从PI3K/Akt/p53通路探讨藤茶总黄酮抗肝癌的作用机制
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摘要
目的:研究藤茶总黄酮(TF)对肝癌裸鼠移植瘤的抑制作用,预测其作用机制可能与调控细胞凋亡肌醇磷脂-3-激酶/蛋白激酶B/p53(PI3K/Akt/p53)通路的相关因子有关。方法:建立BEL-7404肝癌裸鼠移植瘤模型,实验分为模型组,5-氟尿嘧啶(5-FU,1.0 g·L~(-1))组及TF高、中、低质量浓度(30,15,7.5 g·L~(-1))组,给药干预2周后处死裸鼠,剥离出瘤组织,根据瘤重和瘤体积计算抑瘤率(IR)及相对肿瘤增殖率(T/C);采用逆转录聚合酶链式反应(RT-PCR)检测PI3K/Akt/p53中相关基因胞内PI3K,Akt1,p53,半胱氨酸天冬氨酸蛋白酶-3(Caspase-3),B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax)mRNA表达;采用免疫组化检测相关蛋白PI3K,Akt1,p53,Caspase-3,Bcl-2,Bax蛋白的表达。结果:TF高、中、低质量浓度组IR分别为53.26%,35.94%,26.74%,T/C分别为59.74%,69.66%,84.82%;逆转录聚合本科链式反应(RT-PCR)表明,与模型组比较,TF 30 g·L~(-1)能明显下调PI3K,Akt1,Bcl-2 mRNA表达,显著上调抑癌基因p53,Capsase-3,Bax mRNA表达;免疫组化表明,与模型组比较,TF 30,15 g·L~(-1)均明显下调PI3K,Akt1,Bcl-2蛋白的表达,显著上调p53,Capsase-3,Bax蛋白的表达。结论:TF有明显的体内抗肝癌活性,其机制可能与上调p53,Caspase-3的表达,激活细胞凋亡PI3K/Akt/p53通路,从而抑制Bcl-2,提高Bax的表达,促进肝癌细胞凋亡有关。
Objective:To study inhibitory effect of total flavonoids from Ampelopsis grossedentata(TF)on transplanted tumors of human hepatocellular carcinoma in nude mice,and predict that its mechanism may be related to relevant factors regulating phosphatidylinositol 3 kinase(PI3K)/protein kinases B(Akt)/p53pathway in apoptosis.Method:The nude mice transplanted BEL-7404 hepatoma model was established and divided into model group,5-fluorouracil(5-FU)group(1.0 g·L~(-1))and TF(30,15,7.5 g·L~(-1))groups.Nude mice were put to death after two weeks of administration.The tumor tissues were excised,and tumor inhibition rate(IR)and relative tumor proliferation rate(T/C)were calculated.Reverse transcription PCR(RT-PCR)was used to detect PI3K,Akt1,p53 gene(p53),Caspase-3,B cell lymphoma/lewkmia-2(Bcl-2),Bcl-2 associated X protein(Bax)mRNA expressions,immunohistochemical method was used to detect expressions of relevant proteins PI3K,Akt1,p53,Caspase-3,Bcl-2,Bax.Result:The establishment of xenograft tumor in mice showed that TF was administered orally once per day for two consecutive weeks.IRs were 53.26%,35.94%,and 26.74%,respectively.T/Cs were 59.74%,69.66%,and 84.82%,respectively.RT-PCR experiments showed that compared with model group,when TF concentration was 30 g·L~(-1),mRNA expressions of PI3K,Akt1,and Bcl-2 were significantly down-regulated,and mRNA expressions of tumor suppressor genes p53,Capsase-3,and Bax were significantly up-regulated.Immunohistochemical method results showed that compared with model group,at TF concentrations of 30,15 g·L~(-1),all PI3K,Akt1,Bcl-2 protein expressions were significantly down-regulated,while p53,Capsase-3,Bax protein expressions were significantly increased.Conclusion:TF has an obvious antiliver cancer activity in vivo.Its mechanism may be correlated with up-regulation of expressions of p53,Caspase-3,and activation of apoptosis PI3K/Akt/p53 pathway,thereby inhibiting Bcl-2,increasing expression of Bax,and promoting hepatocellular apoptosis.
