The nuclear receptor coactivator 4 regulates ferritinophagy induced by vibrio splendidus in coelomocytes of Apostichopus japonicus

doi:10.1016/j.fsi.2024.109745

Fish & Shellfish Immunology
鱼类和贝类免疫学

Volume 151, August 2024, 109745
第 151 卷，2024 年 8 月，109745

https://doi.org/10.1016/j.fsi.2024.109745IF: 4.1 Q1
https://doi.org/10.1016/j.fsi.2024.109745IF： 4.1 第一季度 Get rights and content 获取权利和内容

Highlights 突出

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AjNCOA4 was characterized and validated in Apostichopus japonicus.
AJNCOA4 在 Apostichopus japonicus 中进行了表征和验证。
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The recombinant AjNCOA4 was expressed in E. coli and purified.
重组 AjNCOA4 在大肠杆菌中表达并纯化。
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AjNCOA4 interacted with AjFerritin in coelomocytes.
AJNCOA4 与腔体细胞中的 Aj铁蛋白相互作用。
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Knockdown of AjNCOA4 reduced intracellular Fe²⁺ levels.
敲除 AjNCOA4 降低了细胞内 Fe²⁺ 水平。
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AjNCOA4 promoted ferritinophagy induced by V. splendidus.
AJNCOA4 促进 V. splendidus 诱导的铁蛋白自噬。

Abstract 抽象

Iron homeostasis is vital for the host's defense against pathogenic invasion and the ferritinophagy is a crucial mechanism in maintaining intracellular iron homeostasis by facilitating the degradation and recycling of stored iron. The nuclear receptor coactivator 4 (NCOA4) serves as a ferritinophagy receptor, facilitating the binding and delivery of ferritin to the autophagosome and lysosome. However, NCOA4 of the sea cucumber Apostichopus japonicus (AjNCOA4) has not been reported until now. In this study, we identified and characterized AjNCOA4 in A. japonicus. This gene encodes a polypeptide containing 597 amino acids with an open reading frame of 1794 bp. The inferred amino acid sequence of AjNCOA4 comprises an ARA70 domain. Furthermore, a multiple sequence alignment demonstrated varying degrees of sequence homology between AjNCOA4 from A. japonicus and other NCOA4 orthologs. The phylogenetic tree of NCOA4 correlates with the established timeline of metazoan evolution. Expression analysis revealed that AjNCOA4 is expressed in all tested tissues, including the body wall, muscle, intestine, respiratory tree, and coelomocytes. Following challenge with Vibrio splendidus, the coelomocytes exhibited a significant increase in AjNCOA4 mRNA levels, peaking at 24 h. We successfully obtained recombinant AjNCOA4 protein through prokaryotic expression and prepared a specific polyclonal antibody. Immunofluorescence and co-immunoprecipitation experiments demonstrated an interaction between AjNCOA4 and AjFerritin in coelomocytes. RNA interference-mediated knockdown of AjNCOA4 expression resulted in elevated iron ion levels in coelomocytes. Bacterial stimulation enhanced ferritinophagy in coelomocytes, while knockdown of AjNCOA4 reduced the occurrence of ferritinophagy. These findings suggest that AjNCOA4 modulates ferritinophagy induced by V. splendidus in coelomocytes of A. japonicus.
铁稳态对于宿主防御病原入侵至关重要，铁蛋白自噬是通过促进储存铁的降解和回收来维持细胞内铁稳态的关键机制。核受体共激活因子 4 （NCOA4）作为铁蛋白自噬受体，促进铁蛋白与自噬体和溶酶体的结合和递送。然而，海参 Apostichopus japonicus （AjNCOA4）的 NCOA4 直到现在才被报道。在这项研究中，我们鉴定并表征了 AjNCOA4 在 A. japonicus 中。该基因编码一个包含 597 个氨基酸的多肽，开放阅读框为 1794 bp。Aj NCOA4 的推断氨基酸序列包含一个 ARA70 结构域。此外，多序列比对表明来自 A. japonicus 的 AjNCOA4 与其他 NCOA4 直系同源物之间存在不同程度的序列同源性。NCOA4 的系统发育树与已建立的后生动物进化时间表相关。表达分析显示 AjNCOA4 在所有测试组织中表达，包括体壁、肌肉、肠道、呼吸树和体腔细胞。在用 Vibrio splendidus 攻击后，腔细胞表现出 AjNCOA4 mRNA 水平的显着增加，在 24 小时达到峰值。我们通过原核表达成功获得重组 AjNCOA4 蛋白，并制备了特异性多克隆抗体。免疫荧光和免疫共沉淀实验表明 AjNCOA4 和 Aj铁蛋白在体腔细胞中存在相互作用。RNA 干扰介导的 AjNCOA4 表达敲低导致腔细胞中铁离子水平升高。细菌刺激增强了体腔细胞中的铁自噬，而敲除 AjNCOA4 减少了铁自噬的发生。这些发现表明 AjNCOA4 调节 V 诱导的铁蛋白自噬。在 A 的腔腔细胞中表现出色。日本人。

Keywords 关键字

Sea cucumber

Vibrio splendidus

NCOA4

Ferritinophagy

Coelomocytes

海参

Vibrio splendidus

NCOA4

铁蛋白自噬

体腔细胞

1. Introduction 1. 引言

In recent years, the economically significant marine organism sea cucumber Apostichopus japonicus, renowned for its high medicinal value, has witnessed a substantial expansion in both cultivation scale and area. However, diseases pose a significant hindrance to the growth of this industry, with the skin ulcer syndrome (SUS) being particularly severe [1]. The Vibrio splendidus bacterium is identified as the primary culprit behind this malady [2]. As iron is essential to many processes that keep life going, maintaining iron homeostasis is critical to the host's defense against pathogenic invasion. Furthermore, it regulates the expression of specific pathogenic genes and is intricately linked to bacterial pathogenicity [3]. By competing with pathogenic microbes for iron in the environment, the host can hinder their growth [4]. Additionally, the host possesses the capability to meticulously regulate iron metabolism, thereby enhancing iron absorption, optimizing iron utilization, and preserving iron balance.
近年来，具有重要经济意义的海洋生物海参 Apostichopus japonicus 以其高药用价值而闻名，在养殖规模和面积上都出现了大幅扩张。然而，疾病对该行业的发展构成了重大阻碍，其中皮肤溃疡综合征（SUS）尤其严重 [1]。Vibrio splendidus 细菌被确定为这种疾病背后的罪魁祸首 [2]。由于铁对于维持生命的许多过程至关重要，因此维持铁稳态对于宿主抵御病原体入侵至关重要。此外，它还调节特定致病基因的表达，并与细菌致病性有着错综复杂的联系 [3]。通过与环境中的病原微生物竞争铁，宿主可以阻碍它们的生长 [4]。此外，宿主具有精心调节铁代谢的能力，从而增强铁的吸收，优化铁的利用，并保持铁的平衡。

Ferritinophagy plays an important role in maintaining intracellular iron homeostasis by facilitating the degradation and recycling of stored iron [5]. This recycling process ensures that iron can be effectively utilized in cellular processes while mitigating oxidative damage caused by excessive iron accumulation [5]. Specifically, ferritinophagy is a selective autophagy that specifically targets the degradation of intracellular ferritin [5]. Ferritin, a major iron storage protein, is essential to iron homeostasis and is involved in a wide range of physiologic and pathologic processes [6]. The mammalian ferritin is composed of ferritin heavy chain (FTH) and ferritin light chain (FTL) subunits [6]. These subunits are transcribed from distinct genes and can be assembled into heteropolymers in varying proportions. The FTH is responsible for oxidizing Fe²⁺, whereas the FTL facilitates the mineralization of Fe³⁺. Notably, in numerous invertebrates, ferritin typically contains a single subunit that exhibits properties similar to those of the vertebrate FTH subunit [7]. The crystal structure analysis revealed that AjFerritin, the ferritin in A. japonicus, exhibits a cage-like, hollow spherical shell comprised of 24 subunits, closely resembling the structure observed in other species [8]. Ferritinophagy is crucial in various physiological processes, including cell differentiation, erythropoiesis, and immune response [9]. When intracellular iron levels become depleted, the transcription of both the FTH and FTL is activated, leading to elevated intracellular ferritin levels and the formation of ferritin nanoparticles. As iron levels rise again, autophagy receptors bind to these ferritin nanoparticles and target them for degradation via the autophagy-lysosome pathway [10]. This degradation process ultimately releases iron ions from the degraded ferritin molecules into the cytoplasm, enabling their utilization for cellular functions such as heme synthesis or ROS production [11]. During host response to infection or stress, ferritinophagy serves as a mechanism to release iron to meet metabolic needs [12]. Additionally, the increase in intracellular active iron modifies the intracellular oxidation-reduction environment, triggering immune system activation.
铁蛋白自噬通过促进储存铁的降解和再循环，在维持细胞内铁稳态中发挥重要作用 [5]。这种回收过程确保铁可以在细胞过程中得到有效利用，同时减轻铁过度积累造成的氧化损伤 [5]。具体来说，铁蛋白自噬是一种选择性自噬，专门针对细胞内铁蛋白的降解 [5]。铁蛋白是一种主要的铁储存蛋白，对铁稳态至关重要，并参与广泛的生理和病理过程 [6]。哺乳动物铁蛋白由铁蛋白重链（FTH）和铁蛋白轻链（FTL）亚基组成 [6]。这些亚基是从不同的基因转录而来的，可以以不同比例组装成杂聚物。FTH 负责氧化 Fe²⁺，而 FTL 促进 Fe 3+ 的矿化。值得注意的是，在许多无脊椎动物中，铁蛋白通常包含一个亚基，其特性与脊椎动物 FTH 亚基相似 [7]。晶体结构分析显示 AjFerritin，即 A. Japonicus 表现出由 24 个亚基组成的笼状空心球形壳，与其他物种观察到的结构非常相似 [8]。铁蛋白自噬在各种生理过程中至关重要，包括细胞分化、红细胞生成和免疫反应 [9]。当细胞内铁水平耗尽时，FTH 和 FTL 的转录被激活，导致细胞内铁蛋白水平升高和铁蛋白纳米颗粒的形成。随着铁水平再次升高，自噬受体与这些铁蛋白纳米颗粒结合，并通过自噬-溶酶体途径靶向它们进行降解 [10]。这个降解过程最终将铁离子从降解的铁蛋白分子中释放到细胞质中，使其能够用于细胞功能，如血红素合成或 ROS 产生 [11]。在宿主对感染或应激的反应期间，铁蛋白自噬是一种释放铁以满足代谢需要的机制 [12]。此外，细胞内活性铁的增加会改变细胞内氧化还原环境，从而触发免疫系统激活。

