由於奈米銀粒子在醫療和消費產品中被廣泛地使用，並且隨後逐漸釋放到環境中，對生態系統及生物體構成了潛在的風險。關於奈米銀的毒理學風險早已引起學術界的關注。目前已有一些相關的研究被發表，這些文獻有助於理解奈米銀所誘導的毒性。從文獻中可以得出結論，免疫功能和氧化壓力是目前為止確立的主要兩個奈米材料毒性機制。然而，大多數關於奈米銀毒性的研究都探討了氧化壓力的影響，較少有關於奈米銀對免疫反應毒性的研究。斑馬魚被認為是用於篩選環境有毒物質、藥物開發、食品添加劑和人造化學藥品理想的毒性替代測試生物模型，並且已被法規機構用於化學品安全性評估。替代方法的使用讓毒性測試更有效且更符合道德。因此，在本項研究中，我們嘗試使用斑馬魚胚胎來評估奈米銀的免疫毒性。此外，先前的研究發現，諸如紫檀芪等植物化學成分具有多種不同的生理活性，包括抗氧化、抗發炎、抗腫瘤增生以及促進細胞凋亡，這些活性可以對生理損傷提供保護作用。因此，本研究的目的是使用斑馬魚胚胎模式來研究奈米銀對先天性免疫系統的影響，並研究紫檀芪對奈米銀所誘導之免疫毒性的保護作用。
實驗中使用了野生種斑馬魚胚胎以及具有螢光蛋白標記的嗜中性白血球和巨噬細胞基因轉殖斑馬魚。合成並表徵了奈米銀粒子和聚乳酸-羥基乙酸共聚物(PLGA)包覆的紫檀芪。根據急性毒性試驗，將斑馬魚胚胎暴露於不同濃度的奈米銀和PLGA包覆的紫檀芪中，以篩選出非致死劑量，進而研究其對免疫系統的影響。在受精後三到四小時內，將斑馬魚胚胎放在12孔板中暴露5天，然後每天更換溶液。觀察斑馬魚的形態和存活率，並且每天記錄。在螢光顯微鏡下分別觀察活性氧化物的累積、免疫細胞的數量和聚集情形。為了瞭解奈米銀處理後免疫細胞的功能，進行了切尾試驗。透過實時定量聚合酶鏈反應以測試免疫調節基因的表達量。VE-鈣黏蛋白的免疫螢光染色用於研究奈米銀對微血管內皮細胞之由’’奈米材料誘導的內皮細胞滲漏（NanoEL）’’作用。
穿透式電子顯微鏡顯示，合成的檸檬酸鹽封端奈米銀均為球形，大小約為8.89±1.68奈米，其介達電位在-28.85 mV有最高峰。根據斑馬魚胚胎存活測試的結果，將濃度為0.3 μg/ml的奈米銀和0.3 μg/ml PLGA包覆之紫檀芪用於後續研究。絨毛膜在胚胎發育初期具有保護作用。在第三天孵化後，奈米銀處理組觀察到嗜中性白血球的螢光表現增加，但在第五天時觀察到表現減少。另外，奈米銀暴露組顯示，在切尾試驗中免疫細胞的反向遷移能力降低，表明免疫細胞的功能受到影響。此外，斑馬魚稚魚在尾部受傷後持續暴露於奈米銀中會很快死亡。紫檀芪的預處理可以增加胚胎的存活率，並減少暴露於奈米銀後胚胎的畸形。與奈米銀處理組相比，紫檀芪還可以激活免疫細胞並促進免疫細胞聚集到受損部位。qPCR的結果表明，隨著奈米銀濃度的增加，IL8、IL10、IL26、溶菌酶、Myd88和TLR2的表達下調，而紫檀芪的添加恢復了基因的表達。相反，在奈米銀處理組中IL6的表達以劑量依賴性方式略微上調。免疫螢光染色結果表明，奈米銀的暴露破壞了微血管內皮細胞屏障的完整性。
結果表明，暴露於奈米銀不僅會導致發育毒性和胚胎死亡，還會影響先天性免疫系統，造成免疫細胞數量和功能的改變。此外，奈米銀也會影響免疫相關細胞因子和趨化因子的表達。 紫檀芪的添加可以激活免疫細胞並促進免疫細胞聚集到受損區域，從而減少由奈米銀引起的損害。總之，研究表明，奈米銀的處理可能會影響免疫系統的調節和免疫細胞的功能。天然化合物紫檀芪的添加可以提供保護作用。

Silver nanoparticles (AgNPs) pose a potential risk to ecosystems and the living organisms due to widespread use in various field, and subsequent gradual releasing into the environment. There are a few studies investigating AgNPs toxicity on immunological function and the toxicity effects are not clear yet. Recent studies indicated that Zebrafish has been considered as a good toxicity alternative testing model and also for evaluating immunological toxicity. Therefore, the purpose of this study was to investigate the toxicity effects of AgNPs on innate immunity using the zebrafish embryo model and to investigate whether natural compound pterostilbene could provide protection against AgNPs-induced immune toxicity. Zebrafish with wild type, neutrophils transgenic line and macrophages transgenic line were used in the experiments. The number and accumulation of immune cells were observed under a fluorescence microscope. The tail transection test, oxidative stress measurement, q-PCR analysis, and immunofluorescence staining were used in the study. The