WT C57BL/6J mice were obtained from the Laboratory Animal Center of Southern Medical University (Guangzhou, China). Fat-1 transgenic mice, which has been reported to carry the gene that encodes the enzyme that coverts n-6 to n-3 PUFAs endogenously (20), were hybridized with WT C57BL/6J mice, and the fat-1 genotypes of each animal were recognized as we previously described (21). A total of 79 WT C57BL/6J and 39 fat-1 mice (male, 8 weeks old, 20-25 g) were included in this study. Mice included in the present study were housed under a 12 h light/dark cycle condition at a constant temperature (19-23°C) and (55±10%) humidity, fed with commercial diet and had free access to food and water. All animal experiments in this study were approved by the Welfare and Ethical Committee for Experimental Animal Care of Southern Medical University (approval no. L2018234). All mice were euthanized with 5% isoflurane. Mice were sacrificed at 0, 2, 6 and 24 h post-APAP injection. Before being sacrificed, blood (50-100 µl) was obtained from the tail vein. After standing for 4 h, blood was centrifugated at 1,400 × g for 10 min at 4°C, and the supernatant was collected as serum.

APAP (cat. no. sc-203425; Santa Cruz Biotechnology, Inc.) was intraperitoneally injected into 8-week-old male WT or fat-1 mice at 400 mg/kg body weight (overdose) (22,23) or at 600 mg/kg body weight (lethal dose) (24,25) as previously described. To demonstrate the effect of exogenous n-3 PUFAs, WT mice were fed with an n-3 PUFA-enriched diet for 3 weeks before APAP administration. n-3 PUFA-enriched diets were modified to contain 60 g/kg oils from DHA ethyl ester-enriched fish oil (Ocean Nutrition Canada) containing 540 g/kg DHA and 50 g/kg EPA. Commercial mouse food was used for the normal diet (ND) control group.

Antibodies (Abs) against phosphorylated (p)STAT3 (rabbit anti-mouse monoclonal; 1:2,000; cat. no. 9145), STAT3 (rabbit anti-mouse monoclonal; 1:1,000; cat. no. 12640), p-p44/42 MAPK (Erk1/2; rabbit anti-mouse monoclonal; 1:1,000; cat. no. 4376), p44/42 MAPK (Erk1/2) (rabbit anti-mouse monoclonal; 1:1,000; cat. no. 4695) and GAPDH (rabbit anti-mouse monoclonal; 1:1,000; cat. no. 5174) were obtained from Cell Signaling Technology, Inc. Anti-IL-11 polyclonal antibody (rabbit anti-mouse; 1:1,000; cat. no. A1902) for immunohistochemistry and immunoblotting was purchased from ABclonal Biotech Co., Ltd. Anti-Fra-1 Ab (mouse anti-mouse monoclonal; 1:100; cat. no. sc-271657) was obtained from Santa Cruz Biotechnology, Inc. The antibody against Bcl-2 (mouse anti-mouse monoclonal; 1:100; cat. no. YM3041) was from ImmunoWay Biotechnology Company. Anti-Bax (rabbit anti-mouse polyclonal; 1:1,000; cat. no. DB123) anti-body was purchased from DB Biotech, spol. s r.o.

Hepatocytes were isolated as previously described (26). Briefly, livers were perfused with calcium-free salt solution and type IV collagenase through the portal vein in situ and then filtered with polyamide mesh. After centrifugation at 50 × g for 1 min at 4°C, the precipitants were collected as hepatocytes for reverse transcription-quantitative PCR (RT-qPCR) and western blotting assays, or for primary hepatocyte culture. The supernatants were harvested for density gradient centrifugation using discontinuous 30/70% (v/v) Percoll gradients to obtain mononuclear cells for RT-qPCR. For primary mouse hepatocyte (PMH) culture, 1×10⁶ hepatocytes were seeded in 6-well dishes coated with mouse tail collagen (cat. no. A1048301; Gibco; Thermo Fisher Scientific, Inc.) in William's E medium (cat. no. A1217601; Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). After incubation for 4 h, the culture was replaced with serum-free RPMI-1640 medium (cat. no. 11875101; Gibco; Thermo Fisher Scientific, Inc.). In some cases, cells were treated with 10 µM Sc144 (cat. no. S7124; Selleck Chemicals), 50 µM U0126 (cat. no. S1102; Selleck Chemicals) or 10 µM C188-9 (cat. no. S8605; Selleck Chemicals) for 2 h before APAP stimulation at 37°C.