The nuclear receptor coactivator 4 (NCOA4) functions as a ferritinophagy receptor, facilitating the binding and delivery of ferritin to the autophagosome and lysosome [13]. Then iron ions are released by degrading ferritin. The flux of ferritinophagy is modulated by NCOA4 levels, which are in turn tightly controlled by intracellular iron levels [14]. Specifically, high intracellular iron levels promote the iron-bound interaction of NCOA4 with the ubiquitin E3 ligase HERC2, leading to proteasomal degradation of NCOA4 [15]. Conversely, low iron levels hinder this interaction, stabilizing NCOA4 and subsequently enhancing ferritinophagy in the lysosome [16]. NCOA4-mediated ferritinophagy plays a crucial role in physiological processes which demands significant iron, such as erythropoiesis. In zebrafish, NCOA4 deletion leads to defects in globin synthesis and hemoglobinization [16]. Similarly, NCOA4-mediated ferritinophagy is essential for iron availability in heme synthesis in the NCOA4-knockout mouse [17]. Additionally, ferritinophagy regulates susceptibility to infectious diseases. For instance, Escherichia coli survive by utilizing ferritin-bound iron from autophagosomes to support their proliferation [18]. The ferritin transport is reliant on NCOA4, and suppressing NCOA4 reduces bacterial load [19]. Autophagy inhibitors and iron chelators can also mitigate bacterial burden and host cell death, indicating therapeutic potential in regulating NCOA4-dependent ferritinophagy during certain bacterial infections [19]. NCOA4 mediated ferritinophagy is an essential part of iron homeostasis under normal and pathological conditions [19]. However, the role of NCOA4 in invertebrates is an area that has not been fully explored. Based on the importance of NCOA4 in systemic iron homeostasis, future research on the function of NCOA4 in diseases with iron homeostasis disorders will be beneficial.
核受体共激活因子 4 （NCOA4）作为铁蛋白自噬受体发挥作用，促进铁蛋白结合并递送至自噬体和溶酶体 [13]。然后通过降解铁蛋白释放铁离子。铁蛋白自噬的通量受 NCOA4 水平的调节，而 NCOA4 水平又受到细胞内铁水平的严格控制 [14]。具体来说，细胞内铁水平高会促进 NCOA4 与泛素 E3 连接酶 HERC2 的铁结合相互作用，从而导致 NCOA4 的蛋白酶体降解 [15]。相反，低铁水平会阻碍这种相互作用，稳定 NCOA4 并随后增强溶酶体中的铁蛋白自噬 [16]。NCOA4 介导的铁蛋白自噬在需要大量铁的生理过程中起着至关重要的作用，例如红细胞生成。在斑马鱼中，NCOA4 缺失导致珠蛋白合成和血红蛋白化缺陷 [16]。同样，NCOA4 介导的铁蛋白自噬对于 NCOA4 敲除小鼠血红素合成中的铁可用性至关重要 [17]。此外，铁蛋白自噬调节对传染病的易感性。例如，大肠杆菌通过利用自噬体中的铁蛋白结合铁来支持其增殖而存活 [18]。铁蛋白的转运依赖于 NCOA4，抑制 NCOA4 可减少细菌载量 [19]。自噬抑制剂和铁螯合剂还可以减轻细菌负荷和宿主细胞死亡，表明在某些细菌感染期间调节 NCOA4 依赖性铁蛋白自噬具有治疗潜力 [19]。NCOA4 介导的铁蛋白自噬是正常和病理条件下铁稳态的重要组成部分 [19]。然而，NCOA4 在无脊椎动物中的作用是一个尚未得到充分探索的领域。基于 NCOA4 在全身性铁稳态中的重要性，未来对 NCOA4 在铁稳态障碍疾病中功能的研究将是有益的。

In this study, we successfully amplified the full-length cDNA sequence of AjNCOA4 from the sea cucumber A. japonicus. To assess its evolutionary status, a phylogenetic tree analysis was conducted. Additionally, the expression levels of AjNCOA4 were examined using real-time quantitative reverse transcription PCR (qRT-PCR). We also obtained the recombinant protein of AjNCOA4 through prokaryotic expression, facilitating the analysis of its interaction with AjFerritin. Furthermore, the functional significance of NCOA4 was elucidated using RNA interference, revealing its regulatory involvement in ferritinophagy. Consequently, our investigation into NCOA4-mediated ferritinophagy offers promising avenues for the prevention and treatment of bacterial infectious diseases.
在这项研究中，我们成功扩增了海参 Aj NCOA4 的全长 cDNA 序列。为了评估其进化状态，进行了系统发育树分析。此外，使用实时定量逆转录 PCR （qRT-PCR）检测 Aj NCOA4 的表达水平。我们还通过原核表达获得了 AjNCOA4 的重组蛋白，有助于分析其与 AjFerritin 的相互作用。此外，使用 RNA 干扰阐明了 NCOA4 的功能意义，揭示了其在铁蛋白自噬中的调节参与。因此，我们对 NCOA4 介导的铁蛋白自噬的研究为预防和治疗细菌传染病提供了有希望的途径。

2. Materials and methods 2. 材料和方法

2.1. Ethics statement 2.1. 道德声明

The animals used in this study, including sea cucumbers and ICR mice, were commercially farmed. The Experimental Animal Ethics Committee of Ningbo University in China has granted approval for the research scheme (No.13127). The laboratory procedures were carried out in strict adherence to the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals.
本研究中使用的动物，包括海参和 ICR 小鼠，都是商业养殖的。中国宁波大学实验动物伦理委员会已批准该研究计划（第 13127 号）。实验室程序严格遵守美国国立卫生研究院的实验动物护理和使用指南。

2.2. Animal and bacterium
2.2. 动物和细菌

The experiment utilized A. japonicus, with an average weight of 125 ± 15 g, purchased from Dalian Pacific Fisheries Company. Prior to the experiment, the A. japonicus was reared in the culture system for a week to ensure stability in its survival state. Every six sea cucumbers were are raised in a single 30 L tank with the condition of recirculating oxygenated seawater, a salinity level of approximately 30 %, and a temperature of 16 °C. The bacterium V. splendidus, which was isolated from diseased A. japonicus, was stored at −80 °C with glycerol in the laboratory. Before its utilization in the experiment, the strain underwent verification through 16S rDNA sequencing.
该实验使用从大连太平洋渔业公司购买的 A. japonicus，平均重量为 125 ± 15 g。在实验之前，日本曲霉在培养系统中饲养一周，以确保其生存状态的稳定性。每 6 个海参在一个 30 升的水箱中饲养，条件是循环含氧海水，盐度约为 30 %，温度为 16 °C。从患病的 A. japonicus 中分离的细菌 V. splendidus 在实验室中与甘油一起储存在 -80 °C 下。在用于实验之前，该菌株通过 16S rDNA 测序进行了验证。

2.3. Cloning and characterization of AjNCOA4 from A. japonicus
2.3. 日本蚜蓿 AjNCOA4 的克隆和表征

Using sterilized scissors, the sea cucumber was dissected, and the coelomic fluid was collected while removing large tissue fragments through a 200-mesh cell screen. The filtered fluid was then centrifuged at 800×g and 16 °C for 5 min, isolating the coelomocytes of A. japonicus. RNAiso Plus (Takara, Dalian, China) was used to extract total RNA. The concentration and purity of RNA were assessed by NanoDrop, and the quality of RNA was assessed by 1.5 % agarose gel electrophoresis detection. 1 μg of total RNA and Oligo dT Primer were used to synthesize the first strand cDNA by PrimeScrip 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). Based on the transcriptome and genome data of A. japonicus, primers (Table 1) were designed for PCR amplification of AjNCOA4 expression sequence tag (EST). The full-length cDNA of AjNCOA4 was amplified using SMARTer RACE 5’/3’ Kit (Clontech, CA, US). The amplified products were purified from the agarose gels, and ligated to the pMD19-T vector (Takara, Dalian, China) for sequencing. The molecular weight and theoretical isoelectric point (pI) were determined by analyzing the protein sequence using the ExPASy ProtParam tool. BLAST software from the National Center for Biotechnology Information (NCBI) was used to compare the sequence, SMART software analyzed its domain composition [20], and MEGA7.0 software constructed an evolutionary tree [21].
使用消毒剪刀解剖海参，收集体液，同时通过 200 目细胞筛选去除大组织碎片。然后将过滤后的液体在 800× g 和 16 °C 下离心 5 分钟，分离日本曲霉的腔细胞。使用 RNAiso Plus （Takara， Dalian， China）提取总 RNA。通过 NanoDrop 评估 RNA 的浓度和纯度，通过 1.5% 琼脂糖凝胶电泳检测评估 RNA 的质量。使用 1 μg 总 RNA 和 Oligo dT 引物通过 PrimeScrip 第一链 cDNA 合成试剂盒（Takara，大连，中国）合成第一链 cDNA。基于日本蚜蓿的转录组和基因组数据，设计了用于 Aj NCOA4 表达序列标签（EST）的 PCR 扩增的引物（表 1）。使用 SMARTer RACE 5'/3' 试剂盒（Clontech， CA， US）扩增 AjNCOA4 的全长 cDNA。从琼脂糖凝胶中纯化扩增产物，并连接到 pMD19-T 载体（Takara， Dalian， China）进行测序。通过使用 ExPASy ProtParam 工具分析蛋白质序列来确定分子量和理论等电点（pI）。使用美国国家生物技术信息中心（NCBI）的 BLAST 软件比较序列，SMART 软件分析其结构域组成 [20]，MEGA7.0 软件构建进化树 [21]。

