The primary antibodies used in this study were as follows: anti-NLRP3 (ab4207), anti-IL-18 (ab68435), anti-IL-1β (ab2105), anti-ASC (ab127537), anti-caspase-1 (ab179515), and anti-GSDMD-N (ab215203) purchased from Abcam (Cambridge, United Kingdom); anti-Caspase-11 (sc-56038) purchased from Santa Cruz Biotechnology (Dallas, United States of America); anti-ubiquitin (10201-2-AP), anti-Nedd4 (21698-1-AP), anti-GAPDH (60004-1-Ig), and horseradish peroxidase (HRP)-conjugated secondary antibodies (SA00001-2) purchased from Proteintech (Wuhan, China); and anti-Neutrophil (bs-19701R) purchased from Biosynthesis Biotechnology Co. (Beijing, China). Cy3 goat anti-mouse IgG (H + L) (A0521) and FITC goat anti-rabbit IgG (H + L) (A0562) used for immunofluorescence analysis were purchased from Beyotime (Shanghai, China). FuGENE-HD transfection reagent (E2311) was purchased from Promega (Madison, United States of America). ELISA kits for IL-1β and IL-18 were purchased from Shanghai Enzyme-linked Biotechnology (Shanghai, China). The LDH activity detection kit (C0016) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Pyroptosis Compound Library (L7400), ST (S8322) were purchased from Selleckchem (Houston, TX). Escherichia coli LPS (L2630) and FITC-conjugated LPS from E. coli 0111:B4 (F3665) were purchased from Sigma-Aldrich (St. Louis, MO).

Six-to-eight-week-old male C57BL/6 mice, weighing 18–21 g, were purchased from Chengdu Dashuo Experimental Animal Company (Chengdu, China). All mice were raised in specific pathogen-free animal housing and provided with bacteria-free food and water. Mice were randomly divided into four groups (group 1: vehicle group; group 2: 10 mg/kg LPS; group 3: 5 mg/kg ST; group 4: 5 mg/kg ST + 10 mg/kg LPS, n = 6 in each group). Then, the mice were sacrificed, and tissues and blood were collected. Sera were isolated from blood using centrifugation at 4000 rpm at room temperature and stored at −80°C for biochemical analysis. Tissues were fixed in a 4% formalin solution and embedded in paraffin for histological analysis. All animal procedures were approved by the Animal Policy and Welfare Committee of Chengdu Medical College (CDYXY-2019036).

RAW264.7 cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37°C with 5% CO₂. All media and supplements were purchased from Invitrogen (Carlsbad, CA, United States).

Flow cytometry was used to measure pyroptosis using an Annexin V-PE/7-AAD Detection Kit (KeyGEN, Jiangsu, China), according to the manufacturer’s instructions. RAW264.7 cells were pre-treated with or without ST at 2.5, 5, and 10 μM, and then harvested, washed with phosphate-buffered saline (PBS), and stained with 7-AAD for 15 min. After the reaction, 450 μl binding buffer was added, followed by incubation with 1 μl Annexin V-PE at 37°C in the dark for 15 min. Finally, the cells were analyzed using a flow cytometer (FACSCalibur, Becton-Dickinson, Franklin Lakes, NJ, United States).

Cells were seeded in 96-well plates and primed with 1 mg/ml Pam3CSK4 in Opti-MEM for 4 h. Subsequently, the cells were treated with the different compounds for specific time periods and then transfected with 1 ug/mL LPS plus FuGENE-HD (2.5%, v/v) in Opti-MEM for 16 h (Ye et al., 2021). Lytic cell death was determined using a lactate dehydrogenase (LDH) activity detection kit.

Liver tissues were weighed, and liver indices calculated as the ratio of liver weight to body weight.

Mice were anaesthetized using 0.6% pentobarbital sodium (40 mg/kg), and liver tissues were collected and fixed with 4% paraformaldehyde (in PBS) for 48 h at room temperature, dehydrated using an ethanol gradient, cleared with xylene, embedded in paraffin, and cut into 5 μm sections.

