2.1. Experimental Rats

We purchased 30 clean-grade Sprague–Dawley male rats, aged 5–7 weeks and weighing 120–150 g, from Shanghai Slack Laboratory Animal Co. Ltd. (license number SCXK (Shanghai) 2017-0005). The animals were housed in individual cages at the animal experiment center of the 900th Hospital of the Joint Logistics Support Force of the Chinese People’s Liberation Army, with five animals per cage. The animals were housed at a room temperature of 25^°C ± 2^°C and humidity of 60% ± 5%. The study was approved by the ethics committee of the 900th Hospital of the People’s Liberation Army Joint Service Support Force.

2.2. Experimental Design

All 30 rats underwent a 7-day adaptive running training with progressively increasing load. During the training, the room temperature was maintained at 25^°C ± 2^°C and the humidity was 60% ± 5%. By Day 7, the running speed was 29 m/min and the training time was 25 min. Twenty four Sprague–Dawley rats that successfully completed the running were selected and randomly divided into two groups, with 12 rats in each group. In the EHS group (EHSG), the rats ran on the treadmill with a speed of 29 m/s under the environment temperature of 35^°C ± 2^°C and humidity of 80% ± 10%. After the core body temperature reached the diagnostic criteria of EHS, running was stopped, while the control group (CG) did not undergo any running.

2.3. Automatic Biochemical Analyzer to Detect Liver Function

The blood samples were collected by gently piercing the rat’s inferior vena cava using a 5-mL syringe. Alanine transaminase (ALT) and aspartate transaminase (AST) levels were measured to assess liver function.

2.4. Preparation and Observation of Liver Tissue Section

The liver tissues were dehydrated using ethanol solution with different concentrations and then embedded in paraffin to create a paraffin block. Tissue sections, 5-μm thick, were sliced from the paraffin block, dewaxed with xylene and ethanol, stained with hematoxylin and eosin (H&E), and then observed under a light microscope.

2.5. IL-1β and IL-18 in Serum Were Detected by Enzyme-Linked Immunosorbent Assay (ELISA)

Serum levels of IL-1β and IL-18 were measured by ELISA kits purchased from Xitang Biological Technology Company. All procedures were performed according to manufacturer’s protocol.

2.6. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) for Caspase-1 and GSDMD in the Liver

Total RNA was extracted using QIAGEN RNeasy Mini Kit (Qiagen, Germany), and cDNA was synthesized with random hexamer primers and RevertAid reverse transcriptase. SYBR Universal PCR Master Mix (Thermo Scientific, United States) was used for amplification. GADPH was used as an internal reference for protein normalization, and the mRNA expression of each group was calculated by the 2^-ΔΔCt method.

2.7. Western Blotting (WB)

Fifty milligrams of liver tissue was ground into powder using liquid nitrogen and then lysed by RIPA lysis solution (containing 5% protease inhibitor). The samples were kept on ice for 15 min to ensure complete lyse, centrifuged at 12,000 rpm for 5 min at 4°C, and then the supernatant was collected. The bicinchoninic acid (BCA) method was used to measure protein concentration. After mixing the protein with 5x sodium dodecyl sulfate (SDS) protein buffer, it was denatured in a water bath at 100°C for 10 min. Twenty-five micrograms of the denatured protein was separated, and the protein was transferred to polyvinylidene fluoride (PVDF) by electrophoresis. The PVDF membrane was blocked at room temperature for 2 h, and primary antibody was incubated overnight. Afterward, the membrane was incubated with an horseradish peroxidase (HRP)–conjugated secondary IgG antibody for 2 h at room temperature. Enhanced chemiluminescence (ECL) method was used to visualize protein bands.

2.8. Statistical Analysis

Statistical analysis was conducted using SPSS 22.0 software. Measurement data from two independent samples with normal distribution were describe as mean ± standard deviation; data with a skewed distribution were described by median and quartile. For normally distributed data, the homogeneity of variances between two independent samples was assessed using the t-test. For data with unequal variances, Welch’s t-test was used. The Mann–Whitney U test was used for data with a skewed distribution. A p value of < 0.05 was considered statistically significant.

