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  

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

作　　者：马成勇 张宝云 邓蓓蕾 姚丽秋 王安安 厉瑶 王丽新 张天嵩 MA Chengyong;ZHANG Baoyun;DENG Beilei;YAO Liqiu;WANG An'an;LI Yao;WANG Lixin;ZHANG Tianshong(Jing'an Hospital of Traditional Chinese Medicine(TCM),Shanghai 200072,China;Shanghai Pulmonary Hospital,Tongji University,Shanghai 200433,China;Jing'an District Central Hospital,Shanghai 200040,China)

机构地区：[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

出　　处：《中国实验方剂学杂志》2023年第17期96-108,共13页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：上海市科委引导类科技支撑项目(18401901500);上海市中医药三年行动计划中医临床重点专科培育项目(ZY-FWTX-7001);静安区名老中医专家传承创新工作室建设项目(JA2021-MLZZ005);静安区医学科研课题项目(2022ZY04,2022ZY05)。

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)-β、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)-β,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,nit

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 

分 类 号：R285[医药卫生—中药学] R289[医药卫生—中医学] R287R22R2-031R33R24