芍药汤调控MDSCs相关免疫抑制微环境防治慢性肠炎癌变的效应机制
Mechanism of Shaoyaotang in Modulating MDSCs-related Immunosuppressive Microenvironment in Prevention and Treatment of Colitis-associated Carcinogenesis
陈雪 1王成磊 2杨冰炜 1翟浩宇 1吴颖 2李卫东1
作者信息
- 1. 中国中医科学院广安门医院,北京 100053
- 2. 陕西中医药大学,陕西咸阳 712000
- 折叠
摘要
目的:基于骨髓源性抑制细胞(MDSCs)相关免疫抑制微环境,探究芍药汤防治结肠炎相关性结直肠癌(CAC)的效应机制.方法:将140只6周龄SPF级FVB雄性小鼠随机分为空白组、芍药汤未造模组(7.12 g·kg-1)、模型组、柳氮磺吡啶组(0.52 g·kg-1)、芍药汤低剂量组(3.56 g·kg-1)、芍药汤中剂量组(7.12 g·kg-1)、芍药汤高剂量组(14.24 g·kg-1)共7组,每组20只.空白组和芍药汤未造模组采用单次腹腔注射生理盐水(10 mg·kg-1),其余5组小鼠采用单次腹腔注射氧化偶氮甲烷(AOM)(10mg·kg-1),1周后,更换含2％葡聚糖硫酸钠(DSS)饮用水1周,再正常饮用水2周,此为循环3次,共14周,构建CAC小鼠模型.各组于实验第14天开始每日1次灌胃2周,此后1周3次灌胃,直至实验结束.记录每周小鼠体质量,分别于实验开始的第28、98天处死部分小鼠,解剖后测量小鼠结肠长度,记录结肠重量、脾脏重量、肿瘤大小、数目;苏木素-伊红(HE)染色检测结肠肿瘤组织病理形态学;流式细胞技术检测小鼠脾脏中MDSCs、调节性T细胞(Tregs)、CD4+T、CD8+T、CD4+/CD8+T细胞比例;免疫组化学检测小鼠结肠中程序性细胞死亡蛋白-1(PD-1)、程序性细胞死亡蛋白1配体1(PD-L1)、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、磷酸化核转录因子-κB(p-NF-κB)、低氧诱导因子-1α(HIF-1α)的表达水平.结果:第14天时,与空白组比较,模型组小鼠体质量显著下降(P＜0.01),第28天时,体质量下降到最低(23.39±0.95)g;第28、98天,与空白组比较,模型组小鼠结肠长度显著缩短(P＜0.01),结肠指数显著升高(P＜0.01),脾脏指数显著升高(P＜0.01),肿瘤负荷显著增加(P＜0.01);HE结果显示,模型组可见肿瘤细胞,大量炎性细胞浸润,杯状细胞消失,隐窝缺失;芍药汤各剂量组结肠黏膜损伤减轻,炎性细胞浸润减少,隐窝结构破坏减轻;与模型组比较,芍药汤各剂量组小鼠体质量升高;第98天时,芍药汤各剂量组结肠长度显著增加(P＜0.01),结肠指数显著降低(P＜0.01),脾脏指数显著降低(P＜0.01),肿瘤负荷显著降低(P＜0.01);第28、98天,芍药汤中、高剂量组小鼠脾脏中MDSCs、Tregs均显著降低(P＜0.01)、CD4+T、CD4+/CD8+T细胞比例显著升高(P＜0.01);芍药汤各剂量组各时间点小鼠脾脏中CD8+T细胞比例均有不同程度的增高(P＜0.05,P＜0.01)、小鼠结肠组织中PD-1、PD-L1表达水平明显升高(P＜0.05,P＜0.01);第28、98天,芍药汤中、高剂量组小鼠结肠组织中p-AMPK阳性细胞表达显著升高(P＜0.01),p-NF-κB和HIF-1α显著降低(P＜0.01).结论:芍药汤可调控MDSCs募集,调节T淋巴细胞亚群的免疫功能,从而抑制AOM/DSS诱导CAC小鼠结肠肿瘤的发生与发展,其作用机制可能与激活AMPK/NF-κB/HIF-1α通路的表达有关.
