核因子白细胞介素 3（NFIL3）参与调控凡纳滨对虾在氨氮胁迫下 NF-κB 介导的炎症和抗氧化系统。
Nuclear factor interleukin 3 (NFIL3) participates in regulation of the NF-κB-mediated inflammation and antioxidant system in Litopenaeus vannamei under ammonia-N stress.
机构信息
College of Fisheries, Guangdong Ocean University, Zhanjiang, 524088, China; Guangdong Provincial Engineering Laboratory for Mariculture Organism Breeding, Guangdong Ocean University, Zhanjiang, 524088, China.
College of Fisheries, Guangdong Ocean University, Zhanjiang, 524088, China; Guangdong Provincial Engineering Laboratory for Mariculture Organism Breeding, Guangdong Ocean University, Zhanjiang, 524088, China.
出版信息
Fish Shellfish Immunol. 2022 Dec;131:1192-1205. doi: 10.1016/j.fsi.2022.11.028. Epub 2022 Nov 17.
Nuclear factor interleukin 3 (NFIL3) is a critical upstream regulator of the NF-κB pathway. Nevertheless, the detailed molecular mechanism of NFIL3 and its function in shrimp have not been well characterized. In the present study, NFIL3 was identified and characterized from Litopenaeus vannamei. Molecular feature analysis revealed that the open reading frame (ORF) of LvNFIL3 was 2963 bp, which codes for a polypeptide of 516 amino acids with a conserved basic region leucine zipper (bZIP) domain. Sequence alignments and phylogenetic tree analysis showed that the amino acid sequence of LvNFIL3 shared 18.82%-98.07% identity with that of NFIL3 in other species, and was closely related to Penaeus monodon NFIL3. A core promoter in the 5' flanking region of LvNFIL3 was essential for regulation of transcription. LvNFIL3 mRNA was highly expressed in gills and hepatopancreas. Subcellular localization of the protein was observed almost exclusively in the nucleus. Amplification of mRNA by RT-qPCR showed that LvNFIL3 was induced in shrimp gills, hepatopancreas, and muscle after ammonia-N stress. Moreover, silencing of LvNFIL3 increased the mortality of shrimp exposed to ammonia-N. Furthermore, dual-luciferase reporter assay data suggested that LvNFIL3 was capable of activating the NF-κB pathway. Conversely, knockdown of LvNFIL3 decreased NF-κB homolog (Dorsal and Relish) and IkB homolog (Cactus) expression, as well as expression of anti-inflammatory cytokine (IL-16) and five antioxidant-related genes (HO-1, Mn-SOD, CAT, GPx, and GST), whereas NF-κB repressing factor (NKRF) and inflammation-related genes (TNFα and Spz) were upregulated. More importantly, LvNFIL3 knockdown exacerbated the pathology in hepatopancreas exposed to ammonia-N, and the total antioxidant capacity (T-AOC) and superoxide dismutase (T-SOD) were significantly decreased, resulting in a significant increased lipid peroxidation and protein carbonization. Taken together, these data suggest that LvNFIL3 was involved in ammonia-N tolerance in L. vannamei by regulating the inflammation and antioxidant system through the NF-κB pathway.
核因子白细胞介素 3（NFIL3）是 NF-κB 途径的关键上游调节剂。然而，NFIL3 的详细分子机制及其在虾中的功能尚未得到很好的表征。在本研究中，从凡纳滨对虾中鉴定并表征了 NFIL3。分子特征分析表明，LvNFIL3 的开放阅读框（ORF）为 2963bp，编码一个 516 个氨基酸的多肽，具有保守的碱性亮氨酸拉链（bZIP）结构域。序列比对和系统发育树分析表明，LvNFIL3 的氨基酸序列与其他物种的 NFIL3 具有 18.82%-98.07%的同一性，与斑节对虾 NFIL3 密切相关。LvNFIL3 5'侧翼区的核心启动子对于转录调控至关重要。LvNFIL3 mRNA 在鳃和肝胰腺中高度表达。蛋白质的亚细胞定位几乎完全在核内。RT-qPCR 扩增显示，氨氮胁迫后虾的鳃、肝胰腺和肌肉中 LvNFIL3 mRNA 被诱导。此外，LvNFIL3 沉默会增加虾对氨氮的死亡率。此外，双荧光素酶报告基因检测数据表明，LvNFIL3 能够激活 NF-κB 途径。相反，LvNFIL3 的敲低降低了 NF-κB 同源物（Dorsal 和 Relish）和 IkB 同源物（Cactus）的表达，以及抗炎细胞因子（IL-16）和五个抗氧化相关基因（HO-1、Mn-SOD、CAT、GPx 和 GST）的表达，而 NF-κB 抑制因子（NKRF）和炎症相关基因（TNFα 和 Spz）则上调。更重要的是，LvNFIL3 的敲低加剧了氨氮暴露的肝胰腺病理，总抗氧化能力（T-AOC）和超氧化物歧化酶（T-SOD）显著降低，导致脂质过氧化和蛋白质碳化显著增加。综上所述，这些数据表明，LvNFIL3 通过 NF-κB 途径调节炎症和抗氧化系统参与凡纳滨对虾对氨氮的耐受。
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