Wild-type (WT) and CB2^−/− male mice on a C57BL/6 background were used at ages ranging from 8 to 10 weeks. The WT rodents were purchased from the animal centre of Guizhou Medical University (approval number SYXK (Guizhou) 2018–0001, Guiyang, China). The CB2^−/− mice (B6.129P2-Cnr2tm1Dgen 005786) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Animals were housed in temperature- and humidity-controlled rooms on a 12-h light–day cycle and provided with unrestricted access to food and water. All animal experiments were approved by the Guizhou Medical University Animal Care Welfare Committee.

ConA was dissolved in a normal saline solution at a concentration of 2 mg/mL. Statistics require at least six available data for each group to be meaningful. Male WT mice were randomly divided into one of five groups: group I (saline only) included 10 mice that were injected with saline via the tail vein; group II (ConA-treated) included 10 mice that were injected with ConA (20 mg/kg) via the tail vein; group III (1 h after treatment with AM1241, 3 mg/kg) included 10 mice that were injected with AM1241 (dissolved in 1% DMSO, 1 mg/mL), which was administered via intraperitoneal injection 1 h before the injection with ConA via the tail vein; group IV (1 h after treatment with AM1241, 12 mg/kg) included 10 mice that were injected with AM1241 (dissolved in 1% DMSO, 1 mg/mL), which was administered via intraperitoneal injection 1 h before the injection with ConA via the tail vein; and group V (prior to AM1241 treatment) included 10 mice that were injected with AM630 (dissolved in 1% DMSO, 1 mg/mL) via intraperitoneal injection 1 h before they were injected with AM1241 (12 mg/kg) and ConA (20 mg/kg) via the tail vein. Male CB2^−/− mice were randomly divided into two groups: group A (saline only) included eight mice that were injected with saline via the tail vein and group B (ConA treatment) included eight mice that were administered ConA (20 mg/kg) via the tail vein. All mice were killed 9 hours after the injection of ConA. Blood was collected for biochemical detection. Some liver samples were harvested for histology while others were frozen and stored at −80°C for further analysis.

Serum was separated by centrifugation at 3500 r/min at 4°C for 10 min. To assess the level of hepatocellular injury after ConA treatment, serum ALT and AST levels were measured using an automated chemistry analyser.

Mouse liver tissue was collected and stored in 4% paraformaldehyde for at least 24 h. Paraffin sections were stained with haematoxylin and eosin (H&E) to visualize the level of inflammation and tissue damage by light microscopy.

Total RNA was extracted from frozen liver samples using the RNeasy Mini Kit (Axygen, USA). cDNA was synthesized from 1 μg of total RNA using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan). Quantitative real-time PCR was carried out on a ViiA 7 system (Applied Biosystems, USA) in 20 μl reactions containing 2 μl of cDNA (1:10 dilution), 400 nM of primers (All primers were obtained from Sangon, China) and PowerUp SYBR Green Master Mix (Applied Biosystems, USA) according to the manufacturer’s instructions. Data are expressed as the ratio of the target gene level to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the treated group relative to the sham group and were quantified using the comparative cycle threshold Ct method (2^-▵▵CT). The gene primer sequences were as follows:

Total protein was isolated from liver tissue and protein concentrations were quantified using the BCA protein estimation kit (Solarbio, China). For each sample, 20–40 μg of protein were loaded. The proteins were transferred onto PVDF membranes using the wet transfer method. Membranes were blocked in Western Blocking Buffer (Beyotime, China) for 1 h at room temperature and incubated with antibodies against P38 (1:1200, CST, USA), p-P38 (1:1200, CST, USA), JNK (1:1200, Abcam, USA), p-JNK(1:1200, Abcam, USA), ERk1/2 (1:1200, CST, USA), p-ERK1/2 (1:1200, CST, USA), NF-κB (1:1000, CST, USA), p-NF-κB (1:500, CST, USA), cleaved caspase-3 (1:1000, CST, USA), Bcl-2 (1:1000, Abcam, USA), Bax (1:1000, Abcam, USA), cAMP response element-binding protein (CREB) (1:1000, CST, USA) and p-CREB (1:1000, CST, USA). Blots were probed with anti-GAPDH (1:10000, Abcam, USA) as the loading control. All antibodies were incubated with the membranes at 4°C overnight. The secondary antibody was an anti-rabbit HRP-conjugated antibody (1:20000, Lianke technology, China). Immunoblots were developed using the Immobilon ECL method (Millipore, Billerica, Massachusetts, USA).

