收稿日期: 2025-04-27
修回日期: 2025-08-04
网络出版日期: 2025-10-28
基金资助
国家自然科学基金(82160031);新疆维吾尔自治区重点研发专项(2024B03038-1);“天山英才”培养计划医药卫生高层次人才-医药卫生领军人才(TSYC202301A002);新疆医科大学第一附属医院创新团队培养项目(cxtd202405);新疆维吾尔自治区研究生科研创新项目(XJ2025G142)
Echinococcus granulosus native antigen B regulating macrophage polarization through the STAT6/PPAR-γ signaling pathway in vitro
Received date: 2025-04-27
Revised date: 2025-08-04
Online published: 2025-10-28
Supported by
National Natural Science Foundation of China(82160031);Special Research Project of Xinjiang Uygur Autonomous Region(2024B03038-1);“Tianshan Talent” Cultivation Plan - High-level Medical and Health Talents - Medical and Health Leading Talent(TSYC202301A002);Innovation Team Cultivation Project of the First Affiliated Hospital of Xinjiang Medical University(cxtd202405);Xinjiang Uygur Autonomous Region Graduate Research Innovation Project(XJ2025G142)
目的 探讨细粒棘球蚴天然抗原B（nAgB）通过信号转导和转录激活子6（STAT6）/过氧化物酶体增殖物激活受体-γ（PPAR-γ）信号通路体外对RAW264.7巨噬细胞极化的调控作用。方法 无菌条件下提取并纯化nAgB。RAW264.7细胞加入0、1、4、16、64 μg/ml浓度的nAgB，分别于0、6、12、24、48 h后用CCK8法检测细胞活力，并于24 h后用5-乙炔基-2-脱氧尿苷（EdU）染色法检测细胞增殖能力。RAW264.7细胞分为对照组、M1诱导组、M2诱导组、nAgB组、nAgB干预M1诱导组和nAgB干预M2诱导组6个组：nAgB组、nAgB干预M1诱导组和nAgB干预M2诱导组分别加入1 μg/ml nAgB；干预1 h后，M1诱导组和nAgB干预M1诱导组分别加入500 ng/ml脂多糖（LPS）和100 ng/ml γ干扰素（IFN-γ），M2诱导组和nAgB干预M2诱导组分别加入100 ng/ml白细胞介素4（IL-4）和100 ng/ml IL-13。收集各组细胞和上清液，实时荧光定量PCR（qPCR）检测细胞极化标志物和STAT6、PPAR-γ的mRNA相对转录水平，Western blotting检测相关蛋白的相对表达水平，ELISA法检测相关细胞因子的含量。结果 CCK8检测结果表明，培养24 h时，1、4、16、64 μg/ml nAgB干预的细胞活力（1.41 ± 0.09、1.62 ± 0.08、1.78 ± 0.04、1.90 ± 0.04）均高于0 μg/ml（1.07 ± 0.05）（t = 8.67、14.57、27.43、31.87，均P < 0.01）。EdU检测结果表明，1、4、16、64 μg/ml nAgB干预的细胞增殖能力（0.65 ± 0.01、0.78 ± 0.02、0.89 ± 0.02、0.99 ± 0.02）均高于0 μg/ml（0.47 ± 0.02）（t = 16.21、20.58、33.47、39.43，均P < 0.01）。qPCR结果显示，nAgB干预M1诱导组细胞的诱导型一氧化氮合酶（iNOS）、IL-6和IL-1β mRNA相对转录水平（3.23 ± 0.92、1.56 ± 0.58、19.87 ± 0.35）均低于M1诱导组（10.62 ± 1.68、3.97 ± 0.25、24.40 ± 0.03）（t = 6.69、6.62、22.07，均P < 0.01），nAgB干预M2诱导组细胞的Arg-1 mRNA相对转录水平（29.30 ± 2.92）高于M2诱导组（14.94 ± 0.77）（t = 8.23，P < 0.01）；nAgB组细胞的STAT6和PPAR-γ mRNA相对转录水平（59.12 ± 3.03、7.82 ± 0.50）均高于对照组（26.38 ± 1.89、3.71 ± 0.17）（t = 15.90、13.40，均P < 0.01），nAgB干预M1诱导组细胞（40.73 ± 2.91、4.19 ± 0.88）均高于M1诱导组（17.93 ± 1.90、1.76 ± 0.08）（t = 11.37、4.75，均P < 0.01），nAgB干预M2诱导组细胞（140.50 ± 5.64、11.67 ± 0.80）均高于M2诱导组（37.55 ± 5.92、6.87 ± 0.28）（t = 21.82、9.84，均P < 0.01）。