[1]赵颖丹,张天嵩,马　骏,等.细颗粒物2.5在肾小管上皮细胞氧化损伤中的作用及其调控机制[J].新乡医学院学报,2020,37(12):1118-1125.[doi:10.7683/xxyxyxb.2020.12.004]
　ZHAO Yingdan,ZHANG Tiansong,MA Jun,et al.Role of particulate matter 2.5 in oxidative damage of renal tubular epithelial cells and its regulatory mechanism[J].Journal of Xinxiang Medical University,2020,37(12):1118-1125.[doi:10.7683/xxyxyxb.2020.12.004]
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细颗粒物2.5在肾小管上皮细胞氧化损伤中的作用及其调控机制
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
37
期数:
2020年12
页码:
1118-1125
栏目:
基础研究
出版日期:
2020-12-05

文章信息/Info

Title:
Role of particulate matter 2.5 in oxidative damage of renal tubular epithelial cells and its regulatory mechanism
作者:
赵颖丹; 张天嵩; 马　骏; 张伟伟; 易　扬; 顾　波; 王汉清; 宣　怡
（上海市静安区中心医院华山医院静安分院肾内科，上海　200040）
Author(s):
ZHAO Yingdan; ZHANG Tiansong; MA Jun; ZHANG Weiwei; YI Yang; GU Bo; WANG Hanqing; XUAN Yi
（Department of Renal Medicine，Jing′an Branch Hospital，Huashan Hospital，Jing′an District Central Hospital，Shanghai 200040，China）
关键词:
细颗粒物2.5; 肾小管上皮细胞; 微RNA-590-3p; 氧化损伤; 核因子-E2相关因子2
Keywords:
particulate matter 2.5; renal tubular epithelial cells; micro RNA-590-3p; oxidative damage; nuclear factor-erythroid-2-related factor-2
分类号:
R692.6
DOI:
10.7683/xxyxyxb.2020.12.004
文献标志码:
A
摘要:
目的　探讨细颗粒物2.5（PM2.5）在肾小管上皮细胞HK-2细胞氧化损伤中的作用及其调控机制。方法　将对数生长期的人肾小管上皮细胞HK-2细胞随机分为空白对照组、阴性对照组、PM2.5低剂量组、PM2.5中剂量组及PM2.5高剂量组。空白对照组HK-2细胞正常培养，无任何处理；阴性对照组HK-2细胞进行磷酸缓冲盐溶液处理；PM2.5低剂量组HK-2细胞给予50 mg·L-1的PM2.5处理24 h；PM2.5中剂量组HK-2细胞给予100 mg·L-1的PM2.5处理24 h；PM2.5高剂量组HK-2细胞给予200 mg·L-1的PM2.5处理24 h。采用流式细胞术和Edu标记实验检测各组HK-2细胞凋亡率和Edu阳性细胞率，酶联免疫吸附实验法检测各组HK-2细胞氧化应激相关指标超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活性及总活性氧（ROS）、丙二醛（MDA）水平，实时荧光定量聚合酶链式反应检测各组HK-2细胞微RNA（miRNA）-590-3p水平变化。另取对数生长期的HK-2细胞随机分为miRNA类似物对照组、miRNA类似物组、miRNA抑制剂对照组和miRNA抑制剂组，miRNA类似物组和miRNA类似物对照组HK-2细胞分别转染miRNA mimics和miRNA mimics对照序列，miRNA抑制剂组和miRNA抑制剂对照组分别转染miRNA inhibitor和miRNA inhibitor对照序列。Targetscan数据库和双荧光素酶报告实验预测和验证miRNA-590-3p的靶点；应用Western blot检测各组HK-2细胞核因子-E2相关因子2（NFE2L2）表达水平，酶联免疫吸附实验法检测各组HK-2细胞中SOD、GSH-Px活性及ROS、MDA水平。结果　PM2.5低剂量组、PM2.5中剂量组及PM2.5高剂量组HK-2细胞凋亡率均显著高于空白对照组及阴性对照组（P<0.05），PM2.5中剂量组、PM2.5高剂量组HK-2细胞凋亡率均显著高于PM2.5低剂量组（P<0.05），PM2.5高剂量组HK-2细胞凋亡率显著高于PM2.5中剂量组（P<0.05）。PM2.5低剂量组、PM2.5中剂量组及PM2.5高剂量组HK-2细胞Edu阳性细胞率均显著低于空白对照组及阴性对照组，PM2.5中剂量组、PM2.5高剂量组HK-2组胞Edu阳性细胞率均显著低于PM2.5低剂量组（P<0.05），PM2.5高剂量组HK-2细胞Edu阳性细胞率显著低于PM2.5中剂量组（P<0.05）。PM2.5低剂量组、PM2.5中剂量组、PM2.5高剂量组HK-2细胞中ROS和MDA水平均显著高于空白对照组和阴性对照组（P<0.05），PM2.5中剂量组、PM2.5高剂量组HK-2细胞中ROS和MDA水平均显著高于PM2.5低剂量组（P<0.05），PM2.5高剂量组HK-2细胞中ROS和MDA水平显著高于PM2.5中剂量组（P<0.05）。PM2.5低剂量组、PM2.5中剂量组、PM2.5高剂量组HK-2细胞中SOD和GSH-Px活性均显著低于空白对照组和阴性对照组（P<0.05），PM2.5中剂量组、PM2.5高剂量HK-2细胞中SOD和GSH-Px活性均显著低于PM2.5低剂量组（P<0.05），PM2.5高剂量组HK-2细胞中SOD和GSH-Px活性显著低于PM2.5中剂量组（P<0.05）。PM2.5低剂量组、PM2.5中剂量组、PM2.5高剂量组细胞中miRNA-590-3p相对表达水平均显著高于空白对照组和阴性对照组（P<0.05），PM2.5中剂量组、PM2.5高剂量组细胞中miRNA-590-3p相对表达水平均显著高于PM2.5低剂量组（P<0.05），PM2.5高剂量组细胞中miRNA-590-3p相对表达水平显著高于PM2.5中剂量组（P<0.05）。miRNA-590-3p与NFE2L2的3′ 非翻译区（UTR）存在结合靶点。miRNA类似物组总NFE2L2水平和核内NFE2L2水平显著低于miRNA类似物对照组（P<0.05），miRNA抑制剂组总NFE2L2水平和核内NFE2L2水平显著高于miRNA抑制剂对照组（P<0.05）。与miRNA类似物对照组比较，miRNA类似物组HK-2细胞中ROS、MDA水平增高，SOD、GSH-Px水平降低（P<0.05）；与miRNA抑制剂对照组比较，miRNA抑制剂组HK-2细胞中ROS和MDA水平降低，SOD和GSH-Px水平升高（P<0.05）。结论　PM2.5可通过上调miRNA-590-3p表达而抑制NFE2L2表达，进而加重肾小管上皮细胞氧化损伤。
Abstract:
Objective　To investigate the role of particulate matter 2.5 （PM2.5） in oxidative damage of renal tubular epithelial cell HK-2 cells and its regulatory mechanism.Methods　Human renal tubular epithelial cell HK-2 cells in logarithmic growth phase were selected，and they were randomly divided into the blank control group，negative control group，low-dose PM2.5 group，medium-dose PM2.5 group and high-dose PM2.5 group.The HK-2 cells in the blank control group were normal cultured without any treatment；the HK-2 cells in the negative control group were treated with phosphate buffer solution；the HK-2 cells in the low-dose PM2.5 