理冲汤加减联合5-氟尿嘧啶对H22荷瘤小鼠移植瘤上皮间质转化的影响
|
摘要
目的:探讨理冲汤加减联合5-氟尿嘧啶(5-FU)对H22荷瘤小鼠皮下移植瘤上皮间质转化(EMT)的影响。方法:构建小鼠移植性肝癌模型。成瘤后,将其随机分为空白组,5-FU组(5-FU,2. 5 mg·kg-1),理冲汤加减联合5-FU组(5-FU+理冲汤加减组),理冲汤加减组(理冲汤加减25 g·kg-1),每组10只。观察理冲汤加减对小鼠皮下移植瘤的作用及与5-FU合用的效果;采用实时荧光定量聚合酶链式反应(Real-time PCR)检测各组瘤体中EMT相关基因上皮型钙黏蛋白(E-cadherin),神经型钙黏蛋白(N-cadherin),锌指转录因子Snail,Twist mRNA表达;采用蛋白免疫印迹法(Western blot)检测各组瘤体内EMT相关蛋白E-cadherin,N-cadherin,Snail,波形蛋白(Vimentin)的表达。结果:5-FU组,5-FU+理冲汤加减组,理冲汤加减组的抑瘤率分别为59. 18%,84. 42%,10. 39%,均能抑制肝癌移植瘤的生长,且5-FU+理冲汤加减组抑瘤率显著高于5-FU组(P <0. 01)。胸腺指数、脾指数显示,5-FU+理冲汤加减组胸腺指数与脾指数均高于5-FU组(P <0. 05,P <0. 01)。PCR显示,与空白组比较,5-FU组,5-FU+理冲汤加减组E-cadherin的表达上调,N-cadherin,Snail,Twist的表达下调(P <0. 05,P <0. 01);与5-FU组比较,5-FU+理冲汤加减组E-cadherin的表达上调,N-cadherin,Snail,Twist的表达下调(P <0. 05,P <0. 01)。Western blot显示,与空白组比较,5-FU组,5-FU+理冲汤加减组,理冲汤加减组E-cadherin的表达上调,N-cadherin,Snail,Vimentin的表达下调(P <0. 05,P <0. 01);与5-FU组比较,5-FU+理冲汤加减组E-cadherin的表达上调,N-cadherin,Snail,Vimentin的表达下调(P <0. 05,P <0. 01)。结论:理冲汤加减与5-FU单独和联合使用均对肝癌小鼠移植瘤的生长有抑制作用,且两药合用能增强化疗效果,且一定程度上抑制了5-FU的毒性,其机制可能与对肝癌EMT的抑制有关。
Objective: To investigate the effect of modified Lichongtang combined with 5-fluorouracil(5-FU) on tumor epithelial-mesenchymal transition(EMT) in H22 tumor-bearing mice. Method: Mouse models of transplanted hepatoma were constructed. After tumor formation,they were randomly divided into 4 groups: blank group,5-FU group(2. 5 mg·kg-15-FU intraperitoneal injection),modified Lichongtang combined with 5-FU group(5-FU + Chinese medicine group), and modified Lichongtang group(Chinese medicine group,25 g·kg-1 gavage),n = 10 in each group. The effect of modified Lichongtang combined with 5-FU on the tumor inhibition rate of subcutaneous transplanted tumor was observed. The gene expression levels of E-cadherin,N-cadherin,Snail,and Twist in transplanted tumor were observed by Real-time PCR. The protein expression levels of E-cadherin,N-cadherin,Snail,and Vimentin were detected by using Western blot. Result: The tumor inhibiting rate was 59. 18%,84. 42%, and 10. 39% respectively in 5-FU group,5-FU + Chinese medicine group,and Chinese medicine group. All of these can inhibit the growth of liver cancer transplantation tumor,and the tumor inhibiting rate of 5-FU + Chinese medicine group was significantly higher than that in 5-FU group(P < 0. 01). Thymus index and spleen index in 5-FU + Chinese medicine group were significantly higher than those in 5-FU group(P < 0. 05,P < 0. 01). Results of Real-time PCR indicated that,as compared with blank group,expression levels of E-cadherin were up-regulated,but expression levels of N-cadherin,Snail,and Twist were down-regulated in 5-FU group,5-FU + Chinese medicine group,and Chinese medicine group(P < 0. 05,P < 0. 01). As compared with the 5-FU group,expression levels of E-cadherin were up-regulated,but expression levels of N-cadherin,Snail,and Twist were down-regulated in the 5-FU + Chinese medicine group(P < 0. 05,P < 0. 01). Results of Western blot showed that,as compared with blank group,expression levels of E-cadherin were up-regulated,but expression levels of N-cadherin,Snail,Vimentin were down-regulated in 5-FU group,5-FU + Chinese medicine group,and Chinese medicine group(P < 0. 05,P < 0. 