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核呼吸因子1调控NF-κB介导LPS诱导的小鼠急性肺损伤

程康, 朱俐, 王雪婷^*

南通大学特种医学研究院，南通 226019

摘要

本文旨在研究核呼吸因子1 (nuclear respiratory factor 1, NRF1)对脂多糖(lipopolysaccharide, LPS)诱导的肺上皮细胞炎症应答中关键分子核因子-κB (nuclear factor kappa B, NF-κB)的影响，以阐明NRF1对肺上皮细胞炎性应答的调控作用及机制。在体内水平上，雄性BALB/c小鼠经呼吸道转染NRF1小干扰RNA，LPS (4 mg/kg)或生理盐水经呼吸道雾化给药，48 h后取肺组织。采用免疫印迹(Western blot, WB)及real-time PCR法检测肺组织NRF1、NF-κB p65及其靶基因表达变化，免疫荧光染色法检测NRF1及NF-κB p65核转位情况。体外培养L132肺上皮细胞，转染NRF1小干扰RNA，或给予BAY 11-7082 (5 μmol/L)处理24 h后，给予1 mg/L LPS刺激6 h，用real-time PCR法检测NRF1、NF-κB p65及其靶基因表达变化。采用染色质免疫沉淀技术(chromatin immunoprecipitation assay, ChIP)验证NRF1的靶基因RELA (NF-κB p65编码基因)。结果显示：LPS处理组小鼠肺组织或L132细胞NRF1表达水平明显上升，炎症因子p65及其磷酸化水平显著升高，靶基因白介素-1β (interleukin-1β, IL-1β)与白介素-6 (IL-6)显著上升。肺组织NRF1与p65均发生明显的核转位现象。在体内水平干扰NRF1后，上述症状显著减轻。在细胞水平干扰NRF1的表达后，p65表达水平显著下调，且NF-κB抑制剂BAY 11-7082有效抑制了LPS诱导的炎症反应，但对NRF1表达无影响。ChIP实验证实，NRF1可有效结合p65编码基因RELA的启动子区。上述结果提示，NRF1有可能介导了LPS诱导的急性肺损伤，其机制为：NRF1转录调控NF-κB p65，并参与LPS诱导的肺上皮细胞炎症。 

关键词： 核呼吸因子1; 转录调控; NF-κB; 急性肺损伤; 肺上皮炎症

Nuclear respiratory factor 1 mediates LPS-induced acute lung injury through NF-κB

CHENG Kang, ZHU Li, WANG Xue-Ting^*

Institute of Special Environmental Medicine, Nantong University, Nantong 226019, China

Abstract

The purpose of this paper was to study the transcriptional regulation of nuclear respiratory factor 1 (NRF1) on nuclear factor kappa B (NF-κB), a key molecule in lipopolysaccharide (LPS)-induced lung epithelial inflammation, and to clarify the mechanism of NRF1-mediated inflammatory response in lung epithelial cells. In vivo, male BALB/c mice were treated with NRF1 siRNA, followed with LPS (4 mg/kg) or 0.9% saline through respiratory tract, and sacrificed 48 h later. Expression levels of NRF1, NF-κB p65 and its target genes were detected by Western blot and real-time PCR. Nuclear translocation of NRF1 or p65 was measured by immunofluorescent technique. In vitro, L132 cells were transfected with NRF1 siRNA or treated with BAY 11-7082 (5 μmol/L) for 24 h, followed with treatment of 1 mg/L LPS for 6 h. Cells were lysed for detections of NRF1, NF-κB p65 and its target genes as well as the binding sites of NRF1 on RELA (encoding NF-κB p65) promoter by chromatin immunoprecipitation assay (ChIP). Results showed that LPS stimulated NRF1 and NF-κB p65. Pro-inflammatory factors including interleukin-1β (IL-1β) and IL-6 were significantly increased both in vivo and in vitro. Obvious nuclear translocations of NRF1 and p65 were observed in LPS-stimulated lung tissue. Silencing NRF1 resulted in a decrease of p65 and its target genes both in vivo and in vitro. In addition, BAY 11-7082, an inhibitor of NF-κB, significantly repressed the inflammatory responses induced by LPS without affecting NRF1 expression. Furthermore, it was proved that NRF1 had three binding sites on RELA promoter region. In summary, NRF1 is involved in LPS-mediated acute lung injury through the transcriptional regulation on NF-κB p65.

Key words: nuclear respiratory factor 1; transcriptional regulation; NF-κB; acute lung injury; lung epithelial inflammation

收稿日期：　　录用日期：

通讯作者：王雪婷　　E-mail: wangxueting@ntu.edu.cn

DOI: 10.13294/j.aps.2021.0084

引用本文：

程康, 朱俐, 王雪婷. 核呼吸因子1调控NF-κB介导LPS诱导的小鼠急性肺损伤[J]. 生理学报 2022; 74 (3): 401-410.

CHENG Kang, ZHU Li, WANG Xue-Ting. Nuclear respiratory factor 1 mediates LPS-induced acute lung injury through NF-κB. Acta Physiol Sin 2022; 74 (3): 401-410 (in Chinese with English abstract).