逍遥散通过调节肿瘤免疫微环境抑制肺转移前微环境形成

doi:10.11958/20220136

天津医药 ›› 2023, Vol. 51 ›› Issue (3): 263-268.doi: 10.11958/20220136

逍遥散通过调节肿瘤免疫微环境抑制肺转移前微环境形成

汤晓霞¹(), 邓皖利¹, 张勇¹, 陈彬¹, 柴妮², 张澍³, 谢曼丽^△()

1 上海中医药大学附属普陀医院（邮编200062）
2 上海中医药大学附属岳阳中西医结合医院
3 上海市普陀区长征镇社区卫生服务中心

收稿日期:2022-01-23 修回日期:2022-09-18 出版日期:2023-03-15 发布日期:2023-03-02
通讯作者: 谢曼丽 E-mail:t417638025@126.com;6274718522@qq.com
作者简介:汤晓霞（1988），女，主治医师，主要从事中医药抗肿瘤的临床和基础方面研究。E-mail：t417638025@126.com
基金资助:
上海市普陀区卫生健康系统科技创新项目(Ptkwws201907);上海市普陀区卫生健康系统科技创新项目(Ptkwws202002);上海市科学技术委员会科研计划项目(19401933800);上海市科学技术委员会科研计划项目(21010504300)

XiaoYaoSan inhibits lung pre-metastatic niche formation through tumor immune microenvironment regulation

TANG Xiaoxia¹(), DENG Wanli¹, ZHANG Yong¹, CHEN Bin¹, CHAI Ni², ZHANG Shu³, XIE Manli^△()

1 Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China
2 Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine
3 Changzheng Town Community Health Service Center, Putuo District

Received:2022-01-23 Revised:2022-09-18 Published:2023-03-15 Online:2023-03-02
Contact: XIE Manli E-mail:t417638025@126.com;6274718522@qq.com

汤晓霞, 邓皖利, 张勇, 陈彬, 柴妮, 张澍, 谢曼丽. 逍遥散通过调节肿瘤免疫微环境抑制肺转移前微环境形成[J]. 天津医药, 2023, 51(3): 263-268.

TANG Xiaoxia, DENG Wanli, ZHANG Yong, CHEN Bin, CHAI Ni, ZHANG Shu, XIE Manli. XiaoYaoSan inhibits lung pre-metastatic niche formation through tumor immune microenvironment regulation[J]. Tianjin Medical Journal, 2023, 51(3): 263-268.

摘要/Abstract

摘要： Objective To investigate the mechanism of XiaoYaoSan in anti-tumor metastasis. Methods A mouse model of CT26.WT colon cancer xenograft and lung metastasis was established. In vivo imaging of small animals, lung tissue anatomy and HE staining were performed to evaluate the lung metastasis under different doses (200, 400 and 800 mg) of XiaoYaoSan intervention. Tumor M2 macrophage markers were detected by immunofluorescence and Western blot assay. Serum levels of transforming growth factor (TGF) -β and tumor necrosis factor (TNF) -α were detected by enzyme-linked immunosorbent assay (ELISA). The recruitment of VEGFR1⁺ myeloid cells in lung tissue was detected by flow cytometry. The expression levels of phosphatidylinositol 3-kinase (STAT3), vascular endothelial growth factor (VEGF) and pre-metastasis microenvironment markers matrix metalloproteinase (MMP) -9, S100A8 and Fibronectin were detected by Western blot assay. Results After XiaoYaoSan intervention, the lung metastasis was significantly reduced, expression levels of MMP-9, S100A8 and Fibronectin in lung tissue were significantly decreased, M1 polarization occurred in tumor-associated macrophages, the serum level of TGF-β was significantly decreased, and TNF-α level was significantly increased. The recruitment of VEGFR1⁺ myeloid cells was significantly decreased in lung tissue, STAT3 phosphorylation was inhibited, and VEGF expression was significantly decreased. Conclusion The antimetastatic activity of XiaoYaoSan is related to its ability to inhibit the pre-metastatic microenvironment, which may be realized by the phenotypic transformation of tumor-associated macrophages and inhibition of STAT3 signal transduction.

