A total of 110 C57BL/6, 28 Nlrp3^fl/fl mice and 28 Nlrp3^Δhep mice were used. C57BL/6 male mice (8-week-old) weighing 20–22 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Charles River Laboratories). Alb-iCre (strain no. T003814) and B6JNju;B6NNju-Nlrp3 (flper)^tm1/Nju [Nlrp3 locus of X-over P1 (Nlrp3^loxp or Nlrp3^fl/fl)] (strain no. T001231,) mice were purchased from GemPharmatech Co., Ltd. Hepatocyte-specific Nlrp3 knockout mice (Albcre⁺Nlrp3^loxp/loxp, Nlrp3^Δhep) were generated by crossing the Nlrp3^loxp mice with Alb-cre mice and the mice were backcrossed for at least 10 generations on a C57BL/6 background. Male mice (age, 8 weeks old, 20–22 g) were used for the experiments. The mice were raised in a standard room with controlled humidity (55–60%) and temperature (23–25°C) under a 12 h light/dark cycle with ad libitum access to food and water. All mice received two daily health observations including water and food intake, weight and general assessment of animal activity. The study employed humane endpoints according to AVMA Guidelines for the Euthanasia of Animals (https://www.avma.org/resources-tools/avma-policies/avma-guidelines-euthanasia-animals), including when the mice showed an inability to obtain food or water on their own, had a weight loss of >20% of their starting body weight, difficulty moving, were depressed in the absence of anesthesia, or their body temperature was persistently below 37°C. All animal experiments were performed in accordance with the regulations of the ARRIVE guidelines and approved by The Ethics Committee Medical College of Qingdao University (Qingdao, China; approval no. QDU-AEC-2022310).

The APAP-induced liver injury model was established as previously described, with minor modifications (25). Briefly, 25 mg/ml APAP solution was prepared by dissolving APAP (Beijing Solarbio Science & Technology Co., Ltd.) in pyrogen-free phosphate-buffered saline (PBS) at 60°C and cooled to 37°C before injection. The mice were fasted for 15–17 h and then intraperitoneally (IP) injected with a single dose of 300 mg/kg of APAP (sublethal dose; n=5 per group) or 500 mg/kg (lethal dose; n=10 per group). To evaluate the therapeutic effects of NLRP3 and GSDMD inhibition, NLRP3 inhibitor MCC950 (TargetMol Chemicals Inc.) was administered IP into mice at 50 mg/kg 2 h after APAP and GSDMD inhibitor disulfiram (DSF; TargetMol Chemicals Inc.) was administered IP into mice at 50 mg/kg 4 and 24 h before APAP (n=5 per group), with PBS or vehicle (sesame oil) as control group respectively. At 0, 6,12, 24, 36 or 48 h), the mice were euthanized using 5% inhalational isoflurane for >3 min and death was verified by a lack of heartbeat and breathing. Liver tissue and serum samples were harvested for subsequent experiments.

The survival rates of Nlrp3^fl/fl and Nlrp3^Δhep mice were evaluated within 72 h after 500 mg/kg APAP treatment (lethal dose; n=10 per group) as previously described (26). Mice were housed under pathogen-free conditions and were monitored every 6 h with free access to food and water. All of the mice were euthanized at the end of the study.

Mouse primary hepatocytes were isolated from C57BL/6 mice (n=5), Nlrp3^fl/fl and Nlrp3^Δhep mice (n=3) as previously described by two-step perfusion methods with minor modifications (27,28). In brief, the liver was perfused with Hank's Balanced Salt Solution (Wuhan Servicebio Technology Co., Ltd.) containing EGTA (Beijing Solarbio Science & Technology Co., Ltd.) and digested with collagenase type IV (Gibco; Thermo Fisher Scientific, Inc.). Then, the liver was dissolved in residual digestive fluid and filtered with a 100 µm cell strainer. Hepatocytes were obtained by low speed centrifugation (100 × g) for 2 min at 4°C. Cell culture plates were coated with 0.03 mg/ml rat tail collagen I 2 h in advance at room temperature and washed three times with sterile PBS before use. The cells were seeded onto 6-well or 96-well plates with 8×10⁴ cells/ml precoated with rat tail tendon collagen I (Shengyou Biotechnology Co., Ltd.). After overnight incubation, the cells were treated with 1, 2.5, 5 and 10 mM APAP for 12 h at 37°C.

