双丹胶囊联合5-FU对肝癌细胞Huh-7及荷瘤小鼠协同抗肿瘤作用研究

Study on synergistic anti-tumor effect of Shuangdan Capsules combined with 5-FU on hepatocellular carcinoma cells Huh-7 and xenograft mice

探讨双丹胶囊联合5-氟尿嘧啶(5-FU)对肝癌细胞Huh-7及荷瘤小鼠协同抗肿瘤的作用及其作用机制。考察双丹胶囊联合5-FU对Huh-7细胞活力和血管内皮生长因子(VEGF)受体蛋白表达的影响;同时验证药物联用对脐静脉内皮细胞(HUVEC)细胞成管的影响。此外,建立Huh-7荷瘤小鼠模型观察药物联用体内抗肿瘤的效果并采用超声分子成像检测荷瘤小鼠肿瘤血流分布情况。高效液相色谱法(HPLC)分析显示双丹胶囊主要成分为丹参素钠、原儿茶醛、芍药苷、迷迭香酸、紫草酸、丹酚酸B、丹皮酚。细胞增殖试验中双丹胶囊(20 mg·L^-1)联合5-FU(1μmol·L^-1)对Huh-7细胞抑制率达60%,协同指数(CI)为0.59,提示两者具有协同抗肝癌细胞的作用。两药联用显著抑制Huh-7细胞内VEGF受体的表达;此外药物联用组HUVEC的成管过程缓慢,管腔分支点数目、长度和形成面积明显减小。体内实验中双丹胶囊联合5-FU可抑制Huh-7细胞裸鼠皮下移植瘤的生长,延长其生存期;血管内皮细胞标志蛋白血小板-内皮细胞黏附分子(CD31)表达减少,凋亡相关蛋白半胱氨酸蛋白酶-3(caspase-3)表达升高,裸鼠血清中VEGF水平降低。同时药物联用明显抑制肿瘤组织中基质金属蛋白酶2(MMP2)和VEGF基因表达。超声成像显示,双丹胶囊联合5-FU亦促进肿瘤血管坏死,降低肿瘤组织血流量。以上结果表明,双丹胶囊联合5-FU可能通过抑制VEGF和MMP2的表达抑制肿瘤血管生成,进而阻滞肿瘤生长。

This paper discussed the synergistic anti-tumor effect of Shuangdan Capsules combined with 5-fluorouracil(5-FU)on human liver cancer cell line Huh-7 and tumor bearing mice.The effects of Shuangdan Capsules combined with 5-FU on the activity and vascular endothelial growth factor(VEGF)receptor protein expression of Huh-7 cells were investigated,and the effects of drug combination on tube formation of HUVEC cell were also verified.In addition,the mice model of Huh-7 was established to observe the anti-tumor effect of drug combination and the distribution of tumor blood flow in tumor bearing mice by using molecular imaging.HPLC analysis showed that Shuangdan Capsules mainly consisted of danshensusodium,protocatechuic aldehyde,paeoniflorin,rosmarinic acid,alkannic acid,salvianolic acid B,and paeonol.In MTT experiment,the inhibition rate of Shuangdan Capsules(20 mg·L^-1)and 5-FU(1μmol·L^-1)on Huh-7 cells was 60%,and the CI value was 0.59,suggesting that these two drugs had synergistic anti-hepatoma cells effect.The expression of VEGF receptor in Huh-7 cells was inhibited by the combination of these two drugs.In addition,the process of HUVEC was slow,and the number,length and area of the lumen branches decreased significantly.In vivo,Shuangdan Capsules combined with 5-FU inhibited the growth and prolongation of survival of Huh-7 cells in subcutaneous transplanted tumor nude mice;serum expression of CD31 and VEGF in nude mice were decreased,while caspase-3 was increased.Meanwhile,the drug combination significantly inhibited the expressions of MMP2 and VEGF in tumor tissues.Ultrasound showed that Shuangdan Capsules combined with 5-FU also inhibited tumor angiogenesis and reduced blood flow of tumor tissue.The results showed that Shuangdan Capsules combined with 5-FU may inhibit tumor angiogenesis by inhibiting VEGF and MMP2 expressions,thereby blocking tumor growth.