解毒消癥饮增强5-FU抗肝癌作用的体内外研究

摘要

目的:目前,中国是世界上肝癌发病率和死亡率持续上升的国家之一.5-fluorouracil(5-FU)是消化道肿瘤治疗的常用化疗药物,其临床用药表明,随着5-FU剂量的增大其药物毒副作用逐渐增大,5-FU的毒副作用已成为其临床应用的瓶颈问题,因此急需开发新型低毒副作用的肝癌临床治疗药物.中药因其具有多靶点、低毒性、整体调节的作用,越来越受到全世界的关注,因而从中药中寻找降低5-FU用量同时增强5-FU抗肿瘤作用的药物或许是潜在的肝癌治疗手段.解毒消癥饮(JXD)属于临床验方,应用于消化道肿瘤特别是肝癌治疗已10年有余.前期研究发现,JXD通过诱导肿瘤细胞凋亡、阻滞细胞周期及抑制血管新生等方式抑制肿瘤生长.本研究中我们评估了解毒消癥饮乙酸乙酯部位(EE-JXD)联合5-FU的抗肿瘤作用,探讨JXD的生物学作用机制,以期为JXD的临床应用提供更多的实验依据.方法:EE-JXD按照本研究曾发表论文的方法制备,再用DMSO稀释为200 mg/mL的储存液用于细胞实验母液,采用生理盐水进一步配制为6 mg/mL动物实验用药.HepG2细胞用含有10％胎牛血清、1％抗生素的RPMI-1640培养液培养,培养条件为37℃、5％ CO2,HepG2正常培养后取对数期细胞种板,采用不同药物浓度的EE-JXD (0.05、0.1、0.2、0.4 mg/mL),5-FU (0.05、0.1、0.2、0.4 mg/mL),EE-JXD +5-FU[(0.05 +0.05)、(0.1+0.1)、(0.2+0.2)、(0.4+0.4)mg/mL]干预24 h后,经MTT检测细胞活力.细胞被分为对照组、EE-JXD组(0.1 mg/mL)、5-FU组(0.1 mg/mL)、EE-JXD +5-FU组[(0.1 +0.1)mg/mL]共4组,药物干预24 h后,Hochest 33258染色后通过荧光显微镜观察HepG2细胞凋亡的发生情况,AnnexinV-FITC/propidium iodide (PI)标记后流式细胞仪检测细胞早期凋亡和晚期凋亡率.在32只BALB/c (nu/nu)体质量为18～22 g小鼠皮下建立HepG2细胞的移植瘤模型,HepG2细胞密度为5×106/mL被注入右前肢皮下,7d后小鼠被随机分为4组[对照组(生理盐水),EE-JXD组[15 g/(kg·d),i.g.],5-FU组[10mg/(kg·d),i.p.],EE-JXD+5-FU组[15 g/(kg·d)+10mg/(kg·d)]],给药30 d,其间每3天测量肿瘤体积和小鼠体质量,30 d后取材称量肿瘤重量,采用TUNEL法检测肿瘤组织的凋亡率.结果:EE-JXD、5-FU和EE-JXD+ 5-FU均以剂量依赖的方式抑制肿瘤细胞活力,EE-JXD+ 5-FU组抑制作用最强,与对照组比较差异具有统计学意义(P＜0.01).Hoechst荧光染色观察表明,EE-JXD和5-FU能够促进HepG2细胞的凋亡.进一步流式细胞术检测早期和晚期凋亡率表明,5-FU组、EE-JXD组和EE-JXD+5-FU组凋亡发生率分别为5％、7.8％和12.9％.体内实验也证实EE-JXD+ 5-FU能够显著抑制小鼠皮下移植瘤的生长,30 d后对照组肿瘤重量为(0.43±0.06)g,5-FU组为(0.08±0.01)g,5-FU组与对照组比较差异有统计学意义(P＜0.01),5-FU+ EE-JXD组的肿瘤重量为(0.05±0.01)g,与对照组比较差异有统计学意义(P＜0.01),表明EE-JXD有效增强5-FU的抗肿瘤作用.TUNEL检测肿瘤组织中凋亡发生率表明,5-FU组显著诱导了肝癌细胞凋亡的发生,与对照组比较差异有统计学意义(P＜0.05),这种诱导凋亡的作用被EE-JXD增强,EE-JXD+ 5-FU组与对照组比较差异有统计学意义(P＜0.01).而4组间小鼠的体质量相近,差异无统计学意义(P＞0.05).结论:EE-JXD体内外增强了5-FU的抗肿瘤作用,且未见明显的毒副作用,其生物学机制有待进一步研究.%Objective:Up to now,morbidity and mortality of hepatocellular carcinoma (HCC) have been increasing all over the world especially in China.5-fluorouracil (5-FU) is a common used chemotherapy drug for many types tumor in clinic including HCC.However with the bigger dosage of 5-FU applying the more severely toxicity and side-effects have limited it's widely clinical use.Therefore,there is a strong demand to find some new drugs of low side-effect for HCC treatment.Accumulating attention has been paid to Traditional Chinese Medicine (TCM) because of the multi-targets,low-toxicity and the whole system regulation for patients.Maybe it is a potential effective way on finding some TCM to enhance anti-tumor effect with low dose 5-FU.Jiedu Xiaozheng Decoction (JXD),a polyherbal formula of TCM,has been used in clinical treatment for digestive tract tumor especially HCC for more than a decade.The previous studies found that JXD inhibited tumor growth by inducing apoptosis,blocking cell cycle and anti-angiogenesis.In this study,we evaluated anti-cancer effects of JXD ethyl acetate extract (EE-JXD) combined with 5-FU in vitro and in vivo in order to explore the biological mechanism and provide more experimental basis for clinical application of JXD.Methods:EE-JXD was prepared according to the describe in the previous study.Then EE-JXD was diluted to 200 mg/mL with dimethyl sulfoxide for in vitro experiment and was dissolved in normal saline to a final concentration of 6 mg/mL in vivo study.HepG2 cell line was cultured in RPMI-1640 medium supplemented with 10％ fetal bovine serum,1％ penicillin and streptomycin at 37 ℃ in an incubator containing 5％ CO2.In vitro,cells