牡荆苷对结肠癌移植瘤小鼠TAMs/CD8^(+)T细胞活性的调控作用

Regulation of Vitexin on TAMs/CD8^(+) T Cell Activity in Colon Cancer Transplanted Tumor-bearing Mice

目的探讨牡荆苷(Vitexin,VIT)作用于肿瘤相关巨噬细胞及细胞毒性T淋巴细胞(TAMs/CD8^(+)T)的抗结肠癌作用机制。方法采用BALB/c雄性小鼠构建CT26结肠癌皮下移植瘤模型,造模成功后按肿瘤体积随机分为对照组及牡荆苷高、低剂量组(40、20 mg·kg^(-1));每天1次,连续灌胃给药14 d,对照组给予等体积蒸馏水,每3 d测量1次肿瘤体积。给药结束后剖取小鼠肿瘤、脾脏和胸腺称定质量,并采用HE染色法对肿瘤和脾脏进行组织病理学检查;采用免疫荧光法分析肿瘤组织中TAMs表型;流式细胞术分析小鼠肿瘤和脾脏组织中的CD8^(+)细胞数量。选择RAW264.7巨噬细胞模型,采用MTT法检测牡荆苷对细胞活性的影响;以牡荆苷单独(5、10μmol·L^(-1))或分别联合脂多糖(LPS,100 ng·mL^(-1))、LPS+γ干扰素(INF-γ,100 ng·mL^(-1)+50 ng·mL^(-1))、白细胞介素4(IL-4,10 ng·mL^(-1))进行干预后,采用流式细胞术分析iNOS^(+)TAMs、CD206^(+)TAMs细胞比例;Griess法测定培养液中NO含量。结果在结肠癌移植瘤小鼠模型上,与对照组比较,牡荆苷高剂量组肿瘤体积增长明显减缓、肿瘤质量明显降低(P<0.05);肿瘤组织中iNOS的表达明显升高(P<0.05),iNOS/F4/80比率显著升高(P<0.01);肿瘤和脾脏组织中CD3^(+)CD8^(+)T细胞占比明显升高(P<0.05)。在RAW264.7细胞模型上,与对照组比较,LPS、LPS+IFN-γ干预组的iNOS^(+)TAMs计数和培养液中NO含量显著升高(P<0.01,P<0.001);IL-4干预组的CD206^(+)TAMs数量显著增加(P<0.001)。与LPS干预组比较,牡荆苷+LPS干预组的iNOS^(+)TAMs计数和培养液中NO明显减少(P<0.05,P<0.01)。与LPS+INF-γ干预组比较,高剂量牡荆苷+LPS+IFN-γ组的iNOS^(+)细胞比例明显升高(P<0.05),牡荆苷高、低剂量+LPS+IFN-γ干预组的NO含量明显增加(P<0.05,P<0.01)。与IL-4干预组比较,牡荆苷高剂量+IL-4组的CD206^(+)TAMs数量明显减少(P<0.05)。结论在CT26结肠癌皮下移植瘤小鼠模型上牡荆苷可作用于肿瘤微环境促进iNOS^(+)TAMs极化,激活CD8^(+)T细胞发挥抗肿瘤作用。

Objective To investigate the mechanism of action of anti-colon cancer of Vitexin(VIT)on tumorassociated macrophages and cytotoxic T-lymphocytes(TAMs/CD8^(+)T)cells in the tumor microenvironment.Methods BALB/c male mice were used to construct a CT26 colon cancer subcutaneous transplanted tumor model.After successful modelling,the mice were randomly divided into the control group,the high-and low-dose groups(40 and 20 mg·kg^(-1))of VIT;both groups were given intragastric administration once a day for 14 continuous days,and the control group was given an equal amount of distilled water and the tumor volume was measured every 3 days.The tumors,spleen and thymus were dissected and weighed at the end of the administration,and the tumors and spleen were examined histopathologically using HE staining;the phenotype of TAMs in the tumor tissues was analyzed by immunofluorescence;the number of CD8^(+) cells in the tumors and spleen tissues was analyzed by flow cytometry.The RAW264.7 macrophage model was selected and the effect of VIT on cell activity was examined by MTT assay;after intervention with VIT alone(5,10μmol·L^(-1))or in combination with LPS(100 ng·mL^(-1)),LPS+INF-γ(100 ng·mL^(-1)+50 ng·mL^(-1))and IL-4(10 ng·mL^(-1)),respectively.The proportion of iNOS^(+) TAMs and CD206^(+) TAMs cells was analyzed by flow cytometry;the Griess method was used to determine the NO content in the culture medium.Results In the colon cancer transplanted tumor mouse model,the tumor volume growth was significantly slower and the tumor mass was significantly lower in the VIT high-dose group compared with the control group(P<0.05);the expression of iNOS was significantly higher in tumor tissues(P<0.05)and the iNOS/F4/80 ratio was significantly higher(P<0.01);the proportion of CD3^(+)CD8^(+)T cells was significantly higher in tumor and spleen tissues(P<0.05).On the RAW264.7 cell model,iNOS^(+) TAMs counts and NO levels in culture medium were significantly higher in the LPS and LPS+IFN-γintervention groups compared to the control group(P<0.01,P<0.001);the number of CD206^(+)TAMs was significantly increased in the IL-4 intervention group(P<0.001).The iNOS^(+)TAMs counts and NO in the culture medium were significantly reduced in the VIT+LPS intervention group compared to the LPS intervention group(P<0.05,P<0.01).Compared with the LPS+INF-γintervention group,the proportion of iNOS^(+) cells was significantly higher in the high-dose VIT+LPS+IFN-γgroup(P<0.05),and NO secretion was significantly increased in the high-and low-doses of VIT+LPS+IFN-γintervention groups(P<0.05,P<0.01).The number of CD206^(+) TAMs was significantly reduced in the VIT high-dose+IL-4 group compared to the IL-4 intervention group(P<0.05).Conclusion VIT can promote the polarization of iNOS^(+) TAMs in tumor microenvironment and activate CD8^(+)T cells to play anti-tumor effect in CT26 colon cancer subcutaneous transplanted tumor mouse model.