Western blotting结果显示，nAgB干预M1诱导组细胞的iNOS蛋白相对表达水平（0.60 ± 0.02）低于M1诱导组（1.02 ± 0.03）（t = 21.86，P < 0.01），nAgB干预M2诱导组细胞的CD206蛋白相对表达水平（1.03 ± 0.04）高于M2诱导组（0.84 ± 0.02）（t = 7.78，P < 0.01）；nAgB组细胞的p-STAT6和PPAR-γ蛋白相对表达水平（0.76 ± 0.03、0.77 ± 0.02）均高于对照组（0.55 ± 0.05、0.37 ± 0.00）（t = 6.11、40.16，P < 0.05、0.01），nAgB干预M1诱导组细胞（0.60 ± 0.01、0.42 ± 0.04）均高于M1诱导组（0.39 ± 0.05、0.18 ± 0.01）（t = 6.64、10.06，均P < 0.01），nAgB干预M2诱导组细胞（1.12 ± 0.11、0.94 ± 0.02）均高于M2诱导组（0.86 ± 0.05、0.66 ± 0.00）（t = 3.71、28.18，均P < 0.01）。ELISA检测结果显示，nAgB组细胞上清中TGF-β1和IL-10的含量[（70.27 ± 4.57）、（167.00 ± 29.27）pg/ml]均高于对照组[（29.87 ± 2.24）、（50.17 ± 8.99）pg/ml]（t = 13.76、6.61，均P < 0.01）；nAgB干预M1诱导组中TNF-α和IL-1β的含量[（523.20 ± 6.72）、（387.80 ± 3.84）pg/ml]均低于M1诱导组[（995.70 ± 9.92）、（680.90 ± 3.33）pg/ml]（t = 68.32、99.90，均P < 0.01），TGF-β1和IL-10的含量[（47.15 ± 0.98）、（137.30 ± 9.80）pg/ml]均高于M1诱导组[（18.05 ± 0.57）、（21.66 ± 0.07）pg/ml]（t = 44.41、20.44，均P < 0.05）；nAgB干预M2诱导组中TNF-α和IL-1β的含量[（398.50 ± 2.57）、（85.18 ± 5.14）pg/ml]均低于M2诱导组[（578.70 ± 12.36）、（157.60 ± 14.25）pg/ml]（t = 24.71、8.28，均P < 0.01），TGF-β1和IL-10的含量[（293.20 ± 15.09）、（341.20 ± 77.94）pg/ml]均高于M2诱导组[（167.20 ± 20.34）、（72.44 ± 5.28）pg/ml]（t = 8.62、5.82，均P < 0.01）。结论 nAgB可促进巨噬细胞向M2型极化并抑制M1型反应，其作用可能与STAT6/PPAR-γ信号通路相关。
曹丽 , 杨雪花 , 焦红杰 , 何白奇枫 , 张云飞 , 岳迎宾 , 程永凤 , 王佳敬 , 宋海辰 , 严媚 . 细粒棘球蚴天然抗原B通过STAT6/PPAR-γ信号通路调控巨噬细胞极化的体外实验研究[J]. 中国寄生虫学与寄生虫病杂志, 2025 , 43(5) : 627 -634 . DOI: 10.12140/j.issn.1000-7423.2025.05.005
Objective To investigate the regulatory effect of native antigen B (nAgB) from Echinococcus granulosus on the polarization of RAW264.7 macrophages in vitro and its potential involvement in the Signal Transducer and Activator of Transcription 6 (STAT6)/peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling pathway. Methods nAgB was extracted and purified under sterile conditions. RAW264.7 cells were treated with nAgB at concentrations of 0, 1, 4, 16, and 64 μg/ml. Cell viability was assessed at 0, 6, 12, 24, and 48 hours using the CCK8 assay, and cell proliferation was evaluated at 24 hours via 5-ethynyl-2-deoxyuridine (EdU) staining. Cells were divided into 6 groups: control group, M1-polarized group, M2-polarized group, nAgB group, nAgB-treated M1-polarized group, and nAgB-treated M2-polarized group. The nAgB, nAgB-treated M1-polarized, and nAgB-treated M2-polarized groups were treated