[牙龈卟啉单胞菌诱导血管内皮细胞铁下垂的作用及机制研究]。

Q4 Medicine
Q Li, C Lu, J Lin
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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). <b>Results:</b> 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)%] (<i>P<</i>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 <i>P<</i>0.05). The Fe²⁺, LPO, and MDA levels in the LPS group [(29.83±4.25) μmol/10<sup>6</sup> 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<sup>6</sup> 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<sup>6</sup> 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 <i>P<</i>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 <i>P<</i>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 (<i>P<</i>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 <i>P<</i>0.05). <b>Conclusions:</b> Pg drives ferroptosis in vascular endothelial cells, with TNF, IL-6, and PTGS2 identified as the potential novel Hub genes in this process.</p>","PeriodicalId":23965,"journal":{"name":"中华口腔医学杂志","volume":"60 9","pages":"1008-1018"},"PeriodicalIF":0.0000,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"[Investigation of the role and mechanism of <i>Porphyromonas gingivalis</i> in inducing ferroptosis in vascular endothelial cells].\",\"authors\":\"Q Li, C Lu, J Lin\",\"doi\":\"10.3760/cma.j.cn112144-20250420-00145\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p><b>Objective:</b> To investigate whether <i>Porphyromonas gingivalis</i> (Pg) induces ferroptosis in vascular endothelial cells and predict the Hub genes. <b>Methods:</b> 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. 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引用次数: 0

摘要

目的:探讨牙龈卟啉单胞菌(Porphyromonas gingivalis, Pg)是否会诱导血管内皮细胞铁下垂并预测Hub基因的表达。方法:首先用Pg (W83)刺激人脐静脉内皮细胞(HUVEC) 4 h,透射电镜观察其凋亡相关形态学特征。随后,从Pg刺激前后的HUVEC中提取RNA进行转录组测序(RNA-seq)。进行富集分析以确定差异表达基因(DEG)是否与铁下垂有关。鉴定出与凋亡相关的DEG (fe -DEG),然后进行基因本体(GO)功能注释、京都基因与基因组百科全书(KEGG)途径富集分析、蛋白-蛋白相互作用(PPI)网络构建和Hub基因预测。接下来,根据RNA-seq结果,用脂多糖(LPS)刺激HUVEC 24小时。检测已建立的铁下垂标志物。检测指标及方法:细胞计数试剂盒-8测定细胞存活率;DCFH-DA探针检测活性氧(ROS);Fe 2 +、脂质过氧化物(LPO)、丙二醛(MDA)、还原性/氧化性谷胱甘肽比值(GSH/GSSG)(商用试剂盒);JC-1探针检测线粒体膜电位(MMP);Western blotting (WB)和实时荧光定量PCR (RT-qPCR)检测溶质载体家族7成员11 (SLC7A11)、溶质载体家族3成员2 (SLC3A2)和谷胱甘肽过氧化物酶4 (GPX4)的表达。最后,采用RT-qPCR验证LPS刺激24 h后HUVEC中预测的Hub基因的表达,包括肿瘤坏死因子(TNF)或TNF-α、白细胞介素(IL)-6和前列腺素内过氧化物合成酶2 (PTGS2)。结果:pg刺激的HUVEC细胞线粒体大小减小,嵴丢失。pg感染组与对照组之间HUVEC的DEG在铁下垂途径中富集,从中鉴定出56个fe -DEG。GO分析显示在TNF、LPS、生物刺激等响应中富集,KEGG分析显示在TNF、c型凝集素受体、IL-17信号通路等中富集。在56个基因的PPI网络中,TNF、IL-6和PTGS2被预测为Hub基因,这些基因与凋亡相关的途径,包括不饱和脂肪酸生物合成和ROS代谢过程调控显著相关。与对照组[(100.00±1.44)%]相比,LPS显著降低HUVEC活力[(66.77±1.80)%],而fe -1可改善HUVEC活力[(84.50±1.47)%](P0.05)。LPS组ROS荧光强度(1 523.00±250.70)显著高于对照组(328.20±38.68)或LPS+Fer-1组(753.30±67.11)(均P0.05)。LPS组Fe +、LPO和MDA水平[分别为(29.83±4.25)μmol/106、(3.58±0.24)μmol/gprot、(5.54±0.33)μmol/gprot]显著高于对照组[(7.29±0.79)μmol/gprot、(1.08±0.05)μmol/gprot、(2.06±0.17)μmol/gprot]和LPS+Fer-1组[(16.33±1.63)μmol/106、(2.01±0.09)μmol/gprot、(3.24±0.26)μmol/gprot]。LPS组GSH/GSSG比值(2.17±0.08)显著低于对照组(6.96±0.20)和LPS+ fe -1组(4.31±0.81)(均p < 0.05)。LPS组JC-1聚集体/单体荧光强度比(0.46±0.07)明显低于对照组(285.60±160.40),而fe -1预处理(1.53±0.17)明显减轻了这种下降(均P0.05)。LPS组SLC7A11、SLC3A2、GPX4蛋白和mRNA表达量均显著低于对照组和fer1 +LPS组(P0.05)。LPS组TNF、IL-6、PTGS2 mRNA表达量较对照组显著上调,且LPS+Fer-1组这3个因子的表达量显著低于LPS组(均P0.05)。结论:Pg驱动血管内皮细胞铁下垂,TNF、IL-6和PTGS2被认为是这一过程中潜在的新型枢纽基因。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
[Investigation of the role and mechanism of Porphyromonas gingivalis in inducing ferroptosis in vascular endothelial cells].

Objective: To investigate whether Porphyromonas gingivalis (Pg) induces ferroptosis in vascular endothelial cells and predict the Hub genes. Methods: 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). Results: 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)%] (P<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 P<0.05). The Fe²⁺, LPO, and MDA levels in the LPS group [(29.83±4.25) μmol/106 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/106 cells, (1.08±0.05) μmol/gprot, (2.06±0.17) μmol/gprot] and the LPS+Fer-1 group [(16.33±1.63) μmol/106 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 P<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 P<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 (P<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 P<0.05). Conclusions: Pg drives ferroptosis in vascular endothelial cells, with TNF, IL-6, and PTGS2 identified as the potential novel Hub genes in this process.

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来源期刊
中华口腔医学杂志
中华口腔医学杂志 Medicine-Medicine (all)
CiteScore
0.90
自引率
0.00%
发文量
9692
期刊介绍: Founded in August 1953, Chinese Journal of Stomatology is a monthly academic journal of stomatology published publicly at home and abroad, sponsored by the Chinese Medical Association and co-sponsored by the Chinese Stomatology Association. It mainly reports the leading scientific research results and clinical diagnosis and treatment experience in the field of oral medicine, as well as the basic theoretical research that has a guiding role in oral clinical practice and is closely combined with oral clinical practice. Chinese Journal of Over the years, Stomatology has been published in Medline, Scopus database, Toxicology Abstracts Database, Chemical Abstracts Database, American Cancer database, Russian Abstracts database, China Core Journal of Science and Technology, Peking University Core Journal, CSCD and other more than 20 important journals at home and abroad Physical medicine database and retrieval system included.
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