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| SET8通过Keap1-Nrf2/HO-1信号通路抑制LPS诱导的BEAS-2B细胞炎症和氧化应激 |
| SET8 inhibited inflammation and oxidative stress of LPS-induced BEAS-2B via Keap1-Nrf2/HO-1 signaling pathway |
| 投稿时间：2024-05-23 |
| DOI：10.3969/j.issn.1000-0399.2025.03.005 |
| 中文关键词: 赖氨酸甲基转移酶8 急性肺损伤 炎症 氧化应激 |
| 英文关键词: Lysine methyltransferase 8 Acute lung injury Inflammation Oxidative stress |
| 基金项目:国家自然科学基金项目(编号:81800076) |
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| 摘要点击次数: 143 |
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| 中文摘要: |
| 目的 探究赖氨酸甲基转移酶8(SET8)在急性肺损伤（ALI）中的作用和可能的机制。方法 购得人正常肺上皮细胞（BEAS-2B）随机分为正常组（不做任何处理）、脂多糖（LPS）组（5 mg/L LPS处理24 h）、LPS+pc-NC组（转染pc-DNA3.1质粒+LPS处理）、LPS+pc-SET8组（转染pcDNA3.1-SET8质粒+LPS处理）、LPS+pc-SET8+ML385组[转染pcDNA3.1-SET8质粒+LPS和核因子E2相关因子2(Nrf2)抑制剂ML385处理]和LPS+pc-SET8+SnPP组[转染pcDNA3.1-SET8+LPS和血红素氧合酶1(HO-1)抑制剂锡原卟啉IX(SnPP)处理]。实时荧光定量聚合酶链反应（RT-qPCR）分析SET8在LPS诱导的BEAS-2B细胞中的表达水平。细胞计数试剂盒8(CCK-8)法检测各组细胞活力。酶联免疫吸附法（ELISA）试剂盒检测各组细胞上清液中白细胞介素-1β(IL-1β)、IL-6和肿瘤坏死因子α(TNF-α)水平。采用超氧化物歧化酶（SOD）试剂盒、谷胱甘肽过氧化物酶（GSH-Px）试剂盒和丙二醛（MDA）试剂盒检测细胞中SOD、GSH-Px和MDA水平。2',7'-二氯荧光素二乙酸酯（DCFH-DA）荧光探针检测细胞中活性氧（ROS）。蛋白免疫印迹（WB）检测SET8、KELCH样ECH关联蛋白1(Keap1)、Nrf2和HO-1蛋白表达水平。结果 与0 mg/L LPS组相比，SET8在不同浓度LPS诱导的BEAS-2B细胞中均表达下调（P<0.01）。与LPS+pc-NC组比较，LPS+pc-SET8组细胞上清液中IL-1β、IL-6和TNF-α水平降低（P<0.01）,ROS和MDA水平降低（P<0.01），而SOD和GSH-Px水平明显升高（P<0.01）。此外，LPS+pc-SET8组中Keap1蛋白表达水平明显降低（P<0.01）,Nrf2和HO-1蛋白表达明显增加，细胞核中Nrf2蛋白表达减少（P<0.01）。与LPS+pc-SET8组比较，LPS+pc-SET8+ML385和LPS+pc-SET8+SnPP组炎症因子IL-1β、IL-6和TNF-α水平明显升高（P<0.01）,ROS和MDA水平显著升高（P<0.01），而SOD和GSH-Px水平明显降低（P<0.01）。结论 SET8通过Keap1-Nrf2/HO-1信号通路抑制LPS诱导的BEAS-2B细胞炎症响应和氧化应激，对LPS诱导的BEAS-2B的细胞损伤有保护作用。 |
| 英文摘要: |
| Objective To explore the role and potential mechanism of lysine methyltransferase 8（SET8） in acute lung injury（ALI）. Methods Human normal lung epithelial cells（BEAS-2B） were divided into normal group（no treatment）, lipopolysaccharide（LPS） group（5mg/L LPS treatment 24 h）, LPS + pc-NC group（transfected pc-DNA3.1 plasmid + LPS treatment）, LPS + pc-SET8 group（transfected pcDNA3.1-SET8 plasmid + LPS treatment）, LPS + pc-SET8 + ML385 group（transfected pcDNA3.1-SET8 plasmid + LPS and nuclear factor E2-related factor 2（Nrf2） inhibitor ML385 treatment） and LPS + pc-SET8 + tin protoporphyrin Ⅸ（SnPP） group（transfected pcDNA3.1-SET8+ LPS and heme oxygenase-1（HO-1） inhibitor SnPP treatment）. Real-time quantitative PCR was used to analyze the expression level of SET8in LPS-induced BEAS-2B cells. Cell counting kit 8（CCK-8） was used to measure cell viability. Enzyme-linked immunosorbent assay（ELISA） kit was employed to detect the levels of interleukin-1β（IL-1β）, IL-6 and tumor necrosis factor-α（TNF-α） in supernatant of cells in each group. Superoxide dismutase（SOD） kit, glutathione peroxidase（GSH-Px） kit and malondialdehyde（MDA） were used to detect the levels of SOD, GSH-Px and MDA in cells. 2’7’-Dichlorofluorescin diacetate（DCFH-DA） fluorescent probe was used to analyze the level of reactive oxygen species（ROS） in cells. Western blotting was utilized to determine the protein expression levels of SET8, Kelch-like ECH associated protein 1（Keap1）, Nrf2 and HO-1. Results Compared with the 0 mg/L LPS group, the expression of SET8 was down-regulated in BEAS-2B cells induced by different concentrations of LPS（P＜0.01）. Compared with LPS + pc-NC group, the levels of IL-1β, IL-6 and TNF-α in cell supernatant were reduced（P＜0.01）, ROS and MDA levels also decreased（P＜0.01）, and the levels of SOD and GSH-Px significant increased（P＜0.01） in LPS + pc-SET8 group. In addition, the Keap1 protein level was reduced, and the Nrf2 and HO-1 protein levels significantly increased and the Nrf2 protein level decreased in nucleus（P＜0.01） in LPS + pc-SET8 group. Compared with LPS + pc-SET8 group, the levels of inflammatory cytokines IL-1β, IL-6 and TNF-α significantly increased（P＜0.01）, and the levels of ROS and MDA markedly increased（P＜0.01）, and the SOD and GSH-Px levels significantly decreased（P＜0.01） in LPS + pc-SET8 + ML385 and LPS + pc-SET8 + SnPP groups. Conclusion SET8 significantly inhibits the inflammatory response and oxidative stress in LPS-stimulated BEAS-2B cells via the Keap1-Nrf2/HO-1 signaling pathway, thus exerting a protective effect on LPS-induced BEAS-2B cell injury. |
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