Paired Box Gene 6 Regulates Heme Oxygenase-1 Expression and Mitigates Hydrogen Peroxide-Induced Oxidative Stress in Lens Epithelial Cells

Abstract

Purpose

This study aimed to investigate the regulation of heme oxygenase-1 (HO-1) by paired box gene 6 (Pax6) and their roles in hydrogen peroxide (H₂O₂)-induced oxidative stress and apoptosis in lens epithelial cells (LECs) (SRA01/04, HLE-B3).

Methods

Lens anterior capsule membranes of mice of different ages were obtained to compare differences in the expression of Pax6 and HO-1 using Western blotting. Pax6-overexpressing plasmid and small interfering RNA were designed to overexpress and silence Pax6, respectively. Cobalt protoporphyrin (CoPP) was used to promote the expression of HO-1. Oxidative damage in LECs was induced by treatment with H₂O₂ (400 µM) for 24 h. Cell viability was measured using the Cell Counting Kit-8 assay. Intracellular reactive oxygen species (ROS) were detected using flow cytometry and immunofluorescence. Superoxide dismutase (SOD) level was measured using SOD Assay Kit and apoptotic cells were quantified using annexin V-fluorescein isothiocyanate/propidium iodide staining.

Results

Pax6 and HO-1 expression levels showed an age-dependent decrease in LECs of mouse. Overexpressing Pax6 upregulated HO-1 expression level. Silencing Pax6 downregulated the HO-1 expression level, resulting in increased generation of ROS, reduced SOD activity, decreased cell viability, and increased apoptotic cells of LECs under H₂O₂-induced oxidative stress. Overexpressing Pax6 and CoPP both mitigates H₂O₂-induced oxidative stress by increasing the expression of HO-1 of LECs.

Conclusion

Pax6 and HO-1 expression levels showed an age-dependent decrease in LECs in mouse anterior capsules. Pax6 could regulate the expression of HO-1 in LECs. The decrease of Pax6 weakened the antioxidant ability of LECs under H₂O₂-induced oxidative stress by downregulating HO-1, which may be a potential mechanism for the formation of age-related cataract.

Keywords:

• Age-related cataract
• lens epithelial cells (SRA01/04 HLE-B3)
• paired box gene 6
• heme oxygenase-1
• oxidative stress

Ethical approval

All procedures involving mice were performed in accordance with the guidelines of the Animal Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University (Guangzhou, China).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data is available within the article or in the supplementary materials.

Additional information

Funding

This work was supported by the Guangdong Natural Science Foundation [Grant number 2017A030313770].