狭基线纹香茶菜水溶性总黄酮对人肝癌裸鼠移植瘤抑制作用及机制研究

Inhibition Effects of Water-Soluble Total Flavonoids from Isodon lophanthoides var. gerardianus (Benth.) H. Hara on Human Hepatocellular Carcinoma Xenografts in Nude Mice and its Mechanism

目的:观察狭基线纹香茶菜水溶性总黄酮对人肝癌裸鼠移植瘤的抑制作用,探讨其机制。方法:建立荷瘤裸鼠动物模型,给药后观察并测量裸鼠瘤体,计算体积大小,并绘制生长曲线。分离瘤体后,计算抑制率,病理组织学检查,TUNEL染色检测移植瘤细胞凋亡。透射电镜观察瘤组织细胞的自噬小体和细胞超微结构,Western blot法检测Beclin1、Atg5、CyclinD1、Caspase-3、Bax、Bcl-2、nm23-Hl和MMP-9蛋白表达。结果:与模型对照组比较,狭基线纹香茶菜水溶性总黄酮50、100、200 mg/kg具有显著抑制移植瘤生长的作用(P<0.01)。与5-氟尿嘧啶20 mg/kg相比,狭基线纹香茶菜水溶性总黄酮100 mg/kg+5-氟尿嘧啶20 mg/kg联合应用能显著地降低肿瘤/体重比(P<0.01)。病理组织检查发现,与模型对照组比较,狭基线纹香茶菜水溶性总黄酮50、100、200 mg/kg能促使癌细胞核分裂相对减少,肿瘤细胞发生凋亡,可见比较明显的重度坏死。TUNEL染色实验发现,与模型对照组比较,5-氟尿嘧啶20 mg/kg、华蟾素624 mg/kg、联合组、狭基线纹香茶菜水溶性总黄酮100、200 mg/kg能诱导细胞凋亡,移植瘤细胞凋亡率均有显著提高(P<0.01)。电镜观察移植瘤细胞超微结构发现,与模型对照组比较,联合用药组、狭基线纹香茶菜水溶性总黄酮各剂量组可抑制细胞自噬体的形成。与模型对照组比较,狭基线纹香茶菜水溶性总黄酮100、200 mg/kg能促使细胞核固缩,碎裂、溶解,出现凋亡小体。Western blot法分析表明,与模型对照组比较,狭基线纹香茶菜水溶性总黄酮三个剂量组、联合组及两个阳性药组能显著上调Bax和Caspase-3蛋白和下调Bcl-2和Survivin蛋白的表达水平,诱导移植瘤HepG2细胞发生凋亡(P<0.01),能抑制Beclin1、Atg5、CyclinD1、MMP-9等蛋白的表达,能促进nm23-Hl蛋白的表达(P<0.01),抑制HepG2细胞转移,进而抑制人肝癌裸鼠移植瘤的生长。结论:狭基线纹香茶菜水溶性总黄酮可以抑制人肝癌裸鼠移植瘤的生长。

Objective:To investigate the inhibitory effect of the water-soluble total flavonoids from Isodon lophanthoides var.gerardianus(Benth.)H.Hara(WSTF)on hepatocellular carcinoma(HCC)and its mechanism.Methods:Tumor bearing nude mice model was established.After administration,the tumor was observed and the volume was measure to draw the growth curve.After the tumor was separated,the inhibition rate was calculated,the histopathological changes were observed,and the tumor cell apoptosis was determined by TUNEL.Cell autophagosome and ultrastructure was observed by TEM.Protein expressions of Caspase-3,Bax,Bc-2,Surviving,Beclin1,Atg5,Cyclin D1,nm23-Hl and MMP-9 were evaluated by western blot.Results:Compared with the model group,the tumor growth was significantly inhibited in 50、100、200 mg/kg WSTF groups(P<0.01).Compared with 20 mg/kg 5 FU group,the tumor weight/body weight ratio was decreased in 100 mg/kg WSTF combined with 5 FU 20 mg/kg group(P<0.01).In pathohistological results,compared with the model group,the tumor cell nuclear division was reduced in 50、100、200 mg/kg WSTF groups,also tumor cell apoptosis and severe necrosis were observed.In TUNEL dyeing results,compared with the model group,the cell apoptosis was promoted and the apoptosis rate was increased in 20 mg/kg 5 FU group,624 mg/kg cinobufagin group,the combination drug group and 100、200 mg/kg WSTF groups(P<0.01).TEM showed that compared with the model group,nucleopyknosis,nuclear fragmentation,nucleolysis were promoted in 100、200 mg/kg WSTF groups,and the apoptosis body was observed.In western blot result,compared with the model group,Bax and Caspase-3 were up regulated,while Bcl-2 and Survivin were down regulated in all doses of WSTF group,the combination drug group and 2 positive groups,HepG2 cell apoptosis was induced(P<0.01),protein expressions of Beclin1、Atg5、Cyclin D1 and MMP-9 were inhibited,and the protein expression of nm23-H1 were promoted(P<0.01).Also,the cell migration of HepG2 was inhibited,the growth of Human hepatocellular carcinoma transplanted was inhibited in nude mice Conclusion:WSTF could inhibit tumors growth in nude mice.