Institute of Brain Science, Southwest Medical University, Luzhou 646000, China.
Department of Endocrinology and Metabolism, The Affiliated Hospital, Southwest Medical University, Luzhou 646000, China; School of Nursing, Southwest Medical University, Luzhou 646000, China; Wound Healing Basic Research and Clinical Application Key Laboratory of Luzhou, School of Nursing, Southwest Medical University, Luzhou 646000, China.
Int Immunopharmacol. 2024 Sep 30;139:112638. doi: 10.1016/j.intimp.2024.112638. Epub 2024 Jul 28.
Diabetic foot ulcers (DFU), affecting a quarter of diabetic patients and leading to high rates of amputation and mortality, pose significant health and economic burdens. Wound healing in DFU is often compromised by chronic inflammation, underscoring the critical role of immune cells. However, the systematic investigation of immune-related genes (IRGs) in DFU pathogenesis remains elusive. To address this gap, our study aims to explore the association between IRGs and DFU.
To explore biological changes in immune related gene expression in DFU, RNA-seq was performed on wound biopsies derived from 10 DFU patients and 11 healthy controls. Differentially expressed genes (DEGs) between DFU and normal samples were obtained by DEseq2. By intersecting the IRG list from the ImmPort database, the immune-related differentially expressed genes were identified. Function enrichment analysis and protein-protein interaction (PPI) analysis were applied by clusterProfiler and STRING database, and the hub genes of the PPI network were calculated by the cytoHubba plug-ins in Cytoscape. CIBERSORT algorithms was applied to analyze immune infiltration in DFU. And the correlation between immune cells infiltration and hub genes was explored by correlation analysis. Finally, to validate our findings, the transcriptional change of hub genes in DFU was confirmed using external scRNA-seq dataset and RT-qPCR.
RNA-seq analysis detected 8,800 DEGs in DFUs, with 2,351 upregulated and 6,449 downregulated.526 differential IRGs were obtained from intersection of DEGs and IRGs. 526 differential IRGs were obtained from intersection of DEGs and IRGs. Enrichment function analysis of DEGs showed that they played a significant role in immune response. The PPI network was constructed, and the most significant module containing 4 hub genes was identified. CIBERSORT analysis showing that there was a significant difference between DFU and normal controls in the infiltration of immune cells. Compared with normal tissue, DFU tissue contained a higher proportion of resting NK cell, M0 macrophages, and activated mast cell, while resting dendritic cell, activated mast cell, and activated NK cell contributed to a relatively lower portion. Additionally, the analysis for M1/M2 polarization of macrophage cells shown that DFU tissue contained a higher M1/M2 ratio than normal group. Finally, the expression levels of 4 hub genes were confirmed by external scRNA-seq dataset and RT-qPCR.
The immune related hub genes and the difference in immune infiltration between DFU tissue and normal controls might provide new insight for understanding DFU healing.
糖尿病足溃疡（DFU）影响了四分之一的糖尿病患者，导致高截肢率和死亡率，给健康和经济带来了巨大负担。DFU 的伤口愈合常常受到慢性炎症的影响，这突显了免疫细胞的关键作用。然而，DFU 发病机制中免疫相关基因（IRGs）的系统研究仍难以捉摸。为了解决这一差距，我们的研究旨在探讨 IRGs 与 DFU 的关系。
为了探讨 DFU 中免疫相关基因表达的生物学变化，我们对 10 名 DFU 患者和 11 名健康对照者的伤口活检进行了 RNA-seq 分析。通过 DEseq2 获得 DFU 与正常样本之间的差异表达基因（DEGs）。通过与 ImmPort 数据库中的 IRG 列表相交，确定了免疫相关差异表达基因。应用 clusterProfiler 和 STRING 数据库进行功能富集分析和蛋白质-蛋白质相互作用（PPI）分析，并通过 Cytoscape 中的 cytoHubba 插件计算 PPI 网络的枢纽基因。应用 CIBERSORT 算法分析 DFU 中的免疫浸润。通过相关性分析探讨免疫细胞浸润与枢纽基因的相关性。最后，通过外部 scRNA-seq 数据集和 RT-qPCR 验证了枢纽基因在 DFU 中的转录变化。
RNA-seq 分析在 DFU 中检测到 8800 个 DEGs，其中 2351 个上调，6449 个下调。通过 DEGs 和 IRGs 的交集获得了 526 个差异 IRGs。DEGs 的富集功能分析表明，它们在免疫反应中发挥了重要作用。构建了 PPI 网络，并确定了包含 4 个枢纽基因的最显著模块。CIBERSORT 分析表明，DFU 与正常对照组之间免疫细胞的浸润存在显著差异。与正常组织相比，DFU 组织中静止 NK 细胞、M0 巨噬细胞和活化肥大细胞的比例较高，而静止树突状细胞、活化肥大细胞和活化 NK 细胞的比例较低。此外，对巨噬细胞 M1/M2 极化的分析表明，DFU 组织中 M1/M2 比值高于正常组。最后，通过外部 scRNA-seq 数据集和 RT-qPCR 验证了 4 个枢纽基因的表达水平。
DFU 组织与正常对照组之间免疫相关枢纽基因和免疫浸润的差异可能为理解 DFU 愈合提供新的见解。