result indicated that the exposure of AgNPs induced developmental toxicity and death in zebrafish. In addition, AgNPs also affect the innate immunity that the number and function of neutrophils and macrophages were affected. Moreover, the expression of immune-related cytokines and chemokines were also affected. The addition of Pterostilbene could activate immune cells and promote the accumulation of immune cells to the injured area thereby reduce the damage caused by AgNPs. In conclusion, the treatment of AgNPs may affect the regulation of the immune system and the function of immune cells.

Ahamed M, AlSalhi MS, Siddiqui M. 2010. Silver nanoparticle applications and human health. Clinica Chimica Acta 411:1841-1848.
Ahmed S, Ahmad M, Swami BL, Ikram S. 2016. A review on plants extract mediated synthesis of silver nanoparticles for antimicrobial applications: A green expertise. Journal of Advanced Research 7:17-28.
Alarcon EI, Udekwu KI, Noel CW, Gagnon LB-P, Taylor PK, Vulesevic B, et al. 2015. Safety and efficacy of composite collagen–silver nanoparticle hydrogels as tissue engineering scaffolds. Nanoscale 7:18789-18798.
Arora S, Jain J, Rajwade J, Paknikar K. 2009. Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells. Toxicology and Applied Pharmacology 236:310-318.
AshaRani P, Sethu S, Lim HK, Balaji G, Valiyaveettil S, Hande MP. 2012. Differential regulation of intracellular factors mediating cell cycle, DNA repair and inflammation following exposure to silver nanoparticles in human cells. Genome Integrity 3:2.
Balla KM, Lugo-Villarino G, Spitsbergen JM, Stachura DL, Hu Y, Bañuelos K, et al. 2010. Eosinophils in the zebrafish: Prospective isolation, characterization, and eosinophilia induction by helminth determinants. Blood 116:3944-3954.
Beliaeva NF, Kashirtseva VN, Medvedeva NV, Khudoklinova I, Ipatova OM, Archakov AI. 2010. zebrafish as a model organism for biomedical studies. Biomeditsinskaia Khimiia 56:120-131.
Biedermann BC. 2001. Vascular endothelium: Checkpoint for inflammation and immunity. Physiology 16:84-88.
Boraschi D, Italiani P, Palomba R, Decuzzi P, Duschl A, Fadeel B, et al. 2017. Nanoparticles and innate immunity: New perspectives on host defence. Seminars in Immunology 34:33-51.
Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. 2004. Neutrophil extracellular traps kill bacteria. Science 303:1532-1535.
Cao Y. 2018. The toxicity of nanoparticles to human endothelial cells. Cellular and Molecular Toxicology of Nanoparticles 1048: 59-69.