Abstract
Objective:To explore the mechanism of Shaoyaotang in the prevention and treatment of colitis-associated carcinogenesis(CAC)based on myeloid-derived suppressor cells(MDSCs)-related immunosuppressive microenvironment.Methods:A total of 140 six-week-old SPF FVB male mice were randomly divided into seven groups:Blank group,Shaoyaotang without model group(7.12 g·kg-1),model group,sulfasalazine group(0.52 g·kg-1),Shaoyaotang low-dose group(3.56 g·kg-1),Shaoyaotang medium-dose group(7.12 g·kg-1)and Shaoyaotang high-dose group(14.24 g·kg-1),with 20 mice in each group.The blank control group and the Shaoyaotang without model group received a single intraperitoneal injection of physiological saline(10 mg·kg-1),while the other five groups were given a single intraperitoneal injection of azoxymethane(AOM)(10 mg·kg-1).After 1 week,the mice were given drinking water containing 2％dextran sulfate sodium(DSS)for 1 week,followed by normal drinking water for 2 weeks.This cycle was repeated three times over a total period of 14 weeks to establish the CAC mouse model.Each group was administered gavage once daily for 2 weeks starting on the 14th day of the experiment,followed by three times a week until the end of the experiment.The body weight of the mice was recorded weekly.Mice were sacrificed on the 28th and 98th days of the experiment.After dissection,the colon length,colon weight,spleen weight,tumor size,and tumor number were measured.Hematoxylin and eosin(HE)staining was used to assess the pathological morphology of colon tumor tissue.Flow cytometry was used to detect MDSCs,regulatory T cells(Tregs),CD4+T cells,CD8+T cells,and the CD4+/CD8+T cell ratio in the spleen.Immunohistochemistry was used to detect the expression levels of programmed cell death protein-1(PD-1),programmed cell death ligand 1(PD-L1),phosphorylated AMP-activated protein kinase(p-AMPK),phosphorylated nuclear factor-κB(p-NF-κB),and hypoxia-inducible factor 1α(HIF-1α)in the colon tissue.Results:On day 14,compared with the blank group,the body weight of the model group was significantly reduced(P＜0.01),reaching its lowest point on day 28(23.39±0.95)g.On days 28 and 98,compared with the blank group,the colon length in the model group was significantly shortened(P＜0.01),the colon index significantly increased(P＜0.01),the spleen index significantly increased(P＜0.01),and the tumor load significantly increased(P＜0.01).HE staining showed that in the model group,tumor cells,a large number of inflammatory cell infiltrates,goblet cell disappearance,and crypt loss were observed.In each dose group of Shaoyaotang,the damage to the colonic mucosa,inflammatory cell infiltration,and crypt structure destruction were alleviated.Compared with the model group,the body weight of mice in each dose group of Shaoyaotang increased.On day 98,the colon length was significantly increased(P＜0.01),the colon index significantly decreased(P＜0.01),the spleen index significantly decreased(P＜0.01),and the tumor burden significantly decreased(P＜0.01)in each Shaoyaotang dose group.On days 28 and 98,MDSCs and Tregs in the spleen of the medium-and high-dose Shaoyaotang groups were significantly reduced(P＜0.01),while CD4+T cells and the CD4+/CD8+T cell ratio were significantly increased(P＜0.01).The proportion of CD8+T cells in the spleen and the expression levels of PD-1 and PD-L1 in the colon tissues of mice in each Shaoyaotang dose group were significantly increased to varying degrees(P＜0.05,P＜0.01).On days 28 and 98,the expression of p-AMPK-positive cells in the colon tissue of the medium-and high-dose Shaoyaotang groups was significantly increased(P＜0.01),while the expression of p-NF-κB and HIF-1α was significantly reduced(P＜0.01).Conclusion:Shaoyaotang can regulate MDSC recruitment and modulate the immune function of T lymphocyte subsets to inhibit the occurrence and development of AOM/DSS-induced CAC in mice.The mechanism may be related to the activation of the AMPK/NF-κB/HIF-1α pathway.
关键词
芍药汤/结肠炎相关性结直肠癌/骨髓源性抑制细胞/T淋巴细胞亚群/免疫抑制微环境Key words
Shaoyaotang/colitis-associated carcinogenesis/myeloid-derived suppressor cells/T lymphocyte subsets/immunosuppressive microenvironment引用本文复制引用
出版年
2025
国家科技期刊平台
NSTL
维普
万方数据