Study on the Effect of Huangqi-Ezhu-Chonglou Combination on Proliferation and Migration of Colorectal Cancer Cells by Regulating Macrophage Polarization
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摘要:
目的 探讨黄芪-莪术-重楼配伍对巨噬细胞极化的影响及其抑制结直肠癌细胞增殖、迁移的作用机制。 方法 使用佛波酯(Phorbol 12-myristate 13-acetate, PMA)和白细胞介素-4(Interleukin-4, IL-4)刺激THP-1细胞建立M2型巨噬细胞极化模型。实验分为M0组(PMA处理)、M2组(PMA+IL-4处理)、M2+黄芪-莪术-重楼组(PMA+IL-4+黄芪-莪术-重楼配伍处理)。CCK-8检测黄芪-莪术-重楼配伍冻干粉对巨噬细胞活力影响; qPCR和Western blot检测巨噬细胞极化标志物、谷氨酰胺酶(Glutaminase, GLS)mRNA和蛋白表达; ELISA检测细胞上清白细胞介素-10(Interleukin-10, IL-10)、转化生长因子-β(Transforming growth factor-β, TGF-β)、肿瘤坏死因子-α(Tumor necrosis factor-α, TNF-α)含量; CCK-8和Transwell实验检测黄芪-莪术-重楼配伍干预巨噬细胞的培养上清, 即条件培养基(Conditioned medium, CM), 对结直肠癌细胞HCT116增殖和迁移能力影响。 结果 与M0组相比, M2组IL-10、甘露糖受体(Mannose receptor, CD206)、精氨酸酶1(Arginase 1, ARG1)、GLS mRNA和蛋白表达显著升高(P＜0.01, P＜0.001), 巨噬细胞分泌IL-10、TGF-β水平显著升高(P＜0.01, P＜0.001);与M2组相比, M2+黄芪-莪术-重楼配伍组IL-10、CD206、ARG1、GLS mRNA和蛋白表达显著降低(P＜0.05, P＜0.01), TNF-α、诱导型一氧化氮合酶(Inducible nitric oxide synthase, iNOS) mRNA和蛋白表达显著升高(P＜0.05, P＜0.01，P＜0.001), 白细胞介素-1β(Interleukin-1β, IL-1β) mRNA表达水平显著升高(P＜0.01)，细胞上清中IL-10、TGF-β水平显著降低(P＜0.05, P＜0.01), TNF-α水平显著升高(P＜0.01)。CCK-8和Transwell结果显示, 与M0-CM组相比, M2-CM组促进HCT116细胞增殖和迁移(P＜0.01, P＜0.001), M2+黄芪-莪术-重楼-CM组较M2-CM组显著抑制HCT116细胞增殖并使细胞迁移数显著减少(P＜0.01, P＜0.001)。 结论 黄芪-莪术-重楼配伍可通过调控巨噬细胞极化, 抑制结直肠癌细胞增殖、迁移, 其机制可能与谷氨酰胺代谢关键酶GLS表达变化有关。 -
关键词:
- 黄芪-莪术-重楼配伍 /
- 结直肠癌 /
- 迁移 /
- 巨噬细胞极化 /
- GLS
Abstract:OBJECTIVE To investigate the effect of Huangqi-Ezhu-Chonglou combination on macrophage polarization and its mechanism of inhibiting colorectal cancer (CRC) cells proliferation and migration. METHODS THP-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and interleukin-4 (IL-4) to establish M2 macrophage polarization model. The experiment was divided into M0 group (PMA treatment), M2 group (PMA+IL-4 treatment), and M2+ Huangqi-Ezhu-Chonglou combination group (PMA+IL-4+Huangqi-Ezhu-Chonglou combination treatment).The effect of Huangqi-Ezhu-Chonglou combination freeze-dried powder on the viability of macrophage was detected by CCK-8 method. The expression of macrophage polarization markers, glutaminase (GLS) mRNA and protein was detected by qPCR and Western blot. The levels of interleukin-10 (IL-10), transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α) in cell supernatant were detected by ELISA. CCK-8 method and Transwell assays were used to detect the proliferation and migration of HCT116 cells intervened by the supernatant of macrophage culture treated with Huangqi-Ezhu-Chonglou combination, namely conditioned medium (CM). RESULTS Compared with the M0 group, the expression levels of IL-10, mannose receptor (CD206), arginase 1 (ARG1), and GLS mRNA and protein in the M2 group were significantly increased (P < 0.01, P < 0.001), the levels of IL-10 and TGF-β secreted by macrophages were significantly increased (P < 0.01, P < 0.001); compared with the M2 group, the M2+ Huangqi-Ezhu-Chonglou combination group had significantly reduced IL-10, CD206, ARG1, and GLS mRNA and protein expression (P < 0.05, P < 0.01), the mRNA and protein levels of TNF-α and inducible nitric oxide synthase (iNOS) were significantly increased (P < 0.05, P < 0.01, P < 0.001), the interleukin-1β (Interleukin-1β, IL-1β) mRNA expression significantly increased (P < 0. 