The murine macrophage cell line RAW 264.7 and the murine microglial cell line BV2 were purchased from Procell Life Science & Technology Co., Ltd. (cat. nos. CL-0190 and CL-0493, respectively). RAW 264.7 and BV2 cells were cultured in RAW 264.7-specific medium (cat. no. CM-0190; Procell Life Science & Technology Co., Ltd.). and BV2-specific medium (cat. no. CM-0493; Procell Life Science & Technology Co., Ltd.) with 10% FBS and 1% P/S antibiotics, respectively. Cells were incubated in a humidified incubator at 37˚C with 5% CO₂.

Cells were treated with TTC (H20084308, Chengdu Tiantaishan Pharmaceutical Co. Ltd.) at a range of concentrations from 100 to 400 µM for 24 h at 37˚C, with or without pretreatment with the caspase-1 inhibitor, Belnacasan (Beln) (10 µM; cat. no. HY-13205; MedChemExpress) and/or the caspase-11 inhibitor, Wedelolactone (Wede) (20 µM; cat. no. HY-N0551; MedChemExpress) for 30 min at 37˚C to induce the macrophages, LPS (10 µg/ml; cat. no. L6529; Sigma-Aldrich; Merck KGaA) was added 6 h before TTC treatment.

RAW 264.7 and BV2 cells were cultured in 100 mm plates (cat. no. 704002; Wuxi NEST Biotechnology Co., Ltd.) in a humidified incubator at 37˚C with 5% CO₂. Once the cells reached the logarithmic growth phase, the medium was replaced with fresh medium containing TTC at concentrations ranging from 100-400 µM for 24 h at 37˚C. The morphology and confluence of the cells were monitored with a light microscope.

The cells were homogenized in RIPA buffer (cat. no. P0013C; Beyotime Institute of Biotechnology), and the protein concentration was determined using a bicinchoninic acid protein assay kit (cat no. PC0020; Beijing Solarbio Science & Technology). Equal quantities of cell lysate (20 µg/lane) were separated by SDS-PAGE on a 10 or 12% gel and then transferred to a 0.45-µm polyvinylidene difluoride membrane. Membranes were blocked with 5% skim milk at room temperature for 1 h, then incubated with primary antibodies against GSDMA (cat no. sc-376318; 1:100; Santa Cruz Biotechnology, Inc.), GSDMC (cat. no. 27630-1-AP; 1:1,000; Proteintech Group, Inc.), GSDMD (cat. no. ab209845; 1:1,000; Abcam), GSDME (cat. no. ab215191; 1:1,000; Abcam), caspase-1 (cat. no. ab179515; 1:1,000; Abcam), caspase-11 (cat. no. ab180673; 1:1,000; Abcam), caspase-3 (cat. no. 9662; 1:1,000; Cell Signaling Technology, Inc.), and β-actin (cat. no. 66009-1-Ig; 1:10,000; Proteintech Group, Inc.) at 4˚C overnight. Subsequently, the membranes were washed with 1X TBST (0.5% Tween) three times for 10 min and incubated with HRP-conjugated Affinipure goat anti-mouse IgG (H+L) secondary antibodies (cat. no. SA00001-1; 1:10,000; Proteintech Group, Inc.) or HRP-conjugated Affinipure goat anti-rabbit IgG (H+L) secondary antibodies (cat. no. SA00001-2; 1:10,000; Proteintech Group, Inc.) for 1 h at room temperature. After washing with 1X TBST three times for 10 min, the bands were visualized using the ECL FemtoLightChemiluminescence kit (cat. no. PE100; Chengdu Wanda Biotechnology Development Co., Ltd.). To better show the expression levels of the full-length and cleaved forms of the detected proteins, both short and long exposure images are presented in the present study. For relative protein expression quantification, the integrated optical density of the protein bands was assessed using ImageJ software (version 1.31; National Institutes of Health).

Cell viability was measured using the CCK-8 assay kit (Dojindo Laboratories, Inc.) according to the manufacturer's instructions. RAW 264.7 and BV2 cells were seeded in 96-well plates (1x10³ cells/well) and treated with 100-400 µM TTC, then incubated at 37˚C with 5% CO₂. After 24 h, 10 µl CCK-8 reagent was added to each well, and the cells were further incubated for 1 h at 37˚C. Subsequently, the absorbance of samples was measured at 450 nm using a microplate reader.

