祛瘀解毒方调节炎症微环境抗结直肠癌肝转移的作用及机制研究

Study on the effect and mechanism of Quyu Jiedu recipe regulating inflammatory microenvironment against liver metastasis of colorectal cancer

目的探讨祛瘀解毒方抗结直肠癌肝转移的作用及其可能机制。方法将20只BALB/c小鼠按体重随机分成模型组、祛瘀解毒方低剂量组、祛瘀解毒方中剂量组、祛瘀解毒方高剂量组,每组5只。4组均采用小鼠脾脏包膜下接种荧光素酶基因标记的同源小鼠肠癌细胞(CT26.WT-luc)的方式建立结直肠癌肝转移模型。术后第3天,祛瘀解毒方低、中、高剂量组小鼠分别给予2.22 mg/(g·d)、4.44 mg/(g·d)、8.88 mg/(g·d)的祛瘀解毒方灌胃,模型组小鼠给予等体积的生理盐水灌胃,均1次/d,连续灌胃14 d。第17天行小鼠活体成像,第18天行小鼠肝脏体外成像,通过检测荧光光子通量以评估小鼠肝脏转移瘤的形成情况;HE染色法观察小鼠肝组织的病理形态;免疫组化法检测肝转移瘤中细胞增殖标志物(Ki67)、E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)、趋化因子C-C-基元受体2(CCR2)蛋白表达情况;ELISA检测肝转移瘤中细胞因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、转化生长因子-β(TGF-β)、白细胞介素-10(IL-10)及趋化因子CXC型趋化因子配体2(CXCL2)、CC型趋化因子配体2(CCL2)、CC型趋化因子配体3样蛋白1(CCL3L1)的含量。结果活体成像显示祛瘀解毒方各组小鼠的肝区荧光光子通量均较模型组低,其中祛瘀解毒方低剂量组与模型组比较差异有统计学意义(P<0.05);肝脏体外成像显示祛瘀解毒方各组小鼠肝脏的荧光光子通量均明显低于模型组(P均<0.05)。HE染色显示各组小鼠肝脏均形成转移瘤,祛瘀解毒方低、高剂量组转移瘤中Ki67面密度及祛瘀解毒方各组CCR2面密度均明显低于模型组(P均<0.05),祛瘀解毒方中、高剂量组E-cadherin面密度均明显高于模型组(P均<0.05)。祛瘀解毒方低剂量组IL-6含量、祛瘀解毒方中剂量组TNF-α和IL-10含量、祛瘀解毒方高剂量组IL-10含量、祛瘀解毒方各组CXCL2含量、祛瘀解毒方中剂量组和高剂量组CCL2含量均明显低于模型组(P均<0.05),各组间IL-1β、TGF-β、CCL3L1含量比较差异均无统计学意义(P均>0.05)。结论祛瘀解毒方具有抑制结直肠癌肝转移的作用,机制与调节肿瘤组织的炎症微环境有关。

Objective It is to observe the effect of Quyu Jiedu decoction on liver metastasis of colorectal cancer and explore its possible mechanism.Methods Twenty BALB/c mice were randomly divided into model group,and low-dose,medium-dose,high-dose groups of Quyu Jiedu decoction according to their body weight,with 5 mice in each group.Colorectal cancer liver metastasis models were established by inoculating luciferase gene-labeled homologous mouse intestinal cancer cells(CT26.WT-luc)under the mouse spleen capsule in all four groups.On the third day after operation,the mice in the low-dose,medium-dose,high-dose groups of Quyu Jiedu decoction were respectively given 2.22 mg/(g·d),4.44 mg/(g·d)and 8.88mg/(g·d)Quyu Jiedu decoction by intragastric administration,and the mice in the model group were given an equal volume of normal saline,all once per day,continuously treated for 14 days.In vivo imaging of mice was performed on the 17th day,and in vitro imaging of the mouse liver was performed on the 18th day.The formation of liver metastases in mice was evaluated by detecting the fluorescence photon flux;the pathological morphology of mouse liver tissue was observed by HE staining.The protein expressions of cell proliferation marker(Ki67),E-cadherin,vimentin and chemokine C-C-motif receptor 2(CCR2)in liver metastases were detected by chemical assay;the contents of cytokines interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),transforming growth factor-β(TGF-β),interleukin-10(IL-10)and chemokines CXC-type chemokine ligand 2(CXCL2),CC-type chemokine ligand 2(CCL2),CC-type chemokine ligand 3-like protein 1(CCL3L1)in liver metastases were detected by ELISA.Results In vivo imaging showed that the fluorescence photon flux in the liver area of the mice in each group of Quyu Jiedu decoction was lower than that of the model group,and the difference between the low-dose Quyu Jiedu decoction group and the model group was statistically significant(P<0.05);in vitro imaging of the liver showed that the fluorescence photon flux in the liver of the mice in each group of Quyu Jiedu decoction was significantly lower than that in the model group(all P<0.05).HE staining showed that metastases were formed in the livers of mice in each group.The Ki67 areal density in the metastases in the low-dose and high-dose Quyu Jiedu decoction groups and the CCR2 areal density in each group of Quyu Jiedu decoction were significantly lower than those in the model group(all P<0.05),the areal density of E-cadherin in the medium-dose and high-dose groups of Quyu Jiedu decoction was significantly higher than that in the model group(all P<0.05).The content of IL-6 in the low-dose Quyu Jiedu decoction group,the contents of TNF-αand IL-10 in the medium-dose Quyu Jiedu decoction group,the content of IL-10 in the high-dose Quyu Jiedu decoction group,the contents of CXCL2 in each group of Quyu Jiedu decoction,the contents of CCL2 in the medium-dose group and high-dose group of Yujiedu decoction were significantly lower than those in the model group(all P<0.05),and there was no significant difference in the contents of IL-1β,TGF-βand CCL3L1 among the groups(all P>0.05).Conclusion Quyu Jiedu decoction can inhibit liver metastasis of colorectal cancer,and the mechanism may be related to regulating the inflammatory microenvironment in tumor tissue.