To evaluate the degree of inflammatory cell infiltration, liver sections were stained using an H&E staining kit (Beyotime Biotechnology, Shanghai, China). The sections were dewaxed, dehydrated, washed with PBS, and then stained with hematoxylin for 6 min and eosin for 2 min. We evaluated the distribution of liver glycogen using a PAS staining kit (Solarbio, Beijing, China). DNA fragmentation was detected by a TUNEL apoptosis detection kit (KeyGen, Nanjing, China).

IHC was performed using an SP link detection kit (ZSGB-BIO Technology, Beijing, China). Tissue sections were dewaxed, dehydrated, and washed with PBS. Subsequently, the samples were boiled in citrate buffer (pH 6.0) for antigen retrieval and blocked with 5% normal goat serum at 37°C for 1 h. The sections were then incubated overnight at 4°C with the primary antibodies (1:200). After washing with PBS, the sections were incubated with the corresponding secondary antibody for 30 min. Finally, diaminobenzidine was used as the chromogen to visualize the immunocomplexes, and the sections were counterstained with hematoxylin. Images of random liver sections were captured at ×40 and ×20 magnifications using a microscope (XI 71 Olympus, Tokyo, Japan).

The liver tissues were embedded in optimal cutting temperature compound and cut into 10 μm sections for IF staining. After washing with PBS, tissue sections were blocked with 5% BSA for 30 min at 37 C and then incubated overnight at 4 C with primary antibodies. After washing, the sections were incubated with secondary antibodies, Cy3 goat anti-rabbit/mouse IgG (H + L) or Alexa Fluor 488 goat anti-mouse/rabbit IgG (H + L) (1:200) for 1 h at 37°C, and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Fluorescent images were captured at a ×40 magnification using a fluorescence microscope.

After LPS injection, the survival status of the mice was monitored at least every 6 h for two consecutive days. Survival curves were constructed using the GraphPad Prism 7.0 software.

Liver samples were lysed on ice with immunoprecipitation lysis buffer (Beyotime) containing a protease inhibitor cocktail (Beyotime). Samples were centrifuged at 15,000 × g for 15 min, and 200 μl supernatant was transferred to a new tube as the input. Approximately 500 to 1,000 μg of protein was incubated with 1 μg of antibody overnight at 4 C. In total, 30 μl protein A/G-agarose beads (Beyotime) were added to each sample and incubated for another 2 h at 4 C the next day. The beads were then washed thrice with immunoprecipitation lysis buffer. After the final centrifugation, the beads were boiled for 5 min with 60 μl SDS loading buffer (Beyotime), and samples were subjected to western blot analysis using standard protocols.

Total RNA was extracted from spinal cord tissue using a total RNA extraction kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. Next, cDNA was synthesized using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, United States). The mRNA levels of Il18, Il1β, Il6, Il10, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein-1 (MCP-1), and Nedd4 were analyzed using qRT-PCR and the SYBR Green Supermix (Bio-Rad, Hercules, CA, United States). The primers were synthesized by Shanghai Shenggong. The 2^−ΔΔCT method was used to calculate the relative mRNA levels.

Liver tissues and cells were lysed in ice-cold RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). Protein concentrations were determined using a BCA reagent kit (Beyotime Biotechnology, Shanghai, China). Total protein (30 μg) was separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). The membranes were blocked in Tris-buffered saline with 5% non-fat milk and 0.5% bovine serum albumin for 2 h at room temperature and then incubated overnight at 4 C with primary antibodies (1:1,000). After washing, the membranes were incubated with secondary antibodies (1:5,000) for 1 h at 37 C. Blots were visualized with the chemiluminescent HRP substrate (Millipore) and quantified using the Quantity 5.2 software System (Bio-Rad).

Cells were cultured in 96-well plates at 10,000 cells/well, and 24 h after irradiation and/or drug treatment, CCK-8 (C0038, Beyotime, Shanghai, China) was used to detect cell viability according to the manufacturer’s instructions.

