淫羊藿素通过PI3K-Akt和Nrf2通路拮抗肺泡Ⅱ型上皮细胞氧化应激的研究

发布时间：2019-01-09 07:30

【摘要】：目的研究补肾组分淫羊藿素对香烟烟雾提取物(CSE)诱导的人肺泡II型上皮细胞(A549细胞)氧化应激的影响,并从固有的抗氧化通路(PI3K-Akt和Nrf2通路)探讨其机制。方.法 (1)以2.5%,5%,10%CSE刺激A549细胞,24小时后于倒置显微镜下观察细胞形态学变化并用CCK-8法检测细胞活力,选出合适浓度的CSE进行试验； (2)以luM,10μM,100μM ICT干预A549细胞,24小时后于倒置显微镜下观察细胞形态学变化并用CCK-8法检测细胞活力,选出适合浓度的ICT进行试验。 (3)以10μM ICT预培养A549细胞1小时后加入5%、10%CSE刺激24小时后于倒置显微镜下观察细胞形态学变化并用CCK-8法检测细胞活力,与单纯CSE刺激组相对比。 (4)培养A549细胞,并分组。 A组：正常对照组； B组：5%CSE刺激模型组； C组：ICT10μM干预组； D组：ICT10μM干预1小时后予5%CSE刺激组； E组：AKT抑制剂LY294002干预1小时后加ICT10μM干预1小时后予5%CSE刺激组； F组：Nrf2siRNA转染48小时后加入ICT10μM干预1小时后予5%CSE刺激组； (5)检测指标如下： 1)倒置显微镜下观察各组细胞形态学变化； 2)流式细胞仪检测各组细胞内ROS变化； 3)GSH检测试剂盒检测细胞内GSH变化； 4) Western blot检测细胞内Nrf2、P-Akt蛋白的变化； 5) Real-time PCR检测细胞内GCLM mRNA变化。结果 (1)不同浓度CSE均可抑制细胞增殖,且该作用呈浓度依赖性。 (2)中、低浓度(1μM,10μM) ICT对细胞增殖具有促进作用；高浓度(100μ M)ICT对细胞增殖无明显影响。 (3)10μM ICT对CSE诱导的细胞损伤具有保护作用。 (4)CSE可显著引起细胞内ROS释放,而ICT提前干预则可降低ROS产生。 (5)与单独CSE刺激组相比,ICT提前干预1小时可增加细胞内GSH含量。 (6)与单独CSE刺激组相比,ICT促进GCLM基因转录,与此同时,Nrf2蛋白核转位和细胞内磷酸化Akt蛋白明显增加。 (7)采用siRNA技术沉默Nrf2后,ICT对Nrf2下游的抗氧化基因GCLM的转录无促进作用。 (8)采用Akt抑制剂LY294002干预后,ICT对Nrf2的核转位及其下游抗氧化基因GCLM的转录无促进作用,同时对ROS的释放无明显抑制作用。结论 1.CSE可诱导A549细胞氧化应激,从而引起细胞损伤和死亡。 2-补肾组分淫羊藿素是一种新型抗氧化剂,对于CSE诱导的肺泡II型上皮细胞损伤具有保护作用,可为COPD的抗氧化治疗提供新选择； 3.补肾组分淫羊藿素可通过激活细胞固有的抗氧化通路,发挥抗氧化应激作用。具体而言,淫羊藿素可激活PI3K-Akt通路,增加Nrf2核转位,继而启动GCL基因转录并最终增加GSH水平从而减少ROS释放。
[Abstract]:Objective to study the effects of epimedium on oxidative stress induced by cigarette smoke extract (CSE) in human alveolar II epithelial cells (A549 cells) and to explore its mechanism from the intrinsic antioxidant pathways (PI3K-Akt and Nrf2 pathways). Fang. Methods (1) A549 cells were stimulated with 2.5 and 10 CSE respectively. After 24 hours, the morphological changes of A549 cells were observed under inverted microscope and the viability of A549 cells was detected by CCK-8 method. The appropriate concentration of CSE was selected for the experiment. (2) A549 cells were treated with 100 渭 M luM,10 渭 M ICT. After 24 hours, the morphological changes of A549 cells were observed under inverted microscope and the viability of A549 cells was detected by CCK-8 assay. The suitable concentration of ICT was selected for the experiment. (3) A549 cells were precultured with 10 渭 M ICT for 1 hour. After 24 hours of stimulation with 10 渭 M ICT, the morphological changes of A549 cells were observed under inverted microscope and the viability of A549 cells was detected by CCK-8 assay. (4) A549 cells were cultured and grouped. Group A: normal control group, group B: 5%CSE stimulation model group, group C: ICT10 渭 M intervention group, group D: 5%CSE stimulation group after 1 hour of ICT10 渭 M intervention; Group E was treated with AKT inhibitor LY294002 for 1 hour and then treated with ICT10 渭 M for 1 hour. Group F was treated with 5%CSE after 48 hours of Nrf2siRNA transfection and ICT10 渭 M for 1 hour. (5) the indexes were as follows: 1) observe the changes of cell morphology under inverted microscope, 2) detect the changes of intracellular ROS by flow cytometry, 3) detect the changes of intracellular GSH by GSH assay kit. ) Western blot was used to detect the changes of Nrf2,P-Akt protein and 5) Real-time PCR was used to detect the changes of intracellular GCLM mRNA. Results (1) CSE could inhibit cell proliferation in a concentration dependent manner. (2) low concentration (1 渭 m ~ (10) 渭 M) ICT) could promote cell proliferation, while high concentration (100 渭 M) ICT) had no effect on cell proliferation. (3) 10 渭 M ICT has protective effect on CSE induced cell damage. (4) CSE could significantly induce the release of intracellular ROS, while early intervention of ICT could decrease the production of ROS. (5) compared with CSE alone, ICT increased intracellular GSH content 1 hour in advance. (6) compared with CSE alone, ICT promoted the transcription of GCLM gene, at the same time, Nrf2 protein nuclear translocation and intracellular phosphorylated Akt protein increased significantly. (7) after silencing Nrf2 with siRNA technique, ICT did not promote the transcription of antioxidant gene GCLM downstream of Nrf2. (8) after the intervention of Akt inhibitor LY294002, ICT did not promote the transcription of Nrf2 nuclear translocation and its downstream antioxidant gene GCLM, but did not inhibit the release of ROS. Conclusion 1.CSE can induce oxidative stress in A549 cells, resulting in cell injury and death. Epimedium, a new component of Epimedium, is a new antioxidant, which can protect alveolar II type epithelial cells from injury induced by CSE, and provide a new choice for the antioxidation therapy of COPD. 3. Epimedium, a component of tonifying kidney, can exert antioxidant stress by activating the intrinsic antioxidant pathway of cells. Specifically, Epimediin activates the PI3K-Akt pathway, increases the Nrf2 nuclear translocation, then initiates the transcription of the GCL gene and ultimately increases the GSH level, thus reducing the release of ROS.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563.9

【参考文献】