重组胞红蛋白调控肝星状细胞LX2铁死亡作用的研究
Research on the regulation of ferroptosis in hepatic stellate cells line LX2 by recombinant cytoglobin
段训威 1肖桂清 2陈怀宇 1张勇 3吴文林 1高壹 4刁勇5
作者信息
- 1. 泉州师范学院海洋与食品学院,福建泉州 362000;福建省海洋藻类活性物质制备与功能开发重点实验室,福建泉州 362000
- 2. 黎明职业大学轻工学院,福建泉州 362000
- 3. 昌吉市人民医院,新疆昌吉 831100
- 4. 华侨大学附属厦门长庚医院,福建厦门 361000
- 5. 华侨大学医学院,福建泉州 362021
- 折叠
摘要
胞红蛋白(cytoglobin,Cygb)过表达己被证实能减少细胞外基质沉积促进肝纤维化恢复,但其机制尚未明确.本研究构建表达细胞穿膜肽TAT与Cygb的融合蛋白(TAT-Cygb),探究其调控活化型肝星状细胞(hepatic stellate cells,HSCs)铁死亡作用.体外培养肝星状细胞系LX2,分别给予不同浓度的TAT-Cygb及铁死亡诱导剂(erastin)处理,台盼蓝染色、透射电镜、普鲁士蓝染色、试剂盒检测等考察TAT-Cygb对铁死亡表型包括细胞活力、细胞形态特征、铁离子含量、脂质过氧化产物水平及抗氧化系统指标的影响;同时采用铁死亡抑制剂(ferrostain-1,Fer-1)进行反向验证,给予TAT-Cygb和Fer-1共同处理,试剂盒法测定Fe2+、活性氧自由基(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)、4-羟基壬烯醛(4-hydroxynonenal,4-HNE)、烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)和还原型谷胱甘肽(glutathione,GSH)的水平,蛋白印迹法检测HSCs活化指标α平滑肌肌动蛋白(alpha smooth actin,α-SMA)、Ⅰ型胶原蛋白(collagen Ⅰ)、纤连蛋白(fibronectin)的表达水平,免疫荧光观察促纤维化关键指标表皮生长因子受体(epidermal growth factor receptor,EGFR)、肌间线蛋白(desmin)的表达情况.结果显示,TAT-Cygb能显著降低LX2细胞活力,并触发了细胞铁死亡相关事件,包括促进胞内Fe2+堆积并诱导线粒体形态改变,加剧脂质过氧化产物堆积,降低抗氧化指标水平,与erastin发挥相似的作用;而Fer-1显著削弱了 TAT-Cygb诱导的Fe2+、ROS、MD A、4-HNE水平的升高以及NADPH和GSH水平的降低,同时还减轻了TAT-Cygb诱导高表达的α-SMA、collagenⅠ和fibronectin水平,下调了TAT-Cygb对EGFR、desmin的表达抑制作用.这一细胞水平研究表明,TAT-Cygb能诱发活化型HSCs铁死亡事件.本研究揭示了 TAT-Cygb抗肝纤维化潜在的作用机制,为深入研究TAT-Cygb通过调控铁死亡途径发挥其生物学功能的分子机制提供实验依据.
Abstract
Intracellular overexpression of cytoglobin(Cygb)has been shown to reduce extracellular matrix deposition and promote liver fibrosis recovery,but its mechanism is not yet clear.This study constructed and expressed a fusion protein(TAT-Cygb)of cell penetrating peptide TAT and Cygb,to investigate the effect of fusion protein TAT-Cygb on regulating hepatic stellate cells(HSCs)ferroptosis.Cultured human hepatic stellate cells line(LX2)were treated with TAT-Cygb and erastin in vitro,respectively.The effects of ferroptosis phenotype in LX2 cells induced by TAT-Cygb,including cell viability,cell morphology,iron ion(Fe2+)content,lipid peroxidation product levels,and antioxidant system indicators,were investigated using trypan blue staining,transmission electron microscopy,Prussian blue staining,and reagent kits detection.After co-treatment with TAT-Cygb and ferrostain-1,the levels of Fe2+,reactive oxygen species(ROS),malondialdehyde(MDA),4-hydroxynonenal(4-HNE),nicotinamide adenine dinucleotide phosphate(NADPH)and glutathione(GSH)were measured by reagent kits.The protein expression levels of alpha smooth actin(α-SMA),collagen Ⅰ and fibronectin were detected by Western blot,and the protein expression level of epidermal growth factor receptor(EGFR)and desmin relevant to fibrosis were observed by immunofluorescence.The results showed that TAT-Cygb could significantly reduce the viability of LX2 cells and trigger events relevant to ferroptosis,including promoting intracellular Fe2+accumulation,and inducing mitochondrial morphological changes,and intensifying lipid peroxidation products accumulation,and decreasing the level of antioxidant indexes,which played a similar role as erastin;Fer-1 significantly weakened the increase in Fe2+,ROS,MDA,4-HNE levels induced by TAT-Cygb,as well as the decrease in NADPH and GSH levels,while also weakening the TAT-Cygb-induced over-expression levels ofα-SMA,collagen Ⅰ and fibronectin,and TAT-Cygb-induced under-expression levels of EGFR and desmin.This cellular level study indicated that TAT-Cygb can induce ferroptosis of activated HSCs.This study revealed the potential mechanism of TAT-Cygb anti-liver fibrosis,and provided the experimental basis for further research on the molecular mechanism of TAT-Cygb realizing biological function by regulating the ferroptosis pathway.
关键词
胞红蛋白/细胞穿膜肽/铁死亡/肝纤维化/肝星状细胞Key words
cytoglobin/cell penetrating peptide/ferroptosis/hepatic fibrosis/hepatic stellate cell引用本文复制引用
基金项目
福建省自然科学基金资助项目(2023J01899)
厦门市科技局联合基金资助项目(3502Z20227371)
出版年
2024
NETL
NSTL
万方数据