fig. 3

HMOX1 upregulation contributed to ferroptosis in ARPE-19 cells. (A) The number of differentially expressed genes (upregulated in red and downregulated in green) was presented in a volcano plot from RNA-seq analysis. (B) Differentially expressed genes closely associated with ferroptosis in blue light-treated ARPE-19 cells were exhibited in heatmap. (C) Western-blot assay, and (D) relative protein quantitation of HMOX1 and SLC7A11 after treatment with blue light. n = 3, two-tailed Student’s t-test, **p < 0.01, data presented as mean ± SD. Original blots are presented in Supplementary Fig. 6. (E) Western-blot assay, and (F) relative protein quantitation of the upstream transcription factors, AP-1, NF-κB, and Nrf2. n = 3, two-tailed Student’s t-test, **p < 0.01, not significant (N.S.) > 0.05, data presented as mean ± SD. Original blots are presented in Supplementary Fig. 7.

Fig. 4

Inhibition of HMOX1 upregulation protected ARPE-19 cells from ferroptosis in vitro. (A) Cell survival and death were detected through live/dead staining, and (B) quantitation of cell viability. Scale bar: 100 μm, n = 3, one-way ANOVA with Tukey’s post hoc test, **p < 0.01, not significant (N.S.) > 0.05, data presented as mean ± SD. (C) FerroOrange staining and (D) relative fluorescence intensity of ferrous ions. Scale bar: 50 μm. n = 3, one-way ANOVA with Tukey’s post hoc test, **p < 0.01, not significant (N.S.) > 0.05, data presented as mean ± SD. (E) CM-H2DCFDA staining and (F) relative quantitation of intracellular ROS level in ARPE-19 cells. Scale bar: 50 μm. n = 3, one-way ANOVA with Tukey’s post hoc test, **p < 0.01, not significant (N.S.) > 0.05, data presented as mean ± SD. (G) BODIPY staining of LOS generation in ARPE-19 cells and (H) ratio of oxidative BODIPY. Scale bar: 50 μm. n = 3, one-way ANOVA with Tukey’s post hoc test, **p < 0.01, not significant (N.S.) > 0.05, data presented as mean ± SD.

Fig. 5

Inhibition of HMOX1 upregulation prevented retinal damage caused by blue light exposure in vivo. (A) H&E staining showed pigmentation and morphological changes in the RPE pretreated with the HMOX1 inhibitor ZnPP, followed by blue light treatment. Scale bar: 25 μm. Black arrows: RPE cells. ONL: outer nuclear layer, INL: inner nuclear layer. (B) Immunofluorescent staining and (C) relative fluorescence intensity of RPE tight junction biomarker ZO-1. Scale bar: 25 μm. n = 5, one-way ANOVA with Tukey’s post hoc test, **p < 0.01, data presented as mean ± SD. (D) The visual functions of mice in the control, blue light and ZnPP groups were detected through ERG. (E) ERG a- and b-wave amplitude values were measured. n = 5, one-way ANOVA with Tukey’s post hoc test, **p < 0.01, data presented as mean ± SD. (F) After mice with different treatment for 14 days, immunofluorescence staining of typical photoreceptor biomarkers, Arrestin in mouse retina was performed. Scale bar: 100 μm. Hoechst (blue): nuclei. Arrestin: red. ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer.