二冬膏抗NF-κB抑制介导的人肺癌细胞效应探讨
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摘要
目的:探讨二冬膏的抗肿瘤效应,并研究核转录因子κB(NF-κB)在二冬膏抗肿瘤效应中的作用。方法:用四甲基偶氮唑盐比色法(MTT法)检测二冬膏对A549细胞的增殖抑制效应(量效和时效关系);逆转录聚合酶链反应(RT-PCR)法检测二冬膏对A549细胞p65 mRNA表达的影响;采用免疫印迹法(Western blot)检测二冬膏对A549细胞p65蛋白表达的影响。结果:不同剂量组二冬膏含药血清均具有对肺腺癌A549细胞的明显增殖抑制作用(P<0.05),且各自表现出一定时间和剂量相关性,给药干预24 h后细胞增殖均出现抑制,其中以二冬膏中剂量组(3.2 g·kg-1,bid)干预48 h后对细胞增殖的抑制作用最明显。二冬膏中剂量组(3.2 g·kg-1,bid)干预A549细胞24 h后明显抑制p65 mRNA的表达;中剂量组(3.2 g·kg-1,bid)干预A549细胞后48 h对p65蛋白有显著抑制效应。结论:二冬膏可显著抑制人肺腺癌细胞系A549的增殖,通过对NF-κB的抑制介导其抗肿瘤活性。
Objective: To investigate the anti-tumor effect of Erdong Gao and study the effect of nuclear factor-κB( NF-κB) in Erdong Gao. Method: The dose-and time-related inhibitory effect of Erdong Gao in proliferation of A549 cells was determined by methyl thiazolyl tetrazolium( MTT) assay. Effect of Erdong Gao on the p65 mRNA expression and protein expression in A549 cells were detected by reverse transcription-PCR( RTPCR) and Western blot respectively. Result: The MTT test indicated that the proliferation of A549 cells can be significantly inhibited by the drug serum of different concentrations of Erdong Gao( P < 0. 05),showing certain dose-dependence and time-dependence respectively. The cell proliferation was inhibited after 24 hours of drug intervention. The most obvious inhibitory effect was detected in Erdong Gao middle-dose group( 3. 2 g·kg- 1,bid)after intervention for 48 hours. The p65 mRNA expression and p65 protein expression in A549 cells were obviously inhibited in Erdong Gao middle-dose group after intervention for 24 hours and 48 hours respectively. Conclusion:Erdong Gao can significantly inhibit the proliferation of A549 cells in human lung adenocarcinoma cells,and its anti-tumor effect might be mediated by inhibiting NF-κB.
Objective: To investigate the anti-tumor effect of Erdong Gao and study the effect of nuclear factor-κB( NF-κB) in Erdong Gao. Method: The dose-and time-related inhibitory effect of Erdong Gao in proliferation of A549 cells was determined by methyl thiazolyl tetrazolium( MTT) assay. Effect of Erdong Gao on the p65 mRNA expression and protein expression in A549 cells were detected by reverse transcription-PCR( RTPCR) and Western blot respectively. Result: The MTT test indicated that the proliferation of A549 cells can be significantly inhibited by the drug serum of different concentrations of Erdong Gao( P < 0. 05),showing certain dose-dependence and time-dependence respectively. The cell proliferation was inhibited after 24 hours of drug intervention. The most obvious inhibitory effect was detected in Erdong Gao middle-dose group( 3. 2 g·kg- 1,bid)after intervention for 48 hours. The p65 mRNA expression and p65 protein expression in A549 cells were obviously inhibited in Erdong Gao middle-dose group after intervention for 24 hours and 48 hours respectively. Conclusion:Erdong Gao can significantly inhibit the proliferation of A549 cells in human lung adenocarcinoma cells,and its anti-tumor effect might be mediated by inhibiting NF-κB.
引文
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[16]孙昊鑫,朱金华,郭慧君,等.二冬膏对小鼠诱发性肺肿瘤发生TNF-α、IL-6、IL-10、Foxp3的影响[J].中药药理与临床,2013,29(6):1-3.
