铁死亡标志物4-HNE在系统性硬化症细胞模型中的表达及意义

doi:10.19723/j.issn.1671-167X.2024.06.002

摘要/Abstract

摘要：

目的: 探究铁死亡标志物4-羟基壬烯醛(4-hydroxynonenal, 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。

关键词: 系统性硬化症, 成纤维细胞, 铁死亡, 纤维化

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 systemic 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 revealed 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 promoted fibrosis-related gene and protein expression. Flow cytometry results indicated that TGF-β1 significantly 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.

Key words: Systemic sclerosis, Fibroblasts, Ferroptosis, Fibrosis

中图分类号:

R593.25

赵柯林, 夏雪, 史乃旭, 周韩, 盖婧雯, 李萍. 铁死亡标志物4-HNE在系统性硬化症细胞模型中的表达及意义[J]. 北京大学学报（医学版）, 2024, 56(6): 950-955.

Kelin ZHAO, Xue XIA, Naixu SHI, Han ZHOU, Jingwen GAI, Ping LI. Expression and significance of ferroptosis marker 4-HNE in in vitro model of systemic sclerosis[J]. Journal of Peking University (Health Sciences), 2024, 56(6): 950-955.

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