The cDNA of large yellow croaker shp was cloned according to a method described previously (16). SMARTer™ RACE cDNA Amplification Kit (Clontech, USA) was used to clone the 3′- and 5′- end sequence. Then amino acid sequences in SHP were deduced from the full-length cDNA sequence. The molecular weight and isoelectric point were calculated by a Compute pI/Mw tool (http://web.expasy.org/compute_pi/). The multiple sequence alignment was performed using DNAman (Lynnon BioSoft, Canada). The phylogenetic analyses were obtained by the neighbor-joining method and the tree was constructed using the MEGA7 program (https://www.megasoftware.net/).

The mRNA expression of shp was determined in the heart, liver, kidney, brain, adipose tissue, intestine, eye, gill, muscle and spleen of large yellow croakers. The fish with weight of 300-400g were obtained from Xiangshan Harbor Nusery Co., Ltd. (Ningbo, China). Tissues were collected from 6 individuals and distributed into three samples. Samples were rapidly frozen in liquid nitrogen and stored at −80°C.

Large yellow croaker SHP-GFP and lamin B-RFP fusion protein were constructed to determine the subcellular localization. The open reading frame of croaker SHP was introduced into vector pcDNA 3.1-EGFP (17). HEK 293T cells (5 × 10⁵ cells/mL) were transfected with GFP, SHP-GFP and lamin B-RFP expression plasmid. After the cells were cultured for 36 h, they were fixed in 4% paraformaldehyde before observing the localization through a laser confocal microscopy (Leica, Germany).

Macrophage cell lines of the large yellow croaker (LCM) cells (13) were cultured in DMEM/F12 supplemented with 15% fetal bovine serum (FBS) (Biological Industries, Israel), 100 U/mL penicillin and 100 μg/mL streptomycin at 27°C and 5% CO₂. HEK 293T cells were cultured in high-glucose DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C and 5% CO₂.

To investigate the response of SHP to LPS stimulation, LCM cells were seeded into 6-well plate (1 × 10⁶ cells) treated with 0, 25, 50 and 100 μg/mL LPS for 6 h. To explore the role of SHP in inflammatory regulation, LCM cells were injected with recombinant adenovirus encoding croakers SHP (advSHP) for 36 h and then stimulated with LPS (50 μg/mL, Sigma, USA) for 0, 0.25, 0.5, 1, 2 and 4 h. In addition, to study the effect of adenosine monophosphate (AMP)-activated protein kinase (AMPK) on the expression of SHP, AICAR (500 µM, AMPK agonist, MCE, USA) was used to treat LCM cells for 3 h. Cells were pretreated with CC (5 µM, Compound C, AMPK inhibitor, MCE) for 1 h and then stimulated with LPS for 3 h. To investigate the effect of nuclear factor-erythroid 2-related factor 2 (NRF2) on the expression of SHP, cells were pretreated with ML385 (5 µM, NRF2 inhibitor, MCE) for 1 h and then stimulated with LPS (50 μg/mL) or AICAR (500 µM) for 3 h.

The mRNA expression of genes was analyzed by quantitative real-time PCR as described by Du et al. (18). Specific primers were designed to detect the expression of cyclooxygenase 2 (cox2), interleukin 1 beta (il-1β), interleukin 6 (il6) and small heterodimer partner (shp). β-actin and glyceraldehyde-3-phosphate dehydrogenase (gapdh) were used as housekeeping genes. All primer sequences are listed in Table 1 . The levels of gene expression were calculated and normalized via the 2^−ΔΔCT method (19).

Western blot experiments were performed as described by Tan et al. (20). Primary antibodies used in this study were against SHP (OM107591, Omnimabs, USA), FLAG (14793, CST, USA), phosphor-AMPKα (Thr172) (2531, CST, USA), phosphor-ACC (Ser79) (3661, CST, USA), NRF2 (ab62352, Abcam, USA), H3 (4499, CST, USA) and GAPDH (309154, ZSGB-Bio, China). The immunoreactive protein was visualized using an electrochemiluminescence kit (Beyotime, China) and scanned by an Epson Perfection V33 scanner.

The promoter of large yellow croaker SHP was cloned into the reporter plasmid (pGL3-basic vector, Promega, USA) by a ClonExpress II One Step Cloning Kit (Vazyme, China) (17). The expression plasmid of large yellow croaker NRF2 was stored in our laboratory. HEK 293T cells were seeded into 24-well plate (5 × 10⁵ cells) and co-transfected with reporter plasmids (200 ng/well), phRL-CMV plasmid (20ng/well, Promega, USA), and expression plasmids (200-600 ng/well). After 24 h transfection, cells were collected and the luciferase activity was measured using a TransDetect double-luciferase reporter assay kit (TransGen Biotech, China).

