Inflammation and oxidative stress induced by lipid peroxidation metabolite 4-hydroxynonenal in human corneal epithelial cells
Inflammation and oxidative stress induced by lipid peroxidation metabolite 4-hydroxynonenal in human corneal epithelial cells
复制标题
脂质过氧化代谢物4-羟基壬烯醛诱导人角膜上皮细胞炎症和氧化应激
DOI：
10.1007/s00417-020-04647-2
复制
发表时间：
2020-05-22
影响因子：
2.7
通讯作者：
Zhao, Shaozhen
中科院分区：
文献类型：
--
作者：
Liu, Hui;Gambino, Frank, Jr.;Zhao, Shaozhen
目的
氧化应激被广泛认为是干眼症（DED）发病机制中的一个主要因素。4 - 羟基壬烯醛（4 - HNE）是一种众所周知的副产物，常被作为氧化应激诱导的脂质过氧化的指标进行检测，已表明在人类和小鼠角膜干眼症样本中其含量均升高。本研究旨在探讨4 - HNE是否是人类角膜上皮细胞（HCECs）中氧化应激的原因，并探索其产生影响的潜在机制。
方法
SV40永生化的HCECs在含有1%青霉素/链霉素和10%胎牛血清的最低必需培养基（MEM）中培养。将HCECs暴露于含或不含4 - HNE的培养基中，并在4小时和24小时收集细胞培养上清液。按照制造商的说明，使用2',7' - 二氯荧光素二乙酸酯（DCFDA）检测试剂盒测量细胞活性氧物质（ROS）。通过蛋白质印迹法分析抗氧化酶铜/锌超氧化物歧化酶1（SOD1）和NAD（P）H醌脱氢酶1（NQO1）的蛋白质水平。使用NF - κB p65总简易步骤酶联免疫吸附测定试剂盒和蛋白质组分析仪人类细胞因子阵列试剂盒测量NF - κB的激活以及白细胞介素 - 6（IL - 6）和白细胞介素 - 8（IL - 8）的表达。通过乳酸脱氢酶（LDH）细胞毒性测定评估细胞活力。
结果
用4 - HNE处理降低了HCECs的细胞活力。与ROS产生水平相对应的条带强度表明，用4 - HNE处理后ROS的产生显著增加。4 - HNE降低了HCECs中SOD1的水平并上调了NQO1的表达。用4 - HNE处理后，观察到NF - κB的激活以及促炎细胞因子IL - 6和IL - 8的产生显著增加。暴露于N - 乙酰半胱氨酸（NAC），一种抗氧化剂和ROS清除剂，可拮抗4 - HNE对HCECs的氧化作用。
结论
4 - HNE通过增加ROS的产生水平和改变抗氧化酶水平的表达，在体外降低HCECs的细胞活力，从而诱导角膜上皮细胞的氧化应激。本研究展示了4 - HNE产生有害影响的一种潜在途径，并为干眼症的治疗提供了一个新的治疗靶点。
PurposeOxidative stress is widely known to be a major contributor in the pathogenesis of dry eye disease (DED). 4-Hydroxynonenal (4-HNE), a well-known byproduct frequently measured as an indicator of oxidative stress-induced lipid peroxidation, has been shown to be elevated in both human and murine corneal DED samples. This study aims to investigate if 4-HNE is responsible for the oxidative stress in human corneal epithelial cells (HCECs) and explores the underlying mechanism by which it confers its effects.MethodsSV40-immortalized HCECs were cultured in minimum essential media (MEM) with 1% penicillin/streptomycin and 10% fetal bovine serum. HCECs were exposed to media with or without 4-HNE and cell culture supernatants were collected at 4 and 24 h. Cellular reactive oxygen species (ROS) measurement was performed using a 2′,7′-dichlorofluorescein diacetate (DCFDA) assay kit according to the manufacturer’s instructions. Protein levels of antioxidant enzymes copper/zinc superoxide dismutase 1 (SOD1) and NAD(P)H quinone dehydrogenase 1 (NQO1) were analyzed by Western blot. NF-κB activation and expression of IL-6 and IL-8 were measured using an NF-κB p65 Total SimpleStep ELISA Kit and Proteome Profiler Human Cytokine Array Kit. Cell viability was evaluated by LDH cytotoxicity assay.ResultsTreatment with 4-HNE decreased cell viability of HCECs. Band intensities corresponding to levels of ROS production showed a significant increase in ROS generation after treatment with 4-HNE. 4-HNE decreased SOD1 levels and upregulated NQO1 expression in HCECs. A significant increase in activation of NF-κB and production of pro-inflammatory cytokines IL-6 and IL-8 was observed after treatment with 4-HNE. Exposure to N-acetylcysteine (NAC), an antioxidant and ROS scavenger, antagonized the oxidative effects of 4-HNE on HCECs.Conclusion4-HNE induces oxidative stress in corneal epithelial cells by increasing levels of ROS generation and modifying the expression of antioxidant enzyme levels, decreasing cell viability of HCECs in vitro.This study demonstrates a potential pathway by which 4-HNE functions to confer its detrimental effects and provides a new therapeutic target for the treatment of DED.