主 办:北 京 中 医 药 大 学
ISSN 1006-2157 CN 11-3574/R

北京中医药大学学报 ›› 2020, Vol. 43 ›› Issue (12): 1018-1026.doi: 10.3969/j.issn.1006-2157.2020.12.008

• 中药药理 • 上一篇    下一篇

芩麻方通过调控髓源抑制细胞抑制非小细胞肺癌的机制研究*

朱杨壮壮1, 侯怡飞1, 张飞2, 焦肖宁1, 苏琳1, 陈晓1, 朱诗国1, 韦璐瑶1, 王万涛1, 王杰1, 朱娴丹3, 邹纯朴1, 胥孜杭1#   

  1. 1 上海中医药大学基础医学院 上海 201203;
    2 上海交通大学医学院附属新华医院;
    3 上海中医药大学科技实验中心
  • 收稿日期:2020-06-11 出版日期:2020-12-30 发布日期:2021-01-05
  • 通讯作者: #胥孜杭,女,博士,副教授,硕士生导师,主要研究方向:中医药抗肿瘤,E-mail: xuzihang_tcm@126.com
  • 作者简介:朱杨壮壮,女,在读博士生
  • 基金资助:
    *国家自然科学基金青年科学基金项目(No.81804017), 上海市青年科技英才扬帆计划项目(No.18YF1423500),中国博士后基金项目(No.2018M640416),上海市进一步加快中医药事业发展三年行动计划项目[No.ZY (2018-2020)-CCCX-2001-01]

Mechanism of Qinma Fang inhibiting non-small cell lung cancer via regulating myeloid-derived suppressor cells*

Zhu Yangzhuangzhuang1, Hou Yifei1, Zhang Fei2, Jiao Xiaoning1, Su Lin1, Chen Xiao1, Zhu Shiguo1, Wei Luyao1, Wang Wantao1, Wang Jie1, Zhu Xiandan3, Zou Chunpu1, Xu Zihang1#   

  1. 1 School of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
    2 Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China;
    3 Science and Technology Experimental Center, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
  • Received:2020-06-11 Online:2020-12-30 Published:2021-01-05
  • Contact: Associate Prof. Xu Zihang, Ph. D.,Master's Supervisor. Shanghai University of Traditional Chinese Medicine. No.1200 Cailun Road, Pudong District, Shanghai 201203. E-mail: xuzihang_tcm@126.com
  • Supported by:
    National Natural Science Foundation for Young Scientists of China (No. 81804017), Shanghai Sailing Plan for Young Scientists (No. 18YF1423500), Postdoctoral Science Foundation of China (No. 2018M640416), Three-year Action Plan for Further Accelerating the Development of Traditional Chinese Medicine in Shanghai (2018-2020) (No. ZY (2018-2020)-CCCX-2001-01)

摘要: 目的 研究芩麻方对肺癌原位模型小鼠的抑瘤功效及可能作用机制。方法 通过给小鼠肺内注射非小细胞癌Lewis肺癌细胞-荧光素酶标记(LLC-luc)建立肺癌原位小鼠模型。将所有造模小鼠随机分为空白对照组,顺铂组(2 mg/kg腹腔注射,每周2次),芩麻方高、中、低剂量组(2.0、1.0、0.5 g/kg,每日1次),连续给药4周。观察小鼠的外观表现(精神、毛发、活动等) 并统计生存期。MTT法检测芩麻方对LLC-luc细胞增殖的抑制作用。流式细胞术检测小鼠脾脏和肿瘤中髓源抑制细胞(MDSCs)和T细胞数量变化,以及CD8+, T细胞CD107α脱颗粒状况。RT-PCR检测小鼠脾脏中精氨酸酶(Arg1)、一氧化氮合酶(iNOS)、信号转导和转录活化因子3(STAT3)、信号转导和转录活化因子1(STAT1)基因表达。Western blot法检测小鼠脾脏中Arg1、STAT3和p-STAT3蛋白表达。HE染色和ELISA检测不同剂量的芩麻方的肝肾毒性。结果 中剂量芩麻方可延长小鼠生存期 (P<0.05);不同质量浓度的芩麻方干预对LLC-luc肺癌细胞增殖并无直接抑制作用;中剂量芩麻方可降低小鼠脾脏、肿瘤中MDSCs的数量(P<0.01),增加T细胞的数量,且以CD8+, T细胞为主(P<0.01),同时增强CD8+, T细胞CD107α脱颗粒作用(P<0.01);中剂量芩麻方干预后,与MDSCs活化相关的Arg1、STAT3 mRNA表达水平降低,Arg1、STAT3和p-STAT3蛋白表达下调(P<0.01);芩麻方干预对小鼠肝肾组织切片及生化指标无显著影响。结论 芩麻方可能通过下调STAT3信号通路抑制MDSCs增殖和活化从而延长肺癌荷瘤小鼠生存期,且具有用药安全性。该研究有利于芩麻方临床应用的拓展,同时也丰富了肺癌痰污染理论的科学内涵。