ELISA was used to measure the levels of IL-1β present in the cell culture medium The cell culture medium was centrifuged at 1,000 x g for 10 min at 4˚C and the levels of IL-1β present were measured using the mouse IL-1β ELISA Kit (cat. no. KE10003; Proteintech Group, Inc.) according to the manufacturer's protocol. A total of 200 µl cell culture medium was loaded into each well of the ELISA plate. The absorbance of each sample was measured at 450 nm using a microplate reader. Standard curves were established using IL-1β standard samples provided in the kit and the concentration of the cytokines in the collected medium was measured.

The release of LDH from the cells was measured using the LDH cytotoxicity assay kit (cat. no. C0016; Beyotime Institute of Biotechnology). RAW 264.7 and BV2 cells were seeded in 96-well plates (1x10³ cells/well) and treated with 100-400 µM TTC for 24 h at 37˚C. After treatment, cells were centrifuged at 400 x g for 5 min at 4˚C, then 120 µl supernatant was collected and analyzed using the assay kit according to the manufacturer's instructions. Briefly, 60 µl LDH determination working solution was added to the supernatant and incubated for 30 min at 37˚C. Subsequently, the absorbance of samples was measured at 490 nm using a microplate reader. All experiments were performed in triplicate.

The study was approved by the Ethical Approval for Research Involving Animals in Southwest Medical University (Luzhou, China; approval no. SWMU20220028) and all animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. A total of 40 male C57BL/6 mice (weight, ~20 g; age, 6-8 weeks) were purchased from Chongqing Tengxin Biotechnology Co., Ltd., and were kept in the Experimental Animal Center of Southwest Medical University. The animals were supplied with food and water ad libitum and maintained in a 12/12-h light/dark cycle for 2 weeks (ambient temperature, 22-26˚C; relative humidity, 40-60%). Mice were intraperitoneally injected with TTC (2.5 mg/kg) or an equal volume of physiological saline once a day for 3 consecutive days. Then, PMs were isolated as previously reported (18). First, the mice were euthanized by cervical dislocation and soaked in 70% ethanol for 3 min, then transferred to a clean bench. Subsequently, a small incision was made along the ventral aspect of the mouse abdomen with a sterile scalpel and the abdominal skin was carefully removed to expose the peritoneum. Subsequently, using a 5-ml syringe and a needle, 5 ml RPMI medium 1640 (Gibco; Thermo Fisher Scientific, Inc) supplemented with 1% penicillin/streptavidin was injected into the abdomen. The abdomen was gently massaged and the medium was recovered into a 15-ml tube. Then, the medium was centrifuged for 5 min at 4˚C at 310 x g to pellet the cells. The supernatant was discarded and the cells were resuspended in 1 ml complete RPMI medium. Next, the collected cells were transferred to cell plates and cultivated in complete RPMI medium in a humidified incubator with 5% CO₂ at 37˚C. After being incubated for 1 h, only macrophages adhered to the culture dish. The culture supernatant containing other cells was removed and replaced with fresh culture medium. Then macrophages were then used for the subsequent experiments.

Statistical analysis was performed using GraphPad Prism software (version 8.0; Dotmatics). Each experiment was performed in triplicate. Data are presented as the mean ± standard error of the mean. Statistically significant differences were determined by one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The cytotoxicity of various concentrations of TTC in RAW 264.7 and BV2 cells was evaluated. Cell viability was measured using a CCK-8 assay and the morphology of cells was observed using a light microscope. The viability of both RAW 264.7 and BV2 cells was significantly decreased by TTC in a dose-dependent manner (Fig. 1A and B). Furthermore, both RAW 264.7 and BV2 cells treated with >200 µM TTC for 24 h underwent morphological alterations. The dying cells showed marked swelling with large bubbles emanating from the plasma membrane (Fig. 1G). These morphological changes were reminiscent of pyroptosis, as reported previously (15). In addition to morphological changes, pyroptosis is characterized by the release of certain inflammatory factors. The levels of secreted LDH and IL-1β were examined in the present study (Fig. 1C-F). These results demonstrated that the secretion of both LDH and IL-1β was significantly increased after TTC treatment and this effect was TTC-dose-dependent. As the release of LDH and IL-1β have been perceived as hallmarks of pyroptosis (15), combined with the morphological changes after TTC treatment, these results suggested that TTC could induce pyroptosis in RAW 264.7 and BV2 cells.

