阴阳攻积丸含药血清抑制HepG2细胞增殖、侵袭及促进其凋亡的作用
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摘要
目的:探讨阴阳攻积丸(Yingyang Gongji Wan,YYGJ)对肝癌细胞株HepG2的体外作用,为临床应用提供依据。方法:制备YYGJ大鼠含药血清;四唑化合物(MTS)比色法检测YYGJ含药血清组和空白组抑制率;原位末端凋亡法(TUNEL)检测空白组,YYGJ含药血清和5-氟尿嘧啶(5-FU)组凋亡;实时荧光定量PCR(Real-time PCR)及蛋白免疫印迹法(Western blot)检测HepG2细胞上皮细胞钙黏蛋白(E-cadherin),波形蛋白(Vimentin),基质金属蛋白酶-2(MMP-2)和Smad4 mRNA和蛋白表达;细胞迁移分析(transwell)检测HepG2细胞侵袭能力。结果:YYGJ含药血清呈浓度时间依赖性抑制HepG2细胞增殖,与空白组比较,第3天YYGJ 5%,10%,20%含药血清抑制率均明显升高(P <0. 05); YYGJ含药血清组凋亡率显著升高(P <0. 05),YYGJ 50%含药血清组凋亡率与5-FU组比较差异不显著。与空白组比较,YYGJ含药血清组E-cadherin,Smad4 mRNA和蛋白表达均明显升高,Vimentin,MMP-2 mRNA和蛋白均明显降低(P <0. 05),与5-FU作用一致;与空白组比较,YYGJ含药血清组HepG2细胞侵袭能力明显降低(P <0. 05),其中YYGJ 50%含药血清组与5-FU组细胞侵袭数目无显著差异。结论:阴阳攻积丸含药血清可抑制HepG2细胞增殖,诱导细胞凋亡,调节肿瘤细胞上皮间质转换(EMT)相关的E-cadherin,Vimentin,MMP-2,Smad4 mRNA和蛋白表达,并降低肿瘤侵袭能力,与化疗药物5-FU作用特点类似。该方可作为治疗肝癌寒湿证型的代表方,应深入研究其抗癌机制。
Objective: To investigate the effect of Yingyang Gongji Wan( YYGJ) on hepatoma cell line HepG2,and provide evidence for clinical application. Method: YYGJ-contained rats serum was prepared. Then the inhibiory rate of cells was detected by methye thiazolye telrazlium( MTS) method in both YYGJ group and blank group. Apoptosis of HepG2 was detected by TdT-mediated d UT nick-end labeling( TUNEL) method in blank group,YYGJ group,and 5-fluorouracil( 5-FU) group. The mRNA expression and protein expression levels of E-cadherin,Vimentin,metalloproteinase-2( MMP-2) and Smad4 were detected by Real-time quantitative PCR( Real-time PCR) and Western blot respectively. The invasion ability of HepG2 cells was detected by cell migration assay( transwell). Result: YYGJ-contained serum inhibited the proliferation of HepG2 cells in a time and concentration-dependent manner. As compared with blank group,the inhibitory rate was increased in 5%,10%,and 20% YYGJ-contained serum groups on the third day( P < 0. 05),and apoptosis rate was also increased significantly in YYGJ and 5-FU groups( P < 0. 05),but there was no significant difference between 50% YYGJ group and 5-FU group in apoptosis rate. As compared with blank group,the mRNA and protein expression levels of E-cadherin and Smad4 were increased significantly,while Vimentin and MMP-2 mRNA and protein expression levels were decreased significantly in YYGJ and 5-FU groups( P < 0. 05). HepG2 cell invasive ability was decreased significantly in YYGJ and 5-FU groups as compared to blank group( P < 0. 05),but there was no significant difference between 50% YYGJ group and 5-FU group. Conclusion: YYGJ-contained serum can inhibit the proliferation of HepG2 cells,induce apoptosis,regulate epithelial-mesenchymal transition( EMT)-related E-cadherin,Vimentin,MMP-2 and Smad4 genes and proteins,and decrease tumor invasion ability. The effect was similar to that of 5-fluorouracil. As a unique prescription,YYGJ can be used as a representative for the treatment of coldness and dampness syndrome of primary liver cancer and its anti-cancer mechanism should be further studied.
