Investigative ophthalmology & visual science · 2023
HO-1-Mediated Autophagic Restoration Protects Lens Epithelial Cells Against Oxidative Stress and Cellular Senescence.
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Wang Lijun, Lou Wei, Zhang Yao, Chen Ziang, Huang Yang, Jin Haiying
Аннотация
PURPOSE: Oxidative stress and cellular senescence are risk factors for age-related cataract. Heme oxygenase 1 (HO-1) is a critical antioxidant enzyme and related to autophagy. Here, we investigate the crosstalk among HO-1, oxidative stress, and cellular senescence in mouse lens epithelial cells (LECs).
METHODS: The gene expression of HO-1, p21, LC3, and p62 was measured in human samples. The protective properties of HO-1 were examined in hydrogen peroxide (H2O2)-damaged LECs. Autophagic flux was examined by Western blot and mRFP-GFP-LC3 assay. Western blotting and lysotracker staining were used to analyze lysosomal function. Flow cytometry was used to detect intracellular reactive oxygen species and analyze cell cycle. Senescence-associated β-galactosidase assay was used to determine cellular senescence. The crosstalk between HO-1 and transcription factor EB (TFEB) was further observed in TFEB-knockdown cells. The TFEB binding site in the promoter region of Hmox1 was predicted by the Jasper website and was confirmed by chromatin immunoprecipitation assay.
RESULTS: HO-1 gene expression decreased in LECs of patients with age-related nuclear cataract, whereas mRNA expression levels of p21, LC3, and p62 increased. Upon H2O2-induced oxidative stress, LECs showed the characteristics of autophagic flux blockade, lysosomal dysfunction, and premature senescence. Interestingly, HO-1 significantly restored the impaired autophagic flux and lysosomal function and delayed cellular senescence. TFEB gene silencing greatly reduced the HO-1-mediated autophagic restoration, leading to a failure to prevent LECs from oxidative stress and premature senescence.
CONCLUSIONS: We demonstrated HO-1 effects on restoring autophagic flux and delaying cellular senescence under oxidative stress in LECs, which are dependent on TFEB.
METHODS: The gene expression of HO-1, p21, LC3, and p62 was measured in human samples. The protective properties of HO-1 were examined in hydrogen peroxide (H2O2)-damaged LECs. Autophagic flux was examined by Western blot and mRFP-GFP-LC3 assay. Western blotting and lysotracker staining were used to analyze lysosomal function. Flow cytometry was used to detect intracellular reactive oxygen species and analyze cell cycle. Senescence-associated β-galactosidase assay was used to determine cellular senescence. The crosstalk between HO-1 and transcription factor EB (TFEB) was further observed in TFEB-knockdown cells. The TFEB binding site in the promoter region of Hmox1 was predicted by the Jasper website and was confirmed by chromatin immunoprecipitation assay.
RESULTS: HO-1 gene expression decreased in LECs of patients with age-related nuclear cataract, whereas mRNA expression levels of p21, LC3, and p62 increased. Upon H2O2-induced oxidative stress, LECs showed the characteristics of autophagic flux blockade, lysosomal dysfunction, and premature senescence. Interestingly, HO-1 significantly restored the impaired autophagic flux and lysosomal function and delayed cellular senescence. TFEB gene silencing greatly reduced the HO-1-mediated autophagic restoration, leading to a failure to prevent LECs from oxidative stress and premature senescence.
CONCLUSIONS: We demonstrated HO-1 effects on restoring autophagic flux and delaying cellular senescence under oxidative stress in LECs, which are dependent on TFEB.