All data are expressed as the mean ± SD. Statistical analysis was performed using the GraphPad Prism 7.0 software (GraphPad, San Diego, CA, United States) with one-way ANOVA, followed by post-hoc multiple comparisons with Tukey’s test. p < 0.05 was considered statistically significant.

To sufficiently activate caspase-11, appropriate PAMP priming is needed to stimulate the expression of pro-caspase-11 protein prior to transfection or delivery of LPS into the cytosol (Hagar et al., 2013). We initially performed this in RAW264.7 macrophages using Pam3CSK4 (a synthetic ligand for Toll-like receptor TLR2/1 as the priming reagent (Kayagaki et al., 2015). LPS transfection markedly increased caspase-11 activation, GSDMD-NT generation, and LDH release (Supplementary Figure S1). To identify compounds with the ability to inhibit caspase-11 activation, we screened a library of 441 compounds (Selleckchem compounds library) in RAW264.7 cells. Compounds were screened for their ability to inhibit caspase-11 activation and LPS-stimulated pyroptosis at a concentration of 10 μM using different detection methods (Figure 1A). Primary screening with LDH release revealed the ability of the compounds to inhibit lytic cell death after LPS transfection (Figure 1A). A total of 15 compounds that induced LDH release of less than 15% were selected for further evaluation in the secondary screening (Figure 1A). Subsequently, we confirmed the toxicity of these compounds using the CCK-8 assay. The six compounds confirmed in the secondary screening inhibited RAW264.7 cell growth by less than 25% at a concentration of 10 μM (Figure 1A). Flow cytometry assays further detected Annexin V-PE and 7-AAD double-positive cells in RAW264.7 cells pre-treated with these six compounds. The results showed that ST (C₂₃H₂₆N₄O₃, Figure 1B) induced the most significant inhibition of intracellular LPS-induced lytic cell death (Figure 1A). Next, we sought to verify the ability of ST to inhibit caspase-11 activation and pyroptosis at the concentrations of 2.5, 5, and 10 μM. Pre-treatment with ST decreased the number of Annexin V-PE and 7-AAD double-positive cells. ST alone did not increase the number of Annexin V-PE and 7-AAD double-positive cells (Figure 1C). Similarly, western blot and LDH release assay analyses showed that ST dose-dependently inhibited intracellular LPS-induced GSDMD-NT generation, caspase-11 activation, and LDH release. ST alone did not induce any such processes (Figures 1D,E). Together, these data indicate that ST is able to inhibit caspase-11 activation and pyroptosis in RAW264.7 cells.

To investigate the protective role of ST in sepsis-induced liver damage, we challenged mice with a lethal dose of LPS. The result confirmed that ST pre-treatment dose-dependently improved the clinical symptoms in the ALI mice (Supplementary Figure S2). The appropriate dose of ST to attenuate the symptoms of ALI was 5 mg/kg, which was used for subsequent experiments. Subsequently, mice were randomly divided into seven groups (n = 6 per group). The experimental groups are shown in Figure 2A. After the establishment of the ALI model, the 48 h survival rate of mice in the control and ST groups was 100%. Mice began to die 12 h after LPS injection, and the survival rate was zero at 48 h. In the ST + LPS group, mice began to die 30 h after LPS injection. ST pre-treatment significantly prolonged the survival time in the ST group compared to that in the LPS group (Figure 2B). Pathological examinations were performed to determine the effects of ST on hepatic injury (Figure 2C). The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) significantly increased in the LPS group compared to those in the control group. ST pre-treatment significantly inhibited ALT and AST activities in the ST group compared to those in the LPS group (Figures 2D,E). The results of liver weight, body weight, and liver index showed that pre-treatment with ST markedly attenuated LPS-induced liver weight and liver index upregulation (Figures 2F–H). PAS staining of liver sections was performed to evaluate the effect of ST pre-treatment on the restoration of functional hepatocytes in the LPS-damaged liver. PAS staining demonstrated that mice treated with ST showed an uneven distribution of liver glycogen caused by LPS (Figure 2I). These results illustrate that ST plays a protective role against LPS-induced liver injury.