viability was detected by MTT assay after HepG2 cells were treated with different concentrations of EE-JXD (0.05,0.1,0.2,0.4 mg/mL),5-FU (0.05,0.1,0.2,0.4 mg/mL),and EE-JXD+5-FU (0.05+0.05,0.1+0.1,0.2+0.2,0.4+0.4 mg/mL) for 24 hours.And then the HepG2 cells were divided into control group,EE-JXD group (0.1 mg/mL),5-FU group (0.1 mg/mL) and EE-JXD+5-FU group (0.1mg/mL+0.1 mg/mL).Cells apoptosis of four groups were evaluated by fluorescence assay and flow cytometry respectively after treated for 24 hours.In fluorescence assay,the HepG2 cells were stained with Hoechst 33258 at room temperature and apoptosis was observed under a fluorescence microscope.The treated HepG2 cells were processed and analyzed using an Annexin V-FITC/propidium iodide (PI) assay with flow cytometry for quantita tive determination of early and late stage apoptotic cells.Thirty-two male BALB/c (nu/nu) mice (body weight,18-22 g) were used to establish the subcutaneous xenotransplanted tumor model of HepG2 cell line.HepG2 cells in density of 5×106 cells/mL were subcutaneously injected in the right flank of each mouse.After seven days thirty-two mice were randomly assigned to four groups:EE-JXD group,administered 15 g/(kg ·d) EE-JXD (i.g.);5-FU group,administered 10 mg/(kg· d) 5-FU (i.p.),a low dose confirmed by pre-experiment;EE-JXD+5-FU group,administered 15 g/(kg· d) EE-JXD and 10 mg/(kg· d) 5-FU;and control group (normal saline).Tumor volume and body weight were measured every three days.Tumor weight was weighed after the tumor was stripped at day 30.The apoptosis rate of tumor was evaluated by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL).Results:EE-JXD,5-FU and EE-JXD+5-FU inhibited proliferation of HepG2 cells in a concentration-dependent manner.The combination of JXD and 5-FU had a strong inhibitory effect on the proliferation of HepG2 (P＜0.01,compared with the control group).Apoptosis was obvious in both 5-FU and EE-JXD groups in fluorescence assay.Apoptosis were enhanced when EE-JXD combined with 5-FU.Further assay using Annexin V-FITC/PI double staining showed HepG2 cells treated with 5-FU,EE-JXD and EE-JXD+5-FU to induce apoptosis in 5％,7.8％ and 12.9％ after 24 hours.In contrast,mice treated with either 5-FU,EE-JXD or 5-FU plus EE-JXD showed slower progression of established tumors.Control mice reached a total tumor weight of (0.43±0.06) g within 30 days.At this time point,the tumor weight of mice treated with 5-FU was significantly smaller (P＜ 0.01) at (0.08±0.01) g.Significant slowdown in tumor progression compared with control mice was observed in the group of mice receiving 5-FU and EE-JXD,reaching only (0.05±0.01) g tumor volume with in 30 days (P＜ 0.01).During 30 days' treatment,the body weight between the control group and the treatment groups were similar (P＞0.05).Apoptosis index was significantly higher in the 5-FU group compared to the control group (P＜ 0.05),and was further increased when EE-JXD combined with 5-FU (P＜ 0.01).Conclusion:The present study shows that low-dose 5-FU combined with EE-JXD has a strong anti-cancer effect than 5-FU alone.The effect is more obvious in vivo than in vitro experiments.No adverse events appeared in mice treated with both 5-FU and EE-JXD,indicating that 5-FU combine with EE-JXD at the indicated dose is safe.The results suggest that EE-JXD may potentially be combined with 5-FU for HCC and warrants additional investigation.