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Introduction

Local anesthetics exert their analgesic effect by blocking Na+ channels, thereby affecting the conduction of nerve impulses. They are widely used in clinics as they are safe, exhibit fewer complications than general anesthetics and have minimal impact on physiological functions. However, side effects of local anesthetics can occur, such as local anesthetic allergy and toxicity, which may impact the cardiovascular and central nervous systems (1). These side effects are a result of a combination of adverse effects on ionotropic and metabotropic cell signaling, as well as energy transduction (2). The neurotoxic effects of local anesthetics may include apoptosis, inhibition of voltage-dependent calcium channels, calcium depletion in the endoplasmic reticulum, mitochondrial dysfunction and DNA damage (3,4). Nevertheless, the mechanism underlying toxicity exerted by local anesthetics requires further study.

Tetracaine hydrochloride (TTC) is a long-lasting local anesthetic commonly used for topical anesthesia, which is water-soluble, has strong penetrability and a good anesthetic effect (5). However, TTC has a higher cytotoxicity compared with other local anesthetics due to its high permeability (6). Previous studies have reported that TTC exerts a cytotoxic effect on certain cell types, such as human corneal stromal cells and human corneal epithelial cells, inducing apoptosis and necrosis (7,8). Koizumi et al (9) reported that large doses of intrathecal TTC increase the concentration of glutamate in cerebrospinal fluid, thereby causing neuronal injury in rabbits. In addition, AMPA receptor activation was revealed to be involved in TTC-induced neurotoxicity in the spinal cord. Song and Fan (7) reported that TTC-induced apoptosis may be triggered through Fas death receptors and mediated by Bcl-2 family proteins, in a mitochondria-dependent pathway. Pang and Fan (8) reported TTC-induced human corneal epithelial (HCEP) cell apoptosis via a death receptor-mediated mitochondrion-dependent pathway. Werdehausen et al (6) reported that low concentrations of TTC induced the apoptosis of human T-lymphoma cells, whereas a higher concentration of TTC induced necrosis. However, the effect of TTC on macrophages has not currently been fully revealed. Furthermore, whether TTC cytotoxicity involves mechanisms such as pyroptosis is also yet to be elucidated.

Macrophages are specialized, long-lived, phagocytic cells of the innate immune system that are widely distributed in certain tissues and organs of the body, such as the lungs, liver and brain. Macrophages have myriad functions, including eliminating pathogens, promoting tissue development and wound repair, and regulating immunity, metabolism and apoptosis. Additionally, they serve a crucial role in environmental homeostasis and the inflammatory microenvironment (10). A number of previous studies on the central nervous system toxicity of local anesthetics focused on neurons, rather than the immune system (11,12). The present study used a central nervous system immune cell line, the murine microglial cell line BV2, which has a dual effect on neurons. First, they can protect neurons by phagocytosing pathogens and other harmful agents present in the brain tissue. Secondly, under the stimulation of inflammatory factors, they can be activated and secrete inflammatory cytokines, thereby exerting a toxic effect on neurons (13). In addition, the murine macrophage cell line RAW 264.7 and mouse peritoneal macrophages (PMs) were used in the present study to investigate the relationship between TTC and macrophages.

Pyroptosis, also known as gasdermin (GSDM)-mediated programmed necrosis, is a form of pro-inflammatory programmed cell death (PCD) triggered by perturbations of extracellular or intracellular homeostasis related to innate immunity (14). It is initiated by inflammasome activation, which serves a critical role in the defense of hosts against danger signals (9). Pyroptosis is induced by members of the GSDM superfamily, which consist of GSDMA, B, C, D and E, and DFNB59; however, there is no GSDMB gene encoded in the mouse genome. Except for DFNB59, all of the GSDM protein family members possess an N-terminal pore-forming domain and a C-terminal auto-inhibitory domain (15,16). The GSDM proteins initiate proinflammatory cell death via their pore-forming domain after its cleavage by upstream inflammatory caspases (15,16). GSDMD and GSDME are GSDM family proteins that have been widely studied in the context of pyroptosis (17,18). GSDMD-dependent pyroptosis is regulated through a canonical inflammasome pathway, which is activated by caspase-1. Similarly, activation of murine caspase-11 and human caspase-4/-5 is involved in the noncanonical inflammasome pathway (18). GSDME-dependent pyroptosis is activated by apoptotic caspase-3(17). Through the release of IL-1β and IL-18, pyroptotic cells recruit additional inflammatory cells and induce a cascade of inflammatory responses (19). Pyroptosis is characterized by the formation of a plasma membrane pore, swelling of the cell, rupture of the plasma membrane and the subsequent release of intracellular contents (15). This form of cell death is mainly observed in professional phagocytes, such as macrophages, monocytes and dendritic cells, but emerging evidence suggests that pyroptosis can also be induced in cancer cells (20). Previous studies have reported that pyroptosis serves a significant role in macrophage-induced inflammation (21,22). Inflammatory responses are beneficial to humans as they help remove pathogenic microorganisms and antagonize infection, and the release of cytokines may contribute to tissue angiogenesis (23). However, hyperactivated pyroptosis can result in the induction of a substantial inflammatory cascade leading to tissue and organ damage, thereby causing inflammatory diseases, such as lung inflammatory disease, cardiovascular disease and kidney disease (24).