Inflammation is one of the main causes of liver damage during sepsis (Feng et al., 2017). Previous reports have shown that the LPS-induced inflammatory response in the liver may be due to pyroptosis (Hu et al., 2020). H&E staining was used to analyze the inflammatory infiltration of the liver. In the control group, liver tissue revealed a normal hepatic architecture (Figure 3A). However, in the LPS group, the inflammatory infiltration of the liver was significantly increased. Mice pre-treated with ST presented less inflammatory infiltration (Figure 3A). The serum levels of IL-1β and IL-18 were significantly increased in the LPS group compared to those in the control group, and these increases were inhibited by ST pre-treatment (Figures 3B,C). We investigated the mRNA levels of inflammatory cytokines that mediate the development of liver injury. RT-qPCR analysis revealed that LPS injection also dramatically upregulated the expression of pro-inflammatory factors in the liver, including IL-18, IL-6, IL-10, MCP-1, and TNF-α. As expected, ST significantly reduced the expression of these inflammatory mediators (Figures 3D–I). ST inhibited the LPS-induced recruitment of neutrophils (Figure 3J). These results indicate that ST could inhibit the hepatic inflammatory response during ALI in mice.

To clarify whether ST suppresses ALI by inhibiting caspase-11 activation and pyroptosis, we evaluated the expression levels of pyroptosis-related proteins in the liver using western blotting. The results showed that the levels of caspase-11, GSDMD-NT, NLRP3, ASC, caspase-1, IL-1β, and IL-18 were increased in sepsis mice and that ST pre-treatment suppressed this effect (Figure 4A). Caspase-11 was highly expressed in the liver of sepsis mice, and ST pre-treatment remarkably inhibited caspase-11 expression (Figure 4B). Furthermore, compared with the control group, LPS remarkably increased the number of TUNEL-positive cells in the liver, whereas ST pre-treatment significantly reduced these numbers (Figure 4C).

ST is an inhibitor of class I PI3K isoforms and mTOR kinase (Smith et al., 2016). ST can inhibit PI3K/AKT/mTOR signaling, which can be reversed by IGF-1 (Figure 5A). The PI3K/AKT/mTOR pathway has attracted extensive attention as a modulator of autophagy (Xu et al., 2020). We first explored whether ST inhibited pyroptosis by inducing autophagy. Interestingly, the western blotting results showed that ST pre-treatment did not increase the expression of autophagy-related proteins (Supplementary Figure S3A), indicating that autophagy does not participate in the inhibitory effect of ST. Studies have shown that protein ubiquitination has a regulatory effect on the activation of inflammasomes and non-classical inflammasomes. To further elucidate the underlying mechanisms, we verified the level of caspase-11 ubiquitination in liver tissues. The ubiquitination levels of caspase-11 were highly increased and were sustained at high levels in the LPS + ST group compared with those in the LPS group (Figure 5B). Hence, ST may be involved in the regulation of related signaling pathways and of ubiquitin-mediated pyroptosis. Further, IGF-1 inhibited the high ubiquitination levels of caspase-11 in the LPS + ST group (Figure 5C). To this end, we performed double IF staining with caspase-11 and ubiquitin antibodies in the liver. Double IF analysis revealed the co-localization of caspase-11 with ubiquitin in the liver (Figure 5D). The above studies demonstrate that ST promotes the ubiquitination of caspase-11 through PI3K/AKT/mTOR signaling.

Nedd4 plays important regulatory roles in adaptive immunity. In particular, it was previously found that Nedd4 controls non-canonical inflammasome activation through polyubiquitination and subsequent degradation of caspase-11 (Liu et al., 2019). IHC, western blot, and RT-qPCR analysis demonstrated that the expression of Nedd4 was upregulated in the LPS + ST group compared with that in the LPS group and that IGF-1 inhibited the increase of Nedd4 (Figures 6A–C). Thus, we hypothesized that caspase-11 ubiquitination by Nedd4 might inhibit non-canonical inflammasome activation. To test this hypothesis, we used co-immunoprecipitation experiments, and double IF staining revealed that Nedd4 interacted with caspase-11 in the liver (Figures 6D,E).