莱菔硫烷对体外培养牛眼小梁细胞硫氧还蛋白表达的影响及其机制

背景 研究证实莱菔硫烷(SFN)可以激活多条通路,促进机体内抗氧化蛋白的表达.硫氧还蛋白(Trx)是维持细胞内氧化还原稳态的重要抗氧化蛋白之一. 目的 研究SFN对体外培养的牛眼小梁细胞内Trx表达的影响及其机制.方法体外培养牛眼小梁细胞并进行形态学观察,取第3代培养的牛眼小梁细胞,分别加入终浓度为0、10、20和30 μmol/L SFN培养30 min,应用实时荧光定量PCR技术检测细胞内Trx mRNA的表达.将细胞按照随机抽样的方法分为6个组,即空白对照组、LY294002组、U0126组、SFN组、LY294002+ SFN组和U0126+SFN组.采用Western blot法检测各组细胞中Nrf2蛋白和Trx蛋白的相对表达量.结果 体外成功培养牛眼小梁细胞.不同浓度SFN干预下,细胞Trx mRNA相对表达量总体比较差异有统计学意义(F=88.090,P＜0.01).LY294002+ SFN组、U0126+SFN组和SFN组细胞中Trx蛋白和Nrf2蛋白相对表达量显著高于空白对照组,差异均有统计学意义(均P＜0.01).LY294002+ SFN组和U0126+SFN组细胞中Trx蛋白和Nrf2蛋白相对表达量显著高于SFN组,差异均有统计学意义(均P＜0.01). 结论 SFN可通过磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)通路与丝裂原活化蛋白激酶(MAPK)/细胞外调节蛋白激酶1/2(ERK1/2)通路活化Nrf2,促进小梁细胞Trx mRNA及其蛋白的合成,保护细胞抵抗氧化应激损伤.

Effects of sulforaphane on thioredoxin expression in bovine trabecular meshwork cells

Background Recent studies have confirmed that sulforaphane (SFN) can activate multiple pathways,and promote the expression of the antioxidants in cells.Thioredoxin (Trx) plays an important role in maintaining the intracellular redox in the steady state.Objective This study was to investigate the effect and mechanism of SFN on Trx expression in bovine trabecular meshwork cells (BTMCs) cultured in vitro.Methods BTMCs were cultured in vitro and identified by morphological evaluation.The third generation of BTMCs were cultured in the medium with 0,10,20 and 30 μmol/L SFN for 30 minutes.Real-time PCR was applied to measure the expression of Trx mRNA in BTMCs.The BTMCs were randomly divided into normal control group,LY294002 group,U0126 group,SFN group,LY294002 +SFN group and U0126+SFN group.The expressions of Nrf2 protein and Trx protein in each group were measured by Western blot.Results The BTMCs was successfully cultured in vitro.The expressions of Trx mRNA were significantly different among the different concentrationss of SFN treatment (F=88.090,P＜0.01).The expressions of Trx protein and Nrf2 protein in the LY294002 +SFN group,U0126 +SFN group and SFN group were significantly higher than those in the normal control group (all at P ＜ 0.01).The expressions of Trx protein and Nrf2 protein in the LY294002+SFN group and U0126+SFN group were significantly higher than those in the SFN group (all at P＜0.01).Conelusions SFN can activate Nrf2 by phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathways,which can increase the expression level of Trx in BTMCs cultured in vitro.