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LV-NPHP1-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,NPHP1KDHK2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录因子.
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.
基金项目
广东省自然科学基金(2022A1515012307)
广东省自然科学基金(2020A1515010286)
北京白求恩公益基金会项目(SCE093DS)
南方医科大学南方医院院长基金青年项目(2020C021)
国家科技期刊平台
NSTL
维普
万方数据