激活核因子E2相关因子2信号通路抑制高糖诱导人肾小管上皮细胞上皮-间充质转化

doi:10.3877/cma.j.issn.2095-3216.2026.01.003

目的

探讨核因子E2相关因子2(nuclear factor-E2-related factor 2，Nrf2)在高糖诱导人肾小管上皮细胞(human kidney-2, HK-2)间充质转化中的作用。

方法

培养的HK-2细胞分组：正常糖组(NG组，5.5 mmol/L葡萄糖)、高糖组(HG组，30 mmol/L葡萄糖)、高糖加Nrf2 siRNA转染组(HG＋siNrf2组)、高糖加阴性对照siRNA转染组(HG＋NC组)、高糖加N-乙酰半胱氨酸组(HG＋NAC组)、高糖加叔丁基对苯二酚组(HG＋TBHQ组)。应用Western印迹、实时荧光定量PCR和免疫荧光方法检测α-平滑肌肌动蛋白、纤维连接蛋白等表达变化，使用试剂盒检测细胞及线粒体活性氧水平。

结果

相较于NG组，HG组的α-平滑肌肌动蛋白表达较高。与HG组相比，HG＋TBHQ组、HG＋NAC组的α-平滑肌肌动蛋白、纤维连接蛋白的蛋白及mRNA表达则均降低(P均<0.05)；与HG＋NC组相比，HG＋siNrf2组的α-平滑肌肌动蛋白、纤维连接蛋白的蛋白及mRNA表达均较高(P均<0.05)。与NG组相比，HG组的细胞和线粒体活性氧水平均较高(P均<0.05)；与HG组相比，HG＋TBHQ组和HG＋NAC组的细胞和线粒体活性氧水平均较低(P均<0.05)。

结论

激活Nrf2信号通路能够抑制高糖诱导人肾小管上皮细胞的活性氧生成和上皮-间充质转化。

关键词: 高糖, 人肾小管上皮细胞, 氧化应激, 核因子E2相关因子2, 上皮-间充质转化

Objective

To investigate the role of nuclear factor-E2-related factor 2 (Nrf2) in high glucose-induced epithelial-mesenchymal transition (EMT) in human renal tubular epithelial cells (human kidney-2, HK-2).

Methods

Cultured HK-2 cells were divided into: normal glucose group (NG group, 5.5 mmol/L glucose), high glucose group (HG group, 30 mmol/L glucose), high glucose plus Nrf2 siRNA transfection group (HG+ siNrf2 group), high glucose plus negative control siRNA transfection group (HG+ NC group), high glucose plus N-acetylcysteine group (HG+ NAC group), and high glucose plus tert-butylhydroquinone group (HG+ TBHQ group). The expression levels of α-smooth muscle actin and fibronectin were examined using Western blotting, quantitative real-time PCR, and immunofluorescence. Intracellular and mitochondrial reactive oxygen species (ROS) levels were measured with specific assay kits.

Results

Compared with the NG group, α-smooth muscle actin expression was significantly upregulated in the HG group. In contrast, the protein and mRNA expression levels of α-smooth muscle actin and fibronectin were markedly downregulated in the HG+ TBHQ and HG+ NAC groups compared to the HG group (all P<0.05). Furthermore, both the protein and mRNA expression levels of α-smooth muscle actin and fibronectin were significantly higher in the HG+ siNrf2 group than in the HG+ NC group (all P<0.05). Compared with the NG group, both intracellular and mitochondrial ROS levels were significantly higher in the HG group (all P<0.05). In contrast, the levels of intracellular and mitochondrial ROS were markedly lower in the HG+ TBHQ and HG+ NAC groups compared to the HG group (all P<0.05).

Conclusion

Activation of the Nrf2 signaling pathway inhibited high glucose-induced ROS production and EMT in human renal tubular epithelial cells.

