背景:肝缺血再灌注损伤(HIRI)是围手术期的主要临床问题,与早期同种异体移植功能障碍(EAD)密切相关,急性排斥反应(AR)和长期移植物存活。中性粒细胞胞外陷阱(NETs)是在中性粒细胞刺激后通过释放去浓缩的染色质和颗粒蛋白而形成的细胞外结构。越来越多的证据表明,NETs参与了各种肝移植并发症的进展,包括缺血再灌注损伤(IRI)。本研究旨在全面分析NET相关基因(NRGs)在HIRI中的表达模式,识别具有不同特征的HIRI亚型,建立了可靠的EAD预测模型。
方法:微阵列,批量RNA-seq,和单细胞测序数据集从GEO数据库获得。最初,使用差异基因表达分析鉴定差异表达的NRG(DE-NRG)。然后,我们利用非负矩阵分解(NMF)算法对HIRI样本进行分类。随后,我们使用机器学习算法来筛选与EAD相关的集线器NRG,并基于这些集线器NRG开发了EAD预测模型。同时,我们使用HIRI在单细胞水平评估了hubNRGs的表达模式。此外,我们在大鼠原位肝移植(OLT)模型中验证了C5AR1的表达及其对HIRI和NETs形成的影响。
结果:在这项研究中,我们在HIRI背景下确定了11个DE-NRG。基于这11个DE-NRG,将HIRI样品分类为两个不同的簇。Cluster1表现出DE-NRGs的低表达,最小的中性粒细胞浸润,轻度炎症,EAD发病率低。相反,Cluster2表现出相反的表型,具有活化的炎症亚型和较高的EAD发病率。此外,使用与EAD相关的四个集线器NRG建立了EAD预测模型。根据风险评分,HIRI样本分为高危组和低危组。OLT模型证实肝组织中C5AR1表达的显著上调,伴随着NET的形成增加。用C5AR1拮抗剂治疗可改善肝功能,减少组织炎症,并减少了NET的形成。
结论:这项研究区分了两种明显的HIRI亚型,建立了EAD的预测模型,并验证了C5AR1对HIRI的影响。这些发现为开发先进的临床策略以提高肝移植受者的预后提供了新的视角。
BACKGROUND: Hepatic ischaemia-reperfusion injury (HIRI) is a major clinical concern during the perioperative period and is closely associated with early allograft dysfunction (EAD), acute rejection (AR) and long-term graft survival. Neutrophil extracellular traps (NETs) are extracellular structures formed by the release of decondensed chromatin and granular proteins following neutrophil stimulation. There is growing evidence that NETs are involved in the progression of various liver transplantation complications, including ischaemia-reperfusion injury (IRI). This study aimed to comprehensively analyse the expression patterns of NET-related genes (NRGs) in HIRI, identify HIRI subtypes with distinct characteristics, and develop a reliable EAD prediction model.
METHODS: Microarray, bulk RNA-seq, and single-cell sequencing datasets were obtained from the GEO database. Initially, differentially expressed NRGs (DE-NRGs) were identified using differential gene expression analyses. We then utilised a non-negative matrix factorisation (NMF) algorithm to classify HIRI samples. Subsequently, we employed machine learning algorithms to screen the hub NRGs related to EAD and developed an EAD prediction model based on these hub NRGs. Concurrently, we assessed the expression patterns of hub NRGs at the single-cell level using the HIRI. Additionally, we validated C5AR1 expression and its effect on HIRI and NETs formation in a rat orthotopic liver transplantation (OLT) model.
RESULTS: In this study, we identified 11 DE-NRGs in the HIRI context. Based on these 11 DE-NRGs, HIRI samples were classified into two distinct clusters. Cluster1 exhibited a low expression of DE-NRGs, minimal neutrophil infiltration, mild inflammation, and a low incidence of EAD. Conversely, Cluster2 displayed the opposite phenotype, with an activated inflammatory subtype and a higher incidence of EAD. Furthermore, an EAD prediction model was developed using the four hub NRGs associated with EAD. Based on risk scores, HIRI samples were classified into high- and low-risk groups. The OLT model confirmed substantial upregulation of C5AR1 expression in the liver tissue, accompanied by increased formation of NETs. Treatment with a C5AR1 antagonist improved liver function, reduced tissue inflammation, and decreased NETs formation.
CONCLUSIONS: This study distinguished two apparent HIRI subtypes, established a predictive model for EAD, and validated the effect of C5AR1 on HIRI. These findings provide novel perspectives for the development of advanced clinical strategies to enhance the outcomes of liver transplant recipients.