探讨肺动脉高压(pulmonary arterial hypertension, PAH)中铁死亡-铜死亡耦合转录特征及其与肺血管免疫微环境重塑的关系。

基于基因表达综合数据库(Gene Expression Omnibus, GEO)数据库获取PAH相关bulk转录组及单细胞RNA测序数据，采用差异表达分析筛选PAH(n＝76)与正常肺组织间差异(n＝42)表达基因，并通过单样本基因集富集分析(single-sample Gene Set Enrichment Analysis, ssGSEA)、基因集变异分析(gene set variation analysis, GSVA)计算铁死亡评分、铜死亡评分及铁死亡-铜死亡交叉评分。将PAH差异表达基因、铁-铜死亡相关基因及交叉评分相关基因取交集，筛选候选耦合基因，并进一步进行表达验证、相关性分析及GO、KEGG功能富集分析。基于单细胞数据进行细胞类型注释，分析不同细胞群中铁死亡-铜死亡评分及核心基因的分布特征，并采用CellChat评估PAH肺组织中细胞通讯变化。进一步通过PAH动物模型(n＝40)，采用血流动力学检测、qRT-PCR和Western blot验证关键分子表达及血流动力学表型。

PAH肺组织中铁死亡、铜死亡及二者交叉相关转录特征发生显著改变，PAH组铁死亡评分、铜死亡评分及铁死亡-铜死亡交叉评分均较正常组显著降低(P＝5.3×10⁵、P＝0.028、P＝0.00029)，且铁死亡评分与铜死亡评分呈正相关(R＝0.42，P＝0.019)。交集分析共获得16个候选基因，进一步筛选获得DLAT、SAT1、MTF1、SLC7A11、GCLM和NCOA4等铁死亡-铜死亡耦合核心候选基因(PAH组DLAT表达上调，其余5个基因表达下调，P分别为8.8×10⁶、2.0×10⁴、3.5×10⁴、0.0073、6.7×10⁵和7.3×10⁴)，其功能主要富集于铁死亡7个基因、硫辛酸代谢4个、谷胱甘肽代谢3个、半胱氨酸和蛋氨酸代谢2个等通路。单细胞分析显示(n＝9)，共注释出16种细胞类型，铁死亡-铜死亡交叉评分在肺血管内皮细胞、平滑肌细胞和巨噬细胞中呈细胞类型特异性变化(log2FC>0.3，min.pct>0.25)，PAH组上述三类细胞的交叉评分均显著低于正常组(P分别<0.05、<0.01和<0.05)。细胞通讯分析提示，PAH组细胞间互作数量由2067增加至2802、互作强度由32.759升高至58.673，巨噬细胞与内皮细胞、成纤维细胞及平滑肌细胞之间的THBS1-CD47、SELPLG-SELE、PECAM1-PECAM1和PPIA-BSG等通讯轴增强。动物实验显示，PAH模型组右心室收缩压(对照组约23 mmHg，PAH组约49 mmHg，P<0.01)和Fulton指数(对照组约0.23，PAH组约0.42，P<0.001)显著升高；同时DLATmRNA及蛋白表达上调(P<0.0001、P<0.001)，而SAT1、MTF1、SLC7A11、GCLM和NCOA4表达下调(P<0.05)。

PAH肺组织中存在铁死亡-铜死亡耦合异常，该异常主要涉及肺血管内皮细胞、平滑肌细胞和巨噬细胞，并可能通过调控谷胱甘肽代谢、硫辛酸代谢及巨噬细胞-血管壁细胞通讯参与肺血管重塑。

To investigate ferroptosis-cuproptosis crosstalk in pulmonary arterial hypertension (PAH) and its potential association with pulmonary vascular immune microenvironment remodeling.

PAH-related bulk transcriptomic and single-cell RNA sequencing datasets were obtained from the GEO database. Differentially expressed genes between PAH(n=76) and normal lung tissues(n=42) were identified, and ferroptosis, cuproptosis and ferroptosis-cuproptosis crosstalk scores were calculated using ssGSEA/GSVA. Candidate crosstalk genes were screened by intersecting PAH differentially expressed genes, ferroptosis/cuproptosis-related genes and crosstalk score-related genes, followed by expression validation, correlation analysis and GO/KEGG functional enrichment analysis. Single-cell RNA sequencing data were further used for cell annotation, cell type-specific score evaluation and core gene distribution analysis. Cell Chat was applied to assess intercellular communication changes in PAH lung tissues. In addition, a PAH animal model(n=40) was used to validate hemodynamic alterations, key molecular expression by qRT-PCR and Western blot.

Ferroptosis-, cuproptosis- and crosstalk-related transcriptional features were markedly altered in PAH lung tissues: the ferroptosis, cuproptosis and crosstalk scores were all significantly lower in PAH than in normal lungs (P=5.3×10^5, 0.028 and 0.00029, respectively), and ferroptosis score was positively correlated with cuproptosis score (R=0.42, P=0.019). Sixteen candidate genes were obtained by intersection analysis, among which DLAT, SAT1, MTF1, SLC7A11, GCLM and NCOA4 were identified as core candidate genes involved in ferroptosis-cuproptosis crosstalk (DLAT was upregulated whereas the other five genes were downregulated in PAH; P=8.8×10^6, 2.0×10^4, 3.5×10^4, 0.0073, 6.7×10⁵ and 7.3×10^4, respectively). Functional enrichment analysis showed that these genes were mainly enriched in ferroptosis 7 genes, lipoic acid metabolism 4 genes, glutathione metabolism 3 genes, and cysteine and methionine metabolism 2 genes. Single-cell analysis (n=9) identified 16 cell types and revealed cell type-specific alterations of the crosstalk score in pulmonary vascular endothelial cells, smooth muscle cells and macrophages, all of which showed significantly lower crosstalk scores in PAH (P<0.05, P<0.01 and P<0.05, respectively). CellChat analysis showed increased interaction number (2 067 vs. 2 802) and interaction strength (32.759 vs. 58.673) in PAH, with enhanced macrophage communication with endothelial cells, fibroblasts and smooth muscle cells through signaling axes including THBS1-CD47, SELPLG-SELE, PECAM1-PECAM1 and FN1-(ITGA5+ ITGB1). Animal experiments showed significantly increased right ventricular systolic pressure (approximately 23 vs. 49 mmHg, P<0.01) and Fulton index (approximately 0.23 vs. 0.42, P<0.001) in the PAH group. Moreover, DLAT was upregulated at both mRNA and protein levels (P<0.0001 and P<0.001), whereas SAT1, MTF1, SLC7A11, GCLM and NCOA4 were downregulated (P<0.05).

Ferroptosis-cuproptosis crosstalk is dysregulated in PAH lung tissues and mainly involves pulmonary vascular endothelial cells, smooth muscle cells and macrophages. This dysregulation may participate in pulmonary vascular remodeling by regulating glutathione metabolism, lipoic acid metabolism and macrophage-vascular wall cell communication.