Objective: To investigate the effect of Yingyang Gongji Wan( YYGJ) on hepatoma cell line HepG2,and provide evidence for clinical application. Method: YYGJ-contained rats serum was prepared. Then the inhibiory rate of cells was detected by methye thiazolye telrazlium( MTS) method in both YYGJ group and blank group. Apoptosis of HepG2 was detected by TdT-mediated d UT nick-end labeling( TUNEL) method in blank group,YYGJ group,and 5-fluorouracil( 5-FU) group. The mRNA expression and protein expression levels of E-cadherin,Vimentin,metalloproteinase-2( MMP-2) and Smad4 were detected by Real-time quantitative PCR( Real-time PCR) and Western blot respectively. The invasion ability of HepG2 cells was detected by cell migration assay( transwell). Result: YYGJ-contained serum inhibited the proliferation of HepG2 cells in a time and concentration-dependent manner. As compared with blank group,the inhibitory rate was increased in 5%,10%,and 20% YYGJ-contained serum groups on the third day( P < 0. 05),and apoptosis rate was also increased significantly in YYGJ and 5-FU groups( P < 0. 05),but there was no significant difference between 50% YYGJ group and 5-FU group in apoptosis rate. As compared with blank group,the mRNA and protein expression levels of E-cadherin and Smad4 were increased significantly,while Vimentin and MMP-2 mRNA and protein expression levels were decreased significantly in YYGJ and 5-FU groups( P < 0. 05). HepG2 cell invasive ability was decreased significantly in YYGJ and 5-FU groups as compared to blank group( P < 0. 05),but there was no significant difference between 50% YYGJ group and 5-FU group. Conclusion: YYGJ-contained serum can inhibit the proliferation of HepG2 cells,induce apoptosis,regulate epithelial-mesenchymal transition( EMT)-related E-cadherin,Vimentin,MMP-2 and Smad4 genes and proteins,and decrease tumor invasion ability. The effect was similar to that of 5-fluorouracil. As a unique prescription,YYGJ can be used as a representative for the treatment of coldness and dampness syndrome of primary liver cancer and its anti-cancer mechanism should be further studied.
引文
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[15]谢金玲,卓少元. Vimentin在原发性肝癌发生发展及凋亡过程中的作用[J].中国肿瘤,2017,26(2):130-134.
[16]何平.探讨基质金属蛋白酶2在肝癌细胞株SMMC-7721上皮间质转化发生中的作用[J].临床医药文献电子杂志,2016,3(14):2682-2684.
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[18]王蓟花,唐静,李端,等.皂荚化学成分和生物活性的研究进展[J].中国野生植物资源,2008,27(6):1-3.
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[20]南京中医药大学.中药大辞典[M].上海:上海科学技术出版社,2006:1592-1597.
[21]郭晨旭,吴迎春,朱元章,等.藏族药翼首草正丁醇部位提取物体外抑制人肝癌Hep3B细胞增殖和侵袭转移[J].中国实验方剂学杂志,2015,21(3):100-105.
[22]龚慕辛,王智民,张启伟,等.吴茱萸有效成分的药理研究进展[J].中药新药与临床药理,2009,20(2):183-187.
[23] Shyu K G,LIN S,Lee C C,et al. Evodiamine inhibits invitro angiogenesis:implication for antitumor genicity[J]. Life Sci,2006,78(19):2234-2243.
[24]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,MMP2表达的影响[J].中国实验方剂学杂志,2015,21(9):141-144.
[25]李玉先,刘晓东,朱照静.半夏药理作用的研究述要[J].辽宁中医学院学报,2004,6(6):459-460.
[26]李佳桓,林海红,杜钢军.巴豆不同给药方式对荷瘤小鼠的影响[J].河南大学学报:医学版,2012,31(1):8-11.