Liver tissues were removed and collected in 1 ml 4% paraformaldehyde for 24 h at room temperature. Sections were cut into 5 µm. Samples were stained with hematoxylin and eosin (H&E) at room temperature for 3 min to observe morphological changes (Nikon Intensilight DS-Ri2; Nikon Corporation). The TUNEL experiment was performed using a commercial kit (cat. no. C1091; Beyotime Institute of Biotechnology) following the manufacturer's protocols. Harris hematoxylin was used at room temperature for 3 min for nuclear staining and sections were mounted with neutral resin. Three fields of view per section were observed (Nikon Intensilight DS-Ri2). For immunohistochemical analysis, specimens were embedded in paraffin and cut into 5-µm thick sections. The sections were deparaffinized in xylene for 15 min at room temperature and rehydrated with a graded series of alcohol, following which a microwave antigen retrieval procedure was performed using 10 mM sodium citrate buffer at 105°C for 10 min. Upon blocking endogenous peroxidase activity, the samples were treated with 5% BSA (cat. no. SRE0096; Sigma-Aldrich; Merck KGaA) at room temperature for 1 h to block non-specific staining. The sections were then incubated with an anti-IL-11 primary antibody (1:50; cat. no. A1902; ABclonal Biotech Co., Ltd.) at 4°C overnight, followed by incubation with the corresponding secondary antibody (HRP-conjugated goat anti-rabbit; 1:200; cat. no. S0001; Affinity Biosciences) at 37°C for 1 h. Peroxidase activity was detected (Nikon Intensilight DS-Ri2) using DAB solution (Beyotime Institute of Biotechnology).

The serum was collected at the indicated time points after APAP injection. Serum ALT activity was measured with a commercial kit (cat. no. C009-2; Nanjing Jiancheng Bioengineering Institute), based on the manufacturer's instructions. Different cytokine levels in the serum and cell culture supernatants were detected using commercial ELISA kits purchased from eBioscience (Thermo Fisher Scientific, Inc.), including IL-6 (cat. no. BMS603-2), IL-11 (cat. no. EMIL11), IL-13 (cat. no. BMS6015) and IL-22 (cat. no. BMS6022).

The Annexin V/PI apoptosis kit was obtained from Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd., cells were stained for 5 min in the dark at room temperature before monitoring. Annexin V⁺/PI⁺ was identified as late apoptosis and Annexin V⁺/PI⁻ was identified as early apoptosis. Cells were stained with JC-1 dye (Nanjing KeyGen Biotech Co., Ltd.) for 30 min at room temperature in the dark to evaluate mitochondrial membrane potential. The cells were analyzed using the FACS LSRFortessa™ flow cytometer (BD Biosciences) with BD FACSDiva™ software (version 8.0.1; BD Biosciences).

Protein samples were obtained from hepatocytes isolated from APAP-treated mice or primary hepatocytes lysed with RIPA buffer (50 mM Tris, 150 mM NaCl and 1% Nonidet P-40, pH 7.4). Protein concentration was measured using a BCA assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc.). Protein samples (20 µg/lane) were separated on 10% SDS-polyacrylamide gels and then transferred onto polyvinylidene fluoride membranes (MilliporeSigma; Merck KGaA). The membranes were blocked with 5% BSA for 1 h at room temperature, and subsequently incubated with the indicated primary antibodies at 4°C overnight. Primary antibodies, including phosphorylated (p)STAT3 (rabbit anti-mouse monoclonal; 1:2,000; cat. no. 9145), STAT3 (rabbit anti-mouse monoclonal; 1:1,000; cat. no. 12640), p-p44/42 MAPK (Erk1/2; rabbit anti-mouse monoclonal; 1:1,000; cat. no. 4376), p44/42 MAPK (Erk1/2) (rabbit anti-mouse monoclonal; 1:1,000; cat. no. 4695) and GAPDH (rabbit anti-mouse monoclonal; 1:1,000; cat. no. 5174) were obtained from Cell Signaling Technology, Inc. Anti-IL-11 polyclonal antibody (rabbit anti-mouse; 1:1,000; cat. no. A1902) was purchased from ABclonal Biotech Co., Ltd. Anti-Fra-1 (mouse anti-mouse monoclonal; 1:100; cat. no. sc-271657) was obtained from Santa Cruz Biotechnology, Inc. The anti-body against Bcl-2 (mouse anti-mouse monoclonal; 1:100; cat. no. YM3041) was from ImmunoWay Biotechnology Company. Anti-Bax (rabbit anti-mouse polyclonal; 1:1,000; cat. no. DB123) antibody was purchased from DB Biotech, spol. s r.o. Subsequently, the membranes were incubated with the corresponding HRP-conjugated secondary antibody (goat anti-rabbit; 1:10,000; cat. no. S0001; Affinity Biosciences) at room temperature for 1 h. The results were visualized with ECL substrate (cat. no. 1705062; Bio-Rad Laboratories, Inc.). Each western blot analysis was performed three times.

