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    PM2.5诱导HK-2细胞损伤的氧化应激-炎症互作机制研究

    Oxidative stress-inflammatory crosstalk mechanism of PM2.5 exposure-induced HK-2 cell injury

      摘要
    • 摘要:
      目的 探讨大气细颗粒物(PM2.5)对人肾小管上皮细胞(HK-2细胞)氧化应激与炎症反应的影响。
      方法 以HK-2细胞为模型, 以不同剂量PM2.5进行24 h染毒。采用吸光度法检测细胞活性; 利用高内涵成像技术测定细胞内活性氧(ROS)水平; 通过酶联免疫法检测超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽(GSH)、白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)含量。结合转录组测序筛选氧化应激与炎症通路, 并采用qRT-PCR验证关键基因(SOD1、SOD2、NOX4、CYP1A1、CYP27A1、CCL2、IL-6、TNF-α)及KIM-1和NGAL的mRNA水平。
      结果 与含0.1% DMSO的对照组相比, PM2.5染毒24 h后, HK-2细胞活性呈剂量依赖性下降。肾损伤标志物KIM-1和NGAL的mRNA表达呈剂量依赖性上升。ROS、SOD、MDA水平随PM2.5剂量增加而上升, 而GSH含量下降, 氧化应激相关基因SOD1、SOD2、NOX4、CYP1A1、CYP27A1上调; 在加入人参皂苷(Rh1)干预剂后, 氧化应激指标恢复。炎症因子IL-6、TNF-α及其相关基因CCL2、IL-6、TNF-α呈剂量依赖性升高; 在加入N-乙酰半胱氨酸(NAC)干预剂后, 炎症因子有所恢复。KEGG通路主要富集于TNF、IL-17信号通路等, 与氧化应激、炎症反应密切相关。
      结论 PM2.5可引起氧化应激和炎症损伤互作, 提示该双重通路可能在PM2.5介导的肾损伤过程中发挥关键调控作用。

       

      Abstract:
      Objective To investigate the effects of fine particulate matter (PM2.5) on oxidative stress and inflammatory responses in human renal tubular epithelial cells (HK-2 cells).
      Methods HK-2 cells were used as an in vitro model and exposed to different concentrations of PM2.5 for 24 h. Cell viability was assessed by absorbance-based assays. Intracellular reactive oxygen species (ROS) levels were quantified using high-content imaging. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay. Transcriptome sequencing analysis was performed to identify oxidative stress- and inflammation-related pathways, and quantitative reverse transcription polymerase chain reaction was employed to validate the mRNA expression levels of key genes (SOD1, SOD2, NOX4, CYP1A1, CYP27A1, CCL2, IL-6, and TNF-α) as well as the renal injury markers KIM-1 and NGAL.
      Results Compared with the control group containing 0.1% DMSO, HK-2 cell viability showed a dose-dependent decrease after 24 h of PM2.5 exposure. The mRNA expression levels of the renal injury markers KIM-1 and NGAL increased in a dose-dependent manner. With the increase in PM2.5 concentration, the levels of ROS, SOD, and MDA were elevated, while the GSH level decreased. Meanwhile, the mRNA expression of oxidative stress-related genes, including SOD1, SOD2, NOX4, CYP1A1, and CYP27A1, was upregulated. After intervention with ginsenoside Rh1, the oxidative stress indicators were restored. Inflammatory cytokines IL-6 and TNF-α, as well as their related genes CCL2, IL-6, and TNF-α, were elevated in a dose-dependent manner. This inflammatory response was attenuated following intervention with N-acetylcysteine. KEGG pathway enrichment analysis revealed significant enrichment in TNF and IL-17 signaling pathways, which are closely associated with oxidative stress and inflammatory responses.
      Conclusion PM2.5 exposure can induce interactive oxidative stress and inflammatory injury, suggesting that the dual pathways may play a key regulatory role in PM2.5-mediated renal injury.

       

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