摘要
探究半夏泻心汤通过重塑色氨酸代谢抑制结肠癌进展的效应及机制。将Balb/c小鼠分为对照(control)组、模型(model)组、半夏泻心汤低剂量(BXD-L)组和半夏泻心汤高剂量(BXD-H)组。除control组外,其余各组皮下注射CT26-Luc细胞建立结肠癌模型,并予以半夏泻心汤进行干预。采用小动物活体成像监测肿瘤生长情况,并测量肿瘤体积和质量;苏木精-伊红(HE)染色法观察小鼠肿瘤组织病理改变;采用免疫组化法检测肿瘤组织Ki67表达;免疫荧光法和流式细胞术检测肿瘤组织中CD3^(+)/CD8^(+)T细胞浸润情况和数目变化,酶联免疫吸附测定(ELISA)法检测肿瘤组织中干扰素-γ(IFN-γ)和白细胞介素2(IL-2)含量变化;靶向代谢组学检测血清中色氨酸(Trp)代谢变化,并测量肿瘤组织中色氨酸含量,采用蛋白免疫印迹(Western blot)法和实时荧光定量PCR(RT-qPCR)法检测肿瘤组织中吲哚胺2,3-双加氧酶1(IDO1)、原癌基因MYC、溶质载体基因家族7成员5(SLC7A5)蛋白及mRNA水平变化。同时,在体外建立“CT26结肠癌细胞+CD8^(+)T细胞”共培养模型,予以半夏泻心汤含药血清干预,采用细胞增殖与活性检测-8(CCK-8)法检测CT26细胞活性,分别测量CT26细胞和CD8^(+)T细胞Trp含量、CD8^(+)T细胞IFN-γ和IL-2的分泌情况;采用RT-qPCR法检测CT26细胞中MYC、SLC7A5 mRNA水平变化。结果显示半夏泻心汤干预后,可明显抑制小鼠结肠癌肿瘤增长,减轻瘤质量、缩小肿瘤体积;组织病理学显示肿瘤细胞排列稀疏,且有不同程度的片状坏死区,且肿瘤组织中增殖标志物Ki67水平表达降低;半夏泻心汤可升高肿瘤组织中IFN-γ、IL-2含量,同时上调CD3^(+)/CD8^(+)T细胞比例,并且降低Trp、IDO1、MYC和SLC7A5含量。体外共培养实验表明半夏泻心汤含药血清可降低CT26细胞对Trp的摄取、增加CD8^(+)T细胞Trp含量,并增强CD8^(+)T细胞IL-2、IFN-γ的分泌,同时降低CT26细胞中MYC、SLC7A5 mRNA含量。综上所述,半夏泻心汤能够抑制MYC/SLC7A5通路重塑结肠癌Trp代谢,调复CD8^(+)T细胞对Trp摄取而增加其细胞毒性,从而抑制结肠癌发展。
This study explores the effect and mechanism of Banxia Xiexin Decoction(BXD)in inhibiting colon cancer progression by reshaping tryptophan metabolism.Balb/c mice were assigned into control,model,low-dose BXD(BXD-L),and high-dose BXD(BXD-H)groups.Except the control group,the other groups were subcutaneously injected with CT26-Luc cells for the modeling of colon cancer,which was followed by the intervention with BXD.Small animal live imaging was employed to monitor tumor growth,and the tumor volume and weight were measured.Hematoxylin-eosin(HE)staining was used to observe the pathological changes in mouse tumors.Immunohistochemistry was used to detect Ki67 expression in tumors.Immunofluorescence and flow cytometry were used to detect the infiltration and number changes of CD3^(+)/CD8^(+)T cells in the tumor tissue.Enzyme-linked immunosorbent assay(ELISA)was employed to measure the levels of interferon-gamma(IFN-γ)and interleukin-2(IL-2)in tumors.Targeted metabolomics was employed to measure the level of tryptophan(Trp)in the serum,and the Trp content in the tumor tissue was measured.Western blot and RT-qPCR were employed to determine the protein and mRNA levels,respectively,of indoleamine 2,3-dioxygenase 1(IDO1),MYC proto-oncogene,and solute carrier family 7 member 5(SLC7A5)in the tumor tissue.Additionally,a co-culture model with CT26 cells and CD8^(+)T cells was established in vitro and treated with the BXD-containing serum.The cell counting kit-8(CCK-8)assay was used to examine the viability of CT26 cells.The content of Trp in CT26 cells and CD8^(+)T cells,as well as the secretion of IFN-γ and IL-2 by CD8^(+)T cells,was measured.RT-qPCR was used to determine the mRNA levels of MYC and SLC7A5 in CT26 cells.The results showed that BXD significantly inhibited the tumor growth,reduced the tumor weight,and decreased the tumor volume in the model mice.In addition,the model mice showed sparse arrangement of tumor cells,varying degrees of patchy necrosis,and downregulated expression of Ki67 in the tumor tissue.BXD elevated the levels of IFN-γ and IL-2 in the tumor tissue,while upregulating the ratio of CD3^(+)/CD8^(+)T cells and lowering the levels of Trp,IDO1,MYC,and SLC7A5.The co-culture experiment showed that BXD-containing serum reduced Trp uptake by CT26 cells,increased Trp content in CD8^(+)T cells,enhanced IL-2 and IFN-γsecretion of CD8^(+)T cells,and down-regulated the mRNA levels of MYC and SLC7A5 in CT26 cells.In summary,BXD can inhibit the MYC/SLC7A5 pathway to reshape Trp metabolism and adjust Trp uptake by CD8^(+)T cells to enhance the cytotoxicity,thereby inhibiting the development of colon cancer.
作者
蒋义芳
黄渝清
赖恒周
李雪珂
龙柳伊
由凤鸣
旷歧轩
JIANG Yi-fang;HUANG Yu-qing;LAI Heng-zhou;LI Xue-ke;LONG Liu-yi;YOU Feng-ming;KUANG Qi-xuan(Traditional Chinese Medicine Regulating Metabolic Diseases Key Laboratory of Sichuan Province,Hospital of Chengdu University of Traditional Chinese Medicine,Chengdu 610075,China;Hospital of Chengdu University of Traditional Chinese Medicine,Chengdu 610075,China;Cancer Institute,Chengdu University of Traditional Chinese Medicine,Chengdu 610072,China)
出处
《中国中药杂志》
北大核心
2025年第5期1310-1320,共11页
China Journal of Chinese Materia Medica
基金
四川省自然科学基金项目(2024NSFSC1849)
国家资助博士后研究人员计划项目(GZC20230334)
四川省中医药管理局科学技术研究专项(2024MS565)。