with 1 μg/ml nAgB. After 1 hour of intervention, the M1-polarized and nAgB-treated M1-polarized groups were stimulated with 500 ng/ml lipopolysaccharide (LPS) and 100 ng/ml interferon-γ (IFN-γ), while the M2-polarized and nAgB-treated M2-polarized groups were stimulated with 100 ng/ml interleukin-4 (IL-4) and 100 ng/ml IL-13. Cells and culture supernatants were collected from all groups. The relative mRNA transcription levels of macrophage polarization markers, STAT6, and PPAR-γ were detected by qPCR. The relative protein expression levels of relevant markers were measured by Western blotting, and the concentrations of related cytokines in the supernatant were determined by ELISA. Results CCK8 assay results at 24 hours showed that cell viability in the 1, 4, 16, and 64 μg/ml nAgB-treated groups (1.41 ± 0.09, 1.62 ± 0.08, 1.78 ± 0.04, 1.90 ± 0.04, respectively) was significantly higher than in the 0 μg/ml group (1.07 ± 0.05) (t = 8.67, 14.57, 27.43, 31.87; all P < 0.01). EdU assay results indicated that the cell proliferation capacity in the 1, 4, 16, and 64 μg/ml nAgB-treated groups (0.65 ± 0.01, 0.78 ± 0.02, 0.89 ± 0.02, 0.99 ± 0.02, respectively) was significantly higher than in the 0 μg/ml group (0.47 ± 0.02) (t = 16.21, 20.58, 33.47, 39.43; all P < 0.01). qPCR results showed that the relative mRNA levels of inducible nitric oxide synthase (iNOS), IL-6, and IL-1β in the nAgB-treated M1-polarized group (3.23 ± 0.92, 1.56 ± 0.58, 19.87 ± 0.35) were significantly lower than those in the M1-polarized group (10.62 ± 1.68, 3.97 ± 0.25, 24.40 ± 0.03) (t = 6.69, 6.62, 22.07; all P < 0.01). Conversely, the relative mRNA level of Arg-1 in the nAgB-treated M2-polarized group (29.30 ± 2.92) was significantly higher than that in the M2-polarized group (14.94 ± 0.77) (t = 8.23, P < 0.01). The relative mRNA levels of STAT6 and PPAR-γ in the nAgB group (59.12 ± 3.03, 7.82 ± 0.50) were significantly higher than those in the control group (26.38 ± 1.89, 3.71 ± 0.17) (t = 15.90, 13.40; both P < 0.01), in the nAgB-treated M1-polarized group (40.73 ± 2.91, 4.19 ± 0.88) were higher than in the M1-polarized group (17.93 ± 1.90, 1.76 ± 0.08) (t = 11.37, 4.75; both P < 0.01), and in the nAgB-treated M2-polarized group (140.50 ± 5.64, 11.67 ± 0.80) were higher than in the M2-polarized group (37.55 ± 5.92, 