Table 1. Primers used in this study.
表 1.本研究中使用的引物。

Name 名字	Sequence (5′–3′) 序列（5′–3′）	Application 应用
AjNCOA4-F	TTCCACGGCGACAACCTAC	EST
AjNCOA4-R	GTCCACCATCCTCATCCCATAA	EST
AjNCOA4-3 AJ非 NCOA4-3	CTCCAGAGAAGCAGCAGGAGGAT	3′ RACE 3' 比赛
AjNCOA4-5 AJ非 NCOA4-5	TATACCAAGTCCTTGAGTGACGG	5′ RACE 5' 比赛
Q-AjNCOA4-F Q-AJNCOA4-F	CCTACTGACGGGTGGAAT	qRT-PCR qRT-PCR 检测
Q-AjNCOA4-R Q-AJNCOA4-R	CATTCTTAGCTCGATTGC
Q-Ajβ-actin-F Q-AJβ-肌动蛋白-F	CCATTCAACCCTAAAGCCAACA
Q-Ajβ-actin-R Q-AJβ-肌动蛋白-R	ACACACCGTCTCCTGAGTCCAT
AjNCOA4- BamH Ⅰ-F AJNCOA4- BamH I.-F	GGATCCATGGCTCAACAAAACAACCCC	recombinant protein 重组蛋白
AjNCOA4-Xho Ⅰ-R AJNCOA4-Xho I.-R	CTCGAGCTAACAAACCGGTCTTTCTGCTAC	recombinant protein 重组蛋白
siAjNCOA4-F	GCAUGCAAGCUUGGCGUAATT	RNA interference RNA 干扰
siAjNCOA4-R 编号AjNCOA4-R	UUACGCCAAGCUUGCAUGCTT
siGFP-F	UUGAACUCCCUCUUGACGGTT
siGFP-R siGFP-R 型	ACGUGACACGUUCGGAGAATT

2.4. Assessment of AjNCOA4 mRNA expressions through qRT-PCR upon bacterial infection
2.4. 细菌感染后通过 qRT-PCR 评估 AjNCOA4 mRNA 的表达

The body wall, muscles, intestine, respiratory tree, and coelomocytes were harvested from healthy A. japonicus. RNAiso Plus was employed to extract total RNA from each tissue. V. splendidus was cultured at 28 °C with shaking at 200 rpm in 2216E medium consisting of 5 g/L tryptone, 1 g/L yeast extract, and 0.01 g/L FePO₄ in filtered seawater. When the bacterial culture medium reached OD₆₀₀ of 1, gradient dilution was carried out and the bacterial concentration was calculated by coating the plate. The sea cucumbers were randomly divided into six groups and cultured in 10 L tank. Then the sea water was added with approximately 100 mL V. splendidus to make the final concentration was 1 × 10⁷ colony forming units (CFU)/mL. Coelomocytes were collected from the sea cucumbers at various time points (0, 12, 24, 48, 72, 96 h), and the total RNA of samples from each group was extracted using RNAiso Plus. The cDNA templates were synthesized using the PrimeScrip RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Specific quantitative primers (Table 1) for AjNCOA4 were designed and synthesized. And qPCR was performed using the TB Green Premix Ex TaqII (Takara, Dalian, China). The reactions were conducted on a Light Cycler 480Ⅱ (Roche, Basel, Switzerland) with the following protocol: 95 °C pre-denaturation for 3 min, followed by 40 cycles of 95 °C for 15 s, 56 °C for 15 s, and 72 °C for 30 s. Each sample was replicated three times. Ajβ-actin served as the reference gene. The relative expression of the target gene was determined using the 2^−ΔΔCt method. SPSS 20.0 software was utilized to perform one-way ANOVA or Student's t-tests on the data, and the results were presented as mean ± SD. P-value of <0.05 was considered statistically significant.
体壁、肌肉、肠道、呼吸树和腔细胞均取自健康的 A. japonicus。使用 RNAiso Plus 从每个组织中提取总 RNA。在 28 °C 下，在 200 rpm 摇动中，在由 5 g/L 胰蛋白胨、1 g/L 酵母提取物和 0.01 g/L FePO 4 组成的 2216E 培养基中，在过滤海水中培养 V. splendidus。当细菌培养基达到 OD₆₀₀ of 1 时，进行梯度稀释，并通过涂布板计算细菌浓度。将海参随机分为 6 组，在 10 L 水箱中培养。然后在海水中加入约 100 mL 的 V. splendidus，使最终浓度为 1 × 10⁷ 菌落形成单位（CFU）/mL。在不同时间点（0 、 12 、 24 、 48 、 72 、 96 h ）从海参中收集体腔细胞，并使用 RNAiso Plus 提取每组样品的总 RNA。使用带有 gDNA Eraser 的 PrimeScrip RT 试剂盒（Takara，Dalian，China）合成 cDNA 模板。设计并合成了 Aj NCOA4 的特异性定量引物（表 1）。并使用 TB Green Premix Ex TaqII（Takara，大连，中国）进行 qPCR。反应在Light Cycler 480II.（Roche，Basel，Switzerland）上进行，实验方案如下：95 °C预变性3 min，然后进行40个循环，分别95 °C 15 s、56 °C15 s和72 °C 30 s。每个样本重复 3 次。 Ajβ-actin 用作参考基因。使用 2^−ΔΔCt 方法测定靶基因的相对表达。使用 SPSS 20.0 软件对数据进行单因素方差分析或学生 t 检验，结果以 SD ±平均值表示。<0.05 的 P 值被认为具有统计学意义。

2.5. Expression of recombinant protein and preparation of polyclonal antibody
2.5. 重组蛋白的表达和多克隆抗体的制备

The ORF sequence of AjNCOA4 was amplified using specific primers (Table 1) then ligated into the pET-28a expression vector via double digestion with restriction enzymes BamH I and XhoI. The recombinant plasmid was subsequently transformed into E. coli BL21 (DE3) by the method of heat shock at 42 °C. A single colony was inoculated into LB medium with 50 μg/mL kanamycin, grown at 37 °C until it reached the logarithmic phase, and induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) to express the recombinant protein. The recombinant protein rAjNCOA4 with His-tagged was purified using a Ni-NTA SeflnoseTM resin (Sangon, shanghai, China). The purified rAjNCOA4 was then combined with an immune-derived adjuvant and injected into four male ICR mice. Blood samples were collected from the immunized mice to generate polyclonal antibodies.
使用特异性引物扩增 AjNCOA4 的 ORF 序列（表 1），然后通过限制性内切酶 Bam H I 和 Xho I 的双重消化连接到 pET-28a 表达载体中。随后在 42 °C 下通过热休克方法将重组质粒转化到大肠杆菌 BL21 （DE3）中。将单个菌落接种到含有 50 μg/mL 卡那霉素的 LB 培养基中，在 37 °C 下生长直至达到对数期，并用 1 mM 异丙基 β-d-1-硫代吡喃半乳糖苷（IPTG）诱导表达重组蛋白。使用 Ni-NTA Seflnose TM 树脂（Sangon，上海，中国）纯化带有 His 标签的重组蛋白 r AjNCOA4。然后将纯化的 rAjNCOA4 与免疫来源的佐剂结合，注射到 4 只雄性 ICR 小鼠中。从免疫小鼠身上采集血样以产生多克隆抗体。

2.6. Western blot analysis
2.6. Western blot 分析

To determine the concentration of purified rAjNCOA4 protein, the BCA Protein Assay Kit (Beyotime, Nanjing, China) was employed, and the loading amount was adjusted to 50 μg of protein. Subsequently, the protein was separated via 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isolated proteins were then transferred onto a 0.45 μm-pored nitrocellulose membrane. Following this, the membrane was blocked using a prepared TBST (50 mM Tris-HCL, 150 mM NaCl, and 0.05 % Tween 20) solution containing 5 % skim milk for 2 h at room temperature. The prepared AjNCOA4 polyclonal antibody was diluted 1:1000 times and incubated overnight at 4 °C. The blot was incubated with anti-mouse IgG conjugated with horseradish peroxidase (HRP) at a dilution of 1:10,000 for 2 h at room temperature. The membrane was rinsed with TBST three times again, incubated with Western Lightning ECL substrate (Perkin Elmer, MA, USA), and the protein signal was detected using the ChemiDoc XRS chemiluminescence imaging system (Bio-Rad, CA, USA).
为了测定纯化的 rAjNCOA4 蛋白的浓度，采用 BCA 蛋白检测试剂盒（Beyotime，南京，中国），并将上样量调整为 50 μg 蛋白质。随后，通过 10 % 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离蛋白质。然后将分离的蛋白质转移到 0.45 μm 孔的硝酸纤维素膜上。在此之后，使用制备的含有 5% 脱脂牛奶的 TBST（50 mM Tris-HCL、150 mM NaCl 和 0.05 % Tween 20）溶液封闭膜 2 小时。将制备的 AjNCOA4 多克隆抗体按 1：1000 倍稀释，并在 4 °C 下孵育过夜。将印迹与偶联辣根过氧化物酶（HRP）的抗小鼠 IgG 以 1：10,000 的稀释度在室温下孵育 2 小时。再次用 TBST 冲洗膜 3 次，与 Western Lightning ECL 底物（Perkin Elmer， MA， USA）一起孵育，并使用 ChemiDoc XRS 化学发光成像系统（Bio-Rad， CA， USA）检测蛋白质信号。

