中国中药杂志

2021, v.46(24) 6502-6510

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甲基丁香酚对人肾小管上皮细胞缺氧/复氧损伤的影响及其机制研究
Effect of methyl eugenol on hypoxia/reoxygenation injury of human renal tubular epithelial cells and its mechanism

匡柏成;侯帅恒;张季;王梦琴;张佳思;孙凯伦;王志恒;李晴雯;宫念樵;
KUANG Bai-cheng;HOU Shuai-heng;ZHAN G Ji;WANG Meng-qin;ZHANG Jia-si;SUN Kai-lun;WANG Zhi-heng;LIQing-wen;GONG Nian-qiao;Key Laboratory of Organ Transplantation, Chinese Academy of Medical Sciences, Key Laboratory of Organ Transplantation, National Health Commission of the People′s Republic of China,Key Laboratory of Organ Transplantation, Ministry of Education, Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology;

摘要(Abstract):

探讨甲基丁香酚对缺氧/复氧(hypoxia/reoxygenation, H/R)致人肾小管上皮细胞HK-2损伤的影响及机制。通过细胞计数试剂盒(cell counting kit-8, CCK-8)检测不同浓度甲基丁香酚对正常培养的及经H/R处理后的HK-2细胞活力;倒置显微镜观察甲基丁香酚对HK-2细胞形态的影响;2′-7′-二氯荧光素乙酸盐(2′,7′-dichlorodihydrofluorescein diacetate, DCFH-DA)荧光染色法检测不同组细胞内活性氧含量;流式细胞术检测细胞凋亡;线粒体膜电位检测试剂盒检测线粒体膜电位的变化;Western blot法检测核转录因子E2相关因子2(nuclear factor erythroid 2 related factor 2, Nrf2)、血红素加氧酶-1(heme oxygenase-1, HO-1)、烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(nicotinamide adenine dinucleotide phosphatase oxidase 4, Nox4)蛋白的表达,共聚焦荧光染色检测Nrf2胞质及胞核浓度改变。结果显示,甲基丁香酚浓度为0~40μmol·L~(-1)时,对HK-2细胞活性无影响;与模型组相比,甲基丁香酚可增加HK-2细胞活性,细胞形态较正常。进一步实验表明,甲基丁香酚可抑制H/R损伤后HK-2细胞内活性氧的释放及线粒体膜电位的下降,促进Nrf2、HO-1表达及Nrf2向细胞核转位,抑制Nox4表达,进而显著降低细胞凋亡,特异性Nrf2抑制剂ML385可逆转甲基丁香酚的保护效果。初步证明甲基丁香酚对HK-2细胞H/R损伤具有明显的保护作用,机制可能与促进Nrf2/HO-1信号通路及抑制Nox4有关。该研究从抗氧化应激的角度探讨甲基丁香酚治疗肾脏缺血再灌注损伤(ischemia-reperfusion injury, IRI)、保护器官功能的可能机制,对相关中药治疗急性肾损伤的研究提供参考。
This study aimed to investigate the effect of methyl eugenol(ME) on hypoxia/reoxygenation(H/R)-induced injury of human renal tubular epithelial HK-2 cells and its mechanism. The viability of HK-2 cells cultured with different concentrations of ME and exposed to H/R was detected by cell counting kit-8(CCK-8) assay. The effect of ME on the morphology of HK-2 cells was observed under an inverted microscope. The content of intracellular reactive oxygen species in different groups was detected after 2′,7′-dichlorodihydrofluorescein diacetate(DCFH-DA) fluorescence staining. Cell apoptosis was determined by flow cytometry. Changes in mitochondrial membrane potential were monitored by JC-1 dye. The concentrations of nuclear factor erythroid 2 related factor 2(Nrf2), heme oxygenase-1(HO-1), and nicotinamide adenine dinucleotide phosphatase oxidase 4(Nox4) were measured by Western blot, followed by the assay of Nrf2 concentration changes in cytoplasm and nucleus by confocal fluorescence staining. The results showed that when the concentration of ME was 0-40 μmol·L~(-1), the activity of HK-2 cells was not affected. Compared with the model group, ME enhanced the activity of HK-2 cells and the cell morphology was normal. As revealed by further experiments, ME inhibited the release of reactive oxygen species and the decline in mitochondrial membrane potential of HK-2 cells after H/R injury, promoted Nrf2/HO-1 expression and Nrf2 translocation to the nucleus, and down-regulated the expression of Nox4, thereby significantly reducing apoptosis. This protective effect of ME could be reversed by the specific Nrf2 inhibitor ML385. These findings have preliminarily proved that ME effectively protected HK-2 cells against H/R injury, which might be related to its promotion of Nrf2/HO-1 signaling pathway and inhibition of Nox4. Such exploration on the possible mechanism of ME in the treatment of renal ischemia-reperfusion injury(IRI) and protection of organ function from the perspective of antioxidant stress has provided reference for related research on the treatment of acute kidney injury with traditional Chinese medicine.

关键词(KeyWords): 甲基丁香酚;缺氧/复氧;HK-2细胞;Nrf2;Nox4
methyl eugenol;hypoxia/reoxygenation;HK-2 cell;Nrf2;Nox4

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81873623)

作者(Author): 匡柏成;侯帅恒;张季;王梦琴;张佳思;孙凯伦;王志恒;李晴雯;宫念樵;
KUANG Bai-cheng;HOU Shuai-heng;ZHAN G Ji;WANG Meng-qin;ZHANG Jia-si;SUN Kai-lun;WANG Zhi-heng;LIQing-wen;GONG Nian-qiao;Key Laboratory of Organ Transplantation, Chinese Academy of Medical Sciences, Key Laboratory of Organ Transplantation, National Health Commission of the People′s Republic of China,Key Laboratory of Organ Transplantation, Ministry of Education, Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology;

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