Previous reports have indicated that TTC can cause allergic reactions and local anesthetics may have antibacterial effects, suggesting a potential effect of TTC on macrophages (25,26). However, the effects of TTC on macrophages and whether pyroptosis is involved in this process have not yet been reported. The present study aimed to find the relationship between TTC and macrophage pyroptosis, and provide a novel mechanism underlying the local anesthetic toxicity of TTC. The findings may point to future novel treatments for local anesthetic toxicity caused by TTC.

Materials and methods

Cell culture

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.

Light microscopy

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.

Western blotting

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 Counting Kit-8 (CCK-8) assay

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

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.

Lactate dehydrogenase (LDH) release assay

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.

Extraction of murine PMs

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

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.

Results

TTC induces pyroptosis in macrophages

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.

Figure 1 Effects of TTC on macrophages. RAW 264.7 and BV2 cells were treated with TTC at a range of concentrations for 24 h. The viability of TTC-treated (A) RAW 264.7 and (B) BV2 cells was determined using the Cell Counting Kit-8 assay. Release of LDH from TTC-treated (C) RAW 264.7 and (D) BV2 cells was measured using an LDH Cytotoxicity Assay Kit. Release of IL-1β from (E) RAW 264.7 and (F) BV2 cells was determined using a Mouse IL-1β ELISA Kit. (G) Morphology of RAW 264.7 and BV2 cells was monitored using light microcopy. The concentrations written on these images refer to the concentration of TTC used. The cells indicated by the red arrows are cells undergoing pyroptosis (magnification, x200). ****P<0.0001 vs. control. C, control; LDH, lactate dehydrogenase; TTC, tetracaine hydrochloride.

TTC induces macrophage pyroptosis mediated by GSDMD cleavage

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.

Figure 2 TTC induces pyroptosis through GSDMD via both canonical and non-canonical inflammatory pathways in macrophages. RAW 264.7 and BV2 cells were treated with TTC at a range of concentrations for 24 h. Protein expression levels of GSDMA, GSDMC, GSDMD, GSDME, caspase-1, caspase-11 and β-actin in (A) RAW 264.7 and (B) BV2 cells were determined by western blotting. TTC, tetracaine hydrochloride; GSDM, gasdermin; C, control.

GSDMD cleavage is regulated by both caspase-1 and caspase-11 in TTC-induced macrophage pyroptosis

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.

Figure 3 Effects of Beln and Wede on TTC-induced activation of caspase-1 and caspase-11 in macrophages. Caspase-1 protein expression levels were examined using western blotting in (A) RAW 264.7 and (B) BV2 cells pretreated with Beln for 30 min followed by 24 h treatment with TTC. Caspase-11 protein expression levels were examined using western blotting in (C) RAW 264.7 and (D) BV2 cells pretreated with Wede for 30 min followed by 24 h treatment with TTC. Protein expression levels were semi-quantified using ImageJ software. *P<0.05 vs. control; #P<0.05 vs. Beln group; &P<0.05 vs. Wede group; %P<0.05 vs. TTC group. TTC, tetracaine hydrochloride; Beln, Belnacasan; Wede, Wedelolaone.

