Department of Ophthalmology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, China.
Invest Ophthalmol Vis Sci. 2023 Dec 1;64(15):6. doi: 10.1167/iovs.64.15.6.
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).
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.
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.
We demonstrated HO-1 effects on restoring autophagic flux and delaying cellular senescence under oxidative stress in LECs, which are dependent on TFEB.
氧化应激和细胞衰老都是与年龄相关的白内障的危险因素。血红素加氧酶 1（HO-1）是一种关键的抗氧化酶，与自噬有关。在这里，我们研究了 HO-1、氧化应激和细胞衰老在小鼠晶状体上皮细胞（LEC）中的相互作用。
在人样本中测量 HO-1、p21、LC3 和 p62 的基因表达。在过氧化氢（H2O2）损伤的 LEC 中检查 HO-1 的保护特性。通过 Western blot 和 mRFP-GFP-LC3 测定法检查自噬流。Western blot 和溶酶体追踪染色用于分析溶酶体功能。流式细胞术用于检测细胞内活性氧并分析细胞周期。衰老相关的β-半乳糖苷酶测定用于确定细胞衰老。在 TFEB 敲低细胞中进一步观察 HO-1 和转录因子 EB（TFEB）之间的串扰。通过 Jasper 网站预测 Hmox1 启动子区域中 TFEB 结合位点，并通过染色质免疫沉淀测定法进行验证。
与年龄相关的核白内障患者的 LEC 中 HO-1 基因表达降低，而 p21、LC3 和 p62 的 mRNA 表达水平增加。在 H2O2 诱导的氧化应激下，LEC 表现出自噬流阻断、溶酶体功能障碍和过早衰老的特征。有趣的是，HO-1 显著恢复了受损的自噬流和溶酶体功能，并延缓了细胞衰老。TFEB 基因沉默大大降低了 HO-1 介导的自噬恢复，导致 LEC 无法抵御氧化应激和过早衰老。
我们证明了 HO-1 在氧化应激下对恢复 LEC 中的自噬流和延迟细胞衰老的作用，这依赖于 TFEB。