01); as compared with 5-FU group,expression levels of E-cadherin were up-regulated, but expression levels of N-cadherin, Snail, and Vimentin were down-regulated in the 5-FU + Chinese medicine group(P < 0. 05, P < 0. 01). Conclusion:Modified Lichongtang,5-FU and their combination have inhibitory effect on the growth of transplanted tumors of hepatocarcinoma mice,and the combination of the two drugs can enhance the effect of chemotherapy and to some extent inhibit the toxicity of 5-FU. The mechanism may be related to the inhibition of liver cancer EMT.
Objective: To investigate the effect of modified Lichongtang combined with 5-fluorouracil(5-FU) on tumor epithelial-mesenchymal transition(EMT) in H22 tumor-bearing mice. Method: Mouse models of transplanted hepatoma were constructed. After tumor formation,they were randomly divided into 4 groups: blank group,5-FU group(2. 5 mg·kg-15-FU intraperitoneal injection),modified Lichongtang combined with 5-FU group(5-FU + Chinese medicine group), and modified Lichongtang group(Chinese medicine group,25 g·kg-1 gavage),n = 10 in each group. The effect of modified Lichongtang combined with 5-FU on the tumor inhibition rate of subcutaneous transplanted tumor was observed. The gene expression levels of E-cadherin,N-cadherin,Snail,and Twist in transplanted tumor were observed by Real-time PCR. The protein expression levels of E-cadherin,N-cadherin,Snail,and Vimentin were detected by using Western blot. Result: The tumor inhibiting rate was 59. 18%,84. 42%, and 10. 39% respectively in 5-FU group,5-FU + Chinese medicine group,and Chinese medicine group. All of these can inhibit the growth of liver cancer transplantation tumor,and the tumor inhibiting rate of 5-FU + Chinese medicine group was significantly higher than that in 5-FU group(P < 0. 01). Thymus index and spleen index in 5-FU + Chinese medicine group were significantly higher than those in 5-FU group(P < 0. 05,P < 0. 01). Results of Real-time PCR indicated that,as compared with blank group,expression levels of E-cadherin were up-regulated,but expression levels of N-cadherin,Snail,and Twist were down-regulated in 5-FU group,5-FU + Chinese medicine group,and Chinese medicine group(P < 0. 05,P < 0. 01). As compared with the 5-FU group,expression levels of E-cadherin were up-regulated,but expression levels of N-cadherin,Snail,and Twist were down-regulated in the 5-FU + Chinese medicine group(P < 0. 05,P < 0. 01). Results of Western blot showed that,as compared with blank group,expression levels of E-cadherin were up-regulated,but expression levels of N-cadherin,Snail,Vimentin were down-regulated in 5-FU group,5-FU + Chinese medicine group,and Chinese medicine group(P < 0. 05,P < 0. 01); as compared with 5-FU group,expression levels of E-cadherin were up-regulated, but expression levels of N-cadherin, Snail, and Vimentin were down-regulated in the 5-FU + Chinese medicine group(P < 0. 05, P < 0. 01). Conclusion:Modified Lichongtang,5-FU and their combination have inhibitory effect on the growth of transplanted tumors of hepatocarcinoma mice,and the combination of the two drugs can enhance the effect of chemotherapy and to some extent inhibit the toxicity of 5-FU. The mechanism may be related to the inhibition of liver cancer EMT.