Figure 4 GSDMD-induced pyroptosis occurs via both canonical and non-canonical inflammatory pathways in TTC-treated macrophages. (A) RAW 264.7 and (B) BV2 cells were treated with 400 µM TTC for 24 h following pretreatment with or without Beln and/or Wede for 30 min. Cell viability was determined using the Cell Counting Kit-8 assay. Release of LDH from (C) RAW 264.7 and (D) BV2 cells was measured using an LDH Cytotoxicity Assay Kit. Release of IL-1β from (E) RAW 264.7 and (F) BV2 cells was determined using a Mouse IL-1β ELISA kit. Protein expression levels of GSDMD and β-actin in (G) RAW 264.7 and (H) BV2 cells was determined by western blotting. (I and J) Protein expression levels were semi-quantified using ImageJ software. *P<0.05 vs. control; #P<0.05 vs. TTC group; &P<0.05 vs. TTC + Beln group; %P<0.05 vs. TTC + Wede group. TTC, tetracaine hydrochloride; Beln, Belnacasan; Wede, Wedelolaone; GSDM, gasdermin.

Regulatory mechanisms underlying TTC-induced pyroptosis differ from LPS-induced pyroptosis 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.

Figure 5 TTC-induced pyroptosis differed from LPS-induced pyroptosis in macrophages. RAW 264.7 and BV2 cells were treated with TTC and/or LPS for 24 h. Cell viability of (A) RAW 264.7 and (B) BV2 cells was determined using a Cell Counting Kit-8. Release of LDH from (C) RAW 264.7 and (D) BV2 cells was measured with LDH Cytotoxicity Assay Kit. Release of IL-1β from (E) RAW 264.7 and (F) BV2 cells was determined with a Mouse IL-1β ELISA Kit. Protein expression levels of GSDMD in (G) RAW 264.7 and (H) BV2 cells and, protein expression levels of caspase-1 and caspase-11 in (I) RAW 264.7 and (J) BV2 cells were determined using western blotting. *P<0.05 vs. control; #P<0.05 vs. LPS group. TTC, tetracaine hydrochloride; LPS, lipopolysaccharide; LDH, lactate dehydrogenase; exp, exposure; GSDM, gasdermin.

TTC induces pyroptosis in macrophages in vivo

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).

Figure 6 TTC induces pyroptosis in macrophages in vivo. Mice were intraperitoneally injected with TTC or PBS, then murine PMs were collected for western blotting. C, control, TTC, tetracaine hydrochloride; PM, peritoneal macrophage; GSDM, gasdermin.

Discussion

Previous studies have reported that TTC causes cytotoxicity in various cell types mainly through the induction of apoptosis, a type of PCD characterized by nuclear fragmentation, plasma membrane blebbing, cell shrinkage and formation of apoptotic bodies (7,8). The present study demonstrated that pyroptosis was the mechanism of cell death involved in TTC cytotoxicity (Fig. 7). Pyroptosis is a form of PCD characterized by the formation of a plasma membrane pore, swelling of the cell, swift plasma membrane disruption, and release of intracellular contents and pro-inflammatory cytokines. Although apoptosis and pyroptosis are different forms of PCD, certain pathological factors can induce both types of PCD as caspase-3 can cause a switch between apoptosis and pyroptosis (28).

Figure 7 Schematic diagram of the mechanism of TTC-induced macrophage pyroptosis. TTC initiates the canonical and non-canonical inflammatory pathways by activating caspase-1 and caspase-11. Activated caspase-1 and caspase-11 cleave GSDMD into GSDMD-C and GSDMD-N. Then, GSDMD-N oligomerizes to form GSDMD pores on the cell membrane to induce pyropotosis in macrophages. IL-1β is activated by caspase-1 and released through the GSDMD pores (solid lines with arrows). The treatment of caspase-1 and caspase-11 inhibtors Beln and Wede could significantly decrease TTC-induced caspase-1 and caspase-11 cleavage, respectively, subsequently abrogating the upregulation of GSDMD-N and macrophagic pyrotosis induced by TTC (dashed line). TTC, tetracaine hydrochloride; GSDM, gasdermin; GSDMD-C, GSDMD C-terminal fragment; GSDMD-N, GSDMD N-terminal fragment.