Data are presented as means ± SEMs and were analyzed using one-way ANOVA, followed by Tukey’s Test. The software used was SPSS 20.0 (SPSS, USA). The significance of the difference between two groups was determined by Student’s t-tests. A P < 0.05 was considered to be statistically significant.

Full-length cDNA of SHP (GenBank Acc. No. KY745777) comprised 1228 bp with the following features: 86 bp 5′-untranslated region (UTR), 774 bp open reading frame (ORF) and 368 bp 3′-UTR. The ORF encodes a polypeptide of 258 amino acids with a predicted molecular weight of 29.25 KDa and theoretical isoelectric point of 6.31 ( Figure 1 ).

The deduced amino acid sequence of the SHP polypeptide in the large yellow croaker, exhibited the highest (89.9%) identity with Barramundi (Lates calcarifer), followed by Atlantic salmon (Salmo salar) (78.3%) and Zebrafish (Danio rerio) (75.91%) ( Figure 2 ). Phylogenetic analysis clustered large yellow croaker SHP with other teleosts and mammals ( Figure 3 ).

The mRNA expression of shp was determined in different tissues of the large yellow croaker such as heart, liver, head kidney, brain, adipose tissue, intestine, eye, gill, muscle and spleen. The highest expression of shp was observed in liver and intestine. The kidney, Gill and spleen had moderate levels while the expression in the brain was lowest (P < 0.05) ( Figure 4 ).

The subcellular localization analysis indicated that SHP-GFP fusion protein in HEK 293T cells could target the nucleus. In contrast, GFP alone was present in the whole cell ( Figure 5 ).

The protein level and mRNA expression of SHP was detected in macrophages after LPS stimulation. A 50 and 100 μg/ml LPS stimulation significantly induced the protein level of SHP (P < 0.05) ( Figure 6A ). LPS treatment significantly increased the mRNA expression of shp in macrophages (P < 0.05) ( Figure 6B ).

To study the role of SHP in response to LPS stimulation, macrophages were infected with recombinant adenovirus encoding croakers SHP ( Figure 7A ). SHP recombinant adenovirus significantly increased the protein level and mRNA expression of SHP in macrophages (P < 0.05) ( Figures 7B, C ). LPS treatment also significantly increased the mRNA expression of pro-inflammatory genes, such as tnfα, il-1β, il-6 and cox2 (P < 0.05) ( Figure 7D ). Overexpression of SHP significantly decreased mRNA expression of tnfα, il-1β, il-6 and cox2 induced by LPS treatment in macrophages (P < 0.05) ( Figure 7D ). To confirm this effect, we detected the expression of il-1β and il-6 in macrophages after LPS treatment at different time points. Increases in the expression of il-1β and il-6 were sustained up to 2 h, followed by a drop at 4 h ( Figure 7E ). SHP overexpression decreased mRNA expression of il-1β and il-6 at 2 h and 4 h ( Figure 7E ).

We studied how LPS induced the expression of SHP. LPS induced the expression of SHP, and a similar trend was obtained in the phosphorylation level of AMPK. LPS treatment significantly increased the phosphorylation level of AMPK in macrophages (P < 0.05) ( Figure 8A ). The AMPK inhibitor (CC) treatment significantly decreased the phosphorylation level of AMPK and protein level of SHP induced by LPS (P < 0.05) ( Figure 8B ). Similarly, CC treatment decreased LPS-induced mRNA expression of shp (P < 0.05) ( Figure 8C ). AICAR, the AMPK activator, significantly increased the phosphorylation of AMPK and ACC ( Figure 8D ), and increased the protein level and mRNA expression of SHP (P < 0.05) ( Figures 8D, E ).

Overexpression of large yellow croaker NRF2 significantly increased the promoter activity of SHP in HEK293T cells (P < 0.05) ( Figure 9A ). In macrophages, LPS stimulation increased the level of nuclear NRF2 ( Figure 9B ). In addition, the level of nuclear NRF2 was increased in macrophages after treatment with AICAR ( Figure 9C ). To confirm the role of NRF2 in the effect of LPS and AMPK on SHP, macrophages were treated with NRF2 inhibitor (ML385), AICAR or LPS. The ML385 treatment significantly decreased mRNA expression of shp induced by LPS and AICAR (P < 0.05) ( Figures 9D, E ).