引文
[1]陆再英,钟南山.内科学[M]. 7版.北京:人民卫生出版社,2012:457.
[2] WANG F S,FAN J G,ZHANG Z,et al. The global burden of liver disease:the major impact of China[J].Hepatology,2014,60(6):2099-2108.
[3]马文杰,蒋春鹤.中医药治疗原发性肝癌的研究进展[J].世界最新医学信息文摘,2015,15(A4):45-46.
[4]邱宇航,孙珏.中医药治疗对提高肿瘤患者生存质量的意义[J].内蒙古中医药,2017,36(16):99-100.
[5]张锡纯.医学衷中参西录[M].太原:山西科学技术出版社,2009:168.
[6]吕玲,张立,李能莲,等.黄芪的抗肿瘤作用[J].世界临床药物,2014,35(5):324-328.
[7]武超,许杜娟,杨翠,等.黄芪皂苷Ⅱ增加5-氟尿嘧啶对人肝癌细胞株Hep G2增殖抑制作用[J].安徽医科大学学报,2016,51(1):78-82.
[8]武静莲,徐强,谢亲建,等.党参抗肿瘤药理作用研究[J].西部中医药,2016,29(8):18-21.
[9]冯娅茹,张文婷,李二文,等.三棱化学成分及药理作用研究进展[J].中草药,2017,48(22):4804-4818.
[10]李学臣,张涛,魏晓东.三棱提取物对H22荷瘤小鼠的抑瘤作用[J].黑龙江医药科学,2010,33(5):78.
[11]赵志梅,张立杰,夏天,等.莪术主要单体成分抗炎、抗肿瘤作用研究进展[J].药物评价研究,2017,40(1):119-124.
[12]卢冬雪,祁明浩,薛恬,等.刘沈林治疗晚期消化道肿瘤用药经验[J].世界中西医结合杂志,2017,12(8):1064-1068.
[13]臧文华,唐德才,尹刚,等.黄芪-莪术配伍及联合顺铂对人肝癌裸鼠原位移植瘤生长的抑制作用[J].中国实验方剂学杂志,2014,20(5):131-136.
[14]臧文华,黄显章,唐德才,等.黄芪-莪术联合顺铂诱导肝癌细胞凋亡及其对miR-122a,miR-221,miR-151表达的影响[J].中国实验方剂学杂志,2016,22(17):87-91.
[15] WU S X,WU G X,HE S,et al. Antitumor effect of volatile oil from sinapis albae semen on H22-bearing mice and its mechanism[J]. Chin Trad Herbal Drugs,2013,44(21):3024-3029.
[16]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,2000:145.
[17] Sanceau J,Poupon M F,Delattre O,et al. Strong inhibition of ewing tumor xenograft growth by combination of human interferon-alpha or interferon-beta with ifosfamide[J]. Oncogene, 2002, 21(50):7700-7709.
[18]金正均.等概率和曲线与“Q50”——估计合并用药效果的一种新方法[J].上海交通大学学报:医学版,1981,1(1):15-18,86.
[19] Hay E D. The mesenchymal cell, its role in the embryo,and the remarkable signaling mechanisms that create it[J]. Dev Dyn,2005,233(3):706-720.
[20] Boyer B, Vallés A M, Edme N. Induction and regulation of epithelial-mesenchymal transitions[J].Biochem Pharmacol,2000,60(8):1091-1099.
[21] KANG Y, Massague J. Epithelial-mesenchymal transitions:twist indevelopment and metastasis[J].Cell,2004,118(3):277-279.
[22] Kalluri R. EMT:When epithelial cells decide to become mesenchymal-like cells[J]. J Clin Invest,2009,119(6):1417-1419.
[23] Guarino M, Rubino B, Ballabio G. The role of epithelial-mesenchymal transition in cancer pathology[J]. Pathology,2007,39(3):305-318.
[24] Tepass U,Truong K,Godt D,Ikura M,et al. Cadherins in embryonic and neural morphogenesis[J]. Nat Rev Mol Cell Biol,2000,1(2):91-100.