6.87 ± 0.28) (t = 21.82, 9.84; both P < 0.01). Western blotting results showed that the relative protein expression of iNOS in the nAgB-treated M1-polarized group (0.60 ± 0.02) was significantly lower than in the M1-polarized group (1.02 ± 0.03) (t = 21.86, P < 0.01), while the expression of CD206 in the nAgB-treated M2-polarized group (1.03 ± 0.04) was significantly higher than in the M2-polarized group (0.84 ± 0.02) (t = 7.78, P < 0.01). The relative protein levels of p-STAT6 and PPAR-γ in the nAgB group (0.76 ± 0.03, 0.77 ± 0.02) were significantly higher than in the control group (0.55 ± 0.05, 0.37 ± 0.00) (t = 6.11, P < 0.05; t = 40.16, P < 0.01), in the nAgB-treated M1-polarized group (0.60 ± 0.01, 0.42 ± 0.04) were higher than in the M1-polarized group (0.39 ± 0.05, 0.18 ± 0.01) (t = 6.64, 10.06; both P < 0.01), and in the nAgB-treated M2-polarized group (1.12 ± 0.11, 0.94 ± 0.02) were higher than in the M2-polarized group (0.86 ± 0.05, 0.66 ± 0.00) (t = 3.71, 28.18; both P < 0.01). ELISA results revealed that the secretion of TGF-β1 and IL-10 in the nAgB group [(70.27 ± 4.57), (167.00 ± 29.27) pg/ml] was significantly higher than in the control group [(29.87 ± 2.24), (50.17 ± 8.99) pg/ml] (t = 13.76, 6.61; both P < 0.01). In the nAgB-treated M1-polarized group, the levels of TNF-α and IL-1β [(523.20 ± 6.72), (387.80 ± 3.84) pg/ml] were significantly lower, while the levels of TGF-β1 and IL-10 [(47.15 ± 0.98), (137.30 ± 9.80) pg/ml] were significantly higher, compared to the M1-polarized group, [(995.70 ± 9.92), (680.90 ± 3.33) pg/ml] and [(18.05 ± 0.57), (21.66 ± 0.07) pg/ml], respectively (t = 68.32, 99.90, 44.41, 20.44; all P < 0.01). Similarly, nAgB-treated M2-polarized group,the levels of TNF-α and IL-1β [(398.50 ± 2.57), (85.18 ± 5.14) pg/ml] were significantly lower, while the levels of TGF-β1 and IL-10 [(293.20 ± 15.09), (341.20 ± 77.94) pg/ml] were significantly higher, compared to the M2-polarized group [(578.70 ± 12.36), (157.60 ± 14.25) pg/ml] and [(167.20 ± 20.34), (72.44 ± 5.28) pg/ml], respectively (t = 24.71, 8.28, 8.62, 5.82; all P < 0.01). Conclusion nAgB promotes macrophage polarization towards the M2 phenotype and suppresses the M1 response. This immunomodulatory effect is potentially mediated through the activation of the STAT6/PPAR-γ signaling pathway.