2.7. Primary coelomocytes culture and RNA interference
2.7. 原代腔细胞培养和 RNA 干扰

The scissors were used to dissect A. japonicus from the cloaca to collect the coelomic fluid using 200-mesh filter. An equal quantity of sterilized anticoagulant solution (0.02 M EGTA, 0.48 M NaCl, 0.019 M KCl, 0.068 M Tris–HCl, pH 7.6) was added and mixed thoroughly. The collected fluid was then centrifuged at 16 °C and 800×g for 10 min. The coelomocytes were resuspended with the sterilized isotonic buffer (0.001 M EGTA, 0.53 M NaCl, and 0.01 M Tris-HCl, pH 7.6). Centrifuge again at the same temperature and speed for 10 min. The coelomocytes were resuspended with the Leibovitz's L-15 medium supplemented with penicillin (10 U/mL), streptomycin (100 μg/mL), gentamicin (50 μg/mL) and NaCl (0.015 g/mL). The coelomocytes were added into a cell culture plate and incubate at a constant temperature of 16 °C for 6 h. The coelomocytes were cultured in 12-well plates at a concentration of 1 × 10⁶ cells/well. Mix the designed and synthesized AjNCOA4-specific small interfering RNA (siRNA) with Lipofectamine 6000 reagent for 15 min to form a transfection mixture. The mixture was added into each well of the plate. The coelomocytes transfected with non-targeted siRNA (siGFP) serve as the negative control. Collect the cells at 24 h post-transfection and analyze the mRNA level of AjNCOA4 using qRT-PCR to assess the knockdown efficiency.
用剪刀从泄殖腔中解剖 A. japonicus ，使用 200 目过滤器收集体液。加入等量的无菌抗凝液溶液（0.02 M EGTA、0.48 M NaCl、0.019 M KCl、0.068 M Tris-HCl，pH 7.6）并充分混合。然后将收集的液体在 16 °C 和 800 × g 下离心 10 分钟。用灭菌的等渗缓冲液（0.001 M EGTA、0.53 M NaCl 和 0.01 M Tris-HCl，pH 7.6）重悬腔细胞。以相同的温度和速度再次离心 10 分钟。用补充有青霉素（10 U/mL）、链霉素（100 μg/mL）、庆大霉素（50 μg/mL）和 NaCl （0.015 g/mL）的 Leibovitz's L-15 培养基重悬腔细胞。将腔细胞加入细胞培养板中，并在 16 °C 的恒温下孵育 6 小时。将体腔细胞以 1 × 10^{6 个细胞}/孔的浓度在 12 孔板中培养。将设计和合成的 AjNCOA4 特异性小干扰 RNA （siRNA）与 Lipofectamine 6000 试剂混合 15 分钟，以形成转染混合物。将混合物加入板的每个孔中。用非靶向 siRNA （siGFP）转染的体腔细胞用作阴性对照。转染后 24 小时收集细胞，并使用 qRT-PCR 分析 AjNCOA4 的 mRNA 水平以评估敲低效率。

2.8. Immunofluorescence (IF) staining
2.8. 免疫荧光（IF）染色

The coelomocytes were seeded onto the glass slides in a 12-well plate at a concentration of 1 × 10⁶ cells/well. Once the cells adhered to the plate's walls, the medium was discarded after 24 h of stimulation with V. splendidus. Subsequently, the cells were fixed with 4 % paraformaldehyde (PFA) for 20 min, followed by permeabilization with 0.1 % Triton X-100 for 10 min. They were then washed three times with PBST (PBS containing 0.05 % Tween-20). To prevent non-specific binding, the cells were blocked with 5 % BSA for 1 h. Afterward, AjNCOA4 polyclonal antibody (1:1000) and commercial rabbit anti-Ferritin antibody (1:1000, 11682-1-AP, Proteintech) were added and incubated with the coelomocytes overnight at 4 °C. The cells were washed with PBST three times. The cells were then incubated for 2 h with Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G (IgG) and Cy3-conjugated goat anti-Rabbit IgG. DAPI was added for 10 min, followed by three washes with PBS. Images were captured using a confocal laser scanning microscope (Leica, Wetzlar, Germany) to observe the co-localization of AjNCOA4 and AjFerritin. The mouse anti-LC3 antibody (1:1000, ab243506, Abcam) was used to detect the co-localization of AjFerritin and AjLC3.
将腔细胞以 1 × 10⁶ 个细胞/孔的浓度接种到 12 孔板中的载玻片上。一旦细胞粘附在板壁上，用 V. splendidus 刺激 24 小时后丢弃培养基。随后，用 4 % 多聚甲醛（PFA）固定细胞 20 分钟，然后用 0.1 % Triton X-100 透化 10 分钟。然后用 PBST（含有 0.05 % Tween-20 的 PBS）洗涤 3 次。为防止非特异性结合，用 5 % BSA 封闭细胞 1 小时。然后，加入 AjNCOA4 多克隆抗体（1：1000）和市售兔抗铁蛋白抗体（1：1000， 11682-1-AP， Proteintech），并在 4 °C 下与体腔细胞孵育过夜。用 PBST 洗涤细胞 3 次。然后将细胞与 Alexa Fluor 488 偶联的山羊抗小鼠免疫球蛋白 G （IgG）和 Cy3 偶联的山羊抗兔 IgG 一起孵育 2 小时。加入 DAPI 10 分钟，然后用 PBS 洗涤 3 次。使用共聚焦激光扫描显微镜（Leica， Wetzlar， Germany）捕获图像，以观察 AjNCOA4 和 AjFerritin 的共定位。小鼠抗 LC3 抗体（1：1000， ab243506， Abcam）检测 Aj铁蛋白和 AjLC3 的共定位。

2.9. Co-immunoprecipitation (Co-IP)
2.9. 免疫共沉淀（Co-IP）

The total protein was extracted by employing a cell lysis buffer (20 mM Tris, 150 mM NaCl, 1 % Triton X-100, pH 7.5) containing PMSF and protease inhibitors, which lysed the coelomocytes of infected A. japonicus following 24 h of immersion in V. splendidus. Subsequently, polyclonal antibodies specific against AjNCOA4 were incubated with protein A/G magnetic beads (P2055, Beyotime) at room temperature for 30 min. These antibody-bound magnetic beads were then incubated with the coelomocyte lysate at 4 °C overnight while shaking gently. To eliminate unbound proteins and impurities, the magnetic beads were washed several times with the cell lysis buffer. The beads were added into SDS-PAGE loading buffer and boiling for 10 min. Finally, the AjFerritin antibody was utilized to investigate the interaction between AjNCOA4 and AjFerritin in coelomocytes of infected A. japonicus by Western blot.
使用含有 PMSF 和蛋白酶抑制剂的细胞裂解缓冲液（20 mM Tris， 150 mM NaCl， 1 % Triton X-100， pH 7.5）提取总蛋白，在中华芹中浸泡 24 小时后裂解感染的日本曲霉的腔细胞。随后，将抗 Aj NCOA4 的特异性多克隆抗体与蛋白 A/G 磁珠（P2055， Beyotime）在室温下孵育 30 分钟。然后将这些抗体结合的磁珠与腔细胞裂解物在 4 °C 下孵育过夜，同时轻轻摇动。为了去除未结合的蛋白质和杂质，用细胞裂解缓冲液洗涤磁珠数次。将珠子加入 SDS-PAGE 上样缓冲液中并煮沸 10 分钟。最后，利用 AjFerritin 抗体通过 Western blot 研究感染的 Aj 日本曲霉体腔细胞中 AjNCOA4 和 AjFerritin 之间的相互作用。

2.10. Iron assay 2.10. 铁测定

The coelomocytes were cultured in a 12-well plate on glass slides, transfected with siRNA for 24 h, and subsequently stained. The Prussian blue method was utilized to detect the accumulation of Fe³⁺ both inside and outside the cells. Initially, the cells were washed three times with ddH₂O followed by the addition of PFA for 15 min of fixation. Subsequently, Prussian stain A (potassium ferrocyanide solution) and B (acid solution) were mixed 1:1 and added onto cells. Blue stain was visible after 30 min. After rinsing three times with ddH₂O, the cells were stained with nuclear fast red solution for 15 min. Imaging was then performed using a light microscope. The presence of hemosiderin or Fe³⁺ resulted in a blue coloration, while the nucleus and other tissues appeared red. The amount of accumulated Fe³⁺ was quantified based on the color intensity measured using ImageJ software. Additionally, FerroOrange fluorescent probes were employed to detect Fe²⁺ levels in the cells. Following three washes with PBS, the FerroOrange fluorescent probe was added to the cells at a final concentration of 1 μM. The cells were incubated in a dark environment for 30 min and then observed under fluorescence microscopy. FerroOrange exhibits an absorption maximum at 542 nm and a fluorescence maximum at 572 nm. Quantitative analysis of FerroOrange fluorescence intensity was achieved using ImageJ software.
将腔细胞在载玻片上的 12 孔板中培养，用 siRNA 转染 24 小时，然后染色。采用普鲁士蓝方法检测细胞内外 Fe ³⁺ 的积累。最初，用 ddH₂O 洗涤细胞 3 次，然后加入 PFA 固定 15 分钟。随后，将普鲁士染色剂 A（亚铁氰化钾溶液）和 B（酸溶液）以 1：1 的比例混合并加入细胞中。30 分钟后可见蓝色染色。用 ddH₂ O 冲洗 3 次后，用细胞核固红溶液染色 15 分钟。然后使用光学显微镜进行成像。含铁血黄素或 Fe³⁺ 的存在导致蓝色，而细胞核和其他组织呈红色。根据使用 ImageJ 软件测量的颜色强度量化积累的 Fe 3+ 的量。此外，使用 FerroOrange 荧光探针检测细胞中的 Fe ²⁺ 水平。用 PBS 洗涤 3 次后，将 FerroOrange 荧光探针以 1 μM 的终浓度添加到细胞中。将细胞在黑暗环境中孵育 30 分钟，然后在荧光显微镜下观察。FerroOrange 在 542 nm 处表现出最大吸收，在 572 nm 处表现出最大荧光。使用 ImageJ 软件实现 FerroOrange 荧光强度的定量分析。