The α mouse liver 12 (AML12) cell line was purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. The cells were cultured in DMEM/F12 (1:1) (Gibco; Thermo Fisher Scientific, Inc.) medium containing 10% fetal bovine serum (Shanghai ExCell Biology, Inc.), Insulin-Transferrin-Selenium (Beyotime Institute of Biotechnology) and 40 ng/ml dexamethasone (MilliporeSigma) with 5% CO₂ at 37°C. Upon reaching a confluency of 70 to 80%, the cells were incubated with 1, 5, 10 and 20 mM APAP for 24 h, subsequently total protein and RNA were extracted for western blotting and reverse transcription-quantitative PCR (RT-qPCR) respectively.

To investigate the therapeutic effects of NLRP3 and GSDMD inhibition in vitro, AML12 cells were seeded onto 96-well plates at a density of 1,000 cells per well, 0, 1, 5, 10 µM MCC950 or 0, 1, 2.5, 5 µM DSF were administrated to the AML12 cell complete medium, with or without 10 mM APAP. To evaluate the toxicity of APAP on hepatocytes, cell viability was measured using a CCK-8 assay (Shanghai Yeasen Biotechnology Co., Ltd.). Briefly, 10 µl of CCK-8 solution was added to each well and incubated for 1 h at 37°C. Absorbance at 450 nm was measured by SpectraMax Absorbance Reader (Molecular Devices, LLC).

Total RNA was extracted from liver tissue, primary hepatocytes or AML12 cells using FastPure Cell/Tissue Total RNA Isolation kit (Vazyme Biotech Co., Ltd.). RNA was reverse transcribed into cDNA using specific primers for Nlrp3, Caspase 1, Il-1β, Tnf-α, Il-6, Mcp-1, Cxcl-1, Cxcl-2, CyclinA2, CyclinD1 and CyclinE1 (Table I) using the PrimeScript First Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. As previously described (29), qPCR was performed using SYBR Premix Ex Taq (Vazyme Biotech Co., Ltd.) on a CFX96 Real-Time Systems (Bio-Rad Laboratories, Inc.). The thermocycling conditions were as follows: Pre-incubation at 95°C for 2 min; amplification at 95°C for 10 sec and 60°C for 20 sec, followed by 40 cycles; melting curve at 65°C for 5 sec and 95°C for 15 sec. The mRNA levels were quantified using the 2^−ΔΔCq method and normalized to the internal reference gene GADPH (30).

Total protein from liver tissue, primary hepatocytes or AML12 cells was extracted using RIPA buffer(Wuhan Servicebio Technology Co., Ltd.) as described previously (31). BCA Protein Assay kit (Epizyme; ZJ102) to confirm the protein concentration. Briefly, the protein samples (40 µg) were separated by 7.5, 10 and 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (MilliporeSigma). After blocking with 5% skimmed milk powder for an hour at room temperature, the membranes were incubated with primary antibodies including: NLRP3 (1:1,000; AG-20B-0014-C100; Adipogen Life Sciences, Inc.), IL-1β (1:1,000; A11369; ABclonal Biotech Co., Ltd.), Caspase-1 (1:1,000; A0964; ABclonal Biotech Co., Ltd.), GSDMD (1:1,000; cat. no. AF4012; Affinity Biosciences), β-actin (1:1,000; AC026; ABclonal Biotech Co., Ltd.), Caspase-3 (1:1,000; 9662S; Cell Signaling Technology, Inc.), PCNA (1:1,000, AF0239; Affinity Biosciences), CyclinD1 (1:1,000; A0310; ABclonal Biotech Co., Ltd.) at 4°C overnight. Next day, these membranes were washed three times using Tris-buffered saline with 0.1% Tween-20 for ten minutes each and then incubated with Goat Anti-Mouse IgG (H+L) HRP or Goat Anti-Rabbit IgG (H+L) HRP (1:4,000; S0002; S0001; Affinity Biosciences,) for 1 h at room temperature. Finally, protein bands were washed three times again and visualized using an ultra sensitive chemiluminescence assay kit (Shanghai Epizyme Biotech Co., Ltd.) and imaged using an automatic chemiluminescence image analysis system (Tanon Science and Technology Co., Ltd.). Protein levels were quantified using ImageJ software (version 1.43; National Institutes of Health) with β-actin as the loading control.