Isolated hepatocytes were homogenized in 1 ml TRIzol^® (Thermo Fisher Scientific, Inc.), and total RNA was extracted based on the manufacturer's instruction. Then, 1,000 ng RNA was synthesized into cDNA using TransScript^® Fly First-Stand cDNA Synthesis SuperMix (TransGen Biotech Co., Ltd.) with RNA removal reagent at 50°C for 10 min and 85°C for 5 sec. SYBR Green Real-Time PCR Master Mix (cat. no. A46112; Applied Biosystems; Thermo Fisher Scientific, Inc.) was applied for qPCR on an ABI Prism 7500 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the following thermocycling conditions: Initial denaturation at 94°C for 30 sec, followed by 40 cycles of denaturation at 94°C for 5 sec, and extension at 60°C for 30 sec. The expression levels of the target genes were normalized to GAPDH gene expression and calculated using the 2^−∆∆Cq method (27). The primer sequences used in this experiment are shown in Table I.

All experiments were independently repeated in triplicate. Statistical analysis was performed using SPSS 12.0 (SPSS, Inc.). The data are presented as the mean ± SD. An unpaired Student's t-test was performed between WT and fat-1 mice or ND control and n-3 PUFA-enriched diet mice at each time point. Comparison of the survival curves was analyzed with the Kaplan-Meier method and log-rank test. P<0.05 was considered to indicate a statistically significant difference.

To confirm the role of n-3 PUFAs on APAP-induced liver injury, WT or fat-1 transgenic mice were challenged with a lethal dose of APAP. Animal mortality was markedly increased in fat-1 transgenic mice compared with that of WT mice (Fig. 1A). To further evaluate the effect of n-3 PUFAs on APAP-induced liver injury, WT or fat-1 transgenic mice were intraperitoneally injected with 400 mg/kg APAP, followed by examination of serum ALT activity and liver pathological changes. Serum samples collected from fat-1 transgenic mice showed markedly elevated levels of ALT activity compared with that of serum samples from WT mice following APAP administration (Fig. 1B). Liver H&E staining revealed severe hepatic damage in APAP-treated fat-1 transgenic mice compared with that of the WT controls (Fig. 1C). In addition, TUNEL-positive cells were markedly more abundant in livers from fat-1 transgenic mice than in livers obtained from APAP-treated WT mice (Fig. 1D). Taken together, these results indicated a potential role of endogenous n-3 PUFAs in aggravating the pathogenesis of APAP-induced liver damage.

As hepatic STAT3 signaling is activated by the IL-6 cytokine family and exerts a protective effect against APAP overdose (6), the present study investigated whether the effect of n-3 PUFAs on APAP hepatotoxicity involves STAT3 signaling. The results revealed attenuated protein levels of pSTAT3 in hepatocytes derived from APAP-treated fat-1 transgenic mice compared with those derived from WT controls (Fig. 2A). Similarly, decreased Bcl-2 and enhanced Bax expression were found in hepatocytes obtained from fat-1 mice at both the transcriptional and translational levels compared with those observed in hepatocytes from WT controls (Fig. 2B and C). Considering that the Bcl family plays an essential role in mitochondrial homeostasis (7,8), JC-1 dye was used to detect the mitochondrial membrane potential by flow cytometry. The results showed an increased JC-1 monomer ratio in hepatocytes from fat-1 transgenic mice compared with that found in hepatocytes from WT mice upon APAP injection, which indicated more severe mitochondrial injury in fat-1 transgenic mice (Fig. 2D). To further validate these results, primary hepatocytes from WT or fat-1 mice were isolated and treated with APAP. As expected, attenuated phosphorylation of STAT3 was observed in the hepatocytes obtained from fat-1 mice (Fig. 2E). Furthermore, APAP treatment induced a higher apoptosis ratio in hepatocytes from fat-1 transgenic mice than that observed in hepatocytes from the WT controls. Of note, pretreatment with SC144, a gp130 inhibitor, abrogated the difference in cell apoptosis between hepatocytes from WT and fat-1 mice during APAP exposure (Fig. 2F). Consistently, the protein levels of Bcl-2 and Bax were comparable between APAP-treated WT and fat-1 mice in the presence of SC144 pretreatment (Fig. 2G). Similarly, C188-9, a STAT3-specific inhibitor, also abrogated the effect of n-3 PUFAs on Bcl-2 and Bax expression (Fig. S1). Collectively, these data suggested that STAT3 inactivation was responsible for the effect of n-3 PUFAs on APAP-induced liver injury.