2.11. Transmission electron microscopy (TEM)
2.11. 透射电子显微镜（TEM）

After undergoing various treatments, the samples were promptly fixed with 2.5 % glutaraldehyde at 4 °C overnight. Following this, a secondary fixation was carried out using 1 % aqueous osmium tetroxide fixative for 2 h. To eliminate residual osmic acid, the samples were washed extensively with PBS. Subsequently, they were dehydrated using a graded series of ethanol and acetone solutions (ranging from 30 % to 100 %) and then embedded in epoxy resin. Ultra-thin sections of the cell samples were obtained and stained with 2 % uranyl acetate and lead citrate. Finally, the ultrastructural images were captured using transmission electron microscopy (H7650, Hitachi, Japan).
经过各种处理后，立即用 2.5 % 戊二醛在 4 °C 下固定过夜。在此之后，使用 1 % 四氧化锇水固定剂进行二次固定 2 小时。为了消除残留的渗透酸，用 PBS 充分洗涤样品。随后，使用一系列分级的乙醇和丙酮溶液（范围从 30 % 到 100 %）对其进行脱水，然后包埋在环氧树脂中。获得细胞样品的超薄切片，并用 2 % 乙酸铀酰和柠檬酸铅染色。最后，使用透射电子显微镜（H7650， Hitachi， Japan）捕获超微结构图像。

2.12. Statistical analysis
2.12. 统计分析

All data were graphed and analyzed utilizing GraphPad Prism. The data are represented as the mean ± SD of three independent experiments. Statistical analyses were performed using Student's t-test or one-way ANOVA. Asterisks indicated significant differences (*, P < 0.05, **, P < 0.01).
所有数据都使用 GraphPad Prism 绘制和分析。数据表示为三个独立实验的平均值 ± SD。使用 Student t 检验或单因素方差分析进行统计分析。星号表示差异显著（*， P < 0.05， **， P < 0.01）。

3. Results 3. 结果

3.1. Identification of AjNCOA4 and phylogenetic analysis
3.1. AjNCOA4 的鉴定和系统发育分析

The full-length cDNA of AjNCOA4, submitted to GenBank with the accession number PP595811, was 2285 bp in length. This cDNA encompassed a 5′-untranslated region (UTR) of 184 bp, a 3′-UTR of 307 bp, and an open reading frame (ORF) of 1794 bp encoding a 597 amino acid residue (Fig. 1A). The predicted molecular weight of AjNCOA4 was 67.74 kDa, with a theoretical pI of 4.47. Analysis using the SMART program identified the presence of an ARA70 domain within AjNCOA4, located between residues 22 and 210 (Fig. 1B). Additionally, a multiple sequence alignment revealed that AjNCOA4 from A. japonicus exhibits varying degrees of sequence homology with other NCOA4 orthologs, including 34.46 % homology with Danio rerio DrNCOA4, 30.70 % with Mytilus galloprovincialis MgNCOA4, 30.29 % with Homo sapiens HsNCOA4, 29.10 % with Aphelenchus avenae AaNCOA4, 28.14 % with Exaiptasia diaphana EdNCOA4, 28.04 % with Mus musculus MmNCOA4, 27.63 % with Gallus gallus GgNCOA4, 26.68 % with Xenopus laevis XlNCOA4, 23.16 % with Strongylocentrotus purpuratus SpNCOA4 (Table 2).
以 PP595811 登录号提交给 GenBank 的 Aj NCOA4 全长 cDNA 长度为 2285 bp。该 cDNA 包括一个 184 bp 的 5′-非翻译区（UTR）、一个 307 bp 的 3′-UTR 和一个 1794 bp 的开放阅读框（ORF），编码一个 597 个氨基酸残基（图 1A）。AjNCOA4 的预测分子量为 67.74 kDa，理论 pI 为 4.47。使用 SMART 程序进行分析发现，AjNCOA4 中存在一个 ARA70 结构域，位于残基 22 和 210 之间（图 1B）。此外，多序列比对显示，来自 A. japonicus 的 AjNCOA4 与其他 NCOA4 直系同源物表现出不同程度的序列同源性，包括与 Danio rerio Dr NCOA4 的同源性为 34.46 %，与 Mytilus galloprovincialis MgNCOA4 的同源性为 30.70%，与智人 Hs NCOA4 的同源性为 30.29%，与 Homo sapiens HsNCOA4 的同源性为 29.10%，与Aphelenchus avenae 机管局NCOA4,28.14 % 与 Exaiptasia diaphana EdNCOA4,28.04 % 与肌肉菌 MMNCOA4,27.63 % 与鸡 GgNCOA4,26.68 % 与非洲爪蟾 XlNCOA4,23.16 % 与紫圆线虫 SpNCOA4（表 2）。

Table 2. The sequence homology of AjNCOA4 with other orthologs.
表 2.AjNCOA4 与其他直系同源物的序列同源性。

Species 物种	Genbank number Genbank 编号	Per. Ident
Danio rerio 丹尼奥·雷里奥	NP_957423.1	34.46 %
Mytilus galloprovincialis	VDI07444.1	30.70 %
Homo sapiens 智人	KAI2555650.1	30.29 %
Aphelenchus avenae	KAH7731057.1	29.10 %
Exaiptasia diaphana	XP_020896187.1	28.14 %
Mus musculus 肌肉麝	AAH85086.1	28.04 %
Gallus gallus 加鲁斯	NP_001006495.1	27.63 %
Xenopus laevis 非洲爪蟾	NP_001089238.1	26.68 %
Strongylocentrotus purpuratus 紫终 Strongylocentrotus purpuratus	XP_030835159.1	23.16 %

A phylogenetic tree analysis was conducted utilizing the amino acid sequences of NCOA4 proteins found in both vertebrate and invertebrate species (Fig. 2). Aside from arthropods, homologues of NCOA4 have been identified in numerous metazoans, extending from cnidarians to mammals. Vertebrates possess two ARA70 family domains, whereas invertebrates harbor just one. Among invertebrates, AjNCOA4 occupies a position between MgNCOA4 and EdNCOA4, and the evolutionary tree of NCOA4 corresponds with the established timeline of metazoan evolution. The conserved ARA70 domain among these homologues suggests functional homology.
利用在脊椎动物和无脊椎动物物种中发现的 NCOA4 蛋白的氨基酸序列进行了系统发育树分析（图 2）。除了节肢动物，NCOA4 的同源物已在许多后生动物中被发现，从刺胞动物延伸到哺乳动物。脊椎动物具有两个 ARA70 家族结构域，而无脊椎动物只有一个。在无脊椎动物中，AjNCOA4 占据介于 MgNCOA4 和 EdNCOA4 之间，NCOA4 的进化树与已建立的后生动物进化时间线相对应。这些同源物中保守的 ARA70 结构域表明功能同源性。

3.2. AjNCOA4 is present and expressed in various tissues of A. japonicus
3.2. AjNCOA4 存在于日本蚜蒲的各种组织中并表达

Using qRT-PCR, we examined the distribution of AjNCOA4 mRNA across multiple tissues, including body wall, muscle, intestine, respiratory tree, and coelomocytes (Fig. 3). Our findings reveal that AjNCOA4 mRNA is ubiquitously expressed in all five tissues, albeit with varying levels. Specifically, AjNCOA4 mRNA expression is relatively low in the body wall, muscle, and respiratory tree. However, expression is higher in the coelomocytes, peaking in the intestines. These observations suggest that AjNCOA4 is widely distributed across the tested tissues.
使用 qRT-PCR，我们检查了 AjNCOA4 mRNA 在多个组织中的分布，包括体壁、肌肉、肠道、呼吸树和体腔细胞（图 3）。我们的研究结果表明，AjNCOA4 mRNA 在所有五个组织中普遍表达，尽管水平不同。具体来说，AjNCOA4 mRNA 在体壁、肌肉和呼吸树中的表达相对较低。然而，在腔细胞中的表达较高，在肠道中达到峰值。这些观察结果表明 AjNCOA4 广泛分布在测试组织中。

3.3. The expression of AjNCOA4 mRNA increased after V. splendidus stimulation
3.3. AjNCOA4 mRNA 的表达在 V. splendidus 刺激后增加

The mRNA levels of AjNCOA4 in the coelomocytes of A. japonicus after challenge with V. splendidus at different time points were determined by qRT-PCR. Significance was compared between the 0 h control group and other time point group (Fig. 4). The mRNA level of AjNCOA4 appeared to be upregulated from 6 h (P < 0.01) during V. splendidus infection. It continued to increase, reached the highest value at 24 h, about 9-fold of the control group (P < 0.01), and began to decrease from 48 h to 72 h, but also was higher than 0 h. The results showed that AjNCOA4 is involved in the immune regulation of the simulated sea cucumber.
通过 qRT-PCR 测定不同时间点中华荟攻击后日本蝇体腔细胞中 AjNCOA4 的 mRNA 水平。比较 0 h 对照组和其他时间点组之间的显着性（图 4）。在 V. splendidus 感染期间，AjNCOA4 的 mRNA 水平似乎从 6 h 开始上调（P < 0.01）。它继续增加，在 24 h 达到最高值，约为对照组的 9 倍（P < 0.01），并从 48 h 开始下降到 72 h，但也高于 0 h。结果表明，AjNCOA4 参与模拟海参的免疫调节。

