| 段桦,王书涵,程孟祺,孙千惠,陈赐慧.和胃消瘤方诱导巨噬细胞极化抑制胃癌细胞增殖和侵袭的机制研究[J].浙江中西医结合杂志,2025,35(8): |
| 和胃消瘤方诱导巨噬细胞极化抑制胃癌细胞增殖和侵袭的机制研究 |
| Study on the Mechanism of Macrophage Polarization Induced by HeweiXiaoliu Formula to Inhibit the Proliferation and Invasion of Gastric Cancer |
| 投稿时间：2025-01-03 修订日期：2025-05-28 |
| DOI： |
| 中文关键词: 胃癌 和胃消瘤方 巨噬细胞极化 同源异型盒蛋白1（PROX-1） 增殖 侵袭 肿瘤微环境 分子机制 |
| 英文关键词:Gastric cancer Hewei Xiaoliu formula (HWXL) Macrophage polarization Prospero homeobox protein 1(PROX-1) Proliferation Invasion Tumor microenvironment Molecular mechanisms |
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| 中文摘要: |
| [目的]探究和胃消瘤方（Hewei Xiaoliu Formula，HWXL)通过诱导巨噬细胞极化抑制胃癌增殖和侵袭的作用机制。[方法] 培养人胃腺癌细胞系（AGS）、人胃癌细胞-27（HGC-27），分成对照组和不同浓度（0%、2%、4%、6%、8%、10%、20%、30%、40%）HWXL组，采用噻唑蓝（MTT）比色法检测细胞增殖率，并进行转录组测序和京都基因与基因组百科全书（KEGG）富集分析。构建人单核细胞白血病细胞（THP-1）孵育的M0型巨噬细胞模型，分成对照组和HWXL组，采用流式细胞仪检测巨噬细胞表达，实时荧光定量逆转录聚合酶链式反应（RT-qPCR）检测肿瘤坏死因子α（TNF-α）、白介素（IL）-1β、IL-10、肿瘤生长因子β（TGF-β）的mRNA表达。构建裸鼠HGC-27细胞皮下移植瘤模型，分成对照组和HWXL组，每组6只，干预2周后处死，采用免疫荧光法检测肿瘤相关巨噬细胞（TAM）表达。构建巨噬细胞培养上清液干预AGS、HGC-27细胞模型，分为对照血清和HWXL组，采用MTT法检测细胞增殖率，Transwell法检测细胞侵袭能力，免疫印迹法检测同源异型盒蛋白1（PROX-1）、磷酸化核因子κB（p-NFκB）、核因子κB（NFκB）、磷酸化细胞外信号调节激酶（p-ERK）、细胞外信号调节激酶（ERK）、磷酸化斯马德蛋白2/3（p-smad2/p-smad3）、斯马德蛋白2/3（smad2/3）、β-连环蛋白（β-Catenin）蛋白表达。[结果] 与对照组比较，不同浓度（2%、4%、6%、8%、10%、20%、30%、40%）的HWXL均可显著抑制AGS、HGC-27细胞增殖（P＜0.05），以20%浓度差异最显著。KEGG富集分析表明机制可能与HWXL调节Wnt、TGF-β、Notch、丝裂原活化蛋白激酶（MAPK）信号通路有关。与对照组比较，HWXL组处理M0型巨噬细胞后M1/M2型比值更高[（0.15±0.04）比(0.68±0.72），P＜0.05]，M1型细胞因子TNF-α[（1.00±0.05）比（6.56±1.36），P＜0.05]、IL-1β[（1.00±0.08）比（4.47±1.00），P＜0.05]mRNA 水平明显升高，M2型细胞因子IL-10[（1.00±0.13）比（0.26±0.04），P＜0.05]、TGF-β[（1.00±0.08）比（0.44±0.09），P＜0.05]mRNA水平明显下降，即TAM向M1方向极化。与对照组比较，HWXL干预裸鼠后肿瘤微环境中M1型标记物诱导型一氧化氮合酶（iNOS）荧光强度显著增加[（10.06±5.74比16.37±1.94，P＜0.05]，M2型TAM标记物精氨酸酶-1（ARG-1）荧光强度显著减少[（59.30±21.69比15.90±15.21]，P＜0.05），M1/M2型比值更高[（0.57±0.28比1.97±1.61），P＜0.05]。与对照组比较，HWXL血清处理TAM后上清液能在第48h显著抑制AGS细胞增殖[（0.38±0.01比0.34±0.03），P＜0.05]和 AGS细胞[（424.00±39.60）比（281.50±23.33），P＜0.05]、HGC27细胞[（281.50±23.33）比(357.67±68.72)，P＜0.05]侵袭。AGS细胞中PROX1[（0.87±0.08）比（0.52±0.18），P＜0.05]、β-catenin[（0.88±0.52）比（0.60±0.29），P＜0.05]、p-NFκB[（0.81±0.26）比（0.28±0.12），P＜0.05]、p-smad2[（0.96±0.18）比（0.64±0.09），P＜0.05]、p-smad3[（1.41±0.70）比（0.62±0.27），P＜0.05]蛋白均显著下降，HGC-27细胞中PROX1[（0.77±0.22）比（0.47±0.09），P＜0.05]、β-catenin[（1.16±0.24）比（0.76±0.05），P＜0.05]、p-NFκB[（0.79±0.31）比（0.37±0.22），P＜0.05]、p-smad2[（0.81±0.04）比（0.52±0.17），P＜0.05]、p-smad3[（0.90±0.37）比（0.45±0.21），P＜0.05]蛋白也显著下降，而 NFκB、p-ERK、ERK、smad2/3蛋白变化均不明显（P＞0.05）。[结论] 和胃消瘤方能够直接抑制胃癌细胞增殖，并诱导TAM向M1方向极化，改善肿瘤微环境。和胃消瘤方还通过诱导TAM极化抑制胃癌增殖和侵袭，作用机制可能与降低PROX1表达或阻断Wnt、TGF-β、NF-κB信号通路有关。 |
| 英文摘要: |
| [Objective] To investigate the effect of Hewei Xiaoliu Formula (HWXL) on proliferation and invasion of gastric cancer (GC) cells by inducing macrophage polarization and the underlying molecular mechanisms. [Methods] The human gastric adenocarcinoma cell line (AGS) and human gastric carcinoma-27 (HGC-27) cells were cultured and divided into control and different concentrations (0%, 2%, 4%, 6%, 8%, 10%, 20%, 30%, 40%) of HWXL groups. Methyl thiazolyl tetrazolium (MTT) colorimetric assay was used to assess the cellular proliferation rates. Subsequently, transcriptome sequencing was performed followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. A model of M0-type macrophages incubated with human monocytic leukaemia cells (THP-1) was constructed and divided into control and HWXL groups. Macrophage expression was detected by flow cytometry, and mRNA expression of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-10, and tumor growth factor beta (TGF-β) was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR). A subcutaneous transplantation