Abstract：In order to explore the immunomodulatory mechanism of Astragalus polysaccharide (APS) on macrophages, head kidney macrophages of 6—7-month-old turbot with body weight of (346.00±95.72) g were isolated in vitro, and 800 μg/mL of APS was used to treat the macrophages for 24 h, and the differential proteins and signaling pathways in the cells were analyzed by proteomics technology. The differentially expressed proteins and signaling pathways in the cells were analyzed using proteomics techniques, and molecular docking was used to evaluate the interaction between APS and pathway receptor proteins. The results showed that no cytotoxic effect, whereas improvement of cell growth to some extent, was observed in the turbot macrophages stimulated by APS at concentrations ranging from 50 to 1000 μg/m for 24 hours, with stimulation of macrophages to produce nitric oxide (NO) at the secretion stabilizing at concentrations above 800 μg/mL. Proteomics analysis revealed upregulation of proteins related to the TNF signaling pathway, PI3K-Akt signaling pathway, and NF-κB signaling pathway at 800 μg/mL. Molecular docking results indicated that APS interacted with Toll-like receptors, specifically TLR2, TLR3, and TLR22, forming one, two, and three hydrogen bonds, respectively. It is therefore speculated that TLR22 may be a core target for APS activation of the head kidney macrophages in turbot.In conclusion, this study preliminarily identifies that APS at 800 μg/mL activates the TNF, PI3K-Akt, and NF-κB signaling pathways to regulate cytokine expression, exerting an immune regulatory effect on the head kidney macrophages of turbot, with a possible involvement of TLR22 in the recognition of APS.
[1] WU X, JIANG B Y, ZHANG Y X, et al. Identification and genomic analysis of a pathogenic circovirus associated with maricultured Scophthalmus maximus L. in China[J]. Virus Research,2024,347:199428. [2] Food and Agriculture Organization of the United Nations. The State of World Fisheries and Aquaculture 2020 Food and Agriculture Organization of the United Nations Sustainability in Action[R]. Rome: FAO, 2022. [3] ZHANG Q F, SU C Z, LUO Y N, et al. Astragalus polysaccharide enhances antitumoral effects of chimeric antigen receptor- engineered (CAR) T cells by increasing CD122+CXCR3+PD-1- memory T cells[J]. Biomedicine & Pharmacotherapy,2024,179:117401. [4] HUANG Z F, YE Y L, KONG L M, et al. Regulatory effects of Astragalus membranaceus polysaccharides on lipid metabolism disorders induced by a high-fat diet in spotted sea bass (Lateolabrax maculatus)[J]. International Journal of Biological Macromolecules,2024,271:132584. [5] HUANG Z F, YE Y L, XU A L, et al. Effects of Astragalus membranaceus polysaccharides on growth performance, physiological and biochemical parameters, and expression of genes related to lipid metabolism of spotted sea bass, Lateolabrax maculatus[J]. Aquaculture Nutrition,2023,2023(1):6191330. [6] 秦蕾,孙玉英,毕可然,等.迟缓爱德华氏菌对大菱鲆巨噬细胞相关生物效应分子产生的影响[J]. 水产科学,2018,37(2):239-243. [7] 孙林浩,程安怡,黄小红,等.黄芪多糖对大黄鱼头肾巨噬细胞的免疫调节作用[J]. 