Immunohistochemistry was carried out using rabbit monoclonal or polyclonal primary antibodies and horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies to detect cleaved caspase-3 (1:500, Wanlei, China), Bax (1:250, Abcam, USA), Bcl-2 (1:400, Abcam, USA) and F4/80 (1:100, Abcam, USA) expression. Expression is represented as a percentage of the total liver area using ImageJ after analysing five fields of stained liver sections per animal.

The levels of cytokines in homogenized liver tissue, including IL-6, were detected by enzyme-linked immunosorbent assay (ELISA) using the Quantikine Elisa kit (R&D, USA), while TNF-α and IFN-γ were detected using the Valukine Elisa kit (Novus, USA) according to the manufacturer’s protocol. The amount of protein in each sample was quantified via BCA, and the final result was expressed as the quantity contained in each milligram of protein.

Hepatocytes (AML-12) were cultured in DMEM/F12 medium while macrophages (RAW264.7) were cultured in high glucose DMEM medium, both of which were supplemented with 10% FBS and 1% cyan-streptomycin and cultured in a 37°C incubator with 5% CO₂. The protein of the cultured cells was extracted and the expression of the CB2 receptor was detected by Western blot. The Kupffer cells were isolated as described earlier.²¹ Cells were processed and treated with TNF-α as described in figure.

Data are expressed as the mean ± SEM and were analysed using a one-way analysis of variance (ANOVA) followed by a Tukey’s post hoc test using GraphPad Prism 5 software. Comparisons between the two groups were analysed using t-tests. In all comparisons, a p < 0.05 was considered to be statistically significant.

We first investigated whether the CB2 receptor had a protective role in the ConA-induced liver injury model. Treatment of mice with ConA for 9 h (20 mg/kg) induced a significant degree of liver injury in CB2^−/− compared with the WT mice, which was characterized by inflammation, necrosis and inflammatory cell infiltration as shown by H&E (Figure 1(a)). Moreover, ALT and AST levels in the CB2-deficient mice were higher than the wild-type mice (Figure 1(b)). These data indicate that the CB2 receptor plays a protective role in ConA-induced liver injury.

When administered after ConA injection, serum levels of ALT and AST increased in the ConA model group (ALT, 5757.5 ± 1036 U/L; AST, 4451 ± 1146 U/L) compared to the NC group (ALT, 28.5 ± 7.35 U/L; AST, 101.6 ± 26.35 U/L). The AM1241 pretreatment groups showed much lower levels of aminotransferase (ALT, 2697 ± 956.3 U/L; AST, 1911 ± 766 U/L and ALT, 500.7 ± 126.9 U/L; AST, 636 ± 99.3 U/L). However, pretreatment with AM630 prior to treatment with AM1241 inhibited the effect of AM1241 (ALT, 3395 ± 1811 U/L; AST, 2326 ± 1054 U/L) (Figure 1(c)). The changes in histopathology were consistent with the ALT and AST results (Figure 1(d)). We observed massive areas of necrosis in the group that was only treated with ConA. In contrast, the AM1241-treated groups showed only minor liver damage, which indicates that AM1241 treatment significantly reduced liver necrosis and the AM1241 (12 mg/kg) pretreatment group showed more effective protection than AM1241 (3 mg/kg) pretreatment group. These results showed that AM1241 treatment could ameliorate ConA-induced acute immune hepatitis in mice.

