淫羊藿素通過PI3K-Akt和Nrf2通路拮抗肺泡Ⅱ型上皮細胞氧化應激的研究
發(fā)布時間：2019-01-09 07:30
【摘要】：目的 研究補腎組分淫羊藿素對香煙煙霧提取物(CSE)誘導的人肺泡II型上皮細胞(A549細胞)氧化應激的影響,并從固有的抗氧化通路(PI3K-Akt和Nrf2通路)探討其機制。 方.法 (1)以2.5%,5%,10%CSE刺激A549細胞,24小時后于倒置顯微鏡下觀察細胞形態(tài)學變化并用CCK-8法檢測細胞活力,選出合適濃度的CSE進行試驗； (2)以luM,10μM,100μM ICT干預A549細胞,24小時后于倒置顯微鏡下觀察細胞形態(tài)學變化并用CCK-8法檢測細胞活力,選出適合濃度的ICT進行試驗。 (3)以10μM ICT預培養(yǎng)A549細胞1小時后加入5%、10%CSE刺激24小時后于倒置顯微鏡下觀察細胞形態(tài)學變化并用CCK-8法檢測細胞活力,與單純CSE刺激組相對比。 (4)培養(yǎng)A549細胞,并分組。 A組：正常對照組； B組：5%CSE刺激模型組； C組：ICT10μM干預組； D組：ICT10μM干預1小時后予5%CSE刺激組； E組：AKT抑制劑LY294002干預1小時后加ICT10μM干預1小時后予5%CSE刺激組； F組：Nrf2siRNA轉染48小時后加入ICT10μM干預1小時后予5%CSE刺激組； (5)檢測指標如下： 1)倒置顯微鏡下觀察各組細胞形態(tài)學變化； 2)流式細胞儀檢測各組細胞內ROS變化； 3)GSH檢測試劑盒檢測細胞內GSH變化； 4) Western blot檢測細胞內Nrf2、P-Akt蛋白的變化； 5) Real-time PCR檢測細胞內GCLM mRNA變化。 結果 (1)不同濃度CSE均可抑制細胞增殖,且該作用呈濃度依賴性。 (2)中、低濃度(1μM,10μM) ICT對細胞增殖具有促進作用；高濃度(100μ M)ICT對細胞增殖無明顯影響。 (3)10μM ICT對CSE誘導的細胞損傷具有保護作用。 (4)CSE可顯著引起細胞內ROS釋放,而ICT提前干預則可降低ROS產生。 (5)與單獨CSE刺激組相比,ICT提前干預1小時可增加細胞內GSH含量。 (6)與單獨CSE刺激組相比,ICT促進GCLM基因轉錄,與此同時,Nrf2蛋白核轉位和細胞內磷酸化Akt蛋白明顯增加。 (7)采用siRNA技術沉默Nrf2后,ICT對Nrf2下游的抗氧化基因GCLM的轉錄無促進作用。 (8)采用Akt抑制劑LY294002干預后,ICT對Nrf2的核轉位及其下游抗氧化基因GCLM的轉錄無促進作用,同時對ROS的釋放無明顯抑制作用。 結論 1.CSE可誘導A549細胞氧化應激,從而引起細胞損傷和死亡。 2-補腎組分淫羊藿素是一種新型抗氧化劑,對于CSE誘導的肺泡II型上皮細胞損傷具有保護作用,可為COPD的抗氧化治療提供新選擇； 3.補腎組分淫羊藿素可通過激活細胞固有的抗氧化通路,發(fā)揮抗氧化應激作用。具體而言,淫羊藿素可激活PI3K-Akt通路,增加Nrf2核轉位,繼而啟動GCL基因轉錄并最終增加GSH水平從而減少ROS釋放。
[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.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R563.9
本文編號：2405311
[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.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R563.9
【參考文獻】
相關期刊論文 前3條
1 徐雯潔;王天芳;王智瑜;于麗麗;呂佳蒼;王慶國;;基于現(xiàn)代文獻的慢性阻塞性肺疾病中醫(yī)證候及證候要素的研究[J];中華中醫(yī)藥雜志;2008年01期
2 呂佳蒼;王智瑜;王天芳;徐雯潔;于麗麗;焦楊;蘇惠萍;劉泉;苗建英;徐艷玲;王明航;陳憲海;;774例慢性阻塞性肺疾病患者常見的中醫(yī)證候類型及其舌象分布特點[J];云南中醫(yī)學院學報;2009年01期
3 董競成;沈自尹;王文健;施賽珠;張玲娟;陳素珍;陳偉華;李婭麗;張耀東;章啟夫;金維岳;汪秀娟;;壽而康和大劑量二丙酸倍氯米松治療慢性阻塞性肺病的臨床研究[J];中西醫(yī)結合雜志;1991年10期
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