A total of 20 healthy men aged 24–66 (37.2±12.0) ingested APAP (500 mg) after fasting for 24 h; as a control a further 20 healthy male volunteers, aged 18–63 (37.8±10.7), who had fasted for 24 but not ingested APAP were enrolled in this study. 5 ml of blood was acquired from the peripheral vein of each patient and the blood samples were then centrifuged at 2,500 × g at 4°C for 5 min to obtain serum. None of the subjects had suffered from viral hepatitis or other autoimmune diseases, and there was no statistically significant difference in ages between the two groups.

CXCR3^−/− mice used in the present study were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). A total of 100 8 week-old male wild type (WT) mice and 50 CXCR3^−/− mice weighing between 18–22 g, were used for experiments, with 6–8 mice in each group. All mice were housed individually in a specific pathogen-free level animal experimental center with free access to water and food.

The 8 week-old male WT and CXCR3^−/− mice were intraperitoneally injected with 250 mg/kg APAP (5). Immediately after this, the experimental group mice were further injected with 100 µg (in 100 µl volume) CXCL9 neutralizing antibody (cat no 50155-R277; Sino Biological Inc., Beijing, China) or 5 µg (in 100 µl volume) recombinant CXCL9 (cat no 50155-MNAE; Sino Biological Inc.). Mice in the control group were injected with an equivalent dose of control IgG or saline. A total of 0.1 ml blood was collected from the tail vein of each mouse before the mice were sacrificed using 5% isoflurane followed by cervical dislocation. Mice were sacrificed and liver tissues were harvested after 8 (n=3) or 24 (n=3) h. Blood samples were centrifuged at 4,500 × g at 4°C for 5 min to obtain serum.

Serum samples from APAP-treated WT and CXCR3^−/− mice were collected for the analysis of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Biochemical analyzer (model AU480; Beckman Coulter, Inc.) was used for ALT and AST colorimetric detection. Caspase-3 expression in liver tissues was determined using a caspase-3 detection kit (Beyotime Institute of Biotechnology; cat. no. C1168S) according to the instructions of the manufacturer.

Mice were sacrificed, then livers were dissected. After overnight incubation in 4% paraformaldehyde, livers were dehydrated and embedded in paraffin. Sections of 5 µm thickness were cut and mounted on poly-lysine-coated slides, and hepatocyte apoptosis was detected using a TUNEL assay (Beyotime Institute of Biotechnology) according to the instructions of the manufacturer.

Mice were sacrificed and their livers were dissected. After overnight incubation at room temperature in 4% paraformaldehyde, livers were dehydrated and embedded in paraffin. Sections of 5 µm thickness were cut and mounted on poly-lysine-coated slides (10 slides per tissue). After H&E staining (hematoxylin, 5 min at room temperature and eosin, 3 min at 20°C) sections were observed (5 views per slide) with a light microscope (magnification ×400).

Total RNA was extracted from cells using the TRIzol^® method (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) then quantified. cDNA was synthesized using the Prime Script™ RT-PCR Kit (Takara Bio, Inc., Otsu, Japan) according to the instructions of the manufacturer. A total of 2 µl cDNA was used for each reaction, amplified by real-time PCR using SYBR^® Premix Ex Taq™ II (Takara Bio, Inc.). Relative mRNA expression was analyzed by qPCR using the IQ™ 5 System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Primers were synthesized by Shanghai ShengGong Co., Ltd. (Shanghai, China). Primer sequences were as follows: CXCL9, forward 5′-CCTAGTGATAAGGAATGCACGATG-3′ and reverse 5′-CTAGGCAGGTTTGATCTCCGTTC-3′; and GAPDH, forward 5′-TGAAGGTCGGAGTCAACGGATT-3′ and reverse 5′-CCTGGAAGATGGTGATGGGATT-3′. The 2^−∆∆Cq method was used to calculate the relative mRNA expression (17). The thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 30 sec, 95°C for 5 sec and 60°C for 31 sec, before a final extension at 94°C for 15 sec. Each experiment was repeated in triplicate.

Cells were lysed with RIPA buffer (Beyotime Institute of Biotechnology) containing protease inhibitors. Protein concentration was quantified using bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Protein samples (10 µg) were separated on 12% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes (Roche, Basel, Switzerland). Membranes were incubated in 5% non-fat milk at 25°C for 1 h to block non-specific binding. Membranes were then membranes incubated overnight at 4°C with primary antibodies targeting phosphorylated (p-) Akt (cat. no. ab81283; 1:500; Abcam, Cambridge, MA, USA), total Akt (cat. no. ab18785; 1:500; Abcam), and β-actin (cat. no. ab8226; 1:500; Abcam). Membranes were washed three times with PBS, then samples were incubated with anti-rabbit or anti-mouse secondary antibodies purchased from Beyotime Institute of Biotechnology (cat. no. A0208/A0216; 1:1,000). Bands were visualized using enhanced chemiluminescence substrate (Thermo Fisher Scientific Inc.). Quantity One (version 4.0; Bio-Rad Laboratories, Inc.) was used for densitometric analysis.