应用海洋学学报,2020,39(1):27-34. [8] SONG X J, JIANG H Y, QI Z, et al. APEC infection affects cytokine-cytokine receptor interaction and cell cycle pathways in chicken Trachea[J]. Research in Veterinary Science,2020,130:144-152. [9] LIU J P, LIU D, GU J F, et al. Shikonin inhibits the cell viability, adhesion, invasion and migration of the human gastric cancer cell line MGC-803 via the Toll-like receptor 2/nuclear factor-kappa B pathway[J]. Journal of Pharmacy and Pharmacology,2015,67(8):1143-1155. [10] CHEN E Y, CHEN C, NIU Z D, et al. Poly(I:C) preconditioning protects the heart against myocardial ischemia/reperfusion injury through TLR3/PI3K/Akt-dependent pathway[J]. Signal Transduction and Targeted Therapy,2020,5(1):216. [11] BARTON G M, MEDZHITOV R. Toll signaling:ripping off the TNF pathway[J]. Nature Immunology,2004,5(5):472-474. [12] BRAUN-NESJE R, KAPLAN G, SELJELID R. Rainbow trout macrophages in vitro:morphology and phagocytic activity[J]. Developmental & Comparative Immunology,1982,6(2):281-291. [13] TELES M, MACKENZIE S, BOLTAÑA S, et al. Gene expression and TNF-alpha secretion profile in rainbow trout macrophages following exposures to copper and bacterial lipopolysaccharide[J]. Fish & Shellfish Immunology,2011,30(1):340-346. [14] MACKENZIE S, PLANAS J V, GOETZ F W. LPS-stimulated expression of a tumor necrosis factor-alpha mRNA in primary trout monocytes and in vitro differentiated macrophages[J]. Developmental & Comparative Immunology,2003,27(5):393-400. [15] NOWAK B F, DANG M, WEBBER C, et al. Changes in the splenic melanomacrophage centre surface area in southern bluefin tuna (Thunnus maccoyii) are associated with blood fluke infections[J]. Pathogens,2021,10(1):79. [16] 王猛,程珂,马春松,等.维生素D3对黄颡鱼头肾极化分型的巨噬细胞影响[J]. 水生生物学报,2023,47(5):693-701. [17] 曾易,白钦如,胡瑞雪,等.基于斑马鱼模型研究天麻多糖的免疫调节作用[J]. 重庆理工大学学报(自然科学),2024,38(8):281-290. [18] 李庆飞,崔坤,艾庆辉,等.培养温度对LPS诱导的离体大黄鱼头肾巨噬细胞抗氧化能力和炎性反应的影响[J]. 水产学报,2019,43(4):810-819. [19] 艾庆辉,崔坤,李庆飞,等.一种大黄鱼头肾巨噬细胞系的建立方法:CN110295136A[P]. 2019-10-01. [20] 罗洪林,甘西,张永德,等.一种永生化罗非鱼巨噬细胞系的建立及鉴定方法:CN106520696A[P]. 2017-03-22. [21] 王宣刚,孔祥福,王欣桐,等.牙鲆头肾巨噬细胞的分离培养与鉴定[J]. 渔业科学进展,2021,42(5):55-61. [22] 胡先勤,周建军,胡骏鹏,等.一种斑点叉尾鮰头肾巨噬细胞原代分离培养方法及应用:CN108192869A[P]. 2018-06-22. [23] SASHINAMI H, TAKAGAKI K, NAKANE A. Salmon cartilage proteoglycan modulates cytokine responses to Escherichia coli in mouse macrophages[J]. Biochemical and Biophysical Research Communications,2006,351(4):1005-1010. [24] TAO Q Y, DU J X, LI X T, et al. Network pharmacology and molecular docking analysis on molecular targets and mechanisms of Huashi Baidu formula in the treatment of COVID-19[J]. Drug Development and Industrial Pharmacy,2020,46(8):1345-1353. [25] HARASHIMA N, INAO T, IMAMURA R, et al. Roles of the PI3K/Akt pathway and autophagy in TLR3 signaling-induced apoptosis and growth arrest of human prostate cancer cells[J]. Cancer Immunology, Immunotherapy,2012,61(5):667-676. [26] BHASKAR S, SUDHAKARAN P R, HELEN A. Quercetin attenuates atherosclerotic inflammation and adhesion molecule expression by modulating TLR-NF-κB signaling pathway[J]. Cellular Immunology,2016,310:131-140. [27] ZHANG B, ZHANG Y F, LI R, et al. MiR-217 inhibits apoptosis of atherosclerotic endothelial cells via the TLR4/PI3K/Akt/NF-κB pathway[J]. European Review for Medical and Pharmacological Sciences,2020,24(24):12867-12877. [28] ZHANG J, XU X, CHEN H B, et al. Construction and analysis for dys-regulated lncRNAs and mRNAs in LPS-induced porcine PBMCs[J]. Innate Immunity,2021,27(2):170-183. [29] WANG Q, SHEN Y L, PAN Y, et al. Tlr2/4 double knockout attenuates the degeneration of primary auditory neurons:potential mechanisms from transcriptomic perspectives[J]. Frontiers in Cell and Developmental Biology,2021,9:750271. [30] 乔延盈,于方媛,张停停,等.