HIF-1α通过调控YAP活性参与干眼症炎症反应

HIF-1α Participates in the Inflammatory Response of Dry Eye Disease by Regulating YAP Activity

摘要: 目的：探讨HIF-1α通过调控yes-相关蛋白(YAP)活性在体外高渗透压诱导的人永生化角膜上皮细胞(HCECs)干眼模型中干预炎症反应的作用及机制。方法：高渗溶液刺激人永生化角膜上皮细胞建立细胞干眼模型，使用CCK-8检测法筛选HIF-1α抑制剂YC-1及YAP抑制剂维替泊芬(verteporfin, VP)的药物安全使用浓度。应用实时荧光定量聚合酶链反应(qRT-PCR)和蛋白印迹法(Western Blotting)检测各组HCECs中缺氧诱导因子-1α (HIF-1α)、YAP、白细胞介素-1β (IL-1β)、肿瘤坏死因子-α (TNF-α)的mRNA表达水平及蛋白的表达水平。结果：YC-1浓度 ≤ 20 μM和VP浓度 ≤ 0.5 μM时，HCECs存活率 ≥ 80%；与正常组相比，高渗应激组HCECs中HIF-1α、YAP、IL-1β、TNF-α的mRNA和蛋白的表达均明显升高；YC-1组HIF-1α、YAP、IL-1β、TNF-α的mRNA和蛋白的表达均较高渗应激组降低；VP和HIF-1α mRNA和蛋白的表达较高渗组无明显差异，YAP、IL-1β、TNF-α和蛋白的表达均明显降低。结论：HIF-1α可能通过调控YAP的活性参与高渗应激诱导的HCECs干眼模型的炎症反应。

Abstract: Objective: To investigate the role and underlying mechanism of HIF-1α in modulating the inflammatory response through regulation of yes-associated protein (YAP) activity in an in vitro dry eye model established in human immortalized corneal epithelial cells (HCECs) under hyperosmotic stress. Methods: An in vitro dry eye model was established by exposing human immortalized corneal epithelial cells to hyperosmotic medium. The Cell Counting Kit-8 (CCK-8) assay was used to determine the safe working concentrations of the HIF-1α inhibitor YC-1 and the YAP inhibitor verteporfin (VP). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to measure the mRNA and protein expression levels of hypoxia-inducible factor-1α (HIF-1α), YAP, interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in HCECs from each group. Results: HCECs viability remained ≥80% when the concentration of YC-1 was ≤20 μM and that of VP was ≤0.5 μM. Compared with the normal group, the hyperosmotic stress group showed significantly increased mRNA and protein expression levels of HIF-1α, YAP, IL-1β, and TNF-α. In the YC-1 group, the mRNA and protein expression levels of HIF-1α, YAP, IL-1β, and TNF-α were all reduced compared with those in the hyperosmotic stress group. In the VP group, no significant difference was observed in HIF-1α mRNA or protein expression compared with the hyperosmotic stress group, whereas the mRNA and protein expression levels of YAP, IL-1β, and TNF-α were significantly decreased. Conclusion: HIF-1α may participate in the inflammatory response in a hyperosmotic stress-induced HCECs dry eye model through regulation of YAP activity.