Proinflammatory cytokines play a vital role in the progression of hepatitis, including TNF-α, IL-6, and IFN-γ. Therefore, we profiled the levels of these cytokines in liver homogenate by ELISA and their corresponding mRNA content in liver tissue by real-time PCR. Our results showed that the levels of these cytokines increased in the ConA-treated group following ConA induction. As expected, the production of TNF-α, IL-6 and IFN-γ was reduced in the AM1241-pretreatment group and AM630 was able to inhibit the action of AM1241 in the AM630 group. Additionally, the changes in mRNA levels were the same as observed for in cytokines, indicating that these changes were statistically significant (Figure 2).

Mitogen-activated protein kinases can change the expression levels of genes by phosphorylating transcription factors, which can mediate various physiological and pathological processes. Therefore, we examined the phosphorylation levels of P38, JNK and ERK1/2 proteins in each group. We found that p-p38 and p-ERK were significantly increased in the model group and decreased after pretreatment with AM1241, and pretreatment with the CB2 inhibitor AM630 was able to inhibit the effects of AM1241. However, we did not observe an increase in p-JNK in the model group despite increased expression after AM1241 was administered. The expression of p-38 and p-ERK1/2 were significantly increased in the AM1241-treated groups compared to the ConA-treated group and the AM630-pretreated group (Figure 3(a)). These data were analysed using ImageJ, which identified statistically significant differences among these changes (Figure 3(b)).

To investigate the possible association between the apoptosis results and the MAPK signalling pathway, we examined protein levels of NF-κB (P-65) and CREB in the liver tissue of each mouse group. As expected, p/T-P65 and p/T-CREB were significantly increased in the model group and pretreatment with AM1241 reduced their phosphorylation. Once again, AM630 reversed this effect. These changes were statistically significant (Figure 3).

ConA-induced liver damage appeared to increase the amount of apoptosis in the liver. Interestingly, pro-apoptotic protein expression did not increase in the model group but increased levels of Bax and cleaved caspase-3 were observed in the AM1241 pretreatment group. Bcl-2 is known as an anti-apoptotic protein but was elevated in both the model group and in the AM630 pretreatment groups. These changes were statistically significant and contrary to our expectations. To further confirm these results, we measured the expression levels of Bax, Bcl-2 and cleaved caspase-3 in liver tissue using immunohistochemistry. As expected, the results were consistent with those of the Western blot. The statistical analyses were carried out using Image-Pro Plus 6.0 software. These data indicate that AM1241 can promote apoptosis in certain liver cells and that this phenomenon is inhibited by the CB2 inhibitor AM630 (Figure 4).

To demonstrate that the target of AM1241 is not hepatocytes but instead some other non-parenchymal cells, including macrophages, we used cultured hepatocytes and macrophages. We found that both liver tissue and macrophages express the CB2 receptor and that the expression of CB2 is increased in the liver tissue of ConA-pretreated mice but hardly expressed in normal liver cells (Figure 5(a)).

After confirming the expression of the CB2 receptor in hepatic macrophages, we detected F4/80, which is a highly specific marker for macrophages, in the liver tissue of each group using immunohistochemistry. These data were analysed using Image-Pro Plus 6.0 software. The results showed a significant increase in F4/80 in the liver tissue of the ConA-treatment group but significant decreases after administration of AM1241, and AM630 once again inhibited the effect of AM1241 (Figures 5(b) and (c)).

To elucidate the specific role of resident macrophage, we examined the comparative expression of CB2 receptor in our RAW macrophage and isolated Kupffer cells from healthy pooled mice. The level of expression is quite similar between those two populations as evident from real-time PCR-based Ct values (Figure 6(a)). We demonstrated a significant correlation of CB2 receptor expression and F4/80 expression in healthy and ConA liver samples (Figure 6(b)). We also induced activation in those isolated Kupffer cells by treatment with LPS in vitro with or without CB2 agonist AM1241 (Figure 6(c)). AM1241 treatment reduced inflammatory cytokine and thus associated hepatocytic cell death.