The molecular mechanism by which TTC could induce macrophage pyroptosis was subsequently examined. It has previously been reported that the GSDM protein family is the dominant effector of pyroptosis (15). To determine which GSDM protein exerts TTC-induced pyroptosis, the protein expression levels and the cleavage status of the GSDM protein family (GSDMA, GSDMC, GSDMD and GSDME) in RAW 264.7 and BV2 cells were determined by western blotting (Fig. 2A and B). The full-length GSDMD protein expression levels were decreased, whereas GSDMD N-terminal fragment (GSDMD-N), the fragment that possesses the pore-forming activity, was increased in macrophages treated with 400 µM TTC. However, GSDMA, GSDMC and GSDME demonstrated no change in protein expression levels upon TTC treatment. These findings suggested that GSDMD, the most frequently reported effector of pyroptosis (18), could be involved in TTC-induced pyroptosis, and TTC may influence the cleavage of GSDMD, rather than its total expression. Therefore, TTC-induced macrophage pyroptosis may be mediated by GSDMD cleavage.

GSDMD-dependent pyroptosis is regulated through the canonical inflammasome pathway by caspase-1 activation, and the noncanonical inflammasome pathway by caspase-11 in mice and caspase-4/-5 in humans (18). In the present study, the involvement of GSDMD in TTC-induced pyroptosis was demonstrated; therefore, further examination of the expression of caspase-1 and caspase-11 by western blotting was performed (Fig. 2A and B). These results showed that the protein expression levels of both cleaved caspase-1 (P20 and P12) and caspase-11 were upregulated after TTC stimulation, suggesting the involvement of both canonical and non-canonical inflammatory pathways. Additionally, expression of cleaved caspase-3 was also upregulated by TTC (Fig. S1A and B), suggesting that TTC-induced cell death may be caused by apoptosis in addition to pyroptosis. This is consistent with a previous study that reported apoptosis as a major mechanism of cell death caused by TTC in HCEP cells (8).

To validate these results in the present study, cells were treated with the caspase-1 inhibitor Beln and the caspase-11 inhibitor Wede. The inhibitory activity of Beln and Wede on functional caspase-1 and caspase-11, respectively, was confirmed. The results demonstrated that Beln treatment alone had no significant effect on the protein expression levels of caspase-1, but significantly decreased TTC-induced caspase-1 cleavage (Fig. 3A and B). Additionally, Wede caused a significant decrease in TTC-induced caspsase-11 cleavage (Fig. 3C and D). Inhibition of TTC-induced pyroptosis by Beln and Wede was examined by pretreating cells with the inhibitors for 30 min, followed by TTC exposure. These results demonstrated that both Beln and Wede significantly abrogated the decrease of cell viability (Fig. 4A and B), LDH release (Fig. 4C and D), IL-1β release (Fig. 4E and F) and the upregulation of GSDMD-N induced by TTC (Fig. 4G and H). Moreover, treatment of cells with both Beln and Wede significantly inhibited the vast majority of GSDMD expression, indicating a synergistic effect in reversing TTC-induced pyroptosis compared with treatment using a single inhibitor (Fig. 4G-J). These results suggested that TTC-induced pyroptosis may be mediated by both canonical and non-canonical inflammatory pathways in macrophages.

LPS is known to induce pyroptosis in macrophages (27). In the present study, the mechanism of induction of macrophage pyroptosis by TTC and LPS were compared. The results showed that TTC or LPS treatment of both RAW 264.7 and BV2 cells demonstrated a significant decrease in cell viability (Fig. 5A and B), significant increases in the secretion of LDH (Fig. 5C and D) and IL-1β (Fig. 5E and F), and GSDMD cleavage (Fig. 5G and H). However, when caspase-1/11 was detected, both TTC and LPS could induce caspase-1/11 cleaveage, but the cleavage fragments were not the same (Fig. 5I and J). Compared with LPS, TTC treatment influenced caspase-1 and caspase-11 cleavage, rather than upregulating the whole protein. In addition, it was demonstrated that the cleaved fragments of caspase-1/-11 induced by TTC differed from those induced by LPS, as the cleavage fragments of caspase-1/-11 generated by TTC and LPS were located at different positions on the western blot membrane, suggesting the mechanism of TTC-induced pyroptosis might not be the same as that of LPS. Further investigation is required to identify the mechanism of action of the different cleavage pattern induced by TTC treatment demonstrated in the present study and how TTC interacts with these inflammatory caspases.

Next, the potential for TTC to induce pyroptosis in vivo was investigated. TTC or an equal volume of physiological saline was intraperitoneally injected into mice and the PMs were extracted to analyze the protein expression levels of GSDMD. GSDMD-N was upregulated in PMs from the TTC group compared with control group, suggesting that TTC induced macrophage pyroptosis in vivo (Fig. 6).