目的 探讨黄腐酚调节核因子红系2相关因子2（Nrf2）/核因子-κB（NF-κB）/哺乳动物雷帕霉素靶蛋白（mTOR）/蛋白激酶B（Akt）信号，抑制铁死亡和氧化应激，缓解干眼症小鼠模型损伤的分子机制。方法 体外研究采用人角膜上皮细胞系HCECs细胞建立高渗透压应激（HOS）模型，并将细胞分组为对照组、模型组、黄腐酚50 μmol/L组。CCK-8法测定HCECs细胞存活能力。FITC-AnnexingV/PI双标记流式细胞术测定细胞凋亡率。DCF-DA荧光染料联合激光共聚焦显微镜和流式细胞术测定细胞内活性氧自由基水平。ELISA测定细胞内铁死亡相关指标丙二醛（MDA）、还原型谷胱甘肽（GSH）和亚铁离子（Fe^2＋）水平。Western blotting法测定各分组HCECs细胞中凋亡相关指标[B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、裂解型-半胱氨酸天冬氨酸蛋白酶-3（cleaved-Caspase-3）、Caspase-3]、铁死亡相关指标[谷胱甘肽过氧化物酶4（GPX4）、溶质载体家族7A11（SLC7A11）、血红素加氧酶-1（HO-1）]。在体动物模型研究，建立干眼症小鼠模型，将32只雄性Balb/c小鼠随机分为对照组、模型组、黄腐酚组、黄腐酚＋ML385组。苏木精–伊红（HE）染色和TUNEL染色小鼠眼角膜组织。Western blotting法测定各组小鼠眼角膜组织中Nrf2、p-p65、p65、p-mTOR、mTOR、p-Akt、Akt的表达水平。结果 与模型组相比，黄腐酚50 μmol/L组细胞存活能力显著增强（P＜0.05）。与模型组相比，黄腐酚组细胞凋亡率显著降低；Bax、cleaved-Caspase-3蛋白相对表达水平显著降低，Bcl-2蛋白相对表达水平显著升高；GPX4、SLC7A11、HO-1蛋白相对表达水平显著升高；MDA和Fe^2＋水平显著降低，GSH水平升高；DCF-DA相对荧光强度值和DCF-DA染色阳性细胞数值显著降低（P＜0.05）。而与黄腐酚组相比，黄腐酚＋ML385组部分逆转了上述检测指标（P＜0.05）。HE染色和组织病理学分析结果显示，与模型组相比，黄腐酚组小鼠角膜总厚度、角膜上皮厚度变厚，角膜上皮细胞层数增加，细胞间隙面积/视野面积的比值降低，每视野下TUNEL阳性细胞数减少（P＜0.05）；黄腐酚组Nrf2蛋白相对表达水平显著增强，p-p65/p65、p-mTOR/mTOR、p-Akt/Akt蛋白相对表达水平显著降低（P＜0.05）。与黄腐酚组相比，黄腐酚＋ML385组部分逆转了上述检测指标（P＜0.05）。结论 黄腐酚调节Nrf2/NF-κB/mTOR/Akt信号降低细胞铁死亡抑制氧化应激，可用于保护干眼症小鼠模型的眼部损伤。

Objective To investigate the molecular mechanisms of xanthohumol in regulating Nrf2/NF-κB/mTOR/Akt signaling pathway to inhibit ferroptosis and oxidative stress, and relieve dry eye disease mice model injury. Methods In vitro studies used human corneal epithelial cell line HCECs cells to establish a hyperosmotic stress (HOS) model, and the cells were divided into control group, model group, and xanthohumol 50 μmol/L group. The viability of HCECs cells was determined by the CCK-8 method. The apoptosis rate of cells was determined by FITC-AnnexingV/PI double-labeled flow cytometry. The levels of reactive oxygen species in cells were determined by DCF-DA fluorescent dye combined with laser confocal microscopy and flow cytometry. ELISA was used to determine the levels of MDA, GSH, and Fe²⁺, which are related to ferroptosis in cells. The apoptosis-related indicators (Bcl-2, Bax, cleaved-Caspase-3, Caspase-3), and ferroptosis-related indicators (GPX4, SLC7A11, HO-1) in HCECs cells of each group were determined by Western blotting. In vivo animal model studies were conducted to establish a mice model of dry eye syndrome. Thirty-two male Balb/c mice were randomly divided into control group, model group, xanthohumol group, and xanthohumol + ML385 group. HE staining and TUNEL staining of mouse corneal tissues. The expression levels of Nrf2, p-p65, p65, p-mTOR, mTOR, p-Akt, and Akt in the corneal tissues of mice in each group were determined by Western blotting. Results Compared with the model group, the cell survival ability of the xanthohumol group was significantly enhanced (P < 0.05). Compared with the model group, the apoptosis rate of cells in the xanthohumol group was significantly reduced, the relative expression levels of Bax and cleaved-Caspase3 proteins were significantly decreased, while the relative expression level of Bcl-2 protein was significantly increased, the relative expression levels of GPX4, SLC7A11, and HO-1 proteins were significantly increased. The levels of MDA and Fe²⁺ decreased significantly, while the level of GSH increased. The relative fluorescence intensity value of DCF-DA and the number of positive cells stained with DCF-DA were significantly decreased (P < 0.05). Compared with xanthohumol group, the xanthohumol +ML385 group partially reversed the above detection indicators (P < 0.05). The results of HE staining and histopathological analysis showed that compared with the model group, the total corneal thickness and corneal epithelial thickness of mice in the xanthohumol group were increased, the number of corneal epithelial cell layers were increased, the ratio of intercellular space area to field area were decreased, and the number of TUNEL-positive cells per field were decreased (P < 0.05). The relative expression level of Nrf2 protein in the xanthohumol group was significantly enhanced, while the relative expression levels of p-P65 /p65, p-mTOR/mTOR, and p-Akt/Akt proteins were significantly decreased (P < 0.05). Compared with the xanthohumol group, the xanthohumol +ML385 group partially reversed the above detection indicators (P < 0.05). Conclusion Xanthohumol regulated the Nrf2/NF-κB/mTOR/Akt signaling pathway to reduce ferroptosis and inhibit oxidative stress. It could be used to protect the ocular damage in a mouse model of dry eye disease.

R285.5

新疆医科大学第五临床医学院（第五附属医院）国自然启航项目（XYDWFY-GQH-2024-14）