tumor model of HGC-27 cells was constructed in nude mice, divided into control and HWXL groups, with six mice in each group and sacrificed after 2 weeks of intervention. Immunofluorescence was used to detect tumor-associated macrophage (TAM) expression. Macrophage supernatants were constructed to intervene in AGS and HGC-27 cell models, which were divided into control and HWXL groups. MTT assay was used to detect cell proliferation rate, Transwell assay was used for cell invasive capacity, and immunoblotting assay was used to detect PROX1, phosphorylated NF-κB (p-NFκB)/total NF-κB, phosphorylated ERK (p-ERK)/total ERK, phosphorylated Smad2 (p-Smad2), phosphorylated Smad3 (p-Smad3), Smad2/3, and β-catenin. [Results] Compared with the control group, different concentrations (2%, 4%, 6%, 8%, 10%, 20%, 30% and 40%) of HWXL significantly inhibited the proliferation of AGS and HGC-27 cells (P < 0.05), with the most significant difference at 20% concentration. KEGG enrichment analysis indicated that the mechanism may be related to the regulation of Wnt, TGF-β, Notch and mitogen-activated protein kinase (MAPK) signalling pathways by HWXL. Compared with the control group, the M1/M2 type ratio was higher in the HWXL group after treatment of M0-type macrophages [(0.15±0.04) vs. (0.68±0.72), P<0.05], and the mRNA levels of M1-type cytokines including TNF-α [(1.00±0.05) vs. (6.56±1.36), P<0.05], IL-1β [(1.00±0.08) vs. (4.47±1.00), P<0.05] were significantly higher, the M2-type cytokines including IL-10 [(1.00±0.13) vs. (0.26±0.04), P<0.05], TGF-β [(1.00±0.08) vs. (0.44±0.09), P<0.05] were significantly decreased. The above suggests that TAM is polarised towards M1. Compared with the control group, the fluorescence intensity of the M1-type marker inducible Nitric Oxide Synthase (iNOS) was significantly increased [(10.06±5.74 vs.16.37±1.94, P<0.05], and the fluorescence intensity of the M2-type TAM marker Arginase-1(ARG-1) was significantly decreased [(59.30±21.69 vs. 15.90±15.21], P<0.05] in the tumour microenvironment of nude mice in the HWXL group, with the M1/M2 ratio was higher [(0.57±0.28 vs. 1.97±1.61), P<0.05]. Compared with the control group, the supernatant of HWXL serum-treated TAM significantly inhibited the proliferation of AGS cells at the 48th h [(0.38±0.01 vs. 0.34±0.03), P<0.05] and decreased the invasive ability of AGS cells [(424.00±39.60) vs. (281.50±23.33), P<0.05] and HGC-27 cells [(281.50±23.33) vs. (357.67±68.72), P<0.05]. The protein levels of PROX1 [(0.87 ± 0.08) vs. (0.52 ± 0.18), P<0.05], β-catenin [(0.88 ± 0.52) vs. (0.60 ± 0.29), P<0.05], p-NFκB [(0.81 ± 0.26) vs. (0.28 ± 0.12), P<0.05], p-smad2 [(0.96 ± 0.18) vs. (0.64 ± 0.09), P<0.05], and p-smad3 [(1.41 ± 0.70) vs. (0.62 ± 0.27), P<0.05] in AGS cells were significantly decreased. In HGC-27 cells, the protein levels of PROX1 [(0.77 ± 0.22) vs. (0.47 ± 0.09), P < 0.05], β-catenin [(1.16 ± 0.24) vs. (0.76 ± 0.05), P<0.05], p-NFκB [(0.79 ± 0.31) vs. (0.37 ± 0.22), P<0.05], p-smad2 [(0.81 ± 0.04) vs. (0.52 ± 0.17), P<0.05], and p-smad3 [(0.90 ± 0.37) vs. (0.45 ± 0.21), P<0.05] were also significantly decreased. However, the changes in NFκB, p-ERK, ERK, and smad2/3 proteins were not significant (P>0.05). [Conclusion] HWXL was able to directly inhibit the proliferation of gastric cancer cells and induced TAM polarisation towards M1, improving the tumor microenvironment. HWXL also inhibited the proliferation and invasion of gastric cancer by inducing TAM polarisation, and the mechanism of action may be related to reducing PROX1 expression or blocking the Wnt, TGF-β, and NF-κB signalling pathways. |
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