南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (1): 156-165.doi: 10.12122/j.issn.1673-4254.2024.01.18

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C/EBPβ介导NPHP1敲低的肾小管上皮细胞TNF-α通路下游炎症因子的表达

黄丹梅，刘雅清，李丹彤，张静兰，杨翌晨，孙良忠   

  1. 南方医科大学南方医院儿科，广东 广州 510515
  • 发布日期:2024-01-19

C/EBPβ mediates expressions of downstream inflammatory factors of the tumor necrosis factor-α signaling pathway in renal tubular epithelial cells with NPHP1 knockdown

HUANG Danmei, LIU Yaqing, LI Dantong, ZHANG Jinglan, YANG Yichen, SUN Liangzhong   

  1. Department of Pediatrics, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
  • Published:2024-01-19

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摘要： 目的 探讨NPHP1基因缺陷肾小管上皮细胞TNF-α信号通路激活及其调控的炎症因子表达。方法 使用重组慢病毒LVNPHP1-RNAi构建敲低NPHP1表达的人近端肾小管细胞株（HK2）细胞（NPHP1KD HK2）。通过实时荧光定量PCR、Western blot、酶联免疫吸附实验法检测各细胞株TNF-α表达、p38和C/EBPβ激活状态及其调控炎症因子CXCL5、CCL20、IL-1β、IL-6、MCP-1等表达情况。通过构建siRNA敲低野生型和NPHP1KD HK2细胞C/EBPβ表达，观察上述指标变化。结果 敲低NPHP1表达后，NPHP1KD HK2细胞TNF-α、C/EBPβ、CXCL5、IL-1β和IL-6的mRNA表达量增加（P<0.05）；Western blotting结果显示，phospho-p38、C/EBPβ表达上调（P<0.05）；培养液上清IL-6水平增加（P<0.05）。使用siRNA敲低C/EBPβ表达后，NPHP1KDHK2细胞CSF2、CCL20、IL-1β和IL-6的mRNA表达水平下调（P<0.05）；Western blot显示phospho-p38表达下调（P<0.05）；培养液上清IL-6水平下降（P<0.001）。结论 NPHP1基因缺陷的NPHP1KDHK2细胞中TNF-α信号通路被激活，其调控的下游印证因子表达上调。C/EBPβ可能是介导NPHP1KDHK2细胞TNF-α信号通路相关炎症因子表达的关键转

关键词: 肾单位肾痨；TNF-α；C/EBPβ；炎症因子；NPHP1

Abstract: Objective To explore the activation of tumor necrosis factor-α (TNF-α) signaling pathway and the expressions of the associated inflammatory factors in NPHP1-defective renal tubular epithelial cells. Methods A human proximal renal tubular cell (HK2) model of lentivirus-mediated NPHP1 knockdown (NPHP1KD) was constructed, and the expressions of TNF-α, p38, and C/EBPβ and the inflammatory factors CXCL5, CCL20, IL-1β, IL-6 and MCP-1 were detected using RT-qPCR, Western blotting or enzyme-linked immunosorbent assay. A small interfering RNA (siRNA) was transfected in wild-type and NPHP1KDHK2 cells, and the changes in the expressions of TNF-α, p38, and C/EBPβ and the inflammatory factors were examined. Results NPHP1KDHK2 cells showed significantly increased mRNA expressions of TNF-α, C/EBPβ, CXCL5, IL-1β, and IL-6 (P<0.05), protein expressions of phospho-p38 and C/EBPβ (P<0.05), and IL-6 level in the culture supernatant (P<0.05), and these changes were significantly blocked by transfection of cells with siRNA-C/EBPβ (P<0.05). Conclusion TNF-α signaling pathway is activated and its associated inflammatory factors are upregulated in NPHP1KDHK2 cells, and C/EBPβ may serve as a key transcription factor to mediate these changes.

Key words: nephronophthisis; tumor necrosis factor-α; C/EBPβ; inflammatory factor; NPHP1