[Investigation of the role and mechanism of in inducing ferroptosis in vascular endothelial cells].

To investigate whether (Pg) induces ferroptosis in vascular endothelial cells and predict the Hub genes. Firstly, human umbilical vein endothelial cells (HUVEC) were stimulated with Pg (W83) for 4 h, and transmission electron microscopy was used to observe ferroptosis-related morphological characteristics. Subsequently, RNA was extracted from HUVEC before and after Pg stimulation for transcriptome sequencing (RNA-seq). Enrichment analysis was performed to determine if differentially expressed genes (DEG) associated with ferroptosis. Ferroptosis-related DEG (Fer-DEG) were identified and then underwent gene ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, protein-protein interaction (PPI) network construction, and Hub gene prediction. Next, based on RNA-seq results, HUVEC were stimulated with lipopolysaccharide (LPS) for 24 h. Established ferroptosis markers were detected. The indices and detection methods were as follows: cell viability via cell counting kit-8; reactive oxygen species (ROS) by the DCFH-DA probe; Fe²⁺, lipid peroxides (LPO), malondialdehyde (MDA), and reduced/oxidized glutathione ratio (GSH/GSSG) with commercial kits; mitochondrial membrane potential (MMP) using the JC-1 probe; solute carrier family 7 member 11 (SLC7A11), solute carrier family 3 member 2 (SLC3A2), and glutathione peroxidase 4 (GPX4) expressions by Western blotting (WB) and real-time fluorescence quantitative PCR (RT-qPCR). Finally, RT-qPCR was used to validate the expression of predicted Hub genes in HUVEC after 24 h LPS stimulation, including tumor necrosis factor (TNF) or TNF-α, interleukin (IL)-6, and prostaglandin-endoperoxide synthase 2 (PTGS2). The mitochondria exhibited size reduction and cristae loss in Pg-stimulated HUVEC. DEG of HUVEC between the Pg-infected and control groups were enriched in the pathway of ferroptosis, and from which 56 Fer-DEG were identified. GO analysis showed enrichment in in responses to TNF, LPS, biotic stimulus, etc. and KEGG analysis revealed enrichment in TNF, C-type lectin receptor, and IL-17 signaling pathways, etc. In the 56-gene PPI network, TNF, IL-6, and PTGS2 were predicted as Hub genes, which were significantly associated with ferroptosis-related pathways, including unsaturated fatty acid biosynthesis and ROS metabolic process regulation. Compared to the control group [(100.00±1.44)%], LPS significantly reduced HUVEC viability [(66.77±1.80)%], which could be ameliorated by Fer-1 [(84.50±1.47)%] (0.05). The ROS fluorescence intensity in the LPS group (1 523.00±250.70) was significantly higher than in the control (328.20±38.68) or LPS+Fer-1 (753.30±67.11) group (all 0.05). The Fe²⁺, LPO, and MDA levels in the LPS group [(29.83±4.25) μmol/10 cells, (3.58±0.24) μmol/gprot, (5.54±0.33) μmol/gprot, respectively] were significantly higher than both the control group [(7.29±0.79) μmol/10 cells, (1.08±0.05) μmol/gprot, (2.06±0.17) μmol/gprot] and the LPS+Fer-1 group [(16.33±1.63) μmol/10 cells, (2.01±0.09) μmol/gprot, (3.24±0.26) μmol/gprot]. Furthermore, the GSH/GSSG ratio in the LPS group (2.17±0.08) was considerably lower than both the control group (6.96±0.20) and the LPS+Fer-1 group (4.31±0.81) (all 0.05). The JC-1 aggregate/monomer fluorescence intensity ratio of the LPS group (0.46±0.07) was markedly lower than the control group (285.60±160.40), while Fer-1 pretreatment (1.53±0.17) obviously mitigated this decrease (all 0.05). SLC7A11, SLC3A2, and GPX4 protein and mRNA expression levels in the LPS group were dramatically lower than both the control group and the Fer-1+LPS group (0.05). The mRNA expression levels of TNF, IL-6, and PTGS2 in the LPS group were strongly upregulated compared to the control group, and the expressions of these three factors in the LPS+Fer-1 group were significantly lower than those in the LPS group (all 0.05). Pg drives ferroptosis in vascular endothelial cells, with TNF, IL-6, and PTGS2 identified as the potential novel Hub genes in this process.