3.1. Animal Models

The rat models of EHS were successfully established. The rectal temperature of the rats exceeded 42.5°C, with an average rectal temperature of 42.6 (42.6°C, 42.8°C). The time required for the rats to reach EHS was 30.7 ± 7.0 min.

3.2. Changes of Hepatic Function

The results are represented in Table 1. Serum ALT levels in the EHSG were higher than those in the CG. The AST levels of the EHSG were significantly higher than those in the CG, with a statistically significant difference (Z = −2.194, p = 0.028).

3.3. Significant Pathological Changes Were Observed in the Livers of the Rats in the EHSG

Under a light microscope, the liver lobule structures in the CG (Figure 1(B)) were intact, with normal size and morphology of liver cells, and no cell signs of degeneration, necrosis, and inflammatory cell infiltration. In the EHSG (Figure 1(A)), hepatic lobules were disorganized; the hepatic sinusoids were congested; some hepatic cells showed necrosis with pyroptosis characteristics of pyroptosis, such as nuclear pyknosis, fragmentation, and lysis. In addition, inflammatory cell infiltration was observed around the central vein.

3.4. Serum Concentrations of Proinflammatory Cytokines Were Significantly Elevated in the EHSG

As shown in Table 2 and Figure 2, the serum concentration of IL-1β in the EHSG was 121.5 ± 51.9 pmol/L, which was significantly increased than in the CG (14.7 ± 11.4 pmol/L) (p < 0.001). The serum concentration of IL-18 in the EHSG was 18.0 ± 6.6 pmol/L, which was obviously elevated compared to the CG (6.1 ± 1.8 pmol/L) (p < 0.001). These differences are statistically significant.

3.5. The mRNA Expression of Pyroptosis-Related Genes Was Significantly Increased in the EHSG

As shown in Table 3 and Figure 3, the mRNA relative expression of Caspase-1 in the EHSG was 10.1 ± 2.6, markedly higher than in the CG (1.3 ± 0.9), with a statistically significant difference (p < 0.001). The relative expression of GSDMD mRNA in the EHSG was 20.8 ± 8.0, which was significantly higher than in the CG (1.3 ± 1.0), and the difference was statistically significant (p < 0.001).

3.6. Marked Increase in the Expression of Pyroptosis-Related Proteins Is Observed in EHS Liver Tissues

As shown in Figure 4 and Table 4, the expression level of Caspase-1 protein in the EHSG increased significantly (5.910 ± 1.620 vs. 1.050 ± 0.360), and the difference was statistically significant compared to the CG (p = 0.01). The expression level of GSDMD protein in the EHSG was markedly elevated (0.620 ± 0.12 vs. 0.370 ± 0.020), with a statistically significant difference compared to the CG (p = 0.009). In Figure 5, the protein expression level of cleaved Caspase-1 and cleaved GSDMD in the EHSG increased significantly.

With the intensification of the greenhouse effect, global climate is gradually getting warmer, and extreme weather events are becoming more frequent. The incidence of heat stress–related diseases, such as heat spasms, heat exhaustion, and heat stroke, has been steadily increasing, particularly among individuals engaged in production labor, military training, and physical exercise in hot climate, especially in tropical and subtropical regions where the summer temperature and humidity prevail. EHS is a severe illness that poses a significant risk to the health and life of patients. It is prone to cause secondary MODs, including rhabdomyolysis, disseminated intravascular coagulation, and acute liver and renal failure, resulting in a mortality rate of up to 35% [10].