Carlson C, Hussain SM, Schrand AM, K. Braydich-Stolle L, Hess KL, Jones RL, et al. 2008. Unique cellular interaction of silver nanoparticles: Size-dependent generation of reactive oxygen species. The Journal of Physical Chemistry B 112:13608-13619.
Carocho M, Barreiro MF, Morales P, Ferreira IC. 2014. Adding molecules to food, pros and cons: A review on synthetic and natural food additives. Comprehensive Reviews in Food Science and Food Safety 13:377-399.
Chen R-J, Lee Y-H, Yeh Y-L, Wu W-S, Ho C-T, Li C-Y, et al. 2017. Autophagy-inducing effect of pterostilbene: A prospective therapeutic/preventive option for skin diseases. Journal of Food and Drug Analysis 25:125-133.
Chi-Hsin H, Zhi-Hong W, Chan-Shing L, Chiranjib C. 2007. The zebrafish model: Use in studying cellular mechanisms for a spectrum of clinical disease entities. Current Neurovascular Research 4:111-120.
Choi JE, Kim S, Ahn JH, Youn P, Kang JS, Park K, et al. 2010. Induction of oxidative stress and apoptosis by silver nanoparticles in the liver of adult zebrafish. Aquatic Toxicology 100:151-159.
Choo Q-Y, Yeo SCM, Ho PC, Tanaka Y, Lin H-S. 2014. Pterostilbene surpassed resveratrol for anti-inflammatory application: Potency consideration and pharmacokinetics perspective. Journal of Functional Foods 11:352-362.
Cook‐Mills JM, Deem TL. 2005. Active participation of endothelial cells in inflammation. Journal of Leukocyte Biology 77:487-495.
Cuervo AM, Dice JF. 1998. Lysosomes, a meeting point of proteins, chaperones, and proteases. Journal of Molecular Medicine 76:6-12.
Danese S, Dejana E, Fiocchi C. 2007. Immune regulation by microvascular endothelial cells: Directing innate and adaptive immunity, coagulation, and inflammation. The Journal of Immunology 178:6017-6022.
De Jong WH, Van Der Ven LT, Sleijffers A, Park MV, Jansen EH, Van Loveren H, et al. 2013. Systemic and immunotoxicity of silver nanoparticles in an intravenous 28 days repeated dose toxicity study in rats. Biomaterials 34:8333-8343.
Drake PL, Hazelwood KJ. 2005. Exposure-related health effects of silver and silver compounds: A review. The Annals of Occupational Hygiene 49:575-585.
Feltis BN, O'Keefe SJ, Harford AJ, Piva TJ, Turney TW, Wright PF. 2012. Independent cytotoxic and inflammatory responses to zinc oxide nanoparticles in human monocytes and macrophages. Nanotoxicology 6:757-765.
Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, et al. 2007. Novel cell death program leads to neutrophil extracellular traps. The Journal of Cell Biology 176:231-241.
Gaillet S, Rouanet J-M. 2015. Silver nanoparticles: Their potential toxic effects after oral exposure and underlying mechanisms–a review. Food and Chemical Toxicology 77:58-63.
Gaiser BK, Hirn S, Kermanizadeh A, Kanase N, Fytianos K, Wenk A, et al. 2012. Effects of silver nanoparticles on the liver and hepatocytes in vitro. Toxicological Sciences 131:537-547.
Gamucci O, Bertero A, Gagliardi M, Bardi G. 2014. Biomedical nanoparticles: Overview of their surface immune-compatibility. Coatings 4:139-159.
Gopalakrishnan V, Helmink BA, Spencer CN, Reuben A, Wargo JA. 2018. The influence of the gut microbiome on cancer, immunity, and cancer immunotherapy. Cancer Cell 33:570-580.
H Meijer A, P Spaink H. 2011. Host-pathogen interactions made transparent with the zebrafish model. Current Drug Targets 12:1000-1017.
Halliwell B, Chirico S. 1993. Lipid peroxidation: Its mechanism, measurement, and significance. The American Journal of Clinical Nutrition 57:715S-724S.
