黄芪甲苷通过调控Keap1-Nrf2-ARE信号通路减轻PM2.5诱导的肾小管上皮细胞氧化损伤
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上海市静安区卫生和计划生育委员会课题(2018MS04)


Astragaloside Alleviates Particulate Matter 2.5-induced Oxidative Injury of Renal Tubular Epithelial Cells by Regulating the Kelch-like ECH-associated Protein 1-Nuclear Factor Erythroid 2-related Factor 2-Antioxidant Response Element Signaling Pathway
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    摘要:

    目的 探讨黄芪甲苷减轻细颗粒物2.5(particulate matter 2.5,PM2.5)所致肾小管上皮细胞氧化损伤的作用及分子机制。方法 将HK-2细胞分为5组:对照组(仅磷酸盐缓冲液处理)、模型组(200 μg/mL PM2.5处理24 h)、黄芪甲苷低剂量组(200 μg/mL PM2.5及10 nmol/L黄芪甲苷共同处理24 h)、黄芪甲苷中剂量组(200 μg/mL PM2.5及100 nmol/L黄芪甲苷共同处理24 h)、黄芪甲苷高剂量组(200 μg/mL PM2.5及200 nmol/L黄芪甲苷共同处理24 h)。采用流式细胞术和TUNEL检测不同方法处理的HK-2细胞凋亡情况,采用EdU实验和CCK8实验检测细胞增殖情况,采用Western blot检测不同方法处理的HK-2细胞中Kelch样环氧氯丙烷相关蛋白1(Kelch-like ECH-associated protein 1,Keap1)-核因子红细胞相关因子2(nuclear factor erythroid 2-related factor 2, Nrf2)-抗氧化反应元件(antioxident response element, ARE)信号通路相关蛋白的表达水平。结果 流式细胞术和TUNEL染色结果显示,黄芪甲苷显著抑制PM2.5导致的肾小管上皮细胞凋亡,其抑制效果随黄芪甲苷浓度的增加而增加。CCK8检测结果和EdU细胞增殖检测结果显示,黄芪甲苷显著促进肾小管上皮细胞增殖,细胞活力随黄芪甲苷浓度的增加而增加。进一步检测显示,黄芪甲苷显著抑制PM2.5导致的肾小管上皮细胞活性氧和丙二醛水平升高,并显著促进超氧化物歧化酶和谷胱甘肽过氧化物酶水平的升高。Western blot结果显示,与模型组比较,黄芪甲苷处理显著提高总Nrf2、核Nrf2、血红素氧化酶-1以及NAD(P)H醌脱氢酶1(NQO1)水平(P<0.05)。结论 黄芪甲苷通过调控Keap1-Nrf2-ARE信号通路减轻PM2.5导致的肾小管上皮细胞氧化损伤。

    Abstract:

    Objective To investigate the effect and molecular mechanism of astragaloside in reducing particulate matter 2.5 (PM2.5)-induced oxidative injury of renal tubular epithelial cells. Methods HK-2 cells were divided into control group (treated with phosphate buffered saline alone), model group (treated with 200 μg/mL PM2.5 for 24 hours), low-dose astragaloside group (treated with 200 μg/mL PM2.5 and 10 nmol/L astragaloside for 24 hours), middle-dose astragaloside group (treated with 200 μg/mL PM2.5 and 100 nmol/L astragaloside for 24 hours), and high-dose astragaloside group (treated with 200 μg/mL PM2.5 and 200 nmol/L astragaloside for 24 hours). Flow cytometry and TUNEL assay were used to evaluate the apoptosis of HK-2 cells treated with different methods; the EdU and CCK-8 assays were used to evaluate the proliferation of HK-2 cells; Western blot was used to measure the expression of the proteins involved in the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway in HK-2 cells treated with different methods. Results Flow cytometry and TUNEL staining showed that astragaloside treatment significantly inhibited the apoptosis of renal tubular epithelial cells induced by PM2.5 in a dose-dependent manner. The CCK-8 and EdU assays showed that astragaloside significantly promoted the proliferation of renal tubular epithelial cells, and cell viability increased with the increase in the concentration of astragaloside. Further tests showed that astragaloside significantly inhibited the increases in reactive oxygen species and malondialdehyde in renal tubular epithelial cells induced by PM2.5 and significantly promoted the increases in superoxide dismutase and glutathione peroxidase. Western blot showed that compared with the model group, the astragaloside treatment groups had significant increases in the levels of total Nrf2, nuclear Nrf2, HO1, and NQO1 (P<0.01). Conclusion Astragaloside alleviates PM2.5-induced oxidative injury of renal tubular epithelial cells by regulating the Keap1-Nrf2-ARE signaling pathway.

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赵颖丹,张天嵩,马 骏,张伟伟,易 扬,顾 波,王汉清,宣 怡.黄芪甲苷通过调控Keap1-Nrf2-ARE信号通路减轻PM2.5诱导的肾小管上皮细胞氧化损伤[J].安徽中医药大学学报,2020,39(2):69-75

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  • 在线发布日期: 2020-04-10