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白术乙醇提取物对秀丽隐杆线虫寿命的影响及其机制研究
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| 篇名： | 白术乙醇提取物对秀丽隐杆线虫寿命的影响及其机制研究 |
| TITLE： | Study on the Effects of Atractylodes macrocephala Ethanol Extract on Life Span of Caenorhabditis elegans and Its Mechanism |
| 摘要： | 目的：研究白术乙醇提取物（AM）对秀丽隐杆线虫（简称“N2线虫”）寿命的影响，并基于转录因子SKN-1/核因子E2相关因子2（Nrf2）信号通路探讨其作用机制。方法：将N2线虫分为空白对照组、阳性对照组（100μmol/L姜黄素，下同）和AM低、中、高剂量组（100、200、300μg/mL，下同），考察正常、氧化应激（40mmol/LH2O2）条件下AM对N2线虫寿命的影响（以平均存活时间计）以及正常条件下AM对N2线虫繁殖能力的影响（以子代数量计）。用700μmol/LH2O2建立小鼠神经瘤母细胞N2a氧化应激模型，采用MTT法考察阳性对照和AM低、中、高剂量对模型细胞存活率的影响。人胚胎肾上皮细胞293T转染Nrf2-抗氧化反应元件（ARE）质粒后，采用荧光素酶报告基因法考察阳性对照和AM低、中、高剂量作用24h以及AM中剂量作用12、18、24h对Nrf2-ARE荧光素酶活性的影响。采用实时定量聚合酶链式反应法考察阳性对照和AM低、中、高剂量对N2a细胞中Nrf2下游抗氧化基因NQO-1、HO-1mRNA表达以及对N2线虫中SKN-1下游抗氧化基因GCS-1、GST-7、GST-10、HSP-60、HSP-16.2、SOD-3mRNA表达的影响。结果：与空白对照组比较，阳性对照组和AM各剂量组N2线虫在正常、氧化应激下的平均存活时间均显著延长，第1天子代数量（除AM高剂量组外）、第2天子代数量（除AM低剂量组外）和总子代数量（除AM低剂量组外）均显著增加（P＜0.05或P＜0.01）。阳性对照组和AM中、高剂量组N2a细胞存活率显著高于模型组（P＜0.05或P＜0.01）。与空白对照组比较，阳性对照组和AM各剂量组293T细胞中Nrf2-ARE荧光素酶相对活性以及AM中剂量组293T细胞培养不同时间的Nrf2-ARE荧光素酶相对活性均显著增强（P＜0.01），并有剂量依赖性和时间依赖性趋势。与空白对照组比较，阳性对照组和AM各剂量组N2a细胞/线虫中HO-1、NQO-1（除阳性对照组外）、GCS-1（除AM低剂量组外）、GST-7（除阳性对照组和AM低剂量组外）、GST-10、HSP-60（除AM低剂量组外）、HSP-16.2（除阳性对照组和AM低剂量组外）、SOD-3（除阳性对照组和AM低剂量组外）mRNA的相对表达量均显著升高（P＜0.05或P＜0.01）。结论：AM可延长N2线虫在正常和氧化应激状态下的寿命，并可提高线虫的繁殖能力，其作用机制可能与激活SKN-1/Nrf2信号通路有关。 |
| ABSTRACT： | OBJECTIVE： To study the effects of Atractylodes macrocephala ethanol extract （AM） on life span of Caenorhabditis elegans（called N 2 nematode for short ），and to investigate its mechanism based on transcription factor SKN- 1/ nuclear factor E 2 related factor 2（Nrf2）. METHODS ：N2 nematode were divided into blank control group ，positive control group （100 μ mol/L curcumin，similarly hereinafter ），AM low-dose ，medium-dose and high-dose groups （100，200，300 μ g/mL， similarly hereinafter ）. The effects of AM on the life span （by average survival time ）of N 2 nematode under normal condition and oxidant stress condition （40 mmol/L H 2O2）as well as its effects on reproductive capability （by the number of filial generation ）of N2 nematode under normal condition were investigated . 700 μmol/L H2O2 was used to establish neuroblastoma cells N 2a oxidant stress model. Effects of positive control ，low-dose，medium-dose and high-dose of AM on the survival rate of model cells were detected by MTT method. After human embryonic renalepithelial cells 293T were transfected with Nrf 2-ARE plasmid ， the effects of positive control and AM on luciferase activity of Nrf2-ARE were detected by luciferase reporter gene method at low，medium and high dose for 24 h and at medium dose for 12，18 and 24 h. RT-PCR was used to detect the effects ofpositive control ，low-dose，medium-dose and high-dose of AM on the mRNA expression of downstream genes NQO- 1 and HO- 1 of Nrf 2 in N 2a cells as well as mRN A expression of en@hactcm.edu.cn downstream genes GCS- 1，GST-7，GST-10，HSP-60，HSP- 16.2 and SOD- 3 of SKN- 1 in N 2 nematode. RESULTS ：Compared with blank control group ，average survival time of N 2 nematode under normal and oxidant stress condition was significantly prolonged in positive control group and AM groups ；the number of filial generation on the first day （except for AM high-dose group ），the number of filial generation on the second day （except for AM low-dose group ） and the total number of filial generation （except for AM low-dose group ） were increased significantly（P＜0.05 or P＜0.01）. The survival rate of N 2a cells in positive control group ，AM medium-dose and high-dose groups were significantly higher than that of model group （P＜0.05 or P＜0.01）. Compared with blank control group ，Nrf2-ARE luciferase relative activity of 293T cells in positive control group and AM groups as well as Nrf 2-ARE luciferase relative activity of 293T cells in AM medium-dose group after different time of treatment were increased significantly （P＜0.01），in dose-dependent and time-dependent trend. Compared with blank control group ，mRNA relative expression of HO- 1 and NQO- 1（except for positive control group ），GCS-1（except for AM low-dose group ），GST-7（except for positive control group and AM low-dose group ）， GST-10 and HSP- 60（except for AM low-dose group ），HSP-16.2（except for positive control group and AM low-dose group ）and SOD-3 （except for positive control group and AM low-dose group ） were increased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS：AM can prolong the life span of N 2 nematode under normal and oxidant stress condition and improve the its reproductive capacity ，the mechanism of which may be associated with the activation of SKN- 1/Nrf2 signaling pathway. |
| 期刊： | 2021年第32卷第04期 |
| 作者： | 赵建平，吴丽敏，鲁晓娜，王辉，王潘，张振强，徐江雁，谢治深 |
| AUTHORS： | ZHAO Jianping，WU Limin，LU Xiaona，WANG Hui，WANG Pan，ZHANG Zhenqiang，XU Jiangyan，XIE Zhishen |
| 关键字： | 白术；乙醇提取物；秀丽隐杆线虫；寿命；氧化应激；核因子E2相关因子2；SKN-1 |
| KEYWORDS： | Atractylodes macrocephala；Ethanol extract ；Caenorhabditis elegans ；Life span ；Oxidant stress ；Nrf2；SKN-1 |
| 阅读数： | 358 次 |
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