Animals

Male C57BL/6J mice aged 8–10 weeks and weighing 20–25 g were obtained from Hunan Slack Jingda Experimental Animal Breeding Co., Ltd (Changsha, China). The mice were housed in the Animal Experimental Center of Central South University, with a controlled temperature range of 22–24 °C, humidity, and a 12-h light/dark cycle. They were provided with standard diet and water ad libitum. All animal protocols were conducted in accordance with the guidelines for experimental animal welfare ethics established by the Chinese Society of Laboratory Animals. The care of animals and experimental procedures were approved by the Animal Experimental Center of Central South University.

Experimental animal models

Polymicrobial sepsis was induced by administering a single intraperitoneal dose of lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Sigma Chemical, MO, United States) at a dosage of 20 mg/kg. To investigate the effects of acute O-GlcNAc increase in mice with LPS-induced sepsis, Thiamet-G (TMG) was administered intravenously at a dosage of 600 µg/kg, 12 h prior to LPS treatment. Appropriate control groups were included, with vehicle administration (for LPS and TMG) at the corresponding time points. The animals were randomly assigned to one of the following four groups: saline control (Control), vehicle administration followed by LPS treatment (LPS), TMG administration followed by LPS treatment (LPS + TMG), and TMG administration followed by saline treatment (TMG).

Measurement of organ blood perfusion

Ten hours after intraperitoneal injection of LPS, we assessed blood flow perfusion in the liver, mesentery, and lower limb using laser Doppler imaging. The procedure for measuring organ blood flow perfusion was conducted as previously described [8]. Mice were anesthetized with intraperitoneal pentobarbital sodium at a dosage of 20 mg/kg body weight. They were then placed on a light-absorbing pad with an electric heating blanket set to maintain a temperature of 37 °C. Laser Doppler imaging was performed using the PeriScan PIM3 system from Somnotec (Singapore), following the manufacturer's instructions.

To measure blood perfusion, a midline abdominal incision was made, and the right lobe of the liver was carefully exposed. Laser Doppler imaging was used to monitor blood flow in this region. Additionally, a selected area of the mesentery was monitored for blood flow. Throughout the procedure, the mice's core temperature was maintained at 37 °C using a heat lamp.

Cell culture and stimulation

Human umbilical vein endothelial cell lines (HUVECs) and human kidney fibroblast HEK 293T cells were obtained from the American Type Culture Collection (ATCC). HUVECs were cultured in DMEM-F12 medium, while HEK 293T cells were cultured in DMEM. Both culture media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. HUVECs were plated on six-well plates and exposed to a series of LPS solutions (0, 0.2, 0.4, 0.6, 0.8, 1.0 µg/ml) obtained from InvivoGen (# tlrl-3pelps) for 24 h. The protein levels of O-GlcNAc and GSDMD were assessed by western blotting.

Lactate dehydrogenase assay

The HUVECs were seeded in a 6-well culture plate one day prior to the experiment and incubated for 24 h. Subsequently, the cells were transferred to 96-well plates. For transfection, the cells were treated with Lipofectamine 2000 reagent (Invitrogen) and transfected with 1 µg/ml of LPS. The transfection was allowed to proceed for 24 h. Cell death was measured using a lactate dehydrogenase (LDH) assay obtained from Abcam, following the instructions provided by the manufacturer.

Immunofluorescence staining

To prepare frozen aorta tissues, an optimum cutting temperature compound was injected gently through the trachea to maintain fluid in the lungs. The aorta tissues were then frozen, and sections of 5 µm thickness were cut using a cryostat microtome. These sections were mounted on microscope slides. The slides were fixed with 4% paraformaldehyde to preserve the tissue structure and permeabilized with 0.1% Triton X-100 to allow antibody penetration. Primary antibodies specific to CD31 were applied to the slides, followed by incubation with fluorescence-conjugated secondary antibodies. The slides were visualized under a Zeiss fluorescence microscope at a magnification of 40×, and images were captured for further analysis and evaluation.

