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Generation of il-6^−/− zebrafish via CRISPR/Cas9 technology. (A) Diagram of the IL-6 single-guide RNA (sgRNA). Boxes depict exons while lines depict introns. Protospacer adjacent motif (PAM) and target sequences are indicated in blue and red, respectively. (B) Sequencing results of wild-type (WT) and il-6 knockout homozygous (il-6^{−/−−/−}) zebrafish. The red frame indicates base deletion. According to the DNA sequencing results, il-6^−/− zebrafish with 7 bp deletion mutation were obtained. (C) Protein domains of the il-6 gene in WT and il-6^−/− zebrafish. The TMD domain is shown in blue, the IL6 domain in green, and the mutated region of the protein in yellow. il-6^−/− zebrafish showed a frameshift mutation in IL-6, resulting in premature termination of translation compared to wild-type zebrafish.

Liver injury in WT and il-6^−/− zebrafish post A. hydrophila infection. (A) Pathological changes in WT and il-6^−/− zebrafish liver tissue after A. hydrophila infection were indicated by Hematoxylin and Eosin (HE) staining. Samples were collected at 12 h after A. hydrophila infection. The black arrow indicates infiltration of red blood cells. Scale bar = 50 μm. il-6^−/− zebrafish had milder liver injury compared to WT zebrafish after A. hydrophila infection. (B) Liver alanine transaminase (ALT) and aspartate transaminase (AST) levels. Samples used for enzyme activity (biological replicates = 5, experimental repetitions = 3) were collected 12 h after A. hydrophila infection. Data were subjected to the Student’s t-test to identify the significance. The levels of AST and ALT in the liver of il-6^−/− zebrafish were significantly lower than those of WT zebrafish. ** p < 0.01. WT-AH: WT zebrafish treated with A. hydrophila; KO-AH: il-6^−/− zebrafish treated with A. hydrophila.

Analysis and qPCR validation of liver transcriptome data from the WT and il-6^−/− zebrafish after A. hydrophila infection. (A) Principal component analysis (PCA) on the liver transcriptome data of WT and il-6^−/− zebrafish after A. hydrophila infection. Volcano plots (B) and heatmap clustering (C) of differentially expressed genes (DEGs). The points of the blue represent down-regulated genes, the points of the red represent up-regulated genes, and the gray points represent genes without significant change. The results showed that the transcriptome of the KO-AH group was significantly different from that of the WT-AH group. (D) Validation of transcriptome data using qPCR. Ten genes were randomly selected. The relative expression levels were identified by qPCR (blue column) and plotted against the expression levels from the transcriptome data (red column). The qPCR results demonstrated strong concordance with the transcriptome data, confirming the reliability of the transcriptome data. apoeb: apolipoprotein Eb; dazap2: azoospermia-associated protein 2; aldh1l1: aldehyde dehydrogenase 1L1; abcc2: ATP-binding cassette-2; ctnmb2: catenin, beta 2; aclya: ATP citrate lyase a; cyp51: sterol 14α-demethylase; dpm3: dolichol-phosphate-mannose-3; lrpap1: low-density lipoprotein receptor-related protein-associated protein 1; tmco1: transmembrane and coiled-coil domains 1. WT-AH: WT zebrafish treated with A. hydrophila; KO-AH: il-6^−/− zebrafish treated with A. hydrophila.

GO and KEGG pathway enrichment analysis of DEGs. (A) GO annotation analysis. Red: biological process; green: cellular component; blue: molecular function. The vertical axis represents the name of the GO term, the horizontal axis represents the gene ratio, the size of the dots indicates the number of differentially expressed genes in the term, and the color of the dots corresponds to different q-value ranges. (B) Top 20 pathways enriched in KEGG of DEGs. The vertical axis represents the name of the pathway, the horizontal axis represents the number of differentially expressed genes in the pathway, and the color corresponds to different q-value ranges.

Effect of il-6 mutation on oxidative stress triggered by A. hydrophila infection. (A) MDA and (B) SOD levels in liver tissues. Samples used for enzyme activity (biological replicates = 5, experimental repetitions = 3) were collected 12 h after A. hydrophila infection. Data were subjected to the Student’s t-test to identify the significance. ** p < 0.01. (C) Representative staining of ROS in liver sections. Samples were collected at 12 h after A. hydrophila infection. Frozen tissue sections were used for immunofluorescence detection of ROS. Red fluorescence corresponds to the presence of ROS, while blue fluorescence represents the cell nucleus. Scale bar = 50 μm. WT-AH: WT zebrafish treated with A. hydrophila; KO-AH: il-6^−/− zebrafish treated with A. hydrophila.

Expression and purification of recombinant zebrafish IL-6 (rDrIL-6) protein. (A) Analysis of rDrIL-6 protein by SDS-PAGE and WB. The utilized expression vector was pET-32a. The expression of the recombinant zebrafish IL-6 (rDrIL-6) protein was induced at 37 °C for 4 h with different concentrations of IPTG (1.0 and 1.5 mM). KDa: kilo Dalton; M: marker; 1: control group; 2: 1.0 mmol/L IPTG-induced group; 3: 1.5 mmol/L IPTG-induced group; (B) 4: assessment of purified rDrIL-6 protein by SDS-PAGE; (C) 5: confirmation of purified rDrIL-6 protein by WB. Anti-His antibody was used as a primary antibody.

Effect of rDrIL-6 protein on liver injury induced by A. hydrophila infection in il-6^−/− zebrafish. (A) The levels of ALT and AST in the liver. Samples used for enzyme activity (biological replicates = 5, experimental repetitions = 3) were collected 12 h after infection. The experimental data was subjected to the multifactor analysis of variance (ANOVA) to identify the significance. Values with the same letter superscripts represent no significant difference, while those with different letter superscripts represent significant differences (p < 0.05). (B) HE staining of liver tissues with different treatments. Samples were collected 12 h after A. hydrophila infection. Scale bar = 50 μm. WT-AH: WT zebrafish treated with A. hydrophila; rDrIL-6: il-6^−/− zebrafish treated with a mixture of A. hydrophila and rDrIL-6 protein; TrxA: il-6^−/− zebrafish treated with a mixture of A. hydrophila and control protein TrxA; KO-AH: il-6^−/− zebrafish treated with A. hydrophila.

Effect of rDrIL-6 protein on liver oxidative stress in il-6^−/− zebrafish post A. hydrophila infection. (A) MDA and SOD levels in liver tissues after A. hydrophila infection. Samples used for enzyme activity (biological replicates = 5, experimental repetitions = 3) were collected 12 h after infection. The experimental data were subjected to the Student’s t-test to identify the significance. Compared with the KO-AH group, the rDrIL-6 group showed significantly higher levels of MDA and lower SOD activities. * p < 0.05; ** p < 0.01. (B) Representative staining of ROS in liver sections. Frozen tissue sections were used for immunofluorescence detection of ROS. Red fluorescence corresponds to the presence of ROS, while blue fluorescence represents the cell nucleus. Scale bar = 50 μm. rDrIL-6: il-6^−/− zebrafish treated with a mixture of A. hydrophila and rDrIL-6 protein; KO-AH: il-6^−/− zebrafish treated with A. hydrophila.