基于DLL4/Notch1信号通路探讨人参皂苷Rg-3对AOM/DSS诱导的炎症相关性结直肠癌小鼠血管形成机制

Study on the mechanism of ginsenoside Rg-3 on angiogenesis of AOM/DSS-induced inflammation-related colorectal cancer mice based on DLL4/Notch signal pathway

目的基于Delta样配体4(delta-like ligand 4,DLL4)/Notch1信号通路探究人参皂苷Rg-3对氧化偶氮甲烷/葡聚糖硫酸钠(azoxymethane/dextran sulphate sodium,AOM/DSS)诱导的炎症相关性结直肠癌小鼠血管形成机制。方法48只C57BL/6J APCMin/+小鼠随机分为对照组、模型组、人参皂苷Rg-3组(10 mg/kg),每组16只。建立AOM/DSS诱导的结直肠癌模型,采集腹主动脉血与完整结直肠组织,检测血常规与炎症指标,HE染色观察肠组织形态,免疫组化、Western blotting检测肠组织DLL4、Notch1表达。肿瘤细胞、人脐静脉内皮细胞(human umbilical vascular endothelial cells,HUVECs)分为对照组、人参皂苷Rg-3组、人参皂苷Rg-3+DLL4组,通过MTT、克隆形成、划痕、Transwell与血管形成实验检测细胞增殖、迁移、血管形成能力,Western blotting检测DLL4/Notch信号通路、血管形成相关分子表达情况。结果与对照组相比,模型组、人参皂苷Rg-3组RBC、Hct、Hb水平降低(P<0.05),IL-1β、IL-6、TNF-α、DLL4、Notch1表达水平升高(P<0.05);与模型组相比,人参皂苷Rg-3组RBC、Hct、Hb水平提高(P<0.05),IL-1β、IL-6、TNF-α、DLL4、Notch1表达水平下降(P<0.05),肿瘤数量、2~3.5 mm及>3.5 mm的肿瘤数量减少(P<0.05)。细胞实验结果显示,与对照组相比,人参皂苷Rg-3组肿瘤细胞、HUVECs增殖、克隆形成、迁移、血管形成能力下降(P<0.05),DLL4、Notch1、VEGF、VEGFR2、转录因子重组信号结合蛋白Jκ(recombination signal binding protein-Jκ,RBP-Jκ)、Hes1表达水平降低(P<0.05)。结论人参皂苷Rg-3通过抑制DLL4/Notch1信号通路促进血管形成,抑制AOM/DSS诱导结直肠癌进展。

Objective To explore the angiogenesis mechanism of ginsenoside Rg-3 on azoxymethane/dextran sul-phate sodium(AOM/DSS)induced inflammation associated colorectal cancer mice based on delta-like ligand 4(DLL4)/Notch1 signaling pathway.Methods Fourty-eight C57BL/6J APCMin/+mice were randomly divided into con-trol group,model group and ginsenoside Rg-3 group(10 mg/kg),with 16 mice in each group.The model of colorectal cancer induced by AOM/DSS was established.Abdominal aorta blood and intact colorectal tissue were collected to de-tect blood routine and inflammatory indexes.The intestinal morphology was observed by HE staining.The expressions of DLL4 and Notch1 in intestinal tissues were detected by immunohistochemistry and Western blotting.Tumor cells and hu-man umbilical vascular endothelial cells(HUVECs)were divided into control group,ginsenoside Rg-3 group and gin-senoside Rg-3+DLL4 group.MTT,clonogenesis,scratches,Transwell and angiogenesis tests were used to detect cell proliferation,migration and angiogenesis,and Western blotting was used to detect DLL4/Notch signaling pathway and expression of molecules related to angiogenesis.Results Compared with the control group,the levels of RBC,Hct and Hb in model group and ginsenoside Rg-3 group were decreased(P<0.05),while the expression levels of IL-1β,IL-6,TNF-α,DLL4 and Notch1 were increased(P<0.05).Compared with the model group,the levels of RBC,Hct and Hb in ginsenoside Rg-3 group were increased(P<0.05),the expression levels of IL-1β,IL-6,TNF-α,DLL4 and Notch1 were decreased(P<0.05),and the number of tumors,2-3.5 mm and>3.5 mm tumors were decreased(P<0.05).Compared with control group,tumor cells and HUVECs in ginsenoside Rg-3 group had decreased proliferation,clonogen-esis,migration and angiogenesis(P<0.05),and decreased expression levels of DLL4,Notch1,VEGF,VEGFR2,re-combination signal binding protein-Jκ(RBP-Jκ)and Hes1(P<0.05).Conclusion Ginsenoside Rg-3 promotes an-giogenesis by inhibiting the DLL4/Notch1 signaling pathway,thus inhibiting the progression of AOM/DSS-induced color-ectal cancer.