In the present study, an increase in the release of IL-1β from macrophages treated with 100, 200 and 400 µM TTC was demonstrated, while the pyroptosis-related proteins GSDMD-N, caspase-1 and caspase-11 were only detected in macrophages treated with 400 µM TTC. However, caspase-1 and caspase-11 inhibitors, Wede and Beln, reduced IL-1β release, which suggests that the release of IL-1β is associated with caspase-1 and caspase-11 expression, but is not wholly dependent on these cytokines. A similar trend was demonstrated by LDH release from TTC-treated cells. These results suggested that TTC could induce multiple cell death patterns in macrophages. In addition, necroptosis, similar to pyroptosis, is a lytic, inflammatory type of PCD, which is mainly mediated by MLKL proteins, and is characterized by cellular swelling, membrane rupture and cytoplasmic and nuclear disintegration (29). Furthermore, ferroptosis is an additional type of PCD that involves lipid peroxidation caused by iron accumulation and is characterized by mitochondrial condensation, reduced mitochondrial cristae and increased membrane density (30). NETosis is a unique form of PCD that occurs in response to various pathogens, cytokines and other physiological stimuli, and is characterized by the release of decondensed chromatin and granular contents into the extracellular space, which can form web-like structures known as neutrophil extracellular traps (31). However, it is currently unknown whether these forms of PCD, including necroptosis, ferroptosis and NETosis, are involved in TTC induced-cytotoxicity, and further in-depth studies are required to elucidate these underlying molecular mechanisms.

Local anesthetics, such as lidocaine, and general anesthetics, such as propofol and sevoflurane, have previously been reported to affect cell viability (32). Certain anesthetics can affect cell pyroptosis and related molecular mechanisms (Table I) (32-41). Combined anesthesia refers to the simultaneous or sequential use of multiple anesthetic drugs or techniques during surgical procedures to achieve the desired anesthetic state; it involves the synergistic effects of different drugs to enhance the effectiveness of anesthesia while minimizing side effects (42). Therefore, the different effects of certain anesthetics on the process of pyroptosis may provide some theoretical support for their use in combined anesthesia.

Table I Effects of different anesthetics on the process of pyroptosis.

Table I

Effects of different anesthetics on the process of pyroptosis.

First author, year	Anesthetic	Tissue/cell type	Disease	Effect	Mechanism of action	(Refs.)
Ding et al, 2021	Lidocaine	Spinal cord	Spinal cord injury	Promotion	Upregulates NLRP3/ASC/caspase-1	(32)
Ding et al, 2021	Dexmedetomidine	Spinal cord	Spinal cord injury	Suppression	Inhibits priming and inflammasome activation, and reduces pyroptosis via protein kinase C-δ phosphorylation	(32)
Shi et al, 2022	Remimazolam	Brain	Cerebral I/R injury	Suppression	Inhibits NLRP3 inflammasome-dependent pyroptosis	(33)
Ye et al, 2018	Ketamine	Primary mouse hippocampal neuron	Hippocampal neurotoxicity	Promotion	Promotes NLRP3/caspase-1 complex recruitment to mitochondria	(34)
Li et al, 2022	Esketamine	Primary mouse astrocyte	POCD	Suppression	Inhibits pyroptosis-associated proteins via inhibition of the STING/TBK1 signaling pathway	(35)
Sun et al, 2019	Propofol	Bone marrow-derived macrophages, J774	Propofol infusion syndrome	Promotion	Activates NLRP3/ASC/caspase-1 pathway via mitochondrial reactive oxygen species	(36)
Liu et al, 2020		NR8383	Acute lung injury	Suppression	Inhibits the expression of pyroptotic proteins and enhances the expression of sirtuin 1	(37)
Dai et al, 2021	Sevoflurane	Primary rat hippocampal neuron	Neuroinflammation, neurocognitive impairment	Promotion	Activates NF-κB-mediated pyroptosis	(38)
Deng et al, 2022		AC16	I/R injury	Suppression	Activates the AMPK/ULK1 pathway to trigger autophagic flux and suppress NLRP3-mediated pyroptotic cell death	(39)
Chen et al, 2022		H19-7	Ischemic brain injury	Suppression	Regulates the Mafb/DUSP14 axis to mitigate oxygen-glucose deprivation-induced pyroptosis	(40)
Zheng et al, 2022		RAW 264.7	Acute lung injury	Suppression	Phosphorylates and activates the GSK-3β to suppress pyroptotic cell death	(41)

[i] I/R, ischemia/reperfusion; POCD, postoperative cognitive dysfunction.