Biochemical analysis and ELISA assay. Blood serum aspartate transaminase (AST) and serum alanine aminotransferase (ALT) levels in mice were measured using a Chemray 800 automatic biochemical analyzer (Rayto Life and Analytical Sciences Co., Ltd.) according to the manufacturer's instructions (S03040; S03030; Rayto Life and Analytical Sciences Co., Ltd.). Additionally, levels of inflammatory cytokines IL-6, IL-18, IL-1β and TNF-α were measured using mouse interleukin-6(IL-6) ELISA kit (ml098430); mouse interleukin-18 (IL-18) ELISA kit (ml002294); mouse interleukin-1β (IL-1β) ELISA kit (ml098416); mouse tumor necrosis factor-α (TNF-α) ELISA kit (ml002095)(Shanghai Enzyme-linked Biotechnology Co., Ltd.) according to the manufacturer's instructions. The absorbance values were measured at 450 nm using a SpectraMax Absorbance Reader (Molecular Devices, LLC).

Histological sectioning and IHC were performed as previously described (32). Briefly, liver samples from the left lateral lobes of mice were fixed with 4% paraformaldehyde (Wuhan Servicebio Technology Co., Ltd., G1101) for 24 h at room temperature and embedded in paraffin or optimal cutting temperature (OCT) compound (Wuhan Servicebio Technology Co., Ltd.) and stored at −80°C. The paraffin was sliced into 5 µm sections and frozen OCT compound samples were sliced into 8 µm sections. Sections were hematoxylin and eosin (H&E) stained to examine the pathological changes in liver tissue after deparaffinization and dehydration through descending ethyl alcohol). For IHC, the sections were subjected to antigen retrieval by microwaving in citrate buffer (pH 6.0, Biogenex, CA) for 20 min at 99–100°C. After washing with PBS for 15 min, the sections were incubated with 3% hydrogen peroxide for 10 min at room temperature. IHC was carried out by incubating the sections with antibodies including: NLRP3 (1:200; DF7834; Affinity Biosciences), IL-1β (1:200; ab9722; Abcam), Caspase-1 (1:200; ab207802; Abcam), GSDMD (1:200; AF4012; Affinity Biosciences), PCNA (1:200; cat. no. ab92552; Abcam), Ki67 (1:200, ab15580; Abcam), CD68 (1:200; GB113109; Wuhan Servicebio Technology Co., Ltd.), Ly6G (1:200; GB11229; Wuhan Servicebio Technology Co., Ltd.) and Caspase-3 (1:200; 9662S; Cell Signaling Technology, Inc.) at 4°C overnight. Next day, according to the manufacturer's instructions for immunochromogenic reagent (EliVision plus mouse/rabbit) (KIT9901, Maixing Company, China), the sections were incubated with R1(reaction enhancement solution) for 20 min, and then incubated with R2(HRP-conjugated anti-mouse/rabbit IgG polymer) in the dark for 30 min at room temperature. Finally, the sections were stained with DAB chromogenic kit (Wuhan Servicebio Technology Co., Ltd.) for 2 min at room temperature. The sections were images of the sections were captured under an BX50 light microscope (Olympus Corporation).

The frozen sections were subjected to TUNEL staining using a TUNEL kit (Shanghai Yeasen Biotechnology Co., Ltd.), following the manufacturer's instructions. Briefly, the sections were fixed with 4% paraformaldehyde at room temperature for 30 min. After washing with PBS for 30 min, Proteinase K (20 µg/ml) were incubated for 10 min at room temperature and TDT incubation buffer were incubated at 4°C overnight. Subsequently, nuclei were counterstained with 2 µg/ml 4′,6-diamidino-2-phenylindole (Beyotime Institute of Biotechnology) for 5 min at room temperature. The sections were sealed with PBS. The TUNEL-positive cells were visualized under a fluorescence microscope (Olympus Corporation) and 5 numbers of field were calculated.

All experiments were conducted at least three times, and statistical analyses were performed using GraphPad Prism 8.0 software (Dotmatics). Differences between groups were assessed using an unpaired Student's t-test for two groups or one-way ANOVA followed by Tukey's post hoc test for multiple groups. Survival curves were compared using the log-rank (Mantel-Cox) test. P<0.05 was considered to indicate a statistically significant difference.