关键词: 逍遥散, 肿瘤免疫微环境, 转移前微环境, 肿瘤相关巨噬细胞, 肿瘤转移

Key words: Xiao Yao San, tumor immune microenvironment, PMNs, TAMs, tumor metastasis

中图分类号:

R73-37

图/表 10

图1 逍遥散灌胃28 d后基于荧光素酶的小鼠活体成像

Fig.1 In vivo luciferase-based bioluminescence imaging after 28 days of intragastric administration of XiaoYaoSan

图2 逍遥散灌胃28 d后各组小鼠的肺脏解剖及HE染色

Fig.2 Lung anatomy and HE staining of mice in each group after 28 days of intragastric administration of XiaoYaoSan

图3 逍遥散灌胃14 d后各组小鼠肺组织中MMP-9、S100A8及Fibronectin的表达情况 A：模型组；B：逍遥散低剂量组；C：逍遥散中剂量组；D：逍遥散高剂量组。

Fig.3 Expression levels of MMP-9, S100A8 and Fibronectin in lung tissue of mice after 14 days of intragastric administration of XiaoYaoSan

表1 各组小鼠肺转移前微环境相关蛋白表达水平比较 (n=5,$\bar{x}$±s)

Tab.1 Comparison of expression levels of microenvironment-related proteins before metastasis between four groups

组别	MMP-9	S100A8	Fibronectin
模型组	1.00±0.00	1.00±0.00	1.00±0.00
逍遥散低剂量组	0.79±0.12^a	0.96±0.14	0.52±0.06^a
逍遥散中剂量组	0.63±0.06^ab	0.55±0.08^ab	0.46+0.05^ab
逍遥散高剂量组	0.26±0.04^abc	0.45±0.06^ab	0.26+0.06^abc
F	99.660^＊＊	53.200^＊＊	202.900^＊＊

图4 小鼠原位肿瘤组织F4/80及iNOS的免疫荧光染色

Fig.4 F4/80 and iNOS immunofluorescence staining of tumor tissue

图5 小鼠原位肿瘤组织CD206、ARG1、PDL2的表达情况 A：模型组；B：逍遥散低剂量组；C：逍遥散中剂量组；D：逍遥散高剂量组。

Fig.5 Expression levels of CD206, ARG1 and PDL2 in tumor tissue

图6 各组小鼠肺组织中VEGFR1+髓系细胞的含量比较

Fig.6 Comparison of VEGFR1+ myeloid cells in lung tissue between the four groups

表2 各组小鼠M2型巨噬细胞标志物及TGF-β、TNF-α含量比较 (n=5,$\bar{x}$±s)

Tab.2 Comparison of M2 macrophage markers, TGF-β and TNF-α between the four groups of mice

组别	F4/80⁺/iNOS⁺细胞（mm²）			CD206		ARG1
模型组	89.00±10.12			1.00±0.00		1.00±0.00
逍遥散低剂量组	102.33±12.57			0.65±0.08^a		0.84±0.07
逍遥散中剂量组	160.67±9.87^ab			0.26±0.06^ab		0.33±0.04^ab
逍遥散高剂量组	174.33±12.99^ab			0.18±0.03^ab		0.25±0.05^abc
F	67.637^＊＊			263.290^＊＊		305.852^＊＊
组别		PDL2	TGF-β（μg/L）		TNF-α（μg/L）
模型组		1.00±0.00	129.10±11.02		31.67±7.97
逍遥散低剂量组		0.76±0.07^a	113.00±7.86		42.33±7.80
逍遥散中剂量组		0.75±0.08^a	84.25±6.65^a		74.54±10.12^ab
逍遥散高剂量组		0.35±0.06^abc	77.67±10.55^ab		90.13±9.64^ab
F		97.404^＊＊	34.610^＊＊		46.580^＊＊

图7 各组小鼠肺组织p-STAT3及VEGF表达水平 A：模型组；B：逍遥散低剂量组；C：逍遥散中剂量组；D：逍遥散高剂量组。

Fig.7 Expression levels of p-STAT3 and VEGF in lung tissue of each group of mice

表3 各组小鼠肿瘤组织中p-STAT3及VEGF表达水平比较 (n=5,$\bar{x}$±s)

Tab.3 Comparison of p-STAT3 and VEGF levels in tumor tissue of each group

组别	p-STAT3	VEGF
模型组	1.00±0.00	1.00±0.00
逍遥散低剂量组	0.78±0.10^a	0.54±0.15^a
逍遥散中剂量组	0.41±0.09^ab	0.53±0.09^a
逍遥散高剂量组	0.39±0.12^ab	0.37±0.12^ab
F	54.260^＊＊	32.741^＊＊

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逍遥散通过调节肿瘤免疫微环境抑制肺转移前微环境形成

XiaoYaoSan inhibits lung pre-metastatic niche formation through tumor immune microenvironment regulation

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