通过多队列转录组整合与单细胞分辨率分析筛选肺动脉高压(pulmonary arterial hypertension, PAH)稳健相关关键基因，阐明其细胞来源及与免疫微环境重塑的潜在联系，为PAH生物标志物与机制研究提供依据。

分别对3个独立转录组队列(GSE217438、GSE117261、GSE33463)进行PAH与对照差异表达分析并取交集获得跨队列一致候选基因；基于相互作用基因/蛋白质检索工具(search tool for the retrieval of interacting genes/proteins, STRING)构建蛋白互作(protein-protein interaction, PPI)网络定位关键节点；采用加权基因共表达网络分析(weighted gene co-expression network analysis, WGCNA)构建共表达网络并进行模块-性状关联与功能富集；进一步分析肺组织单细胞转录组数据，明确G蛋白信号传导调节因子1(regulator of G- protein signaling 1, RGS1)的细胞来源并在单核-巨噬细胞谱系中开展拟时序分析，同时比较对照与PAH的细胞通讯差异并筛选关键配体-受体轴；最后构建野百合碱(monocrotaline, MCT)(60 mg/kg)诱导大鼠PAH模型，通过肺泡巨噬细胞RT-qPCR、Western blot及肺组织免疫组化验证RGS1表达变化。

三队列差异基因交集提示候选基因具有较强跨队列稳定性，其中免疫调控相关基因RGS1被进一步聚焦；在3个独立队列中，RGS1在PAH组显著上调(P<0.05)。蛋白互作网络结果表明，RGS1位于网络中心位置并与多种G蛋白信号及免疫相关分子相互作用，提示其可能参与GPCR免疫相关通路。WGCNA分析结果显示RGS1所属深灰色模块与PAH表型高度相关(cor＝0.43, P＝5e-5)，该模块基因富集适应性免疫反应、免疫球蛋白介导反应及淋巴细胞介导免疫等通路。单细胞分析显示RGS1主要在免疫细胞中高表达，PAH组巨噬细胞比例显著上升并伴随炎症相关通路富集(P<0.05)；拟时序结果表明单核-巨噬细胞沿轨迹发生连续状态迁移，RGS1表达随伪时间动态变化；细胞通讯分析则显示PAH组整体通讯数量和强度增强，并筛选到在PAH组的配体-受体对PPIA-BSG通讯概率升高，提示信号网络重塑。模型组接受野百合碱(monocrotaline, MCT)动物模型中实验组肺泡巨噬细胞RGS1 mRNA与蛋白水平上调(P<0.01)，同时肺组织免疫组织化学(immunohistochemistry, IHC)显示RGS1阳性信号增强。

RGS1可能参与PAH免疫微环境重塑，并有望成为免疫相关生物标志物和潜在干预靶点。

To identify robust pulmonary arterial hypertension (PAH)-associated genes through multi-cohort transcriptomic integration and single-cell resolution analyses, and to elucidate their cellular origins and potential links to immune microenvironment remodeling.

Differential expression analysis of PAH and control was conducted on three independent transcriptome cohorts (GSE217438, GSE117261, GSE33463) respectively, and the intersection was taken to obtain consistent candidate genes across cohorts. Build a protein-protein interaction (PPI) network based on STRING to locate key nodes; The co-expression network was constructed using WGCNA and module-trait association and functional enrichment were carried out. Further analyze the single-cell transcriptome data of lung tissue to clarify the cellular origin of RGS1 and conduct quasi-temporal analysis in the mononuclear macrophage lineage. At the same time, compare the differences in cell communication between the control and PAH and screen the key ligand-receptor axes. Finally, a rat PAH model induced by MCT (60 mg/kg) was constructed. The expression changes of RGS1 were verified by RT-qPCR of alveolar macrophages, Western blot and immunohistochemistry of lung tissue.

The intersection of differentially expressed genes across three cohorts indicates candidate genes exhibit strong cross-cohort stability, with the immune-regulatory gene RGS1 further identified. In all three independent cohorts, RGS1 was significantly upregulated in the PAH group (P<0.05). Protein interaction network analysis revealed RGS1 occupies a central position within the network and interacts with multiple G protein signaling and immune-related molecules, suggesting its potential involvement in GPCR-mediated immune pathways. WGCNA analysis revealed that the dark gray module containing RGS1 was highly correlated with the PAH phenotype (cor=0.43, P=5e-5). Genes in this module were enriched in pathways including adaptive immune response, immunoglobulin-mediated response, and lymphocyte-mediated immunity. Single-cell analysis revealed that RGS1 is predominantly highly expressed in immune cells. The proportion of macrophages significantly increased in the PAH group, accompanied by enrichment of inflammation-related pathways (P<0.05). The results of pseudotime series showed that monocyte-macrophage migration occurred in a continuous state along the trajectory, and the expression of RGS1 changed dynamically with pseudotime. Cell communication analysis showed that the number and intensity of overall communication were increased in PAH group, and the probability of ligand-receptor pair PPIA-BSG communication was significantly increased in PAH group, suggesting that the signal network was remodled. The expressions of RGS1 mRNA and protein in alveolar macrophages in the experimental group were significantly up-regulated in the MCT animal model (P<0.01), and the positive signal of RGS1 in lung tissue was enhanced by IHC.

RGS1 may be involved in the remodeling of the immune microenvironment in PAH and serve as an immune-related biomarker and potential therapeutic target.