Activation of inflammatory caspases, such as murine or human caspase-1, or murine caspase-11 and its human homologs caspase-4/-5, preceding the induction of pyroptosis is a tightly regulated process (43,44). A previous study reported that caspase-1 is activated when the central scaffold of canonical inflammasomes, made of NLRP3, NLRP1, AIM2, NAIP-NLR4 and pyrin, detects its cognate ligands (45). Aberrant or excessive activation of caspase-1 can cause, or be associated with, certain autoinflammatory, autoimmune or metabolic diseases (44). In mice, the caspase-11 non-canonical inflammasome has been reported to sense infections caused by certain bacteria, such as Escherichia coli, Salmonella Typhimurium, Legionella pneumophila and Burkholderia thailandensis, by responding to the presence of cytoplasmic LPS (46). Additionally, caspase-11 activation serves an important role in the mediation of endotoxic shock and sepsis (47). In the present study, TTC-induced macrophage pyroptosis was demonstrated to be mediated through GSDMD cleavage by both caspase-1 and caspase-11. Furthermore, as the cleavage fragments of caspase-1/-11 generated after TTC and LPS treatment showed different patterns, the cleaved caspase-1 and caspase-11 caused by TTC and LPS may be different The mechanism of action of how TTC interacts with inflammatory caspases and whether it participates in other cellular pathways has yet to be explored. In addition, the alternative mechanism of TTC-induced macrophage pyroptosis demonstrated in the present study may influence the future development of therapeutics that target pyroptosis. As GSDMD cleavage is definitive, the development of therapies targeting GSDMD may a better alternative than targeting inflammatory caspases.

Previous studies on the central nervous system toxicity of local anesthetics have focused on the effects of anesthetics on neurons as opposed to the immune system (11,12). The present study used the BV2 central nervous system immune cell line, which exerts a dual effect on neurons. As the present study demonstrated that TTC could cause BV2 pyroptosis, it could be suggested that TTC can indirectly exert toxic effects on neurons by inducing BV2 pyroptosis to release inflammatory factors, which can further trigger central nervous system toxicity Traditionally, benzodiazepines are used to antagonize the symptoms of local anesthetic poisoning by enhancing the inhibitory effects of γ-aminobutyric acid-ergic neurons in the limbic system (48). However, these drugs cause certain side effects, which in severe cases can affect the cognitive function of the patients (48). Presently used drugs such as disulfiram (49), dimethyl fumarate (50) and Prussian blue nanozyme (51) inhibit the pyroptotic pathway to alleviate diseases, such as familial Mediterranean fever, experimental autoimmune encephalitis and neurodegeneration. Hence, novel drugs that target the pyroptosis pathway could potentially provide a new future direction for the prevention and treatment of local anesthetic toxicity.

In conclusion, the present study demonstrated that the local anesthetic TTC induced macrophage pyroptosis through GSDMD cleavage and was mediated by both canonical and non-canonical inflammatory caspases. These results could potentially provide a promising strategy for the future prevention and treatment of local anesthetic toxicity.

Supplementary Material

TTC induces apoptosis in macrophages. RAW 264.7 and BV2 cells were treated with TTC under indicated concentrations for 24 h. (A and B) Caspase-3 and β-actin as an internal control were analyzed by western blotting in (A) RAW 264.7 and (B) BV2 cells.

Acknowledgements

Not applicable.

Funding

Funding: This study was supported by funding from the Sichuan Science and Technology Program (grant nos. 2022NSFSC1594 and 2022YFS0632), the Joint Foundation of Luzhou Government and Southwest Medical University (grant no. 2021LZXNYD-D08) and the Scientific Research Foundation of Southwest Medical University (grant no. 2021ZKZD011).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

RZ and WG performed the experiments and drafted the manuscript. PY, ZQ, DZ, JJ and LL prepared materials, and collected and analyzed data. XJ and JF contributed to the design of the work, revised the manuscript and approved the final version of the manuscript to be published. All authors read and approved the final manuscript. RZ and JF confirm the authenticity of all the raw data.

Ethics approval and consent to participate

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.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References