Total glucosides of paeony inhibit breast cancer growth by inhibiting TAMs infiltration through NF-κB/CCL2 signaling
Total glucosides of paeony inhibit breast cancer growth by inhibiting TAMs infiltration through NF-κB/CCL2 signaling
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白芍总苷通过 NF-κB/CCL2 信号传导抑制 TAM 浸润，从而抑制乳腺癌生长
DOI：
10.1016/j.phymed.2022.154307
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发表时间：
2022-07-13
期刊：
影响因子：
7.9
通讯作者：
Fu, Huiying
中科院分区：
文献类型：
--
作者：
Jin, Lu;Guo, Yingxue;Fu, Huiying
目的：肿瘤相关巨噬细胞（tumor-associated macrophages，TAMs）和炎性因子的高密度表达是肿瘤免疫耐受的重要因素。以往研究发现白芍总苷（TGP）对多种炎症因子的释放具有较强的抑制作用，但其抑制作用是否能改善肿瘤的炎症微环境尚不清楚。因此，在本研究中，我们研究了TGP通过调节TAM浸润来抑制乳腺癌肿瘤生长和转移的机制。方法：观察白芍总苷对多种小鼠肿瘤模型的影响。苏木精-伊红染色检测肺转移。采用流式细胞仪检测肿瘤微环境（TME）中T细胞（CD 3 + CD 4+和CD 3 + CD 8+）效应和记忆亚群以及TAM（CD 45 + CD 11b +F4/80+）群。采用脂多糖（LPS）刺激巨噬细胞实验观察白芍总苷的体外抗炎作用。此外，加入条件培养基（CM）以使用实时细胞分析仪（RTCA）xCELLigence系统检测4 T1乳腺癌细胞生长。使用流式细胞仪微珠阵列（CBA）试剂盒和定量聚合酶链反应（qPCR）测量炎症细胞因子和趋化因子水平。免疫荧光和Western blot分析检测核内NF-κ B的表达。结果如下：TGP可抑制肿瘤生长和肺转移，显著减少肿瘤浸润的CD 4+和CD 8 + T细胞中CD 45 + CD 11b +F4/80+（TAMs）细胞数，增加CD 44 LowCD 62 LHi（T记忆干细胞）和CD 44 HiCD 62 LHi（中央记忆细胞）细胞数。此外，TGP还能降低肿瘤组织中炎性因子的水平，从而抑制TAMs的浸润，改善炎症免疫抑制的微环境。在体外实验中，TGP通过抑制LPS刺激的巨噬细胞分泌IL-10和C-C基序趋化因子配体2（CCL 2）及其mRNA表达，抑制4 T1细胞生长，抑制巨噬细胞M2极化。TGP还可通过抑制CCL 2和IL-10的自分泌而直接抑制4 T1细胞的增殖。进一步的机制研究表明TGP通过抑制NF-κ B B在巨噬细胞核内的积聚而抑制CCL 2的分泌。结论：TGP主要通过NF-κ B B/CCL 2信号通路减少TAM募集，从而促进TME内T细胞浸润。TGP在平衡炎症反应方面具有独特的优势。此外，我们的研究结果提出了新的见解TAM浸润的机制，抑制TGP，与潜在的应用开发新的治疗靶向CCL 2途径。
Purpose: The high density of tumor-associated macrophages (TAMs) and inflammatory factors are crucial ele-ments leading to tumor immune tolerance. Previously, we found that total glucosides of paeony (TGP) have strong inhibitory effects on the release of various inflammatory factors; however, it is unclear whether the inhibitory effects can improve the inflammatory microenvironment of tumors. Therefore, in the present study, we investigated the mechanism via which TGP depresses tumor growth and metastasis via modulation of TAM infiltration in breast cancer. Methods: We assessed the effects of TGP on various mouse models of tumor. Lung metastasis was detected using hematoxylin and eosin staining. T cell (CD3+CD4+ and CD3+CD8+) effector and memory subsets, and TAM (CD45+CD11b+F4/80+) populations in the tumor microenvironment (TME) were examined using flow cytom-etry. Lipopolysaccharide (LPS)-stimulated macrophage experiments were used to investigate the TGP anti-inflammatory effects in vitro. Furthermore, conditional medium (CM) was added to detect 4T1 breast cancer cell growth using a Real-Time Cell Analyzer (RTCA) xCELLigence system. Inflammatory cytokine and chemokine levels were measured using cytometric bead array (CBA) kits and quantitative polymerase chain reaction (qPCR). NF-kappa B expression in the nucleus was examined by immunofluorescence and Western blot analysis. Results: TGP suppressed tumor growth and lung metastasis, decreased CD45+CD11b+F4/80+ (TAMs) population obviously, and increased CD44LowCD62LHi (T memory stem cells) and CD44HiCD62LHi (central memory cells) populations in the tumor-infiltrating CD4+ and CD8+ T cells. In addition, TGP reduced inflammatory factor levels in tumors, thus inhibiting the infiltration of TAMs to improve the inflammation immunosuppressive microen-vironment. In the in vitro experiment, TGP inhibited IL-10 and C-C Motif Chemokine Ligand 2 (CCL2) secretion and mRNA expression in LPS-stimulated macrophages to inhibit 4T1 cell growth and restrain macrophages M2 polarization. In addition, TGP can directly inhibit 4T1 cell proliferation by restraining autocrine CCL2 and IL-10. Further mechanistic studies reavealed that TGP inhibited CCL2 secretion by inhibiting NF-kappa B accumulation in the nucleus in macrophages. Conclusion: TGP reduced TAM recruitment mainly through the NF-kappa B/CCL2 signaling pathway, thereby pro-moting T cell infiltration in the TME. TGP has a unique advantage in balancing the inflammatory response. Furthermore, our results present novel insights on the mechanisms underlying TAM infiltration that were inhibited by TGP, with potential application in development of novel therapies targeting CCL2 pathways.