Human serum samples were collected and CXCL9 levels were determined, in accordance with ELISA kit instructions (cat. no. ab119588; Abcam). The absorbance of each sample was detected at wavelengths 562 and 450 nm.

Isolation and culture of primary hepatocytes were performed as previously described (18). In brief, WT and CXCR3^−/− mice were sacrificed for access to the liver. Liver tissues were digested with digestive buffer containing collagenase, ground, and then filtered for preparation of single cell suspension. After gradient centrifugation and lysis with red blood cell lysis buffer, mouse primary hepatocytes were obtained. Cells were cultured in William's E medium (Gibco; Thermo Fisher Scientific, Inc.) for 14 h, serum-starved for 3 h, and then treated with 100 ng/ml CXCL9. Protein samples were collected at 5 min, 20 min, 1, 4 or 8 h post-treatment.

Graphpad Prism (v6.0) software (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. Data are presented as the mean ± the standard deviation. Differences amongst groups were compared using one-way analysis of variance, followed by Bonferroni correction. P<0.05 was considered to indicate significant difference.

Serum levels of CXCL9 were determined in 20 male patients who took APAP tablets in a fasting state and in 20 fasted male controls. The results demonstrated that serum levels of CXCL9 were significantly higher in patients who took APAP compared with the control group (Fig. 1A).

For the animal experiments, male WT mice were randomly assigned into one of two groups, receiving either intraperitoneal injection of APAP or PBS. The mRNA levels of CXCL9 in liver tissues were detected at 1, 3, 6 and 12 h following injection. Compared with mice injected with PBS, those injected with APAP presented significantly increased mRNA levels of CXCL9, even up to 12 h post-injection (Fig. 1B).

Following intraperitoneal injection of APAP, WT mice received intraperitoneal injection of anti-CXCL9 neutralizing antibody or control immunoglobulin (IgG). Serum and liver samples were collected after 8 and 24 h. Serum levels of ALT and AST were lower in mice injected with anti-CXCL9 neutralizing antibody and CXCR3^−/− mice, compared with the control group of WT mice that received APAP and IgG only (Fig. 2A and B). There were no significant differences in ALT and AST levels between mice injected with anti-CXCL9 neutralizing antibody and CXCR3^−/− mice. (Fig. 2A and B). HE staining revealed that neutralization of CXCL9 attenuated APAP-induced liver injury and knockout of CXCR3 also alleviated APAP-induced liver injury (Fig. 2C). TUNEL assay indicated that hepatocyte apoptosis was alleviated in liver tissues from the anti-CXCL9 neutralizing antibody group and the CXCR3^−/− group (Fig. 2D). These results suggested that neutralization of CXCL9 or knockout of CXCR3 attenuated APAP-induced liver injury.

To further explore the specific mechanism of CXCL9 in APAP-induced liver injury, primary hepatocytes were isolated from WT mice for subsequent experiments. Primary hepatocytes were treated with or without recombinant CXCL9 for different time periods. Activities of caspase-3 and caspase-8 in hepatocytes were significantly elevated following CXCL9 treatment for 4 and 8 h (Fig. 3A and B). The AKT pathway was activated within the first 5 min of CXCL9 treatment in a time-dependent manner, and the activation lasted for up to 8 h (Fig. 3C and D).

Studies have demonstrated that CXCL9 is involved in tumor metastasis and the inflammatory response via binding to the CXCR3 receptor. Hence, it was hypothesized that CXCL9 regulates hepatocyte apoptosis through CXCR3 in APAP-induced liver injury. Primary hepatocytes from CXCR3^−/− mice were treated with recombinant CXCL9 or vehicle control. Activities of caspase-3 and caspase-8 were measured by fluorescence spectrophotometry. There was no significant difference in expression of caspase-3 and caspase-8 after CXCL9 stimulation for 4 and 8 h (Fig. 4A and B). In addition, there was no significant difference in p-AKT (S437) expression for up to 8 h following CXCL9 treatment, compared with control untreated CXCR3^−/− hepatocytes (Fig. 4C and D). The present data demonstrated that CXCL9 exerted its function in APAP-induced liver injury via binding to CXCR3.

Mouse experiments were further performed to confirm the roles of CXCL9 and CXCR3 in APAP-induced hepatocyte apoptosis in vivo. Intraperitoneal injections of CXCL9 were administered to both WT and CXCR3^−/− mice. At 24 h following CXCL9 administration, the caspase-3 activity in the liver tissues of WT mice was significantly higher compared with CXCR3^−/− mice (Fig. 5A). In addition, serum levels of ALT and AST were also significantly higher in WT mice compared with CXCR3^−/− mice, following 24 h of CXCL9 administration (Fig. 5B and C).