复方斑蝥胶囊抑制人肝细胞癌HepG2215小鼠移植瘤的生长

Inhibitory effect of compound cantharides capsule on the proliferation of xenografts of human hepatocellular carcinoma HepG2215 in mice

目的探讨复方斑蝥胶囊对人HepG2215肝细胞癌移植瘤的抑制作用及其作用机制。方法建立人HepG2215肝癌荷瘤小鼠模型，随机分为5组，A组为生理盐水对照组；B组为复方斑蝥胶囊组，其中B1组为12．5mg·kg^-1·d^-1复方斑蝥胶囊，B2组为25mg·kg^-1·d^-1复方斑蝥胶囊，B3组为37．5mg·kg^-1·d^-1复方斑蝥胶囊；C组为环磷酰胺组（25mg·kg^-1·d^-1）。检测各组小鼠肿瘤体积、重量和外周血甲胎蛋白（AFP）浓度、HBVDNA水平；采用荧光定量逆转录聚合酶链反应（RT-PCR）检测肝癌凋亡相关基因mRNA的表达；采用TUNEL方法检测肿瘤细胞凋亡水平；采用流式细胞仪检测CD3^＋、CDl9^＋、CD4^＋、CD8^＋的水平，免疫组化SP法检测肿瘤微血管密度（MVD）。结果用药结束后，Bl组、B2组和B3组的抑瘤率分别为29．8％、38．7％和48．1％，C组抑瘤率为52．4％，各组间抑瘤率比较，差异有统计学意义（P〈0．05）。A组、B1组、B2组、B3组和C组荷瘤小鼠的中位生存时间分别为（30．0±3．2）d、（49．0±5．1）d、（50．0±5．2）d、（57．5±6．5）d和（49．0±4．7）d，B3组小鼠中位生存时间明显高于其他各组（P〈0．05）。A组、B1组、B2组、B3组和C组的小鼠血清AFP浓度分别为（492．7±48．5）．g／ml、（281．2±25．6）ng／ml、（194．3±18．7）ng／ml、（170．1±15．8）ng／ml和（138．7±12．5）ng／ml，C组可显著抑制AFP的表达。B1组、B2组、B3组和C组的HBVDNA抑制率分别为（46．0±5．1）％、（65．5±6．9）％、（81．3±7．8）％和（19．5±2．1）％，复方斑蝥胶囊可明显抑制HBVDNA复制，并有剂量依赖性。A组、B1组、B2组、B3组和C组的细胞凋亡率分别为（0．27±0．03）％、（7．18±2．12）％、（9．17±2．42）％、（11．27±3．03）％和（5．44±2．45）％，B3组的细胞凋亡率明显高于A组、B1组、B2组和C组（P〈0．05）。B3组的baxmRNA表达水平明显高于C组（P〈0．05）。复方斑蝥胶囊可明显下调bcl-2mRNA表达，随着剂量的增加，对bcl-2mRNA表达下调越明显，B3组的bcl-2mRNA显著低于C组（P〈0．05）。B3组的CD4^＋、CD8^＋、CD3^＋和CD19^＋水平明显高于A组和C组，差异有统计学意义（P〈0．05）。B3组肝癌MVD明显小于A组和C组，差异有统计学意义（P〈0．05）。结论复方斑蝥胶囊可抑制HepG2215细胞中的HBVDNA复制，诱导肝癌细胞的凋亡；并通过提高免疫功能抑制肝癌细胞的生长，明显延长小鼠的中位生存时间。

Objective To investigate the inhibitory effect of compound cantharides capsules on the proliferation of xenografts of human hepatocellular carcinoma HepG2215 in mice and their mechanism of action. Methods One hundred healthy Balb/c mice （5-week old, male： female 1：1 ） were used in this study. Mouse models of human HepG2215 hepatocarcinoma were established. The tumor-bearing mice were divided into five groups randomly. The control group A received daily intragastric administration of physiologic saline. The intervention groups B1, B2 and B3 were treated with compound cantharides capsule in a dose of 12.5 mg·kg^-1·d^-1, 25 mg ·kg^-1·d^-1 and 37.5 mg ·kg^-1·d^-1, respectively, for 10 consecutive days. The group C had intraperitoneal injection of cyclophosphamide （25 mg ·kg^-1·d^-1） for 10 consecutive days. The mice were sacrificed after the completion of administration. The tumors were takenout, the tumor volume was measured, the inhibitory rate of body weight was calculated, and the serum AFP concentration and the level of HBV DNA were determined. The survival of each group mice was analyzed. The levels of mRNA expression of apoptosis-related genes were assayed by quantitative RT-PCR. Apoptosis in the tumor cells was assayed with TUNEL staining. Flow cytometry was used to detect the levels of CD3^＋ , CD19^＋, CD4^＋and CD8^＋, and microvessel density （MVD） of the tumors was assessed by immunohistochemistry. Results After completion of the treatment, the inhibition rate of tumor growth of the groups B1, B2 and B3 was 29.8%, 38.7% and 48.1% ,respectively, and that of the group C was 52.4%, with a significant difference among the groups （P 〈 0.05）. The median survival time of the groups A, B1, B2, B3 and C was （30.0±3.2） days, （49.0 ± 5.1 ） days, （50.0± 5.2） days, （57.5±6.5 ） days and （49.0±4.7） days, respectively. The median survival time of the group B3 was significantly longer than that of other groups （P 〈0.05）. The serum AFP level in the groups A, B1, B2, B3 and C was （492.7± 48.5） ng/ml, （281.2±25.6） ng,/ml, （194.3±18.7） ng/ml,（170.1±15.8） ng/ml and （138.7 ±12.5） ng/ml, respectively, indicating that it was significantly inhibited in the group C. The inhibition rate of HBV DNA replication of the groups B1, B2, B3 and C was （46.0 ± 5.1 ） % , （65.5 ± 6.9 ） % , （ 81.3 ± 7.8 ） % and （ 19. 5 ± 2. 1 ） %, respectively, showing that compound cantharides capsules inhibited HBV DNA replication in a dose-dependent manner. The apoptosis rate of the groups A, B1, B2, B3 and C was （0.27 ± 0.03）%, （7.18 ±2.12）%, （9.17±2.42）%, （11.27 ±3.03）% and （5.44 ±2.45）%, respectively, and that of the group B3 was significantly higher than that of the groups A, B1, B2 and C （ P 〈 0.05 ）. The expression level of bax mRNA was significantly higher than that of the group C （ P 〈 0.05 ）. The drug could significantly decrease the bcl-2 mRNA expression level, more remarkably along with the increasing dose of cantharides, and it was significantly lower than that in the group C （ P 〈 0.05 ）. The levels of CIM^＋ , CD8^＋ , CD3^＋ and CD19^＋ were significantly higher than that in the groups A and C （ P 〈 0. 05）. The value of MVD of the group B3 was significantly lower that that of groups A and C （P 〈0.05）. Conclusion Compound cantharides capsules may inhibit the replication of HBV DNA in HepG2215 cells, inducing apoptosis in the tumor cells, enhancing the immune function to inhibit the growth of liver cancer cells in mice, and significantly prolong the median survival time of tumor-bearing mice.