group were treated with PM2.5 at a concentration of 50 mg·L-1 for 24 h；the HK-2 cells in the medium-dose PM2.5 group were treated with PM2.5 at a concentration of 100 mg·L-1 for 24 h；the HK-2 cells in the high-dose PM2.5 group were treated with PM2.5 at a concentration of 200 mg·L-1 for 24 h.The apoptosis rate and Edu positive cell rate of HK-2 cells in each group were detected by flow cytometry and Edu labeling assay.The levels of superoxide dismutase （SOD），glutathione peroxidase （GSH-Px），total reactive oxygen species （ROS） and malondialdehyde （MDA） were measured by enzyme linked immunosorbent assay.The expression of micro RNA（miRNA）-590-3p in HK-2 cells was detected by real-time fluorescent quantitative polymerase chain reaction.In addition，the HK-2 cells in logarithmic phase were selected and randomly divided into the miRNA mimics control group，miRNA mimics group，miRNA inhibitor control group and miRNA inhibitor group.The HK-2 cells in the miRNA mimics group and miRNA mimics control group were transfected with miRNA mimics and miRNA mimics control sequence，respectively.The HK-2 cells in the miRNA inhibitor group and miRNA inhibitor control group were transfected with miRNA inhibitor and miRNA inhibitor control sequence，respectively.The targets of miRNA-590-3p were predicted and verified by targetscan database and double Luciferase report experiment.The expression of nuclear factor-erythroid-2-related factor-2（NFE2L2） was detected by Western blot.Enzyme linked immunosorbent assay was used to detect the levels of SOD，GSH-Px，ROS and MDA in the HK-2 cells.Results　The apoptosis rate of HK-2 cells in the low-dose PM2.5 group，medium-dose PM2.5 group and high-dose PM2.5 group were significantly higher than that in the blank control group and negative control group（P<0.05），the apoptosis rate of HK-2 cells in the medium-dose PM2.5 group and high-dose PM2.5 group were significantly higher than that in the low-dose PM2.5 group（P<0.05），the apoptosis rate of HK-2 cells in the high-dose PM2.5 group were significantly higher than that in the medium-dose PM2.5 group （P<0.05）.The percentage of Edu positive cells in HK-2 cells in the low-dose PM2.5 group，medium-dose PM2.5 group and high-dose PM2.5 group was significantly lower than that in the blank control group and negative control group（P<0.05），the percentage of Edu positive cells in HK-2 cells in the medium-dose PM2.5 group and high-dose PM2.5 group was significantly lower than that in the low-dose PM2.5 group（P<0.05），the percentage of Edu positive cells in HK-2 cells in the high-dose PM2.5 group was significantly lower than that in the medium-dose PM2.5 group（P<0.05）.The levels of ROS and MDA in the low-dose PM2.5 group，medium-dose PM2.5 group and high-dose PM2.5 group were significantly higher than those in the blank control group and negative control group（P<0.05），the levels of ROS and MDA in the medium-dose PM2.5 group and high-dose PM2.5 group were significantly higher than those in the low-dose PM2.5 group（P<0.05），the levels of ROS and MDA in the high-dose PM2.5 group were significantly higher than those in the medium-dose PM2.5 group（P<0.05）.The levels of SOD and GSH-Px in low-dose PM2.5 group，medium-dose PM2.5 group and high-dose PM2.5 group were significantly lower than those in the blank control group and negative control group（P<0.05），the levels of SOD and GSH-Px in the medium-dose PM2.5 group and high-dose PM2.5 group were significantly lower than those in the low-dose PM2.5 group，the levels of SOD and GSH-Px in the high-dose PM2.5 group were significantly lower than those in the medium-dose PM2.5 group（P<0.05）.Conclusion　PM2.5 can inhibit the expression of NFE2L2 by up-regulating the expression of miRNA-590-3p，thus aggravating the oxidative damage of renal tubular epithelial cells.

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更新日期/Last Update: 2020-12-05