[25] Hazan R B,Phillips G R,QIAO R F,et al. Exogenous expression of N-cadherin in breast cancer cells induces cell migration,invasion,and metastasis[J]. J Cell Biol,2000,148(4):779-790.
[26]魏荷花,杨英捷,訾聃. N-cadherin和肿瘤干细胞相关标志物在卵巢上皮性肿瘤中的表达[J].中国妇幼保健,2017,32(5):1055-1057.
[27]申震. E-Cadherin及Vimentin在子宫颈癌中的表达及其与上皮-间质转化的相关性[D].合肥:安徽医科大学,2013.
[28] Peinado H,Olmeda D,Cano A. Snail,Zeb and b HLH factors in tumour progression:an alliance against the epithelial phenotype[J]. Nat Rev Cancer,2007,7(6):415-428.
[29] Grau Y,Carteret C,Simpson P. Mutations and chromosomal rearrangements affecting the expression of Snail,a gene involved in embryonic patterning in DROSOPHILA MELANOGASTER[J]. Genetics,1984,108(2):347-360.
[30] WANG Y,Ngo VN,Marani M,et al. Critical role for transcriptional repressor Snail2 in transformation by oncogenic RAS in colorectal carcinoma cells[J].Oncogene,2010,29(33):4658-4670.
[31] Salerno P,Garcia-Rostan G,Piccinin S,et al. Twist1plays a pleiotropic role in determining the anaplastic thyroidcancer phenotype[J]. J Clin Endocrinol Metab,2011,96(5):772-781.
[32]方雪妮,周天,李泉旺,等. TWIST表达与恶性肿瘤转移关系的研究进展[J].中国肿瘤,2016,25(11):893-897.
[33] HU F W,TSAI L L,YU C H,et al. Impairment of tumor-initiating stem-like property and reversal of epithelial-mesenchymal transdifferentiation in head and neck cancer by resveratrol treatment[J]. Mol Nutr Food Res,2012,56(8):1247-1258.
[34] Fischer K R,Durrans A,Lee S,et al. Epithelial-tomesenchymal transition is not required for lung metastasis but contributes to chemoresistance[J]. Nature,2015,527(7579):472-476.
[2] WANG F S,FAN J G,ZHANG Z,et al. The global burden of liver disease:the major impact of China[J].Hepatology,2014,60(6):2099-2108.
[3]马文杰,蒋春鹤.中医药治疗原发性肝癌的研究进展[J].世界最新医学信息文摘,2015,15(A4):45-46.
[4]邱宇航,孙珏.中医药治疗对提高肿瘤患者生存质量的意义[J].内蒙古中医药,2017,36(16):99-100.
[5]张锡纯.医学衷中参西录[M].太原:山西科学技术出版社,2009:168.
[6]吕玲,张立,李能莲,等.黄芪的抗肿瘤作用[J].世界临床药物,2014,35(5):324-328.
[7]武超,许杜娟,杨翠,等.黄芪皂苷Ⅱ增加5-氟尿嘧啶对人肝癌细胞株Hep G2增殖抑制作用[J].安徽医科大学学报,2016,51(1):78-82.
[8]武静莲,徐强,谢亲建,等.党参抗肿瘤药理作用研究[J].西部中医药,2016,29(8):18-21.
[9]冯娅茹,张文婷,李二文,等.三棱化学成分及药理作用研究进展[J].中草药,2017,48(22):4804-4818.
[10]李学臣,张涛,魏晓东.三棱提取物对H22荷瘤小鼠的抑瘤作用[J].黑龙江医药科学,2010,33(5):78.
[11]赵志梅,张立杰,夏天,等.莪术主要单体成分抗炎、抗肿瘤作用研究进展[J].药物评价研究,2017,40(1):119-124.
[12]卢冬雪,祁明浩,薛恬,等.刘沈林治疗晚期消化道肿瘤用药经验[J].世界中西医结合杂志,2017,12(8):1064-1068.
[13]臧文华,唐德才,尹刚,等.黄芪-莪术配伍及联合顺铂对人肝癌裸鼠原位移植瘤生长的抑制作用[J].中国实验方剂学杂志,2014,20(5):131-136.