[2]周顺. EMT相关蛋白在肝细胞肝癌组织中的表达鉴定及相关microRNA表达谱的研究[D].南京:南京医科大学,2010.
[3]中华人民共和国国家卫生和计划生育委员会.原发性肝癌诊疗规范(2017年版)[J].临床肝胆病杂志,2017,33(8):1419-1431.
[4]何新慧,朱娇玉,吴中平.历代文献癥瘕、积聚证治计算机分析[J].上海中医药杂志,2004,38(11):62-64.
[5]刑汝雯.黄帝内经[M].武汉:华中科技大学出版社,2017:282-286.
[6]柳长华.巢元方·诸病源候论[M].北京:北京科学技术出版社,2016:194-202.
[7]陈玉超,刘沈林.浅谈扶阳法在肿瘤治疗中的应用[J].吉林中医药,2005,25(3):13-15.
[8]封艳艳,马齐襄,隋彤彤,等.斑蝥素对肝癌Hep G2细胞增殖、凋亡的影响及其可能机制[J].中国实验方剂学杂志,2017,23(15):112-117.
[9]潘宇,李顺祥,黄丹,等.基于网络药理学预测肝复乐治疗肝细胞癌的抗癌活性成分[J].中华中医药杂志,2014,29(5):1490-1498.
[10]石芳毓,娄丽丽,谢吻,等.中药注射剂联合肝动脉插管栓塞化疗术治疗肝癌的网状Meta分析[J].中国实验方剂学杂志,2016,22(10):180-187.
[11]包来发.李中梓医学全书[M].北京:中国中医药出版社,2015:206-210.
[12]许兴涛.论《医宗必读》对中医肿瘤学的贡献[J].河南中医药学刊,2000,15(2):2-3.
[13]卓少元,谢金玲.健脾益气方含药血清通过Caspase-3/Vimentin促进人肝癌MHCC-97H细胞凋亡[J].中国实验方剂学杂志,2016,22(24):161-166.
[14]李裕明,韩克强,郑璐,等.上皮间质转化标志物E-cadherin和Vimentin在原发性肝癌中的表达及其临床意义[J].中华肝脏病杂志,2013,21(4):279-284.
[15]谢金玲,卓少元. Vimentin在原发性肝癌发生发展及凋亡过程中的作用[J].中国肿瘤,2017,26(2):130-134.
[16]何平.探讨基质金属蛋白酶2在肝癌细胞株SMMC-7721上皮间质转化发生中的作用[J].临床医药文献电子杂志,2016,3(14):2682-2684.
[17]黄晓丹,张发明,季国忠. Smad4在肿瘤侵袭和转移中的作用[J].世界华人消化杂志,2009,17(9):849-853.
[18]王蓟花,唐静,李端,等.皂荚化学成分和生物活性的研究进展[J].中国野生植物资源,2008,27(6):1-3.
[19]张传清.皂角为主治愈晚期胃癌1例[J].四川中医,1988,1(2):22.
[20]南京中医药大学.中药大辞典[M].上海:上海科学技术出版社,2006:1592-1597.
[21]郭晨旭,吴迎春,朱元章,等.藏族药翼首草正丁醇部位提取物体外抑制人肝癌Hep3B细胞增殖和侵袭转移[J].中国实验方剂学杂志,2015,21(3):100-105.
[22]龚慕辛,王智民,张启伟,等.吴茱萸有效成分的药理研究进展[J].中药新药与临床药理,2009,20(2):183-187.
[23] Shyu K G,LIN S,Lee C C,et al. Evodiamine inhibits invitro angiogenesis:implication for antitumor genicity[J]. Life Sci,2006,78(19):2234-2243.
[24]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,MMP2表达的影响[J].中国实验方剂学杂志,2015,21(9):141-144.
[25]李玉先,刘晓东,朱照静.半夏药理作用的研究述要[J].辽宁中医学院学报,2004,6(6):459-460.
[26]李佳桓,林海红,杜钢军.巴豆不同给药方式对荷瘤小鼠的影响[J].河南大学学报:医学版,2012,31(1):8-11.