Hartung T. 2009. Toxicology for the twenty-first century. Nature 460:208.
Heidemann J, Domschke W, Kucharzik T, Maaser C. 2006. Intestinal microvascular endothelium and innate immunity in inflammatory bowel disease: A second line of defense? Infection and Immunity 74:5425-5432.
Jain A, Ranjan S, Dasgupta N, Ramalingam C. 2018. Nanomaterials in food and agriculture: An overview on their safety concerns and regulatory issues. Critical Reviews in Food Science and Nutrition 58:297-317.
Jim KK, Engelen-Lee J, van der Sar AM, Bitter W, Brouwer MC, van der Ende A, et al. 2016. Infection of zebrafish embryos with live fluorescent streptococcus pneumoniae as a real-time pneumococcal meningitis model. Journal of Neuroinflammation 13:188.
Johnston HJ, Verdon R, Gillies S, Brown DM, Fernandes TF, Henry TB, et al. 2018. Adoption of in vitro systems and zebrafish embryos as alternative models for reducing rodent use in assessments of immunological and oxidative stress responses to nanomaterials. Critical Reviews in Toxicology 48:252-271.
Jorch SK, Kubes P. 2017. An emerging role for neutrophil extracellular traps in noninfectious disease. Nature Medicine 23:279.
Kapetanovic IM, Muzzio M, Huang Z, Thompson TN, McCormick DL. 2011. Pharmacokinetics, oral bioavailability, and metabolic profile of resveratrol and its dimethylether analog, pterostilbene, in rats. Cancer Chemotherapy and Pharmacology 68:593-601.
Kim YS, Kim JS, Cho HS, Rha DS, Kim JM, Park JD, et al. 2008. Twenty-eight-day oral toxicity, genotoxicity, and gender-related tissue distribution of silver nanoparticles in sprague-dawley rats. Inhalation Toxicology 20:575-583.
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. 1995. Stages of embryonic development of the zebrafish. Developmental Dynamics 203:253-310.
Kosuru R, Rai U, Prakash S, Singh A, Singh S. 2016. Promising therapeutic potential of pterostilbene and its mechanistic insight based on preclinical evidence. European Journal of Pharmacology 789:229-243.
Kovvuru P, Mancilla PE, Shirode AB, Murray TM, Begley TJ, Reliene R. 2015. Oral ingestion of silver nanoparticles induces genomic instability and DNA damage in multiple tissues. Nanotoxicology 9:162-171.
Langheinrich U. 2003. Zebrafish: A new model on the pharmaceutical catwalk. Bioessays 25:904-912.
Lanone S, Boczkowski J. 2006. Biomedical applications and potential health risks of nanomaterials: Molecular mechanisms. Current Molecular Medicine 6:651-663.
Li Y, Li Y, Cao X, Jin X, Jin T. 2017. Pattern recognition receptors in zebrafish provide functional and evolutionary insight into innate immune signaling pathways. Cellular & Molecular Immunology 14:80.
Lin S, Zhao Y, Nel AE, Lin S. 2013. Zebrafish: An in vivo model for nano ehs studies. Small 9:1608-1618.
Liz R, Simard J-C, Leonardi LBA, Girard D. 2015. Silver nanoparticles rapidly induce atypical human neutrophil cell death by a process involving inflammatory caspases and reactive oxygen species and induce neutrophil extracellular traps release upon cell adhesion. International Immunopharmacology 28:616-625.
Loynes CA, Martin JS, Robertson A, Trushell DM, Ingham PW, Whyte MK, et al. 2010. Pivotal advance: Pharmacological manipulation of inflammation resolution during spontaneously resolving tissue neutrophilia in the zebrafish. Journal of Leukocyte Biology 87:203-212.
Luster MI, Portier C, Paît DG, White Jr KL, Gennings C, Munson AE, et al. 1992. Risk assessment in immunotoxicology: I. Sensitivity and predictability of immune tests. Toxicological Sciences 18:200-210.
Malhotra JD, Kaufman RJ. 2007. The endoplasmic reticulum and the unfolded protein response. Seminars in Cell & Developmental Biology 18:716-731.