[14]臧文华,黄显章,唐德才,等.黄芪-莪术联合顺铂诱导肝癌细胞凋亡及其对miR-122a,miR-221,miR-151表达的影响[J].中国实验方剂学杂志,2016,22(17):87-91.
[15] WU S X,WU G X,HE S,et al. Antitumor effect of volatile oil from sinapis albae semen on H22-bearing mice and its mechanism[J]. Chin Trad Herbal Drugs,2013,44(21):3024-3029.
[16]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,2000:145.
[17] Sanceau J,Poupon M F,Delattre O,et al. Strong inhibition of ewing tumor xenograft growth by combination of human interferon-alpha or interferon-beta with ifosfamide[J]. Oncogene, 2002, 21(50):7700-7709.
[18]金正均.等概率和曲线与“Q50”——估计合并用药效果的一种新方法[J].上海交通大学学报:医学版,1981,1(1):15-18,86.
[19] Hay E D. The mesenchymal cell, its role in the embryo,and the remarkable signaling mechanisms that create it[J]. Dev Dyn,2005,233(3):706-720.
[20] Boyer B, Vallés A M, Edme N. Induction and regulation of epithelial-mesenchymal transitions[J].Biochem Pharmacol,2000,60(8):1091-1099.
[21] KANG Y, Massague J. Epithelial-mesenchymal transitions:twist indevelopment and metastasis[J].Cell,2004,118(3):277-279.
[22] Kalluri R. EMT:When epithelial cells decide to become mesenchymal-like cells[J]. J Clin Invest,2009,119(6):1417-1419.
[23] Guarino M, Rubino B, Ballabio G. The role of epithelial-mesenchymal transition in cancer pathology[J]. Pathology,2007,39(3):305-318.
[24] Tepass U,Truong K,Godt D,Ikura M,et al. Cadherins in embryonic and neural morphogenesis[J]. Nat Rev Mol Cell Biol,2000,1(2):91-100.
[25] Hazan R B,Phillips G R,QIAO R F,et al. Exogenous expression of N-cadherin in breast cancer cells induces cell migration,invasion,and metastasis[J]. J Cell Biol,2000,148(4):779-790.
[26]魏荷花,杨英捷,訾聃. N-cadherin和肿瘤干细胞相关标志物在卵巢上皮性肿瘤中的表达[J].中国妇幼保健,2017,32(5):1055-1057.
[27]申震. E-Cadherin及Vimentin在子宫颈癌中的表达及其与上皮-间质转化的相关性[D].合肥:安徽医科大学,2013.
[28] Peinado H,Olmeda D,Cano A. Snail,Zeb and b HLH factors in tumour progression:an alliance against the epithelial phenotype[J]. Nat Rev Cancer,2007,7(6):415-428.
[29] Grau Y,Carteret C,Simpson P. Mutations and chromosomal rearrangements affecting the expression of Snail,a gene involved in embryonic patterning in DROSOPHILA MELANOGASTER[J]. Genetics,1984,108(2):347-360.
[30] WANG Y,Ngo VN,Marani M,et al. Critical role for transcriptional repressor Snail2 in transformation by oncogenic RAS in colorectal carcinoma cells[J].Oncogene,2010,29(33):4658-4670.
[31] Salerno P,Garcia-Rostan G,Piccinin S,et al. Twist1plays a pleiotropic role in determining the anaplastic thyroidcancer phenotype[J]. J Clin Endocrinol Metab,2011,96(5):772-781.
[32]方雪妮,周天,李泉旺,等. TWIST表达与恶性肿瘤转移关系的研究进展[J].中国肿瘤,2016,25(11):893-897.
[33] HU F W,TSAI L L,YU C H,et al. Impairment of tumor-initiating stem-like property and reversal of epithelial-mesenchymal transdifferentiation in head and neck cancer by resveratrol treatment[J]. Mol Nutr Food Res,2012,56(8):1247-1258.
[34] Fischer K R,Durrans A,Lee S,et al. Epithelial-tomesenchymal transition is not required for lung metastasis but contributes to chemoresistance[J]. Nature,2015,527(7579):472-476.