摘要
目的:探究铁死亡标志物4-羟基壬烯醛(4-hydroxy nonenal,4-HNE)在转化生长因子β1(transforming growth factor-beta 1,TGF-β1)诱导的肌成纤维细胞模型中的表达及其生理意义,为系统性硬化症(systemic sclerosis,SSc)纤维化进程的诊断和治疗提供理论依据.方法:将饥饿处理12 h后的小鼠胚胎成纤维细胞(NIH3t3)分为两组,对照组以1％(体积分数)血清培养基培养,TGF-β1处理组以10 μg/L TGF-β1+1％血清培养基培养.通过显微镜观察两组细胞形态的变化,使用实时荧光定量逆转录PCR(reverse transcription quantitative real-time PCR,RT-qPCR)和蛋白免疫印迹实验(Western blot)检测纤维化标志物的表达,验证SSc细胞模型的构建.使用流式细胞术分析两组细胞内活性氧(reactive oxygen species,ROS)的水平,通过Western blot和免疫荧光染色检测TGF-β1处理组中4-HNE的表达水平.结果:显微镜下观察到TGF-β1处理的NIH3t3细胞形态从典型的长梭形逐渐转变为多突起的扁平三角形.RT-qPCR和Western blot检测结果表明,TGF-β1组中纤维化标志物波形蛋白(Vimentin)的表达显著高于对照组(P＜0.01),证实了TGF-β1可以促进纤维化标志物的上调.流式细胞术结果显示,TGF-β1处理组中细胞内ROS水平显著升高,表明其诱导了氧化应激的发生.Western blot和免疫荧光分析均显示,TGF-β1处理后细胞中4-HNE的表达显著增加(免疫荧光强度P＜0.05).结论:TGF-β1可诱导成纤维细胞发生纤维化,同时促进ROS的生成,并显著上调4-HNE的表达水平;4-HNE的显著增加提示其在SSc纤维化过程中可能具有重要作用,可作为潜在的纤维化标志物;本研究为未来探讨4-HNE在SSc中的作用机制和其作为诊断和治疗靶点的可行性提供了实验依据.
Abstract
Objective:To investigate the expression and physiological significance of the ferroptosis marker 4-hydroxynonenal(4-HNE)in myofibroblasts induced by transforming growth factor-β1(TGF-β1),providing theoretical evidence for its potential role in the diagnosis and treatment of fibrosis in sys-temic sclerosis(SSc).Methods:Mouse embryonic fibroblasts(NIH3t3)were cultured and divided into two groups after 12 h of starvation:the control group(cultured in 1％serum-containing medium)and the TGF-β1 group(cultured in 10 μg/L TGF-β1 with 1％serum-containing medium).Cell morphology changes in both groups were observed under a microscope.To confirm successful establishment of the SSc cell model,fibrosis markers were analyzed using reverse transcription quantitative real-time PCR(RT-qPCR)and Western blot.Next,flow cytometry was employed to assess the intracellular levels of reactive oxygen species(ROS)in both groups.Finally,Western blot and immunofluorescence staining were used to measure the expression of 4-HNE in the TGF-β1-treated cells.Results:Microscopic observations re-vealed that TGF-β1 treatment caused the NIH3t3 cells to transition from a typical spindle shape to a flat,polygonal shape with multiple protrusions,indicating fibroblast activation.The RT-qPCR and Western blot analyses showed that the expression of the fibrosis marker Vimentin was significantly upregulated in the TGF-β1 group compared with the control group(P＜0.01),confirming that TGF-β1 effectively pro-moted fibrosis-related gene and protein expression.Flow cytometry results indicated that TGF-β1 signifi-cantly elevated intracellular ROS levels,suggesting the induction of oxidative stress.Furthermore,both Western blot and immuno-fluorescence staining demonstrated a significant increase in 4-HNE expression in the TGF-β1-treated cells(immunofluorescence intensity P＜0.05).Conclusion:TGF-β1 promotes fibroblast activation and fibrosis while inducing ROS production,leading to a marked increase in 4-HNE expression.Given the role of 4-HNE as a marker of lipid peroxidation and its elevated levels in the SSc cell model,this study suggests that 4-HNE could serve as a potential biomarker for fibrosis in SSc.The findings highlight the importance of investigating the mechanisms of 4-HNE in fibrosis and suggest that targeting this pathway could offer new therapeutic opportunities for treating SSc.
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