Effect Mechanism of Modified Baitouwengtang Treating Colorectal Cancer by Regulating CSF1R/STING/TBK1 Signaling to Polarize Phenotype of Tumor-associated Macrophages （TAMs）

MA Chengyong; ZHANG Baoyun; DENG Beilei; YAO Liqiu; WANG An'an; LI Yao; WANG Lixin; ZHANG Tianshong

doi:10.13422/j.cnki.syfjx.20230815

您当前的位置：

首页 >

文章列表页 >

Effect Mechanism of Modified Baitouwengtang Treating Colorectal Cancer by Regulating CSF1R/STING/TBK1 Signaling to Polarize Phenotype of Tumor-associated Macrophages （TAMs）

更新时间：2023-08-07

- Effect Mechanism of Modified Baitouwengtang Treating Colorectal Cancer by Regulating CSF1R/STING/TBK1 Signaling to Polarize Phenotype of Tumor-associated Macrophages （TAMs）
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 17, Pages: 96-108(2023)
- 作者机构：
  
  1.上海市静安区中医医院，上海 200072
  2.同济大学附属上海市肺科医院，上海 200433
  3.复旦大学附属静安区中心医院，上海 200040
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20230815
  CLC： R285;R289;R287;R22;R2-031;R33;R24
- Published：05 September 2023，
  
  Published Online：10 July 2023，
  
  Received：04 May 2023，
- 稿件说明：
扫描看全文
马成勇,张宝云,邓蓓蕾等.从CSF1R/STING/TBK1信号调控肿瘤相关巨噬细胞（TAMs）表型极化探究加味白头翁汤治疗结直肠癌的效应机制[J].中国实验方剂学杂志,2023,29(17):96-108.

MA Chengyong,ZHANG Baoyun,DENG Beilei,et al.Effect Mechanism of Modified Baitouwengtang Treating Colorectal Cancer by Regulating CSF1R/STING/TBK1 Signaling to Polarize Phenotype of Tumor-associated Macrophages （TAMs）[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(17):96-108.
马成勇,张宝云,邓蓓蕾等.从CSF1R/STING/TBK1信号调控肿瘤相关巨噬细胞（TAMs）表型极化探究加味白头翁汤治疗结直肠癌的效应机制[J].中国实验方剂学杂志,2023,29(17):96-108. DOI： 10.13422/j.cnki.syfjx.20230815.

MA Chengyong,ZHANG Baoyun,DENG Beilei,et al.Effect Mechanism of Modified Baitouwengtang Treating Colorectal Cancer by Regulating CSF1R/STING/TBK1 Signaling to Polarize Phenotype of Tumor-associated Macrophages （TAMs）[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(17):96-108. DOI： 10.13422/j.cnki.syfjx.20230815.

摘要

目的

观察加味白头翁汤对结直肠癌MC38细胞荷瘤小鼠瘤体生长及肿瘤组织中肿瘤相关巨噬细胞（TAMs）浸润数量的影响，探析加味白头翁汤是否介导TAMs表型重塑以发挥抗肿瘤免疫调节效应。

方法

首先构建鼠源MC38细胞株C57BL/6小鼠皮下移植瘤模型，设立模型组、阳性药奥沙利铂组（3 mg·kg

-1

）及加味白头翁汤高、低剂量组23.43、46.86 g·kg

-1

，每组各10只，另设立10只健康小鼠为空白组，观察各组小鼠体质量、瘤体体积及生存状态变化，摘取肿瘤组织、脾脏及外周血，计算瘤体体积变化、抑瘤率及脾脏质量；苏木素-伊红（HE）染色观察肿瘤组织病理形态学变化，免疫荧光实验检测荷瘤小鼠肿瘤组织中CD4、CD8、CD206表达水平，采用酶联免疫吸附测定法（ELISA）检测荷瘤小鼠外周血清中细胞因子转化生长因子（TGF）-

