[Immune infiltration and clinical characteristics analysis of coagulation-related genes in sepsis]

To analyze the immune infiltration and critical characteristics of coagulation-related genes in sepsis, reveal the role of coagulation-related differentially expressed gene (CRDEG) in sepsis through large-scale data analysis, and explore the relationship between CRDEG and coagulopathy in sepsis as well as the potential targets.

(1) Bioinformatics experiment: based on the Gene Expression Omnibus (GEO) database integrated from sepsis datasets, samples were divided into 989 sepsis samples and 100 control samples for differential expression analysis. The analysis results were intersected with coagulation-related genes obtained from gene-related databases to obtain CRDEG. According to the median expression of risk score in the sepsis diagnosis model, the sepsis samples were divided into a high-risk group and a low-risk group, and the difference of CRDEG expression between the two groups were compared. A CRDEG-related protein-protein interaction (PPI) network was constructed, and key genes were obtained through algorithm analysis, and the correlation between key genes and sepsis was analyzed, and the diagnostic value of key genes expression for sepsis was evaluated by receiver operator characteristic curve (ROC curve) analysis. The relative abundance of immune cells infiltrated in the high- and low-risk groups of sepsis was compared, and the correlation between key genes and immune cells was calculated based on the Spearman correlation analysis. The key gene mRNA-transcription factor (TF) and key gene mRNA-microRNA (miRNA) regulatory network were constructed to explore the changes in the signaling pathways to coagulopathy in sepsis.(2) Animal experiment verification: 15 male SD rats were randomly divided into a sepsis group, a sepsis-disseminated intravascular coulation (DIC) group and a human recombinant thrombopoietin (hrTPO) intervention group, with 5 rats in each group. The rats' critical indicators [platelet/lymphocyte ratio (PLR), prothrombin time (PT), fibrinogen (FIB), oxygenation index (PaO₂/FiO₂), base excess (BE), lactic acid (Lac)] were detected. The level of CD163 [the scavenger that internalizes ADAMTS-13 (von Willebrand factor-cleaving protease) on the surface of monocyte-macrophages] in peripheral blood was detected by flow cytometry. The activity of plasma ADAMTS-13 and von Willebrand factor (vWF) was detected by enzyme-linked immunosorbent assay (ELISA). The expression of microRNA-50 (miR-150) and p65 (nuclear transcription factor) in peripheral blood leukocytes was detected by reverse transcription-polymerase chain reaction (RT-PCR).

(1)Bioinformatics experiment results: finally, a total of 59 CRDEG were obtained. The expression levels of 25 CRDEG were statistically significant between the high-risk and low-risk groups of sepsis (all P<0.05). The PPI network of the 25 CRDEG was constructed, and the intersection of genes obtained based on five algorithms as key genes, which were chemokine CCL5, matrix metalloproteinase-9 (MMP-9), T-cell surface protein CD8A, chemokine receptor 5 (CCR5), and CD163, respectively. The expression levels of five key genes were statistically significant between the sepsis samples and the control samples (all P<0.05), among which key gene CD163 and MMP-9 were positively correlated with sepsis, and the other three genes were negatively correlated with it. ROC curve analysis showed that the CD163 expression level had a high accuracy in the diagnosis of sepsis [area under the ROC curve (AUC) was 0.746]. The immune infiltration abundance of 20 kinds of immune cells was statistically significant between the high- and low-risk groups of sepsis (all P<0.05). Correlation analysis showed that CD163 was strongly positively correlated with macrophages. Regulatory network analysis showed that nuclear factor-κB (NF-κB), which participates in the coagulopathy of sepsis, might regulate the expression of CD163 and the miR-150 related to coagulopathy in sepsis might regulate NF-κB and CD163. (2) Animal experimental verification results: compared with the sepsis group, the rats in the sepsis-DIC group had worse critical indicators, and coagulation function disorders were aggravated, which was manifested by the increased CD163⁺ subset levels in peripheral blood macrophages, decreased plasma ADAMTS-13 activity, increased vWF activity, downregulated miR-150 expression in peripheral blood white blood cells and upregulated p65 expression. The above indicators of the rats in the hrTPO intervention group were improved to varying degrees compared with the sepsis-DIC group, and there were statistically significant differences between the two groups [ADAMTS-13: (6.44±1.24)% vs. (1.87±0.40), vWF: (224.80±26.19)% vs. (394.10±22.54)%, miR-150 (2^-ΔΔCt): 2.47±0.23 vs. 0.97±0.19, p65 (2^-ΔΔCt): 3.90±0.70 vs. 7.71±0.63, all P<0.05].

By integrating the GEO dataset, CRDEG was identified, with CD163 potentially being the core gene involved in sepsis-induced immune-coagulopathy. The upregulation of macrophage CD163 and upstream NF-ΚB signaling molecules, coupled with the downregulation of miR-150, aligns with the deterioration of critical indicators of sepsis and the trend towards coagulopathy. However, hrTPO can ameliorate these phenomena.