Objective:To study inhibitory effect of total flavonoids from Ampelopsis grossedentata(TF)on transplanted tumors of human hepatocellular carcinoma in nude mice,and predict that its mechanism may be related to relevant factors regulating phosphatidylinositol 3 kinase(PI3K)/protein kinases B(Akt)/p53pathway in apoptosis.Method:The nude mice transplanted BEL-7404 hepatoma model was established and divided into model group,5-fluorouracil(5-FU)group(1.0 g·L~(-1))and TF(30,15,7.5 g·L~(-1))groups.Nude mice were put to death after two weeks of administration.The tumor tissues were excised,and tumor inhibition rate(IR)and relative tumor proliferation rate(T/C)were calculated.Reverse transcription PCR(RT-PCR)was used to detect PI3K,Akt1,p53 gene(p53),Caspase-3,B cell lymphoma/lewkmia-2(Bcl-2),Bcl-2 associated X protein(Bax)mRNA expressions,immunohistochemical method was used to detect expressions of relevant proteins PI3K,Akt1,p53,Caspase-3,Bcl-2,Bax.Result:The establishment of xenograft tumor in mice showed that TF was administered orally once per day for two consecutive weeks.IRs were 53.26%,35.94%,and 26.74%,respectively.T/Cs were 59.74%,69.66%,and 84.82%,respectively.RT-PCR experiments showed that compared with model group,when TF concentration was 30 g·L~(-1),mRNA expressions of PI3K,Akt1,and Bcl-2 were significantly down-regulated,and mRNA expressions of tumor suppressor genes p53,Capsase-3,and Bax were significantly up-regulated.Immunohistochemical method results showed that compared with model group,at TF concentrations of 30,15 g·L~(-1),all PI3K,Akt1,Bcl-2 protein expressions were significantly down-regulated,while p53,Capsase-3,Bax protein expressions were significantly increased.Conclusion:TF has an obvious antiliver cancer activity in vivo.Its mechanism may be correlated with up-regulation of expressions of p53,Caspase-3,and activation of apoptosis PI3K/Akt/p53 pathway,thereby inhibiting Bcl-2,increasing expression of Bax,and promoting hepatocellular apoptosis.
引文
[1]陈明,王举涛,吴珍妮,等.半枝莲总黄酮通过PI3K/Akt/m TOR通路诱导肿瘤细胞自噬的体内实验研究[J].中国中药杂志,2017,42(7):1358-1364.
[2]王容容,谭小宁,李勇敏,等.健脾消癌方对大肠癌肝转移裸鼠模型肝组织PI3K/Akt通路相关蛋白表达的影响[J].中国实验方剂学杂志,2018,24(6):177-181.
[3]曾志涛,傅缨,熊耀斌,等.发酵虫草菌粉对人肝癌细胞增殖、凋亡及VEGF,MMP-2表达的影响[J].中国实验方剂学杂志,2015,21(9):141-144.
[4]ZHANG Q Z,YU D. Protection against cerebral infarction by Withaferin A involves inhibition of neuronal apoptosis,activation of PI3K/Akt signaling pathway,and reduced intimal hyperplasia via inhibition of VSMC migration and matrix metalloproteinases[J]. Adv Med Sci,2017,62(1):186-192.
[5]Maliheh A. Epigallocatechin-3-gallate promotes apoptosis in human breast cancer T47D cells through down-regulation of PI3K/Akt and telomerase[J].Pharmacol Rep,2017,69(5):924-928.
[6]孙阳,于水澜,吴勃岩,等.槲皮素自微乳的抗肿瘤作用及其机制分析[J].中国实验方剂学杂志,2018,24(14):97-101.
[7]史桂兰,黄琳,卿海燕,等.槲皮素对人肝癌SMMC-7721细胞生长抑制及诱导凋亡作用[J].中国实验诊断学,2015,19(1):17-19.
[8]张冬青,汪德清,李卉,等.黄芪总黄酮对K562细胞凋亡及细胞周期的影响[J].中国药理学通报,2010,26(1):136-137.
[9]盛连兵,孙凯,刘海萍,等.金雀异黄素调控Egr1/PTEN信号通路诱导卵巢癌SKOV3细胞凋亡的实验研究[J].中国妇产科临床杂志,2017,18(4):341-344.
[10]王晓东.汉黄芩素对人肝癌SMMC-7721的抑制作用及其作用机制研究[J].中国继续医学教育,2016,8(34):180-182.
[11]于海瑶,李石化,孙丽艳,等.异荭草素对胃癌细胞自噬作用的研究[J].中国生化药物杂志,2015,35(12):36-38.
[12]陈帅,郁建平.藤茶总黄酮抗炎及抑菌作用的实验研究[J].贵阳中医学院学报,2013,35(1):1-3.