、白细胞介素（IL）-6、趋化因子配体2（CCL2）的分泌水平；其次，使用MC38细胞株与鼠源单核巨噬细胞系RAW264.7细胞在体外构建IL-4诱导的共培养模型，细胞增殖与活性检测（CCK-8）法检测加味白头翁汤含药血清的细胞增殖抑制作用，Transwell实验检测IL-4诱导的M2型巨噬细胞对MC38细胞侵袭能力的影响，流式细胞术检测加味白头翁汤含药血清对IL-4诱导的M2型巨噬细胞干预后膜上CD86的表达变化，实时荧光定量聚合酶链式反应（Real-time PCR）检测加味白头翁汤含药血清对共培养后M1型巨噬细胞相关极化因子CD86、诱导型一氧化氮合酶（iNOS）、IL-12及M2型巨噬细胞相关极化因子CD206、CD163、IL-10 mRNA表达水平的影响；最后，蛋白免疫印迹法（Western blot）检测荷瘤小鼠肿瘤组织中集落刺激因子1受体（CSF1R）、干扰素基因刺激蛋白（STING）、TANK结合激酶1（TBK1）蛋白表达水平。

结果

体内实验结果表明，与模型组比较，加味白头翁汤能明显抑制荷瘤小鼠瘤体的生长。免疫荧光实验结果表明加味白头翁汤能增加荷瘤小鼠肿瘤组织中CD8

T细胞浸润数量，降低CD206

TAMs浸润数量，且能下调荷瘤小鼠外周血清中细胞因子IL-6、TGF-

、CCL2的分泌水平；在体外实验结果中，加味白头翁汤含药血清无明显的细胞增殖抑制作用，但与RAW264.7细胞共孵育后的细胞上清液却能抑制MC38细胞的增殖活性，且IL-4诱导的M2型巨噬细胞能增强MC38细胞的侵袭能力，这一效应却能被加味白头翁汤含药血清呈浓度依赖性抑制。同时，Real-time PCR检测结果发现加味白头翁汤含药血清能上调M1型巨噬细胞相关极化因子CD86、iNOS、IL-12 mRNA表达水平，下调M2型巨噬细胞相关极化因子CD206、CD163、IL-10 mRNA表达水平，流式细胞术结果也证实了加味白头翁汤含药血清能增加CD86

M1型巨噬细胞复极化数量，这表明加味白头翁汤能诱导M2型巨噬细胞向M1型巨噬细胞复极化以发挥抗肿瘤生长作用。最后，Western blot检测结果表明加味白头翁汤能下调荷瘤小鼠肿瘤组织中CSF1R蛋白表达，上调STING、TBK1蛋白表达。

结论

加味白头翁汤能下调CD206

TAMs浸润数量，增加CD8

T细胞浸润，从而发挥对结直肠癌生长抑制的抗肿瘤免疫调节效应，这可能与其调节CSF1R信号，进而活化STING/TBK1信号通路，诱导TAMs表型重塑有关。

Abstract

Objective

This study aims to investigate the effect of modified Baitouwengtang （MBTWD） on tumor growth and the number of tumor-associated macrophages （TAMs） in tumor tissue of MC38 cell tumor-bearing mice with colorectal cancer and explores whether MBTWD mediates the remodeling of TAM phenotype to play an immunologically antitumor effect.

Method

Firstly， The C57BL/6 mouse tumor model grafted subcutaneously was established， and then model mice were classified into a model group， positive control group（3 mg·kg

-1

）， and MBTWD groups with high and low dosages（23.43、46.86 g·kg

-1

）， with 10 mice in each group. In addition， 10 healthy mice were set as the blank group， and the changes in body weight， tumor volume， and survival status of mice in each group were observed. Tumor tissue， spleen， and peripheral blood were collected to calculate the tumor volume change， tumor inhibition rate， and spleen mass. Hematoxylin-eosin （HE） staining was used to observe the morphological changes of tumor tissue， and an immunofluorescence assay was used to detect the expression levels of CD4， CD8， and CD206 in tumor tissues of tumor-bearing mice. The secretion levels of transforming growth factor （TGF）-