中药单体调控MAPK信号通路治疗宫颈癌[J/OL]. 中医学报.(2024-09-14)[2024-10-10]. http://kns.cnki.net/kcms/detail/41.1411.R.20240914.0924.020.html. [31] 刘利萍,张焱皓,李茂,等.调控巨噬细胞极化的相关信号通路及其调节机制研究进展[J]. 中国免疫学杂志,2021,37(6):747-753. [32] KUMAR V. The complement system, toll-like receptors and inflammasomes in host defense:three musketeers′ one target[J]. International Reviews of Immunology,2019,38(4):131-156. [33] SAHOO B R. Structure of fish toll-like receptors (TLR) and NOD-like receptors (NLR)[J]. International Journal of Biological Macromolecules, 2020,161:1602-1617. [34] SAMANTA M, SWAIN B, BASU M, et al. Molecular characterization of toll-like receptor 2 (TLR2), analysis of its inductive expression and associated down-stream signaling molecules following ligands exposure and bacterial infection in the Indian major carp, rohu (Labeo rohita)[J]. Fish & Shellfish Immunology,2012,32(3):411-425. [35] NAYA-CATALÀ F, DO VALE PEREIRA G, CARLA PIAZZON M, et al. Cross-talk between intestinal microbiota and host gene expression in gilthead sea bream (Sparus aurata) juveniles:insights in fish feeds for increased circularity and resource utilization[J]. Frontiers in Physiology,2021,12:748265. [36] WANG P F, ZHAO C, WANG C Y, et al. TLR3 gene in Japanese sea perch (Lateolabrax japonicus):molecular cloning, characterization and expression analysis after bacterial infection[J]. Fish & Shellfish Immunology,2018,76:347-354. [37] FIERRO-CASTRO C, BARRIOLUENGO L, LÓPEZ-FIERRO P, et al. Fish cell cultures as in vitro models of pro-inflammatory responses elicited by immunostimulants[J]. Fish & Shellfish Immunology,2012,33(2):389-400. [38] LIU F Q, SU B F, FU Q, et al. Identification, characterization and expression analysis of TLR5 in the mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge[J]. Fish & Shellfish Immunology,2017,68:272-279. [39] ZHAN F B, TAN K, SONG X R, et al. Isolation and expression of four Megalobrama amblycephala toll-like receptor genes in response to a bacterial infection[J]. Fish & Shellfish Immunology,2019,93:1028-1040. [40] STENBERG O K, HOLEN E, PIEMONTESE L, et al. Effect of dietary replacement of fish meal with insect meal on in vitro bacterial and viral induced gene response in Atlantic salmon (Salmo salar) head kidney leukocytes[J]. Fish & Shellfish Immunology,2019,91:223-232. [41] REYES-BECERRIL M, ASCENCIO-VALLE F, ALAMILLO E, et al. Molecular cloning and comparative responses of toll-like receptor 22 following ligands stimulation and parasitic infection in yellowtail (Seriola lalandi)[J]. Fish & Shellfish Immunology,2015,46(2):323-333. [42] ROACH J C, GLUSMAN G, ROWEN L, et al. The evolution of vertebrate toll-like receptors[J]. Proceedings of the National Academy of Sciences of the United States of America,2005,102(27):9577-9582.
2025, Vol. 44 
