Wang Hongli, Zhang Miaomiao, Hu Yiping, He Juan, Zhong Yuchao, Dai Yong, Wang Qingwen
Department of Rheumatism and Immunology, Peking University Shenzhen Hospital, Shenzhen, China.
The Key Laboratory of Inflammatory and Immunology Diseases, Shenzhen, China.
Heliyon. 2024 Jun 26;10(13):e33648. doi: 10.1016/j.heliyon.2024.e33648. eCollection 2024 Jul 15.
The pathogenesis of rheumatoid arthritis (RA) remains elusive. The initiation of joint degeneration is characterized by the loss of self-tolerance in peripheral joints. Ferroptosis, a form of regulated cell death, holds significant importance in the pathophysiology of inflammatory arthritis, primarily due to iron accumulation and the subsequent lipid peroxidation. The present study investigated the association between synovial lesions and ferroptosis-related genes using previously published data from rheumatoid patients. Transcriptome differential gene analysis was employed to identify ferroptosis-related differentially expressed genes (FRDEGs). To validate FRDEGs and screen hub genes, we used weighted gene co-expression network analysis (WGCNA) and receiver operating characteristic (ROC) curves. Subsequently, immune infiltration analysis and single cell analysis were conducted to investigate the relationship between various synovial tissues cells and FRDEGs. The findings were further confirmed through reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunohistochemical staining, and immunofluorescence techniques. Upon intersecting DEGs with ferroptosis-related genes, we identified a total of 104 FRDEGs. Through the construction of a protein-protein interaction (PPI) network, we pinpointed the top 20 most highly concentrated genes as hub genes. Subsequent analyses using ROC curve and WGCNA validated eight FRDEGs: TIMP1, JUN, EGFR, SREBF1, ADIPOQ, SCD, AR, and FABP4. Immuno-infiltration analyses revealed significant infiltration of immune cell in RA synovial tissues and their correlations with the FRDEGs. Notably, TIMP1 demonstrated a positive correlation with various immune cell populations. Single-cell sequencing date of RA synovial tissue revealed predominant expression of TIMP1 is in fibroblasts. RT-qPCR, immunohistochemistry, and immunofluorescence analyses confirmed significant upregulation of TIMP1 at both mRNA and protein levels in RA synovial tissues and fibroblast-like synoviocytes (FLS). The findings provide novel insights into pathophysiology of peripheral immune tolerance deficiency in RA. The dysregulation of TIMP1, a gene associated with ferroptosis, was significantly observed in RA patients, suggesting its potential as a promising biomarker and therapeutic target.
类风湿关节炎（RA）的发病机制仍不清楚。关节退变的起始特征是外周关节自身耐受性的丧失。铁死亡作为一种程序性细胞死亡形式，在炎症性关节炎的病理生理学中具有重要意义，主要是由于铁的积累及随后的脂质过氧化。本研究利用先前发表的类风湿患者数据，调查滑膜病变与铁死亡相关基因之间的关联。采用转录组差异基因分析来鉴定铁死亡相关差异表达基因（FRDEGs）。为了验证FRDEGs并筛选核心基因，我们使用了加权基因共表达网络分析（WGCNA）和受试者工作特征（ROC）曲线。随后，进行免疫浸润分析和单细胞分析，以研究各种滑膜组织细胞与FRDEGs之间的关系。通过逆转录定量聚合酶链反应（RT-qPCR）、免疫组织化学染色和免疫荧光技术进一步证实了研究结果。将差异表达基因（DEGs）与铁死亡相关基因进行交叉分析后，我们共鉴定出104个FRDEGs。通过构建蛋白质-蛋白质相互作用（PPI）网络，我们确定了前20个高度集中的基因作为核心基因。随后使用ROC曲线和WGCNA进行的分析验证了8个FRDEGs：TIMP1、JUN、EGFR、SREBF1、ADIPOQ、SCD、AR和FABP4。免疫浸润分析显示免疫细胞在RA滑膜组织中显著浸润，且它们与FRDEGs相关。值得注意的是，TIMP1与各种免疫细胞群体呈正相关。RA滑膜组织的单细胞测序数据显示TIMP1主要在成纤维细胞中表达。RT-qPCR、免疫组织化学和免疫荧光分析证实，RA滑膜组织和成纤维样滑膜细胞（FLS）中TIMP1在mRNA和蛋白质水平均显著上调。这些发现为RA外周免疫耐受缺陷的病理生理学提供了新的见解。在RA患者中显著观察到与铁死亡相关的基因TIMP1失调，表明其作为有前景的生物标志物和治疗靶点的潜力。
