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.
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).
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.