[13]冯涵,李娜,王华林,等.藤茶中黄酮类成分的功效研究进展[J].公共卫生与预防医学,2018,29(1):82-86.
[14]欧贤红,郑作文.藤茶总黄酮对免疫性肝损伤小鼠的保护作用[J].哈尔滨医药,2011,31(1):1-3.
[15]曾宪彪,韦桂宁,何飞,等.藤茶总黄酮对动脉粥样硬化大鼠血脂及血液流变学的影响[J].重庆医学,2014,43(5):518-520.
[16]周春权,林静瑜,姚欣,等.藤茶总黄酮体外抗肿瘤实验研究[J].中国医药科学,2012,2(9):50-51.
[17]李刚,郑作文,唐云丽.藤茶总黄酮体外抗人肝癌细胞作用研究[J].中国药房,2008(9):652-654.
[18]郑作文.藤茶总黄酮在2215细胞培养中对乙型肝炎病毒HBs Ag、HBe Ag和HBV-DNA的抑制作用[J].山东中医杂志,2003,22(9):561-563.
[19]高倩倩,杨秀芬,欧敏.藤茶总黄酮和二氢杨梅素含药血清对肝癌Hep G2细胞增殖及凋亡的影响[J].中国中药杂志,2011,36(4):500-503.
[20]金慧鸣,谭兴和,蔡文,等.响应面法优化藤茶总黄酮的提取工艺[J].食品安全质量检测学报,2015,6(5):1575-1582.
[21]晏容,刘云,朱欣婷,等.斑蝥素酸镁对人肝癌细胞SMMC-7721裸鼠皮下移植瘤凋亡的影响[J].天然产物研究与开发,2015,27(2):236-240.
[22]冯巍巍,黎梨,黄赞松,等.苦参素通过调控MRP1表达提高多柔比星对人肝癌耐药裸鼠移植瘤的抑制作用[J].右江民族医学院学报,2018,40(1):8-12.
[23]曾云想,郁建平.藤茶总黄酮提取方法的优选[J].山地农业生物学报,2013,32(2):153-158.
[24]钟宇,郑学宝,蔡康荣,等.异丁酰紫草素通过PI3K/Akt/m-TOR通路靶向抑制结肠癌细胞增殖的研究[J].中国中药杂志,2018,43(11):2358-2364.
[25]魏蔚,刘玉华.姜黄素通过活化PI3K/Akt信号通路逆转肝癌耐药细胞株Bel7402/ADR的耐药性[J].中国老年学杂志,2018,38(12):3015-3019.
[26]胡泽成.青龙衣多糖对结肠癌HCT-116细胞增殖、凋亡及PI3K/Akt信号通路的影响[J].中国实验方剂学杂志,2016,22(18):136-139.
[27]辛红,徐巍.甘草查尔酮A通过PI3K/Akt/m TOR信号通路诱导肾癌细胞自噬的研究[J].中国中药杂志,2018,43(17):3545-3552.
[28]陈滨,郭洁文,何素,等.叶下珠复方Ⅱ号对肝癌细胞增殖和胰岛素样生长因子-1受体信号通路转录的作用[J].中国实验方剂学杂志,2018,24(14):108-114.
[29]CHEN Y Y,CAO Y Z,LI F Q,et al. Studies on antihepatoma activity of Annona squamosa L. pericarp extract[J]. Bioorg Med Chem Lett,2017,27(9):1907-1910.
[2]王容容,谭小宁,李勇敏,等.健脾消癌方对大肠癌肝转移裸鼠模型肝组织PI3K/Akt通路相关蛋白表达的影响[J].中国实验方剂学杂志,2018,24(6):177-181.
[3]曾志涛,傅缨,熊耀斌,等.发酵虫草菌粉对人肝癌细胞增殖、凋亡及VEGF,MMP-2表达的影响[J].中国实验方剂学杂志,2015,21(9):141-144.
[4]ZHANG Q Z,YU D. Protection against cerebral infarction by Withaferin A involves inhibition of neuronal apoptosis,activation of PI3K/Akt signaling pathway,and reduced intimal hyperplasia via inhibition of VSMC migration and matrix metalloproteinases[J]. Adv Med Sci,2017,62(1):186-192.
[5]Maliheh A. Epigallocatechin-3-gallate promotes apoptosis in human breast cancer T47D cells through down-regulation of PI3K/Akt and telomerase[J].Pharmacol Rep,2017,69(5):924-928.
[6]孙阳,于水澜,吴勃岩,等.槲皮素自微乳的抗肿瘤作用及其机制分析[J].中国实验方剂学杂志,2018,24(14):97-101.