3.4. Expression of rAjNCOA4 and preparation of polyclonal antibody
3.4. rAjNCOA4 的表达和多克隆抗体的制备

To investigate the role of AjNCOA4 in A. japonicus, the commercially available antibody for AjNCOA4 was unsuitable due to its low homology. Therefore, we prepared a specific antibody against AjNCOA4. Initially, we cloned the ORF sequence of AjNCOA4 into the expression vector pET-28a and expressed it in competent cell BL21 (DE3) E. coli. After induction with IPTG, we successfully obtained the recombinant protein rAjNCOA4 with His tag, exhibiting a molecular weight of approximately 71 kDa (Fig. 5). The protein was then purified using Ni-NTA columns and mixed with an immune adjuvant before immunization in mice. Subsequently, mouse serum samples were collected, and the specificity of the antibody was validated through Western blot. The results demonstrated that our prepared mouse anti-AjNCOA4 antibody could specifically detect the purified recombinant protein, paving the way for its use in subsequent experiments.
为了研究 AjNCOA4 在 A. japonicus 中的作用，市售的 AjNCOA4 抗体由于其低同源性而不合适。因此，我们制备了一种针对 AjNCOA4 的特异性抗体。最初，我们将 AjNCOA4 的 ORF 序列克隆到表达载体 pET-28a 中，并在感受态细胞 BL21 （DE3）大肠杆菌中表达。用 IPTG 诱导后，我们成功获得了带有 His 标签的重组蛋白 rAjNCOA4，分子量约为 71 kDa（图 5）。然后使用 Ni-NTA 柱纯化蛋白质，并在小鼠免疫前与免疫佐剂混合。随后，收集小鼠血清样品，并通过 Western blot 验证抗体的特异性。结果表明，我们制备的小鼠抗 AjNCOA4 抗体可以特异性检测纯化的重组蛋白，为其在后续实验中的应用铺平了道路。

3.5. AjNCOA4 interacted with AjFerritin
3.5. AjNCOA4 与 AjFerritin 相互作用

Intracellular iron is primarily stored in ferritin, and the degradation of ferritin is reliant on ferritinophagy. In order to assess whether AjNCOA4 serves as a ferritinophagy receptor and regulates ferritinophagy in coelomocytes, it is crucial to determine the binding affinity between AjNCOA4 and AjFerritin. In mammals, NCOA4 binds to the FTH subunit. However, ferritin in invertebrates like A. japonicus possesses a single subunit exhibiting comparable properties to mammalian FTH. Notably, AjFerritin exhibits higher homology with HsFTH (63.53 %) than with HsFTL (53.76 %) through multiple sequence alignment (Fig. 6A). Consequently, we employed commercial antibody targeting HsFTH to assess the interaction between AjNCOA4 and AjFerritin in A. japonicus. IF assay revealed the co-localization of AjNCOA4 and AjFerritin in V. splendidus-stimulated coelomocytes (Fig. 6B). Lysates from coelomocytes infected with V. splendidus were immunoprecipitated using AjNCOA4 antibodies. The existence of a 21 kDa immunoreactive band corresponding to AjFerritin in AjNCOA4 immunoprecipitates confirmed the interaction between AjNCOA4 and AjFerritin (Fig. 6C). These findings indicate that AjNCOA4 can bind to AjFerritin in coelomocytes.
细胞内铁主要储存在铁蛋白中，铁蛋白的降解依赖于铁蛋白自噬。为了评估 AjNCOA4 是否作为铁蛋白自噬受体并调节体腔细胞中的铁蛋白自噬，确定 AjNCOA4 和 Aj铁蛋白之间的结合亲和力至关重要。在哺乳动物中，NCOA4 与 FTH 亚基结合。然而，像 A. japonicus 这样的无脊椎动物中的铁蛋白具有单个亚基，表现出与哺乳动物 FTH 相当的特性。值得注意的是，通过多个序列比对，Aj铁蛋白与 HsFTH （63.53%）的同源性高于与 HsFTL （53.76 %）的同源性（图 6A）。因此，我们采用靶向 HsFTH 的商业抗体来评估 AjNCOA4 和 Aj铁蛋白在 A. japonicus 中的相互作用。IF 测定揭示了 AjNCOA4 和 Aj铁蛋白在 V. splendidus 刺激的体腔细胞中的共定位（图 6B）。使用 Aj NCOA4 抗体免疫沉淀来自感染 V. splendidus 的体腔细胞的裂解物。在 Aj NCOA4 免疫沉淀物中存在对应于 Aj 铁蛋白的 21 kDa 免疫反应带，证实了 AjNCOA4 和 Aj铁蛋白之间的相互作用（图 6C）。这些发现表明 AjNCOA4 可以与腔细胞中的 Aj铁蛋白结合。

3.6. Iron homeostasis of coelomocytes was regulated by AjNCOA4
3.6. 体腔细胞的铁稳态受 AjNCOA4 调节

FTH efficiently oxidizes Fe²⁺ into Fe³⁺ and stores it securely within the ferritin core. Conversely, ferritinophagy degrades ferritin molecules releasing free-state Fe²⁺ into the cytoplasm and elevating intracellular iron levels. To investigate AjNCOA4's role in iron homeostasis regulation, we employed RNA interference to knockdown AjNCOA4 expression in coelomocytes, subsequently monitoring alterations in iron content. Successfully knocking down AjNCOA4 expression in coelomocytes via siRNA transfection (Fig. 7A), we observed the accumulation of Fe³⁺ both intracellularly and extracellularly through Prussian blue staining (Fig. 7B). FerroOrange is an orange fluorescent probe specifically designed to detect the unstable bivalent iron ion (Fe²⁺). In the presence of trivalent iron ions (Fe³⁺) or other divalent metal ions other than iron, the fluorescence intensity does not increase. The FerroOrange probe method was used to detect intracellular Fe²⁺ in coelomocytes. It was discovered that the fluorescence intensity of AjNCOA4 knockdown group was weaker than the negative control group (Fig. 7C). It was indicated that knockdown AjNCOA4 would reduce intracellular Fe²⁺ levels in coelomocytes. These findings imply that AjNCOA4 plays a crucial role in maintaining iron homeostasis in coelomocytes of A. japonicus.
FTH 将 Fe²⁺ 有效地氧化成 Fe³⁺，并将其安全地储存在铁蛋白核心内。相反，铁蛋白自噬降解铁蛋白分子，将游离态 Fe²⁺ 释放到细胞质中，并提高细胞内铁水平。为了研究 AjNCOA4 在铁稳态调节中的作用，我们采用 RNA 干扰来敲除腔细胞中 AjNCOA4 的表达，随后监测铁含量的变化。通过 siRNA 转染成功敲低腔细胞中 Aj NCOA4 的表达（图 7A），我们通过普鲁士蓝染色观察到 Fe 3+ 在细胞内和细胞外的积累（图 7B）。FerroOrange 是一种橙色荧光探针，专门设计用于检测不稳定的二价铁离子（Fe²⁺）。在三价铁离子（Fe³⁺）或铁以外的其他二价金属离子存在下，荧光强度不会增加。FerroOrange 探针法检测腔细胞中的细胞内 Fe²⁺。结果发现，AjNCOA4 敲低组的荧光强度弱于阴性对照组（图 7C）。结果表明，敲除 AjNCOA4 会降低腔细胞中细胞内 Fe²⁺ 的水平。这些发现表明 AjNCOA4 在维持 A. japonicus 体腔细胞中的铁稳态中起着至关重要的作用。

3.7. AjNCOA4 promoted ferritinophagy induced by V. splendidus in coelomocytes
3.7. AjNCOA4 促进 V. splendidus 在腔细胞中诱导的铁蛋白自噬

To investigate the function of AjNCOA4 in regulating ferritinophagy, we utilized transmission electron microscopy to observe autophagy in coelomocytes. Our findings revealed the presence of numerous autophagy vesicles in the coelomocytes upon stimulation by V. splendidus. However, upon reducing AjNCOA4 expression via RNA interference, we observed a substantial decrease in the number of autophagy vesicles in these cells (Fig. 8A). Furthermore, we examined the co-localization of the autophagy marker LC3 with AjFerritin in coelomocytes. The result indicated that AjLC3 colocalized with AjFerritin upon stimulation by V. splendidus. However, knocking down AjNCOA4 significantly reduced the co-localization of AjLC3 and AjFerritin (Fig. 8B). These findings suggest that NCOA4 plays a crucial role in promoting ferritinophagy induced by V. splendidus.
为了研究 AjNCOA4 在调节铁蛋白自噬中的作用，我们利用透射电子显微镜观察体腔细胞中的自噬。我们的研究结果显示，在 V. splendidus 刺激后，腔细胞中存在许多自噬囊泡。然而，在通过 RNA 干扰降低 AjNCOA4 表达后，我们观察到这些细胞中自噬囊泡的数量显着减少（图 8A）。此外，我们检查了自噬标志物 LC3 与 Aj铁蛋白在体腔细胞中的共定位。结果表明，AjLC3 在 V. splendidus 刺激下与 Aj铁蛋白共定位。然而，敲低 AjNCOA4 显著降低了 AjLC3 和 Aj铁蛋白的共定位（图 8B）。这些发现表明 NCOA4 在促进 V. splendidus 诱导的铁蛋白自噬中起关键作用。

