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
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