金雀异黄素通过ROS-NLRP3-IL-1β途径保护角膜上皮细胞免受高渗刺激诱导的损伤  

Genistein Protects Corneal Epithelial Cells from Hyperosmotic Stimuli Induced by ROS-NLRP3-IL-1β Pathway

作　　者：周洋[1] 陈婷妍 美丽巴努·玉素甫[1] Zhou Yang;Chen Tingyan;Meilibanu·Yusufu.(Ophthalmology Department,Fifth Affiliated Hospital of Xinjiang Medical University,Xinjiang 830011,China)

机构地区：[1]新疆医科大学第五附属医院眼科,乌鲁木齐830011 [2]成都华厦眼科医院小儿斜弱视科,610041

出　　处：《医学研究杂志》2020年第4期80-86,共7页Journal of Medical Research

基　　金：新疆维吾尔自治区自然科学基金资助项目(2019D01C271)。

摘　　要：目的高渗应激(HS)诱导的ROS-NLRP3-IL-1β信号通路激活被认为是干眼发病机制中上皮细胞炎症的关键步骤。本研究旨在研究金雀异黄素是否可以通过调节这一通路来保护人角膜上皮细胞抑制HS诱导的炎症发生。方法将人角膜上皮细胞(iHCEC和priHCEC)在各渗透压培养基(312、350、400、450mOsm/L)和50μmol/L金雀异黄素中按分组进行不同处理培养。采用CCK-8检测各处理iHCEC和priHCEC的活性。使用小干扰RNA(siRNA)来抑制iHCEC中NRF2的表达。通过RT-PCR检测各处理后iHCEC中NLRP3炎性体相关基因表达水平。ELISA检测各处理后iHCEC和priHCEC中IL-1β分泌水平。通过免疫荧光检测分析各处理后iHCEC中氧化应激标志物,相关试剂盒来检测不同处理iHCEC的酶活性。Western blot法检测各处理后iHCEC中NRF2的蛋白表达情况。结果金雀异黄素可通过抑制ROS-NLRP3-IL-1β信号通路保护人角膜上皮细胞免受HS诱导的损伤。金雀异黄素显著抑制HS诱导的iHCEC中NLRP3炎性体相关基因的表达和IL-1β的分泌。金雀异黄素可显著减弱HS诱导的氧化应激,表现为金雀异黄素预处理后iHCEC内ROS生成量减少和8-OHdG阳性细胞数目减少。金雀异黄素诱导NRF2表达,从而触发几种抗氧化酶的表达。结论金雀异黄素可以抑制HS诱导的人角膜上皮细胞炎症的发生,其可能具有在较早阶段预防和减轻与干眼相关的角膜炎症的作用。Objective Hyperosmotic stress(HS)induced activation of ROS-NLRP3-IL-1βsignaling pathway is considered to be a critical stage in epithelial inflammation in the pathogenesis of dry eye.This study was designed to investigate whether genistein can protect human corneal epithelial cells against HS-induced inflammation by regulating this pathway.Methods Human corneal epithelial cells(iHCEC and priHCEC)were cultured in different osmotic medium(312,350,400,450mOsm/L)and 50μmol/Lgenistein according to different groups.cHC-8 was used to detect each treatment of iHCEC and PriHCEC activity.Small interfering RNA(siRNA)was used to inhibit the expression of NRF2 in iHCEC.The expression level of NLRP3 inflammatory corpus-related genes in iHCEC was detected by RT-PCR.ELISA detected iHCEC and priHCEC after each treatment IL-1βsecretion level.Oxidative stress markers in iHCEC after treatment were detected by immunofluorescence assay,and the related enzyme kits were used to detect the enzymatic activity of iHCEC;the expression of NRF2 in iHCEC was detected by Western blot.Results Genistein can protect human corneal epithelial cells from HS-induced injury by inhibiting ROS-NLRP3-IL-1βsignaling pathway.Genistein significantly inhibits HS-induced expression of NLRP3 inflammasome-related genes in iHCEC and secretion of IL-1β.Genistein can significantly attenuate HS-induced oxidative stress,which is shown by the reduction of ROS production in iHCEC and the decrease in the number of 8-OHdG positive cells after pretreatment with genistein.Flavin induces NRF2 expression,triggering the expression of several antioxidant enzymes.Conclusion This study demonstrates that genistein can inhibit HS-induced inflammation of human corneal epithelial cells,which may have the effect of preventing and alleviating corneal inflammation associated with dry eye at an earlier stage.

关 键 词：干眼 金雀异黄素 高渗刺激 人角膜上皮细胞 ROS NLRP3 IL-1Β 

分 类 号：R774.1[医药卫生—眼科]