蛇床子素对高糖缺氧复氧诱导的肾小管上皮细胞损伤及Nrf2/HO-1信号通路的影响

    Effects of osthole on renal tubular epithelial cell injury and Nrf2/HO-1 signal pathway induced by high glucose, hypoxia and reoxygenation

      摘要
    • 摘要: 目的 探讨蛇床子素对高糖缺氧复氧诱导的肾小管上皮细胞损伤及Nrf2/HO-1信号通路的影响。方法 以高糖培养液培养人肾小管上皮细胞HK-2，并进行缺氧复氧处理建立细胞模型，CCK-8实验检测0、10、20、40、80、160 μg/mL浓度蛇床子素对其生长的影响，筛选最佳作用浓度。将HK-2细胞分为对照组、模型组、蛇床子素（80 μg/mL）组、ML385（Nrf2抑制剂，20 nmol/mL）组、蛇床子素（80 μg/mL）+ML385 （20 nmol/mL）组，除对照组外其余各组以高糖缺氧复氧诱导建立细胞模型，以药物分组处理，CCK-8实验检测各组细胞活力；试剂盒测定各组细胞活性氧（reactive oxygenspecies，ROS）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）、丙二醛（malondialdehyde，MDA）水平及各组细胞释放乳酸脱氢酶（lactate dehydrogenase，LDH）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-6（interleukin-6，IL-6）、白细胞介素-1β（interleukin-1β，IL-1β）水平；免疫荧光实验检测各组细胞Nrf2/HO-1通路蛋白表达；蛋白质印迹法检测各组细胞Nrf2/HO-1通路蛋白表达。结果 与对照组相比，模型组HK-2细胞活力（100±0比43.47±6.14）、细胞GSH-Px水平（9.53±1.64比3.25±0.32）、细胞Nrf2（1.78±0.30比0.89±0.12）蛋白表达明显降低（P均<0.05），HK-2细胞ROS （1.13±0.17比6.52±1.03）及TNF-α水平明显升高（P均<0.05）。与模型组、蛇床子素+ML385组分别相比，蛇床子素组HK-2细胞活力（43.47±6.14、44.83±7.06比87.76±9.42）、细胞GSH-Px水平（3.25±0.32、3.27±0.38比9.07±1.78）、细胞Nrf2（0.89±0.12、0.90±0.18比1.74±0.26）蛋白表达升高（P均<0.05），HK-2细胞ROS（6.52±1.03、6.47±0.96比1.59±0.21）及TNF-α水平降低（P均<0.05）； ML385组HK-2细胞活力（43.47±6.14、44.83±7.06比24.80±3.79）、细胞GSH-Px水平（3.25±0.32、3.27±0.38比0.88±0.15）、细胞Nrf2（0.89±0.12、0.90±0.18比0.30±0.05）蛋白表达降低（P均<0.05），HK-2细胞ROS（6.52±1.03、6.47±0.96比9.76±2.02）及TNF-α水平升高（P均<0.05）。结论 蛇床子素可上调Nrf2/HO-1信号通路蛋白表达，减少高糖缺氧复氧诱导的ROS和炎性因子生成，进而抑制氧化应激及炎症反应，减轻肾小管上皮细胞损伤。

       

      Abstract: Objective To explore the effects of osthole on renal tubular epithelial cell damage and Nrf2/HO-1 signal pathway induced by high glucose, hypoxia and reoxygenation. Methods Human renal tubular epithelial cells HK-2 were cultured in high-glucose medium and subjected to hypoxiareoxygenation treatment to establish a cellular model. CCK-8 experiment was utilized for detecting the effects of 0, 10, 20, 40, 80, 160 μg/ml osthole on its growth and screen the optimal concentration. HK-2 cells were divided into control, model, osthole(80 μg/ml), ML385(Nrf2 inhibitor, 20 nmol/ml) and osthole(80 μg/ml) +ML385(20 nmol/ml) groups. Except for control group, the other groups were induced for establishing cellular models. After high glucose, hypoxia and reoxygenation, CCK-8 experiment was utilized for detecting cellular viability in each group. Kits were employed for measuring the levels of reactive oxygen species(reactive oxygen species, ROS), glutathione peroxidase(GSH-Px), malondialdehyde(MDA), lactate dehydrogenase(LDH), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and interleukin-1β(IL-1β);Immunofluorescence for detecting the expression of Nrf2/HO-1 pathway protein;Western blot for detecting the expression of Nrf2/HO-1 pathway protein. Results Compared with control group, HK-2 cellular viability(100±0 vs 43.47±6.14), cellular GSH-Px level(9.53±1.64 vs 3.25±0.32) and cellular Nrf2 protein(1.78±0.30 vs 0.89±0.12) were markedly down-regulated in model group(all P<0.05);the levels of ROS(1.13±0.17 vs 6.52±1.03) and TNF-α spiked markedly (all P<0.05). Compared with model and osthole + ML385 groups,HK-2 cellular viability (43.47±6.14,44.83±7.06 vs 87.76±9.42),cellular GSH-Px level(3.25±0.32,3.27±0.38 vs 9.07±1.78) and cellular Nrf2 protein(0.89±0.12, 0.90±0.18 vs 1.74±0.26) rose in osthole group (P<0.05);ROS(6.52±1.03, 6.47±0.96 vs 1.59±0.21) and TNF-α declined(both P<0.05); HK-2 cellular viability(43.47±6.14, 44.83±7.06 vs 24.80±3.79), cellular GSH-Px level(3.25±0.32, 3.27±0.38 vs 0.88±0.15) and cellular Nrf2 protein(0.89±0.12, 0.90±0.18 vs 0.30±0.05) decreased in ML385 group(all P<0.05);ROS(6.52±1.03, 6.47±0.96 vs 9.76±2.02) and TNF-α increased(all P<0.05). Conclusion Osthole can up-regulate the expression of Nrf2/HO-1 signaling pathway protein, suppress ROS and inflammatory factors production induced by high glucose, hypoxia and reoxygenation, thereby relieving oxidative stress and inflammation and lessening the damage of renal tubular epithelial cells.

       

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