， interleukin （IL）-6， and chemokine （C-C Motif） ligand 2 （CCL2） in peripheral serum were measured by using enzyme-linked immunosorbent assay （ELISA）. Secondly， a co-culture model induced by IL-4

in vitro

of MC38 cells and murine monocytic macrophage RAW264.7 cells was established. Cell proliferation and activity assay （CCK-8） was used to detect the inhibitory effect of MBTWD containing serum on cell proliferation. A transwell experiment was used to detect the effect of IL-4-induced M2 macrophages on the invasion of MC38 cells. Flow cytometry was used to detect the expression of CD86 on the membrane of M2 macrophages induced by IL-4 with MBTWD containing serum. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the effect of MBTWD containing serum on the mRNA expression levels of M1 macrophage-related polarization factors CD86， nitric oxide synthase （iNOS）， and IL-12， as well as M2 macrophage-related polarization factors CD206， CD163， and IL-10 after co-cultivation. Finally， the protein expression levels of colony-stimulating factor 1 receptor （CSF1R）， stimulator of interferon genes （STING）， and TANK binding kinase 1 （TBK1） in tumor tissues of tumor-bearing mice were detected by Western blot.

Result

In vivo

experimental results show that compared with the model group， the MBTWD can significantly inhibit the tumor growth of tumor-bearing mice. Immunofluorescence experiments show that the MBTWD can increase the number of CD8

T cell infiltration in tumor tissue of tumor-bearing mice， reduce the number of CD206

TAMs infiltration， and down-regulate the secretion levels of cytokines IL-6， TGF-

， and CCL2 in peripheral blood of tumor-bearing mice. The results of in vitro experiments show that the MBTWD containing serum has no obvious inhibitory effect on cell proliferation， but the cell supernatant after co-cultivation with RAW264.7 cells can inhibit the proliferation activity of MC38 cells， and the invasion ability of MC38 cells is enhanced by IL-4-induced M2 macrophages. However， this effect can be inhibited in a concentration-dependent manner by the MBTWD containing serum. At the same time， the results of Real-time PCR show that the MBTWD containing serum can up-regulate the mRNA expression levels of M1 macrophage-related polarization factors CD86， iNOS， and IL-12 and down-regulate those of M2 macrophage-related polarization factors CD206， CD163， and IL-10. Flow cytometry results also confirm that the MBTWD containing serum can increase the number of repolarized CD86

M1 macrophages， indicating that MBTWD can induce M2 macrophages to repolarized M1 macrophages to play an anti-tumor growth role. Finally， Western blot results show that MBTWD can down-regulate the expression of CSF1R protein and up-regulate that of STING and TBK1 proteins in tumor tissue of tumor-bearing mice.

Conclusion

MBTWD can down-regulate the infiltration number of CD206

TAMs and increase the infiltration of CD8

T cells， thereby playing an immunologically antitumor effect on the growth inhibition of colorectal cancer， which may be related to regulating CSF1R signaling and then activating STING/TBK1 signaling pathway to induce phenotypic remodeling of TAMs.

关键词

结直肠癌加味白头翁汤肿瘤免疫微环境肿瘤相关巨噬细胞表型极化集落刺激因子1受体（CSF1R）/干扰素基因刺激蛋白（STING）/TANK结合激酶1（TBK1）信号通路免疫调节效应

Keywords

colorectal cancermodified Baitouwengtangtumor immune microenvironmenttumor-associated macrophagesphenotypic polarizationcolony-stimulating factor 1 receptor （CSF1R）/stimulator of interferon genes （STING）/TANK binding kinase 1 （TBK1） signaling pathwayimmunoregulatory effect

references

SUNG H，FERLAY J，SIEGEL R L，et al.Global cancer statistics 2020： GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries［J］.CA Cancer J Clin，2021，71（3）：209-249.

SIEGEL R L，MILLER K D，JEMAL A.Cancer statistics，2019［J］.CA Cancer J Clin，2019，69（1）：7-34.

CHANGFA XIA， XUESI DONG， HE LI， et al. Cancer statistics in China and United States， 2022： Profiles， trends， and determinants［J］.Chin Med J （Engl），2022，135（5）： 584-590.

NASSOUR J，RADFORD R，CORREIA A，et al.Autophagic cell death restricts chromosomal instability during replicative crisis［J］.Nature，2019，565（7741）：659-663.