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[19]Kleemann R,Verschuren L,de Rooij B J,et al.Evidence for anti-inflammatory activity of statins and PPAR alpha activators in human C-reactive protein transgenic mice in vivo and in cultured human hepatocytes in vitro[J].Blood,2004,103(11):4188-4194.
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[2]包素珍,郑小伟,宋红,等.黄芪建中汤对脾气虚证肺癌转移小鼠TIMP-1基因转录的影响[J].中华中医药学刊,2012,30(4):686-688.
[3]赵连梅,孙佳讳,颜晰,等.中药升陷汤抑制肺癌A549细胞增殖和侵袭转移作用研究[J].中华中医药杂志,2011,26(9):2147-2150.
[4]胡春萍,蔡雪婷,胡婷婷,等.木犀草素诱导非小细胞肺癌细胞株A549凋亡和G2周期阻滞[J].中国中药杂志,2012,37(9):1259-1264.
[5]郑里翔,刘红宁,乔玉丹,等.六味地黄丸对自发乳腺癌小鼠瘤块中血管内皮生长因子和周期蛋D3基因表达的影响[J].中国实验方剂学杂志,2010,16(11):117-119.
[6]郑里翔,王萍,邓佳卉,等.阴虚,阳虚体质血清对肺癌细NF-κB,p16基因表达的影响[J].中华中医药杂志,2013,28(9):2563-2566.
[7]郑里翔,刘红宁,舒洋,等.不同体质血清对A549肺癌细胞周期蛋白表达的影响[J].中华中医药杂志,2012,27(2):324-327.
[8]郑里翔,林栋美,刘红宁,等.滋阴经方经TGF-β途径抑制小鼠自发乳腺癌生长的机理[J].中成药,2011,33(10):1793-1795.
[9]郑里翔,张玉仁,邓科穗,等.二至汤对阴虚阳虚荷瘤小鼠肿瘤生长的影响[J].陕西中医,2008,29(12):1678-1680.
[10]张玉仁,郑里翔,朱卫丰,等.二至丸对阴虚荷瘤小鼠抑瘤作用的研究[J].江西中医学院学报,2007,19(6):58-59.
[11]卫生部中医研究院中药研究所.中药成药制剂手册[M].北京:人民卫生出版社,1965:432.
[12]陈馥馨.新编中成药手册[M].北京:中国中医药科技出版社,1991:3.
[13]高建平,许旭,吴耀平,等.二冬膏祛痰、抗炎及免疫作用的研究[J].中成药,2003,25(9):762-763.
[14]赵益,罗蓉,尚广彬,等.二冬汤含药血清对肺癌细胞A549的作用研究[J].新中医,2012,44(9):113-115.
[15]郭慧君,朱金华,刘春花,等.不同滋阴中药对小鼠诱发性肺肿瘤发生及抗肿瘤免疫功能的影响[J].中国实验方剂学杂志,2012,18(13):226-229.
[16]孙昊鑫,朱金华,郭慧君,等.二冬膏对小鼠诱发性肺肿瘤发生TNF-α、IL-6、IL-10、Foxp3的影响[J].中药药理与临床,2013,29(6):1-3.
[17]Sen R,Baltimore D.Inducibility of kappa immunoglobulin enhancer-binding protein NF-κB by a posttranslational mechanism[J].Cell,1986,47(6):921-928.
[18]Tang X,Liu D,Shishodia S,et al.Nuclear factor-kappa B(NF-κB)is frequently expressed in lung cancer and preneoplastic lesions[J].Cancer,2006,107(11):2637-2646.
[19]Kleemann R,Verschuren L,de Rooij B J,et al.Evidence for anti-inflammatory activity of statins and PPAR alpha activators in human C-reactive protein transgenic mice in vivo and in cultured human hepatocytes in vitro[J].Blood,2004,103(11):4188-4194.
[20]Sethi G,Sung B,Aggarwal B B.Nuclear factor-kappa B activation:from bench to bedside[J].Exp Biol Med(Maywood),2008,233(1):21-23.
[21]Hayden M S,Ghosh S.Shared principles in NF-kappa B signaling[J].Cell,2008,132(3):344-362.