McShan D, Ray PC, Yu H. 2014. Molecular toxicity mechanism of nanosilver. Journal of Food and Drug Analysis 22:116-127.
Pandey RK, Prajapati VK. 2017. Molecular and immunological toxic effects of nanoparticles. International Journal of Biological Macromolecules 107:1278-1293.
Perecko T, Drabikova K, Lojek A, Ciz M, Ponist S, Bauerova K, et al. 2013. The effects of pterostilbene on neutrophil activity in experimental model of arthritis. BioMed Research International, Article ID 106041, 7 pages.
Perrelli G, Piolatto G. 1992. Tentative reference values for gold, silver and platinum: Literature data analysis. Science of the Total Environment 120:93-96.
Planchart A, Mattingly CJ, Allen D, Ceger P, Casey W, Hinton D, et al. 2016. Advancing toxicology research using in vivo high throughput toxicology with small fish models. Alternatives to Animal Experimentation, ALTEX 33:435-452.
Ribas L, Piferrer F. 2014. The zebrafish (danio rerio) as a model organism, with emphasis on applications for finfish aquaculture research. Reviews in Aquaculture 6:209-240.
Santiago-Delpin E, Juan S. 2004. The endothelium and early immune activation: New perspective and interactions. Proceedings of the Transplantation Proceedings 36: 1709-1713.
Scapini P, Lapinet‐Vera JA, Gasperini S, Calzetti F, Bazzoni F, Cassatella MA. 2000. The neutrophil as a cellular source of chemokines. Immunological Reviews 177:195-203.
Schmidt EP, Lee WL, Zemans RL, Yamashita C, Downey GP. 2011. On, around, and through: Neutrophil-endothelial interactions in innate immunity. Physiology 26:334-347.
Scown TM, Santos EM, Johnston BD, Gaiser B, Baalousha M, Mitov S, et al. 2010. Effects of aqueous exposure to silver nanoparticles of different sizes in rainbow trout. Toxicological Sciences 115:521-534.
Setyawati M, Tay CY, Chia S, Goh S, Fang W, Neo M, et al. 2013. Titanium dioxide nanomaterials cause endothelial cell leakiness by disrupting the homophilic interaction of ve–cadherin. Nature Communications 4:1673.
Setyawati MI, Tay CY, Bay BH, Leong DT. 2017. Gold nanoparticles induced endothelial leakiness depends on particle size and endothelial cell origin. ACS Nano 11:5020-5030.
Shvedova AA, Pietroiusti A, Fadeel B, Kagan VE. 2012. Mechanisms of carbon nanotube-induced toxicity: Focus on oxidative stress. Toxicology and Applied Pharmacology 261:121-133.
Spiljar M, Merkler D, Trajkovski M. 2017. The immune system bridges the gut microbiota with systemic energy homeostasis: Focus on tlrs, mucosal barrier, and scfas. Frontiers in Immunology 8:1353.
Spitsbergen JM, Kent ML. 2003. The state of the art of the zebrafish model for toxicology and toxicologic pathology research—advantages and current limitations. Toxicologic Pathology 31:62-87.
Spitsbergen JM, Kent ML. 2003. The state of the art of the zebrafish model for toxicology and toxicologic pathology research—advantages and current limitations. Toxicol Pathol 31:62-87.
Sullivan C, Kim CH. 2008. Zebrafish as a model for infectious disease and immune function. Fish & Shellfish Immunology 25:341-350.
Sun X, Shi J, Zou X, Wang C, Yang Y, Zhang H. 2016. Silver nanoparticles interact with the cell membrane and increase endothelial permeability by promoting ve-cadherin internalization. Journal of Hazardous Materials 317:570-578.
Thiruvengadam M, Rajakumar G, Chung I-M. 2018. Nanotechnology: Current uses and future applications in the food industry. 3 Biotech 8:74.
Thisse C, Thisse B. 2008. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nature Protocols 3:59.