MORIOKA S，PERRY J，RAYMOND M H，et al.Efferocytosis induces a novel SLC program to promote glucose uptake and lactate release［J］.Nature，2018，563（7733）：714-718.

CLOUGHESY T F，MOCHIZUKI A Y，ORPILLA J R，et al.Neoadjuvant anti-PD-1 immunotherapy promotes a survival benefit with intratumoral and systemic immune responses in recurrent glioblastoma［J］.Nat Med，2019，25（3）：477-486.

SCHALPER K A，RODRIGUEZ-RUIZ M E，DIEZ-VALLE R，et al.Neoadjuvant nivolumab modifies the tumor immune microenvironment in resectable glioblastoma［J］.Nat Med，2019，25（3）：470-476.

田建辉，席志超，罗斌，等.“扶正治癌”理论的科学内涵［J］.世界科学技术—中医药现代化，2019，21（5）：943-948.

魏小曼，耿雪尘，李柳，等.基于癌毒病机理论的结直肠癌全程防治体系构建［J］.中华中医药杂志，2023，38（2）：572-575.

田建辉，罗斌，阙祖俊，等.癌症转移亚临床阶段核心病机“正虚伏毒”学说［J］.上海中医药杂志，2021，55（10）：1-3，13.

杨娜娜，吴迪，胡扬，等.中药方剂白头翁汤历史沿革及研究概况［J］.辽宁中医药大学学报，2022，24（12）：146-152.

熊礼凤，孙杭.白头翁汤加味联合化疗治疗湿热蕴结型晚期结直肠癌临床观察［J］.浙江中医杂志，2021，56（7）：509.

陈叶青，李晓琳，陈子睿，等.基于数据挖掘的结直肠癌中医证型与中药应用规律研究［J］.中国中医基础医学杂志，2022，28（3）：412-416.

SONG C H，KIM N，NAM R H，et al.Combination treatment with 17β-estradiol and anti-PD-L1 suppresses MC38 tumor growth by reducing PD-L1 expression and enhancing M1 macrophage population in MC38 colon tumor model［J］.Cancer Lett，2022，543：215780.

YANG T，ZHANG S，YUAN H，et al.Platinum-based TREM2 inhibitor suppresses tumors by remodeling the immunosuppressive microenvironment［J］.Angew Chem Int Ed Engl，2023，62（2）：e202213337.

HAN Y，SUN H，ZHANG A，et al.Chinmedomics，a new strategy for evaluating the therapeutic efficacy of herbal medicines［J］.Pharmacol Ther，2020，216：107680.

YURDAGUL A J R，SUBRAMANIAN M，WANG X，et al.Macrophage metabolism of apoptotic cell-derived arginine promotes continual efferocytosis and resolution of injury［J］.Cell Metab，2020，31（3）：518-533.

刘祎，胡送娇，朱锐秋，等.中医药调节免疫应答抗结直肠癌的研究状况［J］.中国临床药理学杂志，2022，38（13）：1539-1543.

张葛，花宝金.中医与中西医结合治疗晚期结直肠癌回顾性研究［J］.世界中医药，2023，18（4）：518-522.

华永丽，马琪，张晓松，等.基于尿液代谢组学和网络药理学整合策略研究白头翁汤治疗湿热泄泻的作用机制［J］.中国中药杂志，2022，47（14）：3887-3897.

徐世一，刘秀波，陆佳欣，等.黄芪活性成分抗肿瘤作用机制的研究进展［J］.中草药，2022，53（23）：7613-7623.

陈晓霞，路晓庆，刘向荣，等.女贞子—黄芪抗胃癌研究及作用机制的探讨［J］.中国药物与临床，2021，21（23）：3801-3804.

邬世威，陈锦芳，胡兵.藤梨根抗癌成分及其作用机制［J］.中华中医药学刊，2023，41（5）：160-163.

HICKS K C，CHARIOU P L，OZAWA Y，et al.Tumour-targeted interleukin-12 and entinostat combination therapy improves cancer survival by reprogramming the tumour immune cell landscape［J］.Nat Commun，2021，12（1）：5151.

SUN Y，WU L，ZHONG Y，et al.Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma［J］.Cell，2021，184（2）：404-421.

