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

胞红蛋白(cytoglobin, Cygb)过表达已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Fe^(2+)、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Fe^(2+)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Fe^(2+)、ROS、MDA、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、collagen I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。

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(Fe^(2+))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 Fe^(2+),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 Fe^(2+)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 Fe^(2+),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.