In MODs caused by EHS, liver failure is not only common but also closely linked to the prognosis. Pyroptosis, also known as inflammatory necrosis, is another form of programmed cell death distinct from apoptosis and is mediated by Caspase-1. Pyroptosis is characterized by cytolytic phenomena such as cell swelling and membrane blistering, causing inflammatory responses—changes consistent with the pathological changes in the livers of EHS rats in our previous animal experiments [11]. Other animal experiments have also demonstrated that high levels of IL-1β were detected in both plasma and the central nervous system when core body temperature reached 42.5°C, and the trends in core body temperature change were consistent with the plasma IL-1β levels in the heat stroke mice [12–14]. These studies all suggest that cytokines play an important role in the occurrence and progression of EHS. IL-1β and IL-18 are the characteristic cytokines released in the pyroptosis pathway [15–17]. Therefore, we speculated that pyroptosis may be an important form of injury in the pathophysiology of EHS. To test this hypothesis, we established a rat model of EHS in a stimulated hot climate and conducted an exploratory study. The transcript and protein expression levels of Caspase-1 and GSDMD, key molecules in the pyroptosis pathway, as well as the serum levels of inflammatory cytokines including IL-1β and IL-18 in EHS rats were detected in liver tissues. The results showed that Caspase-1 and GSDMD expressions levels were significantly elevated in the liver tissues of EHS rats, and the serum levels of IL-1β and IL-18 were also significantly elevated. This finding suggests that the pyroptosis pathways plays an important role in EHS-induced liver injury and that pyroptosis may be one of the important mechanisms of EHS-induced liver injury.

Pyroptosis can be divided into canonical and noncanonical inflammatory pathways. The canonical inflammatory pathway is triggered when pattern recognition receptors (PRRs) detect pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) [18]. This activates the inflammasome, which in turn activates Caspase-1. Activated Caspase-1 cleaves GSDMD, releasing it from its autoinhibition state and forming the active GSDMD-N-terminal. This active form migrates to the plasma membrane and forms a pore approximately10–14 nm in size, leading to increased cell osmotic pressure and rupture due to swelling. At the same time, Caspase-1 promotes the maturation of IL-1β and IL-18, which together with other inflammatory factors are released through the GSDMD-N-terminal–formed membrane pores [7, 8, 19]. The noncanonical inflammatory pathway is activated by Gram-negative bacterial endotoxin lipopolysaccharide, which directly cleaves Caspase-4 or Caspase-5 (mediated by Caspase-11 in mice). This activation subsequently leads to the cleavage of GSDMD, further mediating pyroptosis [20].

Caspase-1 is activated from an inactive precursor with size of 45 kDa, which consists of three subunits: an N-terminal subunit of 15 kDa, a central subunit of 20 kDa, and a C-terminal subunit of 10 kDa [21, 22]. The pathogenesis of EHS is generally considered as a sepsis-like syndrome, leading to MOD, a concept accepted widely by scholars. Chen et al. [23] found that during septic acute liver injury, hepatocyte pyroptosis can aggravate acute liver injury and inhibit hepatocyte pyroptosis with NLRP3 and Caspase-1 inhibitors which can reduce the severity of septic liver injury. In our experiment, both the mRNA and protein expressions levels of Caspase-1 in the EHSG were significantly higher than those in the CG.

Numerous studies have identified GSDMD as the executor of pyroptosis [24–26]. Full-length GSDMD consists of an N-terminal domain and a C-terminal domain, connected by a flexible region in the middle [25]. In the resting state, intracellular GSDMD is an autoinhibited state due to the presence of the GSDMD-C terminal. Upon stimulated, activated Caspase-1 or Caspase-4/5/11 cleaves GSDMD, releasing it from this autoinhibitory state [24]. The GSDMD-N-terminal domain (cleaved GSDMD) then combines with phosphatidylinositol and cardiolipin on the cell membrane, forming pores in the plasma membrane [27, 28]. At the same time, GSDMD can further activate the inflammasome and execute the pyroptotic program. Our experiment results showed that the mRNA and protein expression levels of GSDMD in EHS rats were significantly higher than those in the CG. However, some studies have pointed out that the activation of GSDMD is not solely dependent on the cleavage of GSDMD-C-terminus; neutrophil elastase can also hydrolyze GSDMD to produce fragments with pore-forming function that mediate cell death [29, 30]. At the same time, it has been noted that other proteins of the gasdermin family, such as GSDME, can also mediate pyroptosis. Therefore, it is necessary to further explore other molecular mechanisms of GSDMD pore formation in pyroptosis, as well as other proteins involved in mediating pyroptosis.