Key words: High glucose, Human renal tubular epithelial cells, Oxidative stress, Nuclear factor-E2-related factor 2, Epithelial-mesenchymal transition

表1 引物序列

检测指标	上游引物序列(5′-3′)	下游引物序列(5′-3′)
Nrf2	5′-TCAGCG ACGGAAAGAGTATGA-3′	5′-CCACTGGTTTCTGACTGGATGT-3′
α-SMA	5′-TGCACAAACGAGGGGAATATAT-3′	5′-CGTTCCTTGTAGCCAATAAAGG-3′
FN	5′-TGGACCAAATCTCCACGGTCT-3′	5′-CTCTCCCCAGGTGATGCCTT-3′
β-actin	5′-TGACGTGGACATCCGCAAAG-3′	5′-CTGGAAGGTGGACAGCGAGG-3′

表1 引物序列

检测指标	上游引物序列(5′-3′)	下游引物序列(5′-3′)
Nrf2	5′-TCAGCG ACGGAAAGAGTATGA-3′	5′-CCACTGGTTTCTGACTGGATGT-3′
α-SMA	5′-TGCACAAACGAGGGGAATATAT-3′	5′-CGTTCCTTGTAGCCAATAAAGG-3′
FN	5′-TGGACCAAATCTCCACGGTCT-3′	5′-CTCTCCCCAGGTGATGCCTT-3′
β-actin	5′-TGACGTGGACATCCGCAAAG-3′	5′-CTGGAAGGTGGACAGCGAGG-3′

图1 高糖对HK-2细胞Nrf2信号通路相关因子的影响注：Nrf2: nuclear factor-E2-related factor 2,核因子E2相关因子2；HO-1: hemeoxygenase-1,血红素氧合酶-1；β-肌动蛋白为内参照；A、B和C：Western印迹法检测各组HK-2细胞中Nrf2、HO-1蛋白表达及比较；D：qPCR法检测各组HK-2细胞中Nrf2 mRNA表达水平；与NG组相比，^aP<0.05

图2 高糖对HK-2细胞上皮-间充质转化相关标志物表达的影响注：α-SMA: α-smooth muscle actin，α-平滑肌肌动蛋白；p-Smad3：磷酸化Smad3；TGF-β1: transforming growth factor-β1，转化生长因子-β1；FN: fibronectin,纤维连接蛋白；A：Western印迹法检测各组HK-2细胞中α-SMA、p-Smad3、TGF-β1、FN蛋白表达(β-肌动蛋白为内参照)；B、C、D和E：各组HK-2细胞中α-SMA、p-Smad3、TGF-β1、FN蛋白相对表达的比较；F和G：qPCR法检测各组HK-2细胞中α-SMA的mRNA表达水平；H：免疫荧光法检测HK-2细胞中α-SMA、TGF-β1的表达(绿色荧光×400)；与NG组相比，^aP<0.05

图3 抑制Nrf2基因表达对高糖诱导HK-2细胞上皮-间充质转化的影响注：α-SMA: α-smooth muscle actin, α-平滑肌肌动蛋白；FN: fibronectin,纤维连接蛋白；A：Western印迹法检测各组HK-2细胞中α-SMA、FN蛋白的表达；B和C：各组HK-2细胞中α-SMA、FN蛋白蛋白相对表达量的比较(β-肌动蛋白为内参照)；D和E：qPCR法检测各组HK-2细胞中α-SMA、FN的mRNA表达水平的比较；与NG组相比，^aP<0.05；与HG＋NC组相比，^bP<0.05

图4 激活Nrf2信号通路对高糖诱导HK-2细胞上皮-间充质转化的影响注：ROS: reactive oxygen species,活性氧；Nrf2: nuclear factor-E2-related factor 2,核因子E2相关因子2；α-SMA: α-smooth muscle actin，α-平滑肌肌动蛋白；FN: fibronectin,纤维连接蛋白；A、B和C：细胞内及线粒体ROS检测及比较；D、E、F和G：Western印迹法检测各组HK-2细胞中Nrf2、α-SMA、FN蛋白的表达及比较(β-肌动蛋白为内参照)；H、I和J：qPCR法检测各组HK-2细胞中Nrf2、α-SMA、FN的mRNA表达水平比较；K：免疫荧光法检测HK-2细胞中α-SMA的表达(绿色荧光×400)；与NG组相比，^aP<0.05；与HG组相比，^bP<0.05

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Activation of nuclear factor-E2-related factor 2 signaling pathway inhibited high glucose-induced epithelial-mesenchymal transition in human renal tubular epithelial cells

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Activation of nuclear factor-E2-related factor 2 signaling pathway inhibited high glucose-induced epithelial-mesenchymal transition in human renal tubular epithelial cells