[7]史桂兰,黄琳,卿海燕,等.槲皮素对人肝癌SMMC-7721细胞生长抑制及诱导凋亡作用[J].中国实验诊断学,2015,19(1):17-19.
[8]张冬青,汪德清,李卉,等.黄芪总黄酮对K562细胞凋亡及细胞周期的影响[J].中国药理学通报,2010,26(1):136-137.
[9]盛连兵,孙凯,刘海萍,等.金雀异黄素调控Egr1/PTEN信号通路诱导卵巢癌SKOV3细胞凋亡的实验研究[J].中国妇产科临床杂志,2017,18(4):341-344.
[10]王晓东.汉黄芩素对人肝癌SMMC-7721的抑制作用及其作用机制研究[J].中国继续医学教育,2016,8(34):180-182.
[11]于海瑶,李石化,孙丽艳,等.异荭草素对胃癌细胞自噬作用的研究[J].中国生化药物杂志,2015,35(12):36-38.
[12]陈帅,郁建平.藤茶总黄酮抗炎及抑菌作用的实验研究[J].贵阳中医学院学报,2013,35(1):1-3.
[13]冯涵,李娜,王华林,等.藤茶中黄酮类成分的功效研究进展[J].公共卫生与预防医学,2018,29(1):82-86.
[14]欧贤红,郑作文.藤茶总黄酮对免疫性肝损伤小鼠的保护作用[J].哈尔滨医药,2011,31(1):1-3.
[15]曾宪彪,韦桂宁,何飞,等.藤茶总黄酮对动脉粥样硬化大鼠血脂及血液流变学的影响[J].重庆医学,2014,43(5):518-520.
[16]周春权,林静瑜,姚欣,等.藤茶总黄酮体外抗肿瘤实验研究[J].中国医药科学,2012,2(9):50-51.
[17]李刚,郑作文,唐云丽.藤茶总黄酮体外抗人肝癌细胞作用研究[J].中国药房,2008(9):652-654.
[18]郑作文.藤茶总黄酮在2215细胞培养中对乙型肝炎病毒HBs Ag、HBe Ag和HBV-DNA的抑制作用[J].山东中医杂志,2003,22(9):561-563.
[19]高倩倩,杨秀芬,欧敏.藤茶总黄酮和二氢杨梅素含药血清对肝癌Hep G2细胞增殖及凋亡的影响[J].中国中药杂志,2011,36(4):500-503.
[20]金慧鸣,谭兴和,蔡文,等.响应面法优化藤茶总黄酮的提取工艺[J].食品安全质量检测学报,2015,6(5):1575-1582.
[21]晏容,刘云,朱欣婷,等.斑蝥素酸镁对人肝癌细胞SMMC-7721裸鼠皮下移植瘤凋亡的影响[J].天然产物研究与开发,2015,27(2):236-240.
[22]冯巍巍,黎梨,黄赞松,等.苦参素通过调控MRP1表达提高多柔比星对人肝癌耐药裸鼠移植瘤的抑制作用[J].右江民族医学院学报,2018,40(1):8-12.
[23]曾云想,郁建平.藤茶总黄酮提取方法的优选[J].山地农业生物学报,2013,32(2):153-158.
[24]钟宇,郑学宝,蔡康荣,等.异丁酰紫草素通过PI3K/Akt/m-TOR通路靶向抑制结肠癌细胞增殖的研究[J].中国中药杂志,2018,43(11):2358-2364.
[25]魏蔚,刘玉华.姜黄素通过活化PI3K/Akt信号通路逆转肝癌耐药细胞株Bel7402/ADR的耐药性[J].中国老年学杂志,2018,38(12):3015-3019.
[26]胡泽成.青龙衣多糖对结肠癌HCT-116细胞增殖、凋亡及PI3K/Akt信号通路的影响[J].中国实验方剂学杂志,2016,22(18):136-139.
[27]辛红,徐巍.甘草查尔酮A通过PI3K/Akt/m TOR信号通路诱导肾癌细胞自噬的研究[J].中国中药杂志,2018,43(17):3545-3552.
[28]陈滨,郭洁文,何素,等.叶下珠复方Ⅱ号对肝癌细胞增殖和胰岛素样生长因子-1受体信号通路转录的作用[J].中国实验方剂学杂志,2018,24(14):108-114.
[29]CHEN Y Y,CAO Y Z,LI F Q,et al. Studies on antihepatoma activity of Annona squamosa L. pericarp extract[J]. Bioorg Med Chem Lett,2017,27(9):1907-1910.
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