As previously described (25), an APAP-induced liver injury mouse model was established by IP injection of a single dose of 300 mg/kg APAP. APAP treatment resulted in liver injury characterized by increased hepatic necrosis (Fig. 1A and B) and elevated serum ALT (Fig. 1C) and AST levels (Fig. 1D). To investigate the potential role of NLRP3 activation in APAP-induced hepatotoxicity, expression levels of NLRP3 in AILI mice were examined. IHC demonstrated an increase in NLRP3, Caspase-1, IL-1β and GSDMD in mice liver after APAP treatment compared with control group (sham) (Fig. 1E). Likewise, increased protein levels of NLRP3, cleaved IL-1β and GSDMD were shown by western blotting compared with the control group (sham) (Fig. 1F). Additionally, APAP treatment increased the levels of proinflammatory cytokines IL-1β (Fig. 1G) and IL-18 (Fig. 1H) in mouse serum compared with 0 h, indicating the activation of NLRP3 and GSDMD. To determine the cellular source of NLRP3 expression after 24 or 48 h post-treatment with APAP, mouse liver parenchymal cells were isolated for western blotting, which showed increased levels of NLRP3, cleaved caspase-1, cleaved IL-1β and GSDMD-N at 24 h post APAP treatment (Fig. 1I).

To investigate whether APAP overdose induces NLRP3 activation in mouse hepatocytes in vitro, AML12 cells and primary hepatocytes were subjected to APAP treatment for 24 and 12 h, respectively. These results showed that both cell types exhibited dose-dependent decrease in cell viability with APAP treatment (Fig. 2A). Likewise, APAP treatment increased the mRNA expression levels of Nlrp3 (Fig. 2B), Caspase-1 (Fig. 2C) and Il-1β (Fig. 2D) in AML12 cells and primary hepatocytes compared with the control. Furthermore, the protein levels of NLRP3 and the active fragments of pyroptosis-related proteins, cleaved caspase-1, cleaved IL-1β and GSDMD-N were elevated upon APAP treatment in both AML12 and primary hepatocytes compared with the control (Fig. 2E-F). Taken together, the aforementioned findings suggested that APAP overdose triggers NLRP3 activation leading to hepatocyte pyroptosis both in vivo and in vitro.

As NLRP3 is crucial for APAP-induced hepatocyte pyroptosis, Nlrp3^Δhep mice were generated by crossing Nlrp3^fl/fl mice with Albcre⁺ mice. Western blotting confirmed the specific knockout of Nlrp3 in hepatocytes and not in non-parenchymal cells (Fig. 3A). To investigate the impact of hepatic Nlrp3 deficiency on APAP-induced liver injury, AILI models were created using Nlrp3^fl/fl and Nlrp3^Δhep mice. Survival assays were conducted through IP injection of a lethal dose (500 mg/kg) of APAP, revealing significantly lower mortality in Nlrp3^Δhep mice compared with Nlrp3^fl/fl mice (Fig. 3B). Liver damage was assessed by injecting 300 mg/kg APAP and measuring alanine aminotransferase (ALT) (Fig. 3C) and aspartate aminotransferase (AST) levels (Fig. 3D), as well as the level of necrosis in liver tissue (Fig. 3E), all of which showed significant reductions in Nlrp3^Δhep mice when compared with Nlrp3^fl/fl mice. Consistently, livers from Nlrp3^Δhep mice exhibited lower TUNEL-positive staining after APAP treatment compared with the Nlrp3^fl/fl mice (Fig. 3F). These findings indicate that hepatic blockade of NLRP3 not only protects against APAP-induced liver injury but also improves survival.