Tiwari DK, Jin T, Behari J. 2011. Dose-dependent in-vivo toxicity assessment of silver nanoparticle in wistar rats. Toxicology Mechanisms and Methods 21:13-24.
Tiwari SB, Amiji MM. 2006. A review of nanocarrier-based cns delivery systems. Current Drug Delivery 3:219-232.
Tran QH, Le A-T. 2013. Silver nanoparticles: Synthesis, properties, toxicology, applications and perspectives. Advances in Natural Sciences: Nanoscience and Nanotechnology 4:033001.
Trickler WJ, Lantz SM, Murdock RC, Schrand AM, Robinson BL, Newport GD, et al. 2010. Silver nanoparticle induced blood-brain barrier inflammation and increased permeability in primary rat brain microvessel endothelial cells. Toxicological Sciences 118:160-170.
Tsai H-Y, Ho C-T, Chen Y-K. 2017. Biological actions and molecular effects of resveratrol, pterostilbene, and 3′-hydroxypterostilbene. Journal of Food and Drug Analysis 25:134-147.
Van Den Brûle S, Ambroise J, Lecloux H, Levard C, Soulas R, De Temmerman P-J, et al. 2015. Dietary silver nanoparticles can disturb the gut microbiota in mice. Particle and Fibre Toxicology 13:38.
Vance ME, Kuiken T, Vejerano EP, McGinnis SP, Hochella Jr MF, Rejeski D, et al. 2015. Nanotechnology in the real world: Redeveloping the nanomaterial consumer products inventory. Beilstein Journal of Nanotechnology 6:1769.
Veldman MB, Lin S. 2008. Zebrafish as a developmental model organism for pediatric research. Pediatric Research 64:470-476.
Villeneuve D, Volz DC, Embry MR, Ankley GT, Belanger SE, Leonard M, et al. 2014. Investigating alternatives to the fish early-life stage test: A strategy for discovering and annotating adverse outcome pathways for early fish development. Environ Toxicol Chem 33:158-169.
Wang RL, Biales A, Bencic D, Lattier D, Kostich M, Villeneuve D, et al. 2008. DNA microarray application in ecotoxicology: Experimental design, microarray scanning, and factors affecting transcriptional profiles in a small fish species. Environmental Toxicology and Chemistry 27:652-663.
Wei L, Lu J, Xu H, Patel A, Chen Z-S, Chen G. 2015. Silver nanoparticles: Synthesis, properties, and therapeutic applications. Drug Discovery Today 20:595-601.
Wu J, Li M, He J, Lv K, Wang M, Guan W, et al. 2019. Protective effect of pterostilbene on concanvalin a-induced acute liver injury. Food & Function 11:7308-7314.
Xu L, Li X, Takemura T, Hanagata N, Wu G, Chou LL. 2012. Genotoxicity and molecular response of silver nanoparticle (np)-based hydrogel. Journal of Nanobiotechnology 10:16.
Yang E-J, Kim S, Kim JS, Choi I-H. 2012. Inflammasome formation and il-1β release by human blood monocytes in response to silver nanoparticles. Biomaterials 33:6858-6867.
Yang H, Biermann MH, Brauner JM, Liu Y, Zhao Y, Herrmann M. 2016. New insights into neutrophil extracellular traps: Mechanisms of formation and role in inflammation. Frontiers in Immunology 7:302.
Yang H, Marion TN, Liu Y, Zhang L, Cao X, Hu H, et al. 2019. Nanomaterial exposure induced neutrophil extracellular traps: A new target in inflammation and innate immunity. Journal of Immunology Research, Article ID 3560180, 8 pages.
Yoon H-S, Hyun C-G, Lee N-H, Park S-S, Shin D-B. 2016. Comparative depigmentation effects of resveratrol and its two methyl analogues in α-melanocyte stimulating hormone-triggered b16/f10 murine melanoma cells. Preventive Nutrition and Food Science 21:155-159.
Zhang X, Zhang J, Xu L, Ma Z, Di S, Gao Y, et al. 2018. Emerging actions of pterostilebene on cancer research. Chinese Journal of Lung Cancer 21:931-936.