Co-immunoprecipitation assay

To lyse HUVECs, cell lysis buffer for Western blot and IP (P0013, Beyotime Biotechnology, Shanghai, China) was used. The cell extracts were treated with Protein A/G agarose beads (Santa Cruz Biotechnology, sc-2003) to remove nonspecifically bound proteins during the immunoprecipitation process. The beads were pelleted by centrifugation, and the supernatant (cell lysate) was transferred to a fresh centrifuge tube kept on ice. Protein A/G agarose beads were added to the cell lysate again, and immunoprecipitation was performed using either anti-GSDMD or anti-caspase-4 antibodies overnight at 4 ºC. The immunoprecipitates were collected by centrifugation, and the supernatant was carefully aspirated and discarded. The pellet was washed with 200 µl of wash buffer. After the final wash, the supernatant was aspirated and discarded, and the pellet was resuspended in 40–60 µl of 1 × electrophoresis sample buffer. The resuspended pellet was then subjected to Western blot analysis using primary antibodies specific to O-GlcNAc or GSDMD, respectively.

GSDMD O-GlcNAcylation site prediction

To predict the O-GlcNAcylation site of GSDMD, four O-GlcNAcylation site prediction sites were utilized. These prediction sites include the DictyOGlyc 1.1 server (http://www.cbs.dtu.dk/services/DictyOGlyc/) [35], NetOGlyc 4.0 server (http://www.cbs.dtu.dk/services/NetOGlyc/) [36], GPP prediction server (https://comp.chem.nottingham.ac.uk/cgi-bin/glyco/bin/getparams.cgi) [37], and GlycoMine (https://glycomine.erc.monash.edu/Lab/GlycoMine/#webserver) [38]. The raw data obtained from these four prediction sites underwent statistical analysis. A Venn diagram was created to determine the predicted set of sites that were identified as the most likely potential O-GlcNAcylation sites for GSDMD. This analysis allowed for a comprehensive evaluation of the predictions made by each site and the identification of common sites predicted by multiple prediction methods.

Plasmid and molecular cloning

The pCMV vector expressing Myc-tagged human OGT (PPL00887-2a) was purchased from PPL. To generate the GSDMD constructs, the gene sequence of GSDMD was obtained from the GenBank database. The template for cloning and construction experiments was obtained, and the necessary modifications were made. The FLAG-tagged GSDMD, FLAG-tagged human GSDMD-NT, FLAG-tagged human GSDMD (S338A) and human GSDMD (S480A) constructs were generated by GENECHEM. The cloning and construction experiments allowed for the creation of the desired mutant plasmids.

Cell transfection

HEK293T and HUVECs were cultured in DMEM or F-12 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, respectively. The cells were incubated in a humidified atmosphere containing 5% CO2. For transfection, cells were grown in 6-well plates until they reached 90% confluence. Plasmid DNA was added to 500 µl of medium, while Lipofectamine 2000 transfection reagent (Invitrogen) was added to another 500 µl of medium. After incubating for 5 min at room temperature, the two media were mixed and incubated for an additional 20 min. The cell culture medium was then replaced with the mixture of media containing the plasmid and Lipofectamine 2000.

Immunoblotting

Cultured HUVECs were collected and lysed with lysis buffer containing 98% RIPA lysis buffer, 1% protease inhibitor, and 1% phosphatase inhibitor. The cells were incubated with the lysis buffer on ice for 30 min. The total protein concentration in the lysate was measured using the BCA protein assay kit from Beyotime Biotechnology, Shanghai, China. Equal amounts of protein from each sample were loaded onto SDS-PAGE gels and separated by electrophoresis. The proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes and blocked with PBST containing 5% skim milk blocking buffer at room temperature for 2 h.

Primary antibodies, including O-GlcNAc (Cell Signaling Technology, #9875), GSDMD (Santa Cruz Biotechnology, sc-393581), GSDMD (Abcam, ab209845), β-actin (SAB, #21338), GAPDH (Proteintech), OGT (Santa Cruz Biotechnology, sc-74546), Myc Tag (Invitrogen, # R951-25), OGA (Proteintech, 14711-1-AP), were incubated with the membranes followed by incubation with HRP-conjugated secondary antibodies (anti-rabbit IgG from Bioword, and anti-mouse IgG from Bioword). Protein bands were detected using an Enhanced Chemiluminescent (ECL) reagent from Thermo Scientific, and the images were acquired with a ChemiDoc MP System from Bio-Rad. Densitometric analysis was performed using ImageJ Software to quantify the protein bands.

Statistics

All experiments were conducted with a minimum of three independent replicates to ensure robustness and reliability of the results. Statistical analysis was performed using GraphPad Prism 8 software. The data are presented as mean ± standard deviation (SD). To compare the mean values of biochemical data between different groups, a two-tailed Student’s t test was employed. For comparisons involving multiple time points, repeated-measures analysis of variance (ANOVA) was used, followed by Bonferroni post-tests. A significance level of P < 0.05 was considered statistically significant for all analyses.