Several STAT3-activating cytokines, including IL-6, IL-11, IL-13 and IL-22, are able to directly target hepatocytes (6,7). Therefore, the present study first assessed the levels of cytokines in the serum of APAP-treated mice. Compared with those of WT mice, APAP-challenged fat-1 transgenic mice showed significantly lower levels of IL-6 and IL-11, but not of IL-13 or IL-22 (Fig. 3A). Next, the mRNA expression of cytokines in hepatocytes and mononuclear cells was evaluated. The results showed that the levels of IL-11 and IL-6 were lower in hepatic mononuclear cells from APAP-treated mice compared with those observed in their WT counterparts (Fig. 3B). Notably, hepatocytes from APAP-treated fat-1 transgenic mice produced less IL-11 than those from WT controls (Fig. 3C), while there was no significant difference in the mRNA level of IL-6 (Fig. 3C). Similarly, immunohistochemical staining showed that the hepatic level of IL-11 was lower in fat-1 transgenic mice than in WT mice upon APAP administration (Fig. 3D). In addition, decreased IL-11 expression was found in hepatocytes obtained from fat-1 mice compared with that found in hepatocytes from WT mice (Fig. 3E). Furthermore, ELISA showed decreased IL-11 production in primary hepatocytes from fat-1 transgenic mice compared with that observed in their WT counterparts (Fig. 3F). Taken together, these results suggested that endogenous n-3 PUFAs limited the expression of IL-11 in hepatocytes during APAP-induced liver damage.

It has been reported that Fra-1, which is recruited to the IL-11 promoter and enhances IL-11 gene transcription, is essential for the production of IL-11 (3). The present results revealed attenuated expression of Fra-1 at the mRNA and protein level in hepatocytes isolated from APAP-treated fat-1 transgenic mice compared with that observed in WT controls (Fig. 4A and B). Considering that Fra-1 expression is mainly dependent on ERK1/2 activity, ERK1/2 activation in hepatocytes from APAP-injected mice was assessed. The results showed that APAP induced a lower level of phosphorylation of ERK1/2 in hepatocytes from APAP-treated fat-1 transgenic mice than that observed in hepatocytes derived from their WT counterparts (Fig. 4C). To identify whether ERK1/2 signaling is involved in the effect of n-3 PUFAs on APAP hepatotoxicity, hepatocytes from WT and fat-1 transgenic mice were treated with U0126, a widely used ERK1/2 inhibitor, to block ERK1/2 activation prior to incubation with APAP. After ERK1/2 inhibition, the level of cell apoptosis was comparable between WT and fat-1 transgenic hepatocytes with U0126 pretreatment (Fig. 4D). Notably, the production of IL-11 (Fig. 4E) and the expression of Fra-1 (Fig. 4F) were similar between hepatocytes from WT mice and those from fat-1 transgenic mice upon APAP stimulation when ERK1/2 signaling was blocked. Taken together, these data suggested that n-3 PUFAs inhibited ERK1/2 phosphorylation following APAP stimulation, which resulted in limited expression of Fra-1 and consequently reduced IL-11 production.

To further demonstrate the effect of n-3 PUFAs on IL-11 production in APAP-induced liver damage, WT mice were fed daily with a n-3 PUFA-enriched diet for 3 weeks prior to APAP administration. As expected, it was found that dietary n-3 PUFAs efficiently enhanced the severity of APAP-induced liver damage, as demonstrated by hepatic pathology (Fig. 5A) and serum ALT levels (Fig. 5B). In addition, a markedly reduced IL-11 level was determined in the serum (Fig. 5C) and liver tissue (Fig. 5D) of mice with a n-3 PUFA-enriched diet compared with that of mice in the ND group. Furthermore, exogenous n-3 PUFAs suppressed ERK1/2 phosphorylation and Fra-1 expression in hepatocytes stimulated with APAP, as evaluated by immunoblotting (Fig. 5E). Notably, exogenous n-3 PUFAs reduced STAT3 phosphorylation and Bcl-2 expression while enhancing Bax expression during the APAP challenge (Fig. 5F). Collectively, these findings indicated that exogenous n-3 PUFAs also exacerbated APAP-induced liver damage and share the same underlying mechanism as endogenous n-3 PUFAs.