4. Discussion 4. 讨论

Due to its rich nutritional value, the sea cucumber stands as a crucial marine economic species in Asia, and its aquaculture industry is rapidly developing to meet the huge demand for this product in Southeast Asia. Nevertheless, the rapid expansion of A. japonicus aquaculture has resulted in significant disease challenges. SUS is the most common and harmful disease which is characterized by the symptoms of shaking head, viscera excretion, and large area ulceration of the body wall. Notably, the immune defense mechanism of A. japonicus largely relies on its innate immune system [22]. Consequently, examining the innate immune system of A. japonicus may offer a novel avenue for understanding the defense mechanisms of this species. Essential for nearly all cellular life forms, iron plays a crucial role in various biological processes, thus maintaining iron homeostasis is fundamental for organismal cell growth [23]. Recently, research has revealed that NCOA4 regulates iron homeostasis by binding to ferritin and facilitating its autophagy degradation, a mechanism referred to as ferritinophagy [13]. In this study, we identified AjNCOA4 in the sea cucumber A. japonicus and investigated its role in regulating ferritinophagy induced by V. splendidus, a primary pathogen of SUS.
由于其丰富的营养价值，海参是亚洲重要的海洋经济物种，其水产养殖业正在迅速发展，以满足东南亚对该产品的巨大需求。然而，日本蚜蒿水产养殖的迅速扩大带来了重大的疾病挑战。SUS 是最常见和最有害的疾病，其特征是摇头、内脏排泄和体壁大面积溃疡的症状。值得注意的是，日本蚜蒲的免疫防御机制在很大程度上依赖于其先天免疫系统[22]。因此，检查 A. japonicus 的先天免疫系统可能为了解该物种的防御机制提供一条新的途径。铁对几乎所有细胞生命形式都是必不可少的，在各种生物过程中起着至关重要的作用，因此维持铁稳态是有机体细胞生长的基础 [23]。最近，研究表明，NCOA4 通过与铁蛋白结合并促进其自噬降解来调节铁稳态，这种机制称为铁蛋白自噬 [13]。在这项研究中，我们在海参 A. japonicus 中鉴定了 AjNCOA4，并研究了其在调节 SUS 主要病原体 V. splendidus 诱导的铁蛋白吞噬中的作用。

NCOA4 was initially discovered as a protein that interacts with the androgen receptor (AR), and its overexpression has been documented to stimulate the transcription of AR-regulating genes [24]. This protein forms a multifaceted interaction by facilitating the self-oligomerization of NCOA4 via its N-terminal coiled helical domain, while also binding to FTH1 through an intrinsically disordered region [25,26]. Both of these interactions play a crucial role in the formation of ferritin-NCOA4 particles. In our study, it was discovered that AjNCOA4 encodes a protein consisting of 597 amino acids, with a predicted molecular weight of 67.74 kDa, and possesses an ARA70 domain (Fig. 1). Through phylogenetic analysis, it was discovered that the evolutionary lineage of NCOA4 aligns with the established evolutionary timeline of metazoans (Fig. 2). Specifically, in vertebrates, NCOA4 encompasses two ARA70 family domains: ARA70-I and ARA70-II. Notably, the ARA70-I domain, situated at the N-terminus and overlapping with the helix-helix domain, exhibits evolutionary conservation across metazoans, ranging from cnidarians to mammals. This conservation of the ARA70 domain among homologues suggests a preserved functionality. However, the ARA70-II domain is exclusively found in vertebrates and lacks obvious sequence homology with the ARA70-I domain. Intriguingly, no protein homologous to NCOA4 was identified in arthropods [27], potentially indicating the existence of alternative proteins within this phylum that compensate for this absence. In fact, certain primitive organisms harbor genes that are absent in arthropods but present in mammals, a phenomenon frequently observed in genome comparisons among highly evolved organisms, including fruit flies. For instance, fibrous collagen, which is encoded by genomes of higher organisms like cnidarians and mammals, is absent from insect genomes [28].
NCOA4 最初被发现是一种与雄激素受体（AR）相互作用的蛋白质，其过表达已被证明可以刺激 AR 调节基因的转录 [24]。这种蛋白质通过其 N 末端卷曲螺旋结构域促进 NCOA4 的自寡聚化，同时还通过固有无序区域与 FTH1 结合，从而形成多方面相互作用 [25,26]。这两种相互作用在铁蛋白-NCOA4 颗粒的形成中起着至关重要的作用。在我们的研究中，发现 AjNCOA4 编码一种由 597 个氨基酸组成的蛋白质，预测分子量为 67.74 kDa，并具有一个 ARA70 结构域（图 1）。通过系统发育分析，发现 NCOA4 的进化谱系与已建立的后生动物进化时间线一致（图 2）。具体来说，在脊椎动物中，NCOA4 包含两个 ARA70 家族结构域：ARA70-I 和 ARA70-II。值得注意的是，位于 N 端并与螺旋-螺旋结构域重叠的 ARA70-I 结构域在后生动物（从刺胞动物到哺乳动物）中表现出进化保守性。ARA70 结构域在同源物中的这种保守性表明功能保留。然而，ARA70-II 结构域仅存在于脊椎动物中，与 ARA70-I 结构域缺乏明显的序列同源性。有趣的是，在节肢动物中没有发现与 NCOA4 同源的蛋白质 [27]，这可能表明该门中存在替代蛋白质来补偿这种缺失。事实上，某些原始生物携带的基因在节肢动物中不存在，但存在于哺乳动物中，这种现象在高度进化的生物（包括果蝇）的基因组比较中经常观察到。例如，由刺胞动物和哺乳动物等高等生物的基因组编码的纤维胶原在昆虫基因组中不存在[28]。

In this study, we discovered that AjNCOA4 is abundantly present across all tested tissues of A. japonicus including body wall, muscle, intestine, respiratory tree, and coelomocytes (Fig. 3). In humans, NCOA4 serves as an intracellular protein that is abundantly expressed across multiple organs, encompassing the adrenal glands, heart, kidneys, lungs, intestines, spleen, and skeletal muscle [29]. The maintenance of the aforementioned tissues' respective functions is significantly influenced by the localization and expression patterns of NCOA4. Numerous studies have demonstrated a strong association between the heightened expression of NCOA4 in mouse bronchial epithelial cells and the pathological progression of chronic obstructive pulmonary disease [30]. Furthermore, in degenerative disorders like disc degeneration, an increase in NCOA4 expression is observed in nucleus pulposus cells [31].
在这项研究中，我们发现 AjNCOA4 大量存在于日本曲霉的所有测试组织中，包括体壁、肌肉、肠道、呼吸树和体腔细胞（图 3）。在人类中，NCOA4 是一种细胞内蛋白，在多个器官中大量表达，包括肾上腺、心脏、肾脏、肺、肠道、脾脏和骨骼肌 [29]。上述组织各自功能的维持受到 NCOA4 定位和表达模式的显着影响。大量研究表明，小鼠支气管上皮细胞中 NCOA4 表达升高与慢性阻塞性肺疾病的病理进展之间存在密切关联 [30]。此外，在椎间盘退化等退行性疾病中，观察到髓核细胞中 NCOA4 表达增加 [31]。

Research has revealed that NCOA4-mediated ferritinophagy significantly enhances the availability of free iron during Mycobacterium tuberculosis (Mtb) infection, thereby promoting the bacterium's growth [32]. Mice with defective NCOA4 in bone marrow cells exhibited significantly increased resistance to Mtb infection, indicating that NCOA4 is a key regulator of ferritin's effect on Mtb infection [33]. Additionally, iron overload in cells induces NCOA4-dependent ferritinophagy, leading to uropathogenic Escherichia coli overreplication and host cell death [18]. The virulence factor Etf-3 of Ehrlichia chaffeensis binds directly to the ferritin light chain, inducing ferritinophagy and enhancing intracellular growth [34]. Furthermore, we demonstrated that V. splendidus infection can up-regulate NCOA4 expression in coelomocytes of A. japonicus (Fig. 4). Collectively, these findings suggest that harnessing host ferritinophagy to acquire iron represents a fundamental escape strategy employed by diverse pathogens.
研究表明，NCOA4 介导的铁蛋白自噬显着提高了结核分枝杆菌（Mtb）感染期间游离铁的可用性，从而促进了细菌的生长 [32]。骨髓细胞中 NCOA4 缺陷的小鼠对 Mtb 感染的抵抗力显著增加，表明 NCOA4 是铁蛋白对 Mtb 感染影响的关键调节因子 [33]。此外，细胞中的铁过载会诱导 NCOA4 依赖性铁蛋白自噬，导致尿路致病性大肠杆菌过度复制和宿主细胞死亡 [18]。 Ehrlichia chaffeensis 的毒力因子 Etf-3 直接与铁蛋白轻链结合，诱导铁蛋白自噬并增强细胞内生长 [34]。此外，我们证明 V. splendidus 感染可以上调 A. japonicus 体腔细胞中 NCOA4 的表达（图 4）。总的来说，这些发现表明，利用宿主铁蛋白自噬来获取铁代表了多种病原体采用的基本逃逸策略。

Ferritin plays a vital role in maintaining iron homeostasis across various organisms due to its conserved nature. It comprises 24 subunits of FTH and FTL, capable of storing up to 4500 ions of Fe²⁺. These ions are subsequently oxidized to Fe³⁺ and stored as a mineral core within the cavity. In times of iron deficiency, ferritin particles are transported to lysosomes to release the stored iron atoms [35]. Ferritinophagy is a process facilitated by NCOA4, which functions as the cargo receptor for ferritin. This process encompasses two stages: NCOA4-ferritin complex formation and ferritin degradation. Specifically, the FTH1 subunit binds to the C-terminal domain of NCOA4, forming the NCOA4-ferritin complex. Some studies have demonstrated that human NCOA4 can bind to mouse FTH, but not to mouse FTL, despite their high degree of identity with humans [36]. Notably, most invertebrates, plants, and microorganisms possess only one ferritin subunit. These subunits share characteristics with both the FTH and FTL, including iron oxidase centers and nucleation sites [37]. In A. japonicus, AjFerritin exhibits higher homology with HsFTH. AjNCOA4 has been shown to bind to AjFerritin in coelomocytes stimulated by V. splendidus (Fig. 6). This suggests the important role of AjNCOA4 in regulating iron homeostasis through ferritinophagy.
由于其保守的性质，铁蛋白在维持各种生物体的铁稳态方面起着至关重要的作用。它包含 FTH 和 FTL 的 24 个亚基，能够储存多达 4500 个 Fe²⁺ 离子。这些离子随后被氧化成 Fe³⁺，并作为矿物核心储存在空腔内。在缺铁时，铁蛋白颗粒被转运到溶酶体以释放储存的铁原子 [35]。铁蛋白自噬是 NCOA4 促进的过程，NCOA4 充当铁蛋白的转运受体。这个过程包括两个阶段：NCOA4-铁蛋白复合物形成和铁蛋白降解。具体而言，FTH1 亚基与 NCOA4 的 C 端结构域结合，形成 NCOA4-铁蛋白复合物。一些研究表明，尽管人 NCOA4 与人类高度相同，但它们可以与小鼠 FTH 结合，但不能与小鼠 FTL 结合 [36]。值得注意的是，大多数无脊椎动物、植物和微生物只有一个铁蛋白亚基。这些亚基与 FTH 和 FTL 具有共同特征，包括铁氧化酶中心和成核位点 [37]。在 A. japonicus 中，Aj铁蛋白与 HsFTH 表现出更高的同源性。AjNCOA4 已被证明与 V. splendidus 刺激的体腔细胞中的 Aj铁蛋白结合（图 6）。这表明 AjNCOA4 在通过铁蛋白自噬调节铁稳态中的重要作用。