During the pathogenesis of AILI, the immune response serves a crucial role, and a timely decrease in inflammation is believed to be important in repairing acute liver injury in mice (33). To investigate whether the protective effect from liver injury in Nlrp3^Δhep mice is related to hepatic decrease in inflammation, the levels of inflammation between the two groups were analyzed after treatment with 300 mg/kg APAP. IHC staining for Ly6G and CD68, which represent infiltration of liver neutrophils and macrophages, respectively, were significantly reduced in Nlrp3^Δhep mice at 24 and 48 h compared with the Nlrp3^fl/fl mice (Fig. 4A). Furthermore, there was a significant decrease in mRNA expression levels of pro-inflammatory cytokines Tnf-α and Il-6 at 24 h, and for Il-1β at both 24 and 48 h post-APAP treatment compared with the Nlrp3^fl/fl mice. Furthermore, a significant decrease in proinflammatory chemokine mRNA levels of Mcp-1 and Cxcl-1 at 24 h and of Cxcl-2 at 24 and 48 h post APAP treatment was observed in Nlrp3^Δhep mice compared with Nlrp3^fl/fl mice (Fig. 4B).

Additionally, compared with Nlrp3^fl/fl mice, significantly decreased protein levels of TNF-α (Fig. 4C), IL-6 (Fig. 4D) at 24 and 48 h, and IL-1β (Fig. 4E) at 24 h post-APAP treatment were measured in Nlrp3^Δhep serum samples. These findings suggest that ablation of hepatic NLRP3 may alleviate APAP-induced liver injury by reducing inflammation.

To assess the therapeutic potential of NLRP3 inhibition in APAP-induced liver injury, MCC950, an NLRP3 inhibitor, was used as previously described (34). In vivo, C57BL/6 mice were administered with MCC950 or PBS following APAP treatment and serum ALT, AST were evaluated at 24 and 48 h post-APAP treatment. The proportion of liver necrosis (Fig. 5A) and TUNEL-positive cell labeling (Fig. 5B) were significantly decreased with MCC950 treatment at 24 and 48 h compared with the PBS controls. Likewise, serum ALT (Fig. 5C) and AST (Fig. 5D) levels were significantly reduced in the MCC950 treated group at 24 h compared with the PBS treatment. In vitro experiments using AML12 cells treated with or without APAP showed that MCC950 improved cell viability, although the increase was small (Fig. 5E).

Additionally, western blotting demonstrated that 1 µM MCC950 treatment significantly decreased the levels of the active fragments from pyroptosis-related proteins including cleaved caspase-1, cleaved IL-1β and GSDMD-N in AML12 cells compared with the 0 µM MCC950 control (Fig. 5F).

To further validate the association between NLRP3-mediated pyroptosis and its protective effect on AILI, DSF, a GSDMD inhibitor, was used as previously described (35,36). Mice were administered with either DSF or vehicle prior to APAP treatment. Area of liver necrosis in the DSF group was decreased compared with vehicle (Fig. 6A). The results also demonstrated a significant reduction in serum ALT/AST levels in the group treated with DSF compared with the vehicle controls. (Fig. 6C and D). Moreover, IHC revealed a significant decrease in caspase-3 positive cells at 24 and 48 h post APAP treatment compared with the vehicle controls. (Fig. 6B), which is considered an indicator of cell death. Moreover, DSF significantly decreased the levels of GSDMD-N and cleaved caspase-3 with 1 µM DSF treatment with compared with the 0 µM DSF control suggesting that this treatment prevented pyroptosis of AML12 cells (Fig. 6E).

Evidence suggests that the timely initiation of liver regeneration is crucial for recovery from APAP-induced hepatotoxicity (6). In the present study, the regenerative capacity of Nlrp3^Δhep and NLRP3^fl/fl mice at 24 and 48 h post APAP treatment were assessed. A significant increase in the number of PCNA-positive and Ki67-positive hepatocytes in Nlrp3^Δhep mice at 24 and 48 h compared with the Nlrp3^fl/fl mice were observed (Fig. 7A).

Moreover, mRNA expression levels of Cyclin A2 (Fig. 7B), Cyclin D1 (Fig. 7C) and Cyclin E1 (Fig. 7D), which are pivotal regulators of cell proliferation, were significantly increased at 24 and 48 h post APAP treatment in Nlrp3^Δhep mice compared with the NLRP3^fl/fl mice.

Furthermore, western blotting confirmed elevated protein levels of CCND1 and PCNA in Nlrp3^Δhep mice at 24 and 48 h post APAP treatment compared with the NLRP3^fl/fl mice (Fig. 7E), consistent with the aforementioned results (Fig. 7B-D). These findings suggest that hepatic NLRP3 deficiency promotes liver repair following APAP-induced liver injury.