基于Hedgehog-Gli1信号通路探讨中药复方肠复康胶囊干预结肠癌血管生成的分子机制

Molecular Mechanism of TCM compound Chang Fu Kang Capsule in Angiogenesis of Colon Cancer Based on Hedgehog-Gli1 Signaling Pathway

目的:观察中药复方肠复康胶囊对人结肠癌Lo Vo细胞裸鼠模型血管生成、肿瘤转移的抑制作用,并探讨其对Hedgehog-Gli1信号通路的干预及对VEGF表达的影响。方法:采用裸小鼠结肠癌原位种植转移模型,将荷瘤鼠50只随机分为5组,每组10只,种植后第1周开始,分别给予生理盐水(模型组)、5-FU(5-FU组)、肠复康胶囊低剂量及高剂量(肠复康胶囊低剂量组、肠复康胶囊高剂量组)、5-FU与肠复康胶囊联合应用(肠复康胶囊+5-FU组),每天1次,共用4周。种植后第5周末处死动物,测量原位肿瘤瘤体质量、抑瘤率,应用免疫组化检测各组结肠癌组织中肿瘤微血管密度(microvessel density,MVD),应用Western blot法检测Gli1、VEGF蛋白表达。结果:与模型组比较,5-FU组、肠复康胶囊低剂量组、肠复康胶囊高剂量组、肠复康胶囊+5-FU组抑瘤率分别为34.96%、27.97%、52.44%、58.74%,平均瘤体质量分别为(0.93±0.27)g、(1.03±0.29)g、(0.68±0.28)g、(0.59±0.22)g,差异均有统计学意义(P<0.05);肠复康胶囊低剂量组、肠复康胶囊高剂量组、肠复康胶囊+5-FU组小鼠MVD分别为(5.33±2.1)、(2.3±1.5)、(0.66±0.5),明显低于模型组与5-FU组,差异均有统计学意义(P<0.05)。模型组区域淋巴结转移率达100.00%,腹膜转移率达91.66%,肝转移率75.00%,肺转移率为58.33%。而肠复康胶囊高剂量组及肠复康胶囊+5-FU组各脏器转移率均较模型组明显降低,差异均有统计学意义(P<0.05)。Western blot检测显示,5-FU组及肠复康胶囊高剂量组Gli1蛋白表达分别(0.660±0.155)、(0.632±0.147),VEGF蛋白表达分别为(1.011±0.481)、(0.981±0.509),与模型组相比,Gli1和VEGF蛋白的表达水平均明显下降,差异均有统计学意义(P<0.05)。结论:肠复康胶囊对结肠癌肿瘤新生血管生成具有抑制作用,其机制与其抑制Hedgehog-Gli1信号通路激活,下调VEGF表达而发挥抗肿瘤血管形成作用有关。

Objective： To observe the effect of compound Chang Fu Kang capsules（ CFK） on human colon cancer cells in nude mice Lo Vo angiogenesis,inhibition of tumor metastasis and explore its effects on the expression of Gli1 and VEGF. Methods： Using 5-fluorouracil（ 5-FU） as positive control,metastatic model of human colon cancer was established by orthotropic implantation of human tumor tissue into colon wall of nude mice. Mice were randomly divided into model control group,5-FU group,low dose CFK group,high dose CFK group,and combined treatment group（ both CFK and 5-FU） respectively. After five weeks,tumor weight,inhibition rates,the expressions of MVD were detected with immunohistochemical technique,Applications Western blot assay Gli1,VEGF protein expression. Results： After treating with the CFK,the tumor growth was significantly inhibited in mice treated respectively with 5-FU,low dose CFK,high dose CFK,and CFK plus 5-FU with an inhibition rate of 34. 96%,27. 97%,52. 44% and58. 74%,MVD decreased significantly in treated groups（ 13. 66 ± 4. 5）,（ 11. 66 ± 4. 1）,（ 5. 33 ± 2. 1）,（ 2. 3 ± 1. 5） and（ 0. 66 ± 0. 5）,（ P〈0. 01,P〈0. 05）,Gli1 and VEGF expression were decreased,Compared with the control group there were significant differences（ P〈0. 01,P〈0. 05）. Conclusion： CFK can prevent angiogenesis of human colon cancer. The mechanism may be the inhibiting of the Hedgehog-Gli1 signaling pathway,which down-regulating the protein expression of VEGF.