01), and the contents of IL-10 and TGF-β in the cell supernatant significantly decreased (P < 0.05, P < 0.01), while TNF-α content significantly increased (P < 0.01). CCK-8 and Transwell results showed that compared with the M0-CM group, the M2-CM promoted the proliferation and migration of HCT116 cells (P < 0.01, P < 0.001), the M2+ Huangqi-Ezhu-Chonglou-CM group significantly inhibited HCT116 cell proliferation and reduced cell migration compared to the M2-CM group (P < 0.01, P < 0.001). CONCLUSION Huangqi-Ezhu-Chonglou combination can inhibit colorectal cancer cells proliferation and migration by regulating macrophage polarization, and its mechanism may be related to the changes in the expression of GLS, a key enzyme in glutamine metabolism. -
Key words:
- Huangqi-Ezhu-Chonglou combination /
- colorectal cancer /
- migration /
- macrophage polarization /
- GLS
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图 1 不同浓度黄芪-莪术-重楼配伍对巨噬细胞活力影响
注: 与空白对照组相比, * *P＜0.01, * * *P＜0.001。x±s，n=4。
Figure 1. Effect of Huangqi-Ezhu-Chonglou combination on the viability of macrophages
图 2 黄芪-莪术-重楼配伍对巨噬细胞IL-10、CD206、ARG1、TNF-α、iNOS、IL-1β mRNA水平的影响
注: 与M0组相比, * *P＜0.01, * * *P＜0.001;与M2组相比, #P＜0.05, ##P＜0.01。x±s，n=3。
Figure 2. Effect of Huangqi-Ezhu-Chonglou combination on the mRNA levels of IL-10, CD206, ARG1, TNF-α, iNOS, IL-1β in macrophage
图 3 黄芪-莪术-重楼配伍对巨噬细胞IL-10、CD206、ARG1、TNF-α、iNOS蛋白表达水平的影响
注: 与M0组相比, * *P＜0.01, * * *P＜0.001;与M2组相比, #P＜0.05, ##P＜0.01, ###P＜0.001。x±s，n=3。
Figure 3. Effect of Huangqi-Ezhu-Chonglou combination on the protein levels of IL-10, CD206, ARG1, TNF-α, iNOS in macrophage
图 4 黄芪-莪术-重楼配伍对巨噬细胞上清液中IL-10、TGF-β、TNF-α水平的影响
注: 与M0组相比, * *P＜0.01, * * *P＜0.001;与M2组相比, #P＜0.05, ##P＜0.01。x±s，n=3。
Figure 4. Effect of Huangqi-Ezhu-Chonglou combination on the levels of IL-10, TGF-β and TNF-α in the supernatant of macrophage
图 5 黄芪-莪术-重楼配伍抑制M2型巨噬细胞极化对结直肠癌细胞增殖、迁移的影响
注: A.CCK-8检测HCT116细胞增殖能力, n=4;B.Transwell检测HCT116迁移能力(×100), n=3;与M0-CM组相比, * *P＜0.01, * * *P＜0.001;与M2-CM组相比, ##P＜0.01, ###P＜0.001。x±s。
Figure 5. Effect of Huangqi-Ezhu-Chonglou combination on the proliferation and migration of colorectal cancer cells by inhibiting M2 macrophage polarization
图 6 黄芪-莪术-重楼配伍对巨噬细胞GLS mRNA和蛋白表达水平的影响
注: A.qPCR检测GLS mRNA表达水平; B.Western blot检测GLS蛋白表达水平; 与M0组相比, * *P＜0.01, * * *P＜0.001;与M2组相比, #P＜0.05, ##P＜0.01。x±s，n=3。
Figure 6. Effect of Huangqi-Ezhu-Chonglou combination on the mRNA and protein levels of GLS in macrophage
表 1 引物序列
Table 1. Primer sequences
基因 序列(5'→3') 长度/bp IL-10 F: TGCCTTCAGCAGAGTGAAGA 20 R: GGTCTTGGTTCTCAGCTTGG 20 CD206 F: GATCCGACCCTTCCTTGACTAAT 23 R: GCGATCCACACACGTTCATTAG 22 ARG1 F: TGGACAGACTAGGAATTGGCA 21 R: CCAGTCCGTCAACATCAAAACT 22 TNF-α F: CGAGTGACAAGCCTGTAGCC 20 R: GTTGACCTTGGTGTGTGGTAGG 22 iNOS F: TCAGCTGTGCCTTCAACCC 19 R: CCGAGGCCAAACACAGCGTA 20 IL-1β F: AAGCTGAGGAAGATGCTGGT 20 R: CGTTATCCCATGTGTCGAAG 20 GLS F: ACCAAAGTTCCCTTCTGTCTTCA 23 R: TGAAGTCACAACAATTGCTCCAG 23 GAPDH F: GAAGGTCGGAGTCAACGGAT 20 R: TGGAAGATGGTGATGGGATT 20 
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