CHEN H，YAO J，BAO R，et al.Cross-talk of four types of RNA modification writers defines tumor microenvironment and pharmacogenomic landscape in colorectal cancer［J］.Mol Cancer，2021，20（1）：29.

GUO F，FENG Y C，ZHAO G，et al.Tumor-associated CD163（+） M2 macrophage infiltration is highly associated with PD-L1 expression in cervical cancer［J］.Cancer Manag Res，2020，12：5831-5843.

SUAREZ-CARMONA M，CHAORENTONG P，KATHER J N，et al.CCR5 status and metastatic progression in colorectal cancer［J］.Oncoimmunology，2019，8（9）：e1626193.

CZIMMERER Z，HALASZ L，DANIEL B，et al.The epigenetic state of IL-4-polarized macrophages enables inflammatory cistromic expansion and extended synergistic response to TLR ligands［J］.Immunity，2022，55（11）：2006-2026.

ZHANG L，LI Z，SKRZYPCZYNSKA K M，et al.Single-cell analyses inform mechanisms of myeloid-targeted therapies in colon cancer［J］.Cell，2020，181（2）：442-459.

KERSTEN K，HU K H，COMBES A J，et al.Spatiotemporal co-dependency between macrophages and exhausted CD8（+） T cells in cancer［J］.Cancer Cell，2022，40（6）：624-638.

XIANG X，WANG J，LU D，et al.Targeting tumor-associated macrophages to synergize tumor immunotherapy［J］.Signal Transduct Target Ther，2021，6（1）：75.

MOTWANI M，PESIRIDIS S，FITZGERALD K A.DNA sensing by the cGAS-STING pathway in health and disease［J］.Nat Rev Genet，2019，20（11）：657-674.

ZHANG D，LIU Y，ZHU Y，et al.A non-canonical cGAS-STING-PERK pathway facilitates the translational program critical for senescence and organ fibrosis［J］.Nat Cell Biol，2022，24（5）：766-782.

LIU H，SU H，WANG F，et al.Pharmacological boosting of cGAS activation sensitizes chemotherapy by enhancing antitumor immunity［J］.Cell Rep，2023，42（3）：112275.

HU S Q，FANG Y，CHEN X，et al.cGAS restricts colon cancer development by protecting intestinal barrier integrity［J］.Proc Natl Acad Sci USA，2021，118（23）：e2105747118.

TIAN M，WANG X，SUN J，et al.IRF3 prevents colorectal tumorigenesis via inhibiting the nuclear translocation of β-catenin［J］.Nat Commun，2020，11（1）：5762.

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Mechanisms of Si Junzitang and Its Main Active Components in Treating Colorectal Cancer： A Review

Antitumor Effect of Fagopyri Dibotryis Rhizoma Extract on Colorectal Cancer Cells Through ROS/MAPK Signaling Pathway

Intervention Mechanism of Huangqintang on Intestinal Inflammation and Proliferation in Colitis-associated Colon Cancer

Anti-colorectal Cancer Mechanism of Zuojinwan and Its Main Monomer Components： A Review

Mechanism of Polyphyllin Ⅰ Extract in Activating Hippo Signal to Induce Apoptosis and Autophagy of Colorectal Cancer Cells

Related Author

SHEN Jialin

ZHAO Xiaoying

XIAO Haijuan

XU Guangya

ZHOU Wenyue

ZHOU Senlin

SHI Zheng

ZHU Lin

Related Institution

Shaanxi University of Chinese Medicine

Affiliated Hospital of Shaanxi University of Chinese Medicine

College of Food and Bioengineering， School of Preclinical Medicine， Clinical Medical College/Affiliated Hospital， Chengdu University

Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences

Heilongjiang University of Chinese Medicine

Postal code：100700
Tel：010-84076882 Email：syfjx_2010@188.com
Technical support is provided by Beijing Founder electronics co., LTD 京ICP备11006657号-10 京公网安备11010802024621
It is recommended to read the content of this site in Chrome&IE9+. Please switch to extreme mode in browser 360.
Cookies We use cookies to help provide and enhance our service and tailor content. By continuing, you agree to the use of cookies.

⁰