关键词: 芩麻方, 髓源抑制细胞, 肺癌原位模型, Lewis肺癌细胞-荧光素酶标记, 痰污染, 小鼠

Abstract: Objective To investigate the antitumor effect of Qinma Fang (Scutellaria Root and Ephedra Herb Formula, QMF) on orthotopic lung cancer mice and explore its potential mechanism. Methods Mouse models of orthotopic lung cancer were established through intrapulmonary injection with LLC-luc cells. All mice were randomly divided into four groups: blank control group, cisplatin group (2 mg/kg cisplatin twice a week by intraperitoneal injection for 4 weeks), QMF group of high, medium, and low doses (QMF at 2.0, 1.0, and 0.5 g/kg once daily with intragastrical administration for 4 weeks). The general conditions of the mice (i.e. spirit, hair, activity, etc.) were observed and the survival length was recorded. MTT was used to detect the inhibitory effects on the proliferation of LLC-luc cells. Flow cytometry was used to detect the change in the numbers of MDSCs and T cells in the spleen and tumor of the model mice and assess the CD107α degranulation efficacy of CD8+, T cells. RT-PCR was applied to exeamine the Arg1, iNOS, STAT3 and STAT1 mRNA expressions in the mouse spleen. Western blot was performed to detecd the protein expressions of Arg1, STAT3 and pSTAT3 in mouse spleen. HE staining and ELISA were used to determine the liver and kidney toxicity of QMF. Results QMF at medium dose could significantly prolong the survival of tumor bearing mice (P<0.05). Intervention of QMF at different concentrations has no direct inhibitory effect on the proliferation of LLC-luc cells. However, QMF at medium dose significantly reduced the quantity of MDSCs (P<0.01) and increased the amount of T cells (especially CD8 +, T cells) (P<0.01) in the spleen and tumor of model mice. Meanwhile, the CD107α degranulation ability of CD8+, T cells was also remarkably enhanced (P<0. 01). In addition, the mRNA expressions of Arg1 and STAT3 related to the activation of MDSCs were obviously decreased after medium-dose QMF administration, and the protein levels of Arg1, STAT3 and pSTAT3 were also down-regulated accordingly (P<0.01). Moreover, no abnormalities were found in the pathological examination of liver and kidney tissue slides and the biochemical parameters. Conclusion QMF may inhibit the proliferation and activation of MDSCs by down-regulating the STAT3 signaling pathway, thus prolonging the survival of tumor-bearing mice with lung cancer. In addition, it is safe to use with minimal toxicity on kidney and liver. This study is conducive to the expansion of clinical application of QMF, and validate the argument that lung cancer might be induced by phlegm.

Key words: Qinma Fang, MDSCs, orthotopic lung cancer model, LLC-luc, phegm-polluted, mice

中图分类号: 

  • R285.5