Data_Sheet_2_Urolithin A alleviates cell senescence by inhibiting ferroptosis and enhances corneal epithelial wound healing.docx

PurposeTo analyze the therapeutic effect and mechanism of Urolithin A (UA) on delayed corneal epithelial wound healing.
MethodsThe C57BL/6 mice were continuously exposed to hyperosmotic stress (HS) for 7 days followed by the removal of central corneal epithelium to establish a delayed corneal epithelial wound healing model in vivo. In vitro, the human corneal epithelial cell line (HCE-T) was also incubated under HS. UA was administered in vivo and in vitro to study its effects on corneal epithelial cells. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to detect the level of cell senescence. Transcriptome sequencing (RNA-seq) was conducted to elucidate the molecular mechanism underlying the effect of UA on corneal epithelial repair. Additionally, the expression of senescence-related and ferroptosis-related genes and the levels of lipid peroxides (LPO) and malondialdehyde (MDA) were measured.
ResultsHyperosmotic stress (HS) significantly increased the proportion of SA-β-gal staining positive cells in corneal epithelial cells and upregulated the expression of p16 and p21 (p < 0.0001). Topical application of UA decreased the accumulation of senescent cells in corneal epithelial wounds and promoted epithelial wound healing. The results of RNA-seq of HS-induced corneal epithelial cells showed that the ferroptosis pathway was significantly dysregulated. Further investigation revealed that UA decreased the level of oxidative stress in HCE-T cells, including the levels of LPO and MDA (p < 0.05). Inhibition of ferroptosis significantly prevented cellular senescence in HS-induced HCE-T cells.
ConclusionIn this study, UA promoted HS-induced delayed epithelial wound healing by reducing the senescence of corneal epithelial cells through the inhibition of ferroptosis.

研究目的：旨在分析尿石素A（Urolithin A，UA）对角膜上皮创伤延迟愈合的治疗作用及机制。
研究方法：将C57BL/6小鼠持续暴露于高渗应激（hyperosmotic stress，HS）环境7天，随后去除中央角膜上皮，构建体内角膜上皮创伤延迟愈合模型。体外实验中，将人角膜上皮细胞系（human corneal epithelial cell line，HCE-T）置于高渗应激环境中培养。分别在体内及体外给予尿石素A干预，以探究其对角膜上皮细胞的作用。采用衰老相关β-半乳糖苷酶（senescence-associated β-galactosidase，SA-β-gal）染色检测细胞衰老水平；通过转录组测序（RNA-seq）阐明尿石素A调控角膜上皮修复的分子机制。此外，还检测了衰老相关及铁死亡相关基因的表达水平，以及脂质过氧化物（lipid peroxides，LPO）和丙二醛（malondialdehyde，MDA）的含量。
研究结果：高渗应激可显著升高角膜上皮细胞中SA-β-gal染色阳性细胞比例，并上调p16、p21的表达（p < 0.0001）。局部外用尿石素A可减少角膜上皮创伤处衰老细胞的蓄积，并促进上皮创伤愈合。高渗应激诱导的角膜上皮细胞转录组测序结果显示，铁死亡通路存在显著异常调控。进一步研究发现，尿石素A可降低HCE-T细胞的氧化应激水平，包括降低LPO及MDA的含量（p < 0.05）。抑制铁死亡可显著阻断高渗应激诱导的HCE-T细胞衰老。
研究结论：本研究表明，尿石素A可通过抑制铁死亡、减轻角膜上皮细胞衰老，从而促进高渗应激诱导的角膜上皮创伤延迟愈合。