Key words： Echinococcus granulosus antigen B; Macrophage polarization; STAT6; PPAR-γ
| [1] | Nobs SP, Kopf M. Tissue-resident macrophages: Guardians of organ homeostasis[J]. Trends Immunol, 2021, 42(6): 495-507. |
| [2] | Xu MM, Wei SY, Su T, et al. Visualizing macrophage polarization through fluorescent mRNA profiling[J]. Biosensors (Basel), 2024, 14(10): 475. |
| [3] | Luo M, Zhao FK, Cheng H, et al. Macrophage polarization: An important role in inflammatory diseases[J]. Front Immunol, 2024, 15: 1352946. |
| [4] | Strizova Z, Benesova I, Bartolini R, et al. M1/M2 macrophages and their overlaps - myth or reality?[J]. Clin Sci (Lond), 2023, 137(15): 1067-1093. |
| [5] | Yan LF, Wang J, Cai X, et al. Macrophage plasticity: Signaling pathways, tissue repair, and regeneration[J]. Med Comm (2020), 2024, 5(8): e658. |
| [6] | Li M, Wang MJ, Wen YJ, et al. Signaling pathways in macrophages: Molecular mechanisms and therapeutic targets[J]. Med Comm (2020), 2023, 4(5): e349. |
| [7] | Lee YJ, Kim BM, Ahn YH, et al. STAT6 signaling mediates PPARγ activation and resolution of acute sterile inflammation in mice[J]. Cells, 2021, 10(3): 501. |
| [8] | 刘白雪, 蒉嫣, 薛垂召, 等. 2023年全国棘球蚴病防治工作进展[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(1): 6-13. |
| Liu BX, Kui Y, Xue CZ, et al. Progress of the national echinococcosis control programme in China, 2023[J]. Chin J Parasitol Parasit Dis, 2025, 43(1): 6-13. (in Chinese) | |
| [9] | Zhou HR, Wang XL, Han S, et al. Advances and challenges in the prevention, control and research of echinococcosis in China[J]. Decod Infect Transm, 2025, 3: 100041. |
| [10] | Wang Y, Ma BC, Wang LY, et al. Effects of management of infection source of echinococcosis in Linzhi, Tibet Autonomous Region of China[J]. Infect Dis Poverty, 2021, 10: 25. |
| [11] | Qian YY, Huang FF, Chen SY, et al. Therapeutic effect of recombinant Echinococcus granulosus antigen B subunit 2 protein on sepsis in a mouse model[J]. Parasit Vectors, 2024, 17(1): 467. |
| [12] | da Silva ED, Cancela M, Monteiro KM, et al. Antigen B from Echinococcus granulosus enters mammalian cells by endocytic pathways[J]. PLoS Negl Trop Dis, 2018, 12(5): e0006473. |
| [13] | 焦红杰, 齐文静, 郭刚, 等. 细粒棘球蚴抗原B对小鼠巨噬细胞RAW264.7的极化作用[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(1): 23-28. |
| Jiao HJ, Qi WJ, Guo G, et al. Polarization effect of Echinococcus granulosus antigen B on the mouse macrophage RAW264.7[J]. Chin J Parasitol Parasit Dis, 2023, 41(1): 23-28. (in Chinese) | |
| [14] | Tawfeek GM, Elwakil HS, El-Hoseiny L, et al. Comparative analysis of the diagnostic performance of crude sheep hydatid cyst fluid, purified antigen B and its subunit (12 Kda), assessed by ELISA, in the diagnosis of human cystic echinococcosis[J]. Parasitol Res, 2011, 108(2): 371-376. |
| [15] | Zhang YF, Yue YB, Cheng YF, et al. Antigen B from Echinococcus granulosus regulates macrophage phagocytosis by controlling TLR4 endocytosis in immune thrombocytopenia[J]. Chem Biol Interact, 2025, 406: 111350. |
| [16] | Silva-álvarez V, Folle AM, Ramos AL, et al. Echinococcus granulosus Antigen B binds to monocytes and macrophages modulating cell response to inflammation[J]. Parasit Vectors, 2016, 9: 69. |
| [17] | Bao JL, Qi WJ, Sun C, et al. Echinococcus granulosus sensu stricto and antigen B may decrease inflammatory bowel disease through regulation of M1/2 polarization[J]. Parasit Vectors, 2022, 15(1): 391. |
| [18] | Lee KY. M1 and M2 polarization of macrophages: A mini-review[J]. Med Biol Sci Eng, 2019, 2(1): 1-5. |