Iron is an essential element in a variety of biological processes, including oxygen binding and transport, ATP production, and DNA biosynthesis and repair [38]. Therefore, cells must maintain a delicate balance between iron availability and storage. Iron enters ferritin in the form of Fe²⁺ through the ferritin iron pore and is subsequently oxidized to Fe³⁺ by FTH1 within the ferritin cage, resulting in an inert deposition of Fe³⁺ unusable by the cell. To utilize ferritin, iron must be released from ferritin and reduced back to Fe²⁺. The flux of the ferritinophagy pathway is dependent on NCOA4 levels, which are tightly regulated by intracellular iron levels. Under iron-replete conditions, increased binding of NCOA4 to the E3 ubiquitin ligase HERC2 leads to proteasomal degradation of NCOA4. Lower NCOA4 levels inhibit ferritinophagy and enhance ferritin iron storage [15]. We also found that AjNCOA4 can regulate the iron levels in the coelomocytes of A. japonicus (Fig. 7). This suggests that NCOA4 can alter iron homeostasis in sea cucumber.
铁是多种生物过程中的必需元素，包括氧结合和运输、ATP 产生以及 DNA 生物合成和修复 [38]。因此，细胞必须在铁的可用性和储存之间保持微妙的平衡。铁通过铁蛋白铁孔以 Fe²⁺ 的形式进入铁蛋白，随后在铁蛋白笼内被 FTH1 氧化成 Fe³⁺，导致细胞无法使用的 Fe³⁺ 惰性沉积。要利用铁蛋白，必须从铁蛋白中释放铁并还原回 Fe²⁺。铁自噬途径的通量取决于 NCOA4 水平，而 NCOA4 水平受细胞内铁水平的严格调节。在铁充满的条件下，NCOA4 与 E3 泛素连接酶 HERC2 的结合增加导致 NCOA4 的蛋白酶体降解。较低的 NCOA4 水平会抑制铁蛋白自噬并增强铁蛋白铁的储存 [15]。我们还发现 AjNCOA4 可以调节 A. japonicus 体腔细胞中的铁水平（图 7）。这表明 NCOA4 可以改变海参中的铁稳态。

Autophagy, a conserved cellular process, involves the degradation of misfolded proteins, damaged or redundant organelles, intracellular pathogens, and other cytoplasmic components by delivering them to lysosomes [39]. This process recycles the breakdown products of certain substrates into cells, serving as nutrients for energy production or macromolecular biosynthesis. Autophagy is thus often described as a mechanism for maintaining cellular homeostasis and survival [40]. During autophagy, the substrate is transported to the lysosome via double-membrane vesicles known as autophagosomes [40]. This process can be either selective or non-selective. In selective autophagy, specific receptors bind to and target certain substrates, such as mitochondria (mitophagy), pathogens (xenophagy), endoplasmic reticulum (ER-phagy), and protein aggregates (aggrephagy), for degradation [40]. In iron-deficient cells, autophagy plays a crucial role in the lysosomal degradation of ferritin. NCOA4, a cargo receptor, specifically targets ferritin to autophagosomes during selective autophagy. Our study demonstrates that AjNCOA4 mediates V. splendidus-induced ferritinophagy in coelomocytes of A. japonicus (Fig. 8). This finding aligns with studies conducted in mammals, indicating that the function of NCOA4 is conserved across different species. Ferritinophagy serves as a vital mechanism for regulating iron content in the body and is closely linked to neurological diseases, tumors, and infectious diseases. Therefore, elucidating the molecular mechanism underlying ferritinophagy is expected to provide a theoretical framework for the treatment of diseases characterized by abnormal iron homeostasis.
自噬是一种保守的细胞过程，涉及通过将错误折叠的蛋白质、受损或多余的细胞器、细胞内病原体和其他细胞质成分输送到溶酶体来降解它们 [39]。这个过程将某些底物的分解产物回收到细胞中，作为能量产生或大分子生物合成的营养物质。因此，自噬通常被描述为维持细胞稳态和存活的一种机制[40]。在自噬过程中，底物通过称为自噬体的双膜囊泡转运到溶酶体 [40]。此过程可以是选择性的，也可以是非选择性的。在选择性自噬中，特异性受体结合并靶向某些底物，如线粒体（线粒体自噬）、病原体（异种自噬）、内质网（ER自噬）和蛋白质聚集体（聚集自噬）进行降解[40]。在缺铁细胞中，自噬在铁蛋白的溶酶体降解中起着至关重要的作用。NCOA4 是一种转运受体，在选择性自噬过程中特异性地将铁蛋白靶向自噬体。我们的研究表明，AjNCOA4 介导 V. splendidus 诱导的日本曲霉体腔细胞中的铁蛋白自噬（图 8）。这一发现与在哺乳动物中进行的研究一致，表明 NCOA4 的功能在不同物种中是保守的。铁蛋白自噬是调节体内铁含量的重要机制，与神经系统疾病、肿瘤和传染病密切相关。因此，阐明铁蛋白自噬的分子机制有望为治疗以异常铁稳态为特征的疾病提供理论框架。

In conclusion, this study revealed that AjNCOA4 is widely expressed across tissues in A. japonicus and its expression is upregulated in coelomocytes following V. splendidus infection. IF and Co-IP experiments further confirmed the association of AjNCOA4 with AjFerritin in A. japonicus. Utilizing RNAi technology, our findings clarify AjNCOA4's regulatory influence on iron homeostasis in coelomocytes, highlighting its function in ferritinophagy during V. splendidus infection of A. japonicus. By elucidating the crucial function of AjNCOA4 in ferritinophagy and iron homeostasis regulation in A. japonicus, our findings offer promising targets and avenues for the development of novel therapeutic interventions against diseases affecting this species. Additionally, these outcomes contribute to enhancing the understanding of NCOA4's role in other animals, thereby advancing the research in the fields of autophagy and iron metabolism.
总之，本研究揭示了 AjNCOA4 在 A. japonicus 组织中广泛表达，并且在 V. splendidus 感染后的体腔细胞中表达上调。IF 和 Co-IP 实验进一步证实了 AjNCOA4 与 Aj 铁蛋白在日本的相关性。利用 RNAi 技术，我们的研究结果阐明了 AjNCOA4 对腔细胞中铁稳态的调节影响，突出了其在 A. japonicus 的 V. splendidus 感染期间的铁蛋白自噬中的作用。通过阐明 AjNCOA4 在 A. japonicus 铁蛋白自噬和铁稳态调节中的关键功能，我们的研究结果为开发针对影响该物种的疾病的新型治疗干预措施提供了有希望的目标和途径。此外，这些结果有助于增强对 NCOA4 在其他动物中的作用的理解，从而推进自噬和铁代谢领域的研究。

CRediT authorship contribution statement
CRediT 作者贡献声明

Zhiqun Yin: performed the experiments and interpreted the data. Zhimeng Lv: performed the partial experiment. Lei Yang: interpreted the data and wrote the manuscript. Chenghua Li: participated in the experimental design, interpreted the data, contributed new reagents, analytic tools, and revised the manuscript. Fei Teng: participated in the experimental design. Weikang Liang: participated in the experimental design, interpreted the data and performed the partial experiment. All authors read and approved the final version of the manuscript.
尹志群：进行实验并解释数据。Zhimeng Lv： 进行部分实验。杨磊：解读数据并撰写手稿。李成华：参与实验设计，解读数据，贡献新试剂、分析工具，并修改手稿。飞腾：参与实验设计。梁伟康：参与实验设计，解读数据，进行部分实验。所有作者都阅读并批准了手稿的最终版本。

Declaration of competing interest
利益争夺声明

The authors declare no competing financial interest.
作者声明没有竞争性的经济利益。

Acknowledgments 确认

The work was supported by grants from National Natural Science Foundation of China (32325050), the Natural Science Foundation of Zhejiang Province (Z24C190006, Q24C190015, LQ23C190004), the Fujian Aquatic Seed Industry Innovation and Industrialization Project (2021FJSC2Y03), and K. C. Wong Magna Fund in Ningbo University
这项工作得到了中国国家自然科学基金（32325050）、浙江省自然科学基金（Z24C190006、Q24C190015、LQ23C190004）、福建省水产种业创新与产业化项目（2021FJSC2Y03）和 KC 的资助。宁波大学 Wong Magna Fund

Data availability 数据可用性

Data will be made available on request.
数据将应要求提供。

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Outline 大纲

Figures (8) 手办（8）

Tables (2) 桌子（2）

Fish & Shellfish Immunology
鱼类和贝类免疫学

Highlights 突出

Abstract 抽象

Keywords 关键字

1. Introduction 1. 引言