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

Effect of Yingyang Gongji Wan Contained Serum on Inhibiting HepG2 Cell Proliferation and Invasion,and Inducing Apoptosis

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.