| [19] | Yao YL, Xu XH, Jin LP. Macrophage polarization in physiological and pathological pregnancy[J]. Front Immunol, 2019, 10: 792. |
| [20] | Ishida K, Nagatake T, Saika A, et al. Induction of unique macrophage subset by simultaneous stimulation with LPS and IL-4[J]. Front Immunol, 2023, 14: 1111729. |
| [21] | Zhu JF, Xu ZP, Chen XJ, et al. Parasitic antigens alter macrophage polarization during Schistosoma japonicum infection in mice[J]. Parasit Vectors, 2014, 7: 122. |
| [22] | Zhou L, Dang Z, Wang SY, et al. Transcription factor STAT enhanced antimicrobial activities in Bombyx mori[J]. Int J Biol Macromol, 2024, 254(Pt 2): 127637. |
| [23] | Xue C, Yao QF, Gu XY, et al. Evolving cognition of the JAK-STAT signaling pathway: Autoimmune disorders and cancer[J]. Signal Transduct Target Ther, 2023, 8(1): 204. |
| [24] | Shi JH, Liu LN, Song DD, et al. TRAF3/STAT6 axis regulates macrophage polarization and tumor progression[J]. Cell Death Differ, 2023, 30(8): 2005-2016. |
| [25] | Blackler G, Yue LZ, Klapak J, et al. Targeting STAT6-mediated synovial macrophage activation improves pain in experimental knee osteoarthritis[J]. Arthritis Res Ther, 2024, 26(1): 73. |
| [26] | Zhang YQ, Li B, Gu WH, et al. Hepatoma cell-derived exosomal SNORD52 mediates M2 macrophage polarization by activating the JAK2/STAT6 pathway[J]. Discov Oncol, 2025, 16(1): 36. |
| [27] | Yan SW, Zhang R, Guo X, et al. Trichinella spiralis dipeptidyl peptidase 1 suppressed macrophage cytotoxicity by promoting M2 polarization via the STAT6/PPARγ pathway[J]. Vet Res, 2023, 54(1): 77. |
| [28] | Szanto A, Balint BL, Nagy ZS, et al. STAT6 transcription factor is a facilitator of the nuclear receptor PPARγ-regulated gene expression in macrophages and dendritic cells[J]. Immunity, 2010, 33(5): 699-712. |
| [29] | Qiu YH, Gan ML, Wang XY, et al. The global perspective on peroxisome proliferator-activated receptor γ (PPARγ) in ectopic fat deposition: A review[J]. Int J Biol Macromol, 2023, 253(Pt 5): 127042. |
| [30] | Okreglicka K, Iten I, Pohlmeier L, et al. PPARγ is essential for the development of bone marrow erythroblastic island macrophages and splenic red pulp macrophages[J]. J Exp Med, 2021, 218(5): e20191314. |
| [31] | Shao X, Xu PC, Ji LL, et al. Low-dose decitabine promotes M2 macrophage polarization in patients with primary immune thrombocytopenia via enhancing KLF4 binding to PPARγ promoter[J]. Clin Transl Med, 2023, 13(7): e1344. |
| [32] | Gopalakrishnan A, Joseph J, Shirey KA, et al. Protection against influenza-induced Acute Lung Injury (ALI) by enhanced induction of M2a macrophages: Possible role of PPARγ/RXR ligands in IL-4-induced M2a macrophage differentiation[J]. Front Immunol, 2022, 13: 968336. |
| [33] | Reddy SS, Agarwal H, Jaiswal A, et al. Macrophage p47phox regulates pressure overload-induced left ventricular remodeling by modulating IL-4/STAT6/PPARγ signaling[J]. Free Radic Biol Med, 2021, 168: 168-179. |
| [34] | Xue L, Wu YY. Activation of PPARγ regulates M1/M2 macrophage polarization and attenuates dextran sulfate sodium salt-induced inflammatory bowel disease via the STAT-1/STAT-6 pathway[J]. Kaohsiung J Med Sci, 2025, 41(2): e12927. |
| [35] | Lee YJ, Kim K, Kim M, et al. Inhibition of STAT6 activation by AS1517499 inhibits expression and activity of PPARγ in macrophages to resolve acute inflammation in mice[J]. Biomolecules, 2022, 12(3): 447. |
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