Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
Invest Ophthalmol Vis Sci. 2012 Jul 27;53(8):5033-43. doi: 10.1167/iovs.12-9729.
We recently reported that SERPINA3K (SA3K), a member of the serine proteinase inhibitor (SERPIN) family, has antiangiogenic and anti-inflammatory activities. Here we investigated the antioxidant effects of SA3K in the corneal epithelium and the mechanism underlying its action.
We established the oxidative stress models induced by hydrogen peroxide (H₂O₂) in cultured human corneal epithelial (HCE) cells and in rat corneal epithelium in vivo. Cell viability, flow cytometry, and TUNEL analysis were conducted to detect viable cells and cell death; reactive oxygen species (ROS) and 3-Nitrotyrosine fluorescent assay was applied to measure ROS levels. Activity assay, immunostaining, Western blot, and quantitative RT-PCR were performed to analyze the factors of the ROS generation/degradation system and pathway.
SA3K protected the HCE cells from H₂O₂-induced oxidative stress in a dose- and time-dependent manner. SA3K also significantly reduced the production of ROS. Regarding the mechanism underlying these effects, SA3K downregulated ROS generation by inhibiting NOX4 and upregulated ROS degradation by increasing the activity of superoxide dismutases and catalase. Furthermore, H₂O₂ induced activation of the Kelch-like ECH-associated protein 1 (KEAP1)/NF-E2-related factor-2 (NRF2) pathway, while SA3K inhibited H₂O₂-induced activation of KEAP1 and NRF2 and their downstream factors, including NAD(P)H quinone oxidoreductase and glutathione S-transferase. In the H₂O₂-induced rat corneal epithelium, SA3K alleviated the oxidative stress and downregulated NOX4 and NRF2.
Collectively, SA3K protects against oxidative stress by targeting the ROS generation/degradation system and modulating the KEAP1-NRF2 signaling pathway.
我们最近报道，丝氨酸蛋白酶抑制剂（SERPIN）家族成员 SERPINA3K（SA3K）具有抗血管生成和抗炎作用。在此，我们研究了 SA3K 在角膜上皮中的抗氧化作用及其作用机制。
我们建立了过氧化氢（H₂O₂）诱导的体外培养人角膜上皮（HCE）细胞和体内大鼠角膜上皮氧化应激模型。通过细胞活力、流式细胞术和 TUNEL 分析检测存活细胞和细胞死亡；通过活性氧（ROS）和 3-硝基酪氨酸荧光测定法检测 ROS 水平。通过活性测定、免疫染色、Western blot 和定量 RT-PCR 分析 ROS 产生/降解系统和途径的相关因子。
SA3K 以剂量和时间依赖的方式保护 HCE 细胞免受 H₂O₂诱导的氧化应激。SA3K 还显著降低了 ROS 的产生。关于这些作用的机制，SA3K 通过抑制 NOX4 下调 ROS 生成，通过增加超氧化物歧化酶和过氧化氢酶的活性来上调 ROS 降解。此外，H₂O₂诱导 Kelch-like ECH-associated protein 1（KEAP1）/NF-E2-related factor-2（NRF2）通路的激活，而 SA3K 抑制 H₂O₂诱导的 KEAP1 和 NRF2 及其下游因子（包括 NAD(P)H 醌氧化还原酶和谷胱甘肽 S-转移酶）的激活。在 H₂O₂诱导的大鼠角膜上皮中，SA3K 减轻了氧化应激并下调了 NOX4 和 NRF2。
总之，SA3K 通过靶向 ROS 产生/降解系统和调节 KEAP1-NRF2 信号通路来抵抗氧化应激。
