Pathogen-free male Wistar rats approximately 7 weeks of age with an initial weight of 200–250 g were purchased from Sipeifu (Beijing, China) Biotechnology Co., Ltd. The rats were kept at an ambient temperature of 24 $\pm$ 1 °C and a relative humidity of 50 $\pm$ 10%, and a 12 h:12 h light/dark period was implemented. Adequate food and water were provided. To rule out the effects of circadian rhythms, all the experiments were conducted at roughly the same time each day (between 12:00 and 14:00). The ambient temperature, humidity and light conditions were kept consistent for each experiment, and noise was excluded as much as possible. All the experiments were double-blind and randomized using a random number table. Rats found to have health problems or abnormal behavior and rats that died before or during the experiment were excluded. No animals were excluded from this study. All experiments were conducted according to the ARRIVE 2.0 guidelines (https://arriveguidelines.org/arrive-guidelines) (Table 1). The experimental procedures were approved by Institutional Animal Care and Use Committee of Beijing Institute of Radiation Medicine (Ethics number: IACUCDWZX- 2022-742) and conducted according to the guidelines and regulations for the use and care of experimental animals in China.

In contrast to the heat acclimation model, which involves only a high-temperature environment without exercise, we propose a new HA training scheme in which rats are subjected to exercise simultaneously in a hot environment and verify the feasibility of this scheme for use with rats. The rats were randomly divided into a control group and an HA model group (n = 5). Sterile Body Cape pills (e-Celsius Performance, Bodycap company, Paris, France) were inserted into the abdominal cavity of anesthetized rats at least one week before heat acclimation training. The rat’s core body temperature (T${}_{\text{core}}$) is wirelessly delivered by the pills to the receivers via e-viewer performance devices (e-Celsius Performance, Bodycap company) during the establishment of the 21-day heat-exercise acclimation model. T${}_{\text{core}}$ recordings were sampled every five minutes. Buprenorphine (Specifications: 1 mL: 0.15 mg, Tianjin Pharmaceutical Research Institute Pharmaceutical Co., Ltd., Tianjin, China) (0.05–0.1 mg/kg intraperitoneally) was administered before the operation to minimize pain from the outset. Following the procedure, carprofen (C153919, Aladdin Biochemical Technology Co., Ltd., Shanghai, China) was given to manage pain during recovery. For pain scoring, we used a combination of behavioral and physiological indicators. These include changes in normal behavior (e.g., reduced activity, altered gait, changes in social interaction); physical signs (e.g., swelling, redness at the site of surgery); physiological measures (e.g., changes in respiratory rate); and specific pain-related behaviors (e.g., guarding the affected area) [31, 32]. The rats had at most mild pain. The rats then underwent two hours of heat-exercise training each day for 21 days in a row to reach the stage of heat acclimation. During the training, the rats were first placed in the prewarmed chamber (36 $\pm$ 1 °C and 55 $\pm$ 5%) for 30 min, then they ran on the chamber treadmill at a certain speed (slope 0) for 30 min and subsequently left in the chamber for 1 h while they moved freely. On the first day after HA training, the heat endurance of the rats was tested; that is, the rats in the control group and the HA group were placed in an environment of 39.5 °C for 30 minutes and then transferred to room temperature. The T${}_{\text{core}}$ changes in the rats were monitored. The changes in T${}_{\text{core}}$ and body weight during training, basal T${}_{\text{core}}$ before and after HA, and heat tolerance after HA were used as the evaluation indices for HA model construction [33].

The EHS rat model was established according to our previously reported method [33]. The rats were placed in a prewarmed chamber that was maintained at 39.5 $\pm$ 0.5 ℃ with a relative humidity of 55 $\pm$ 5% and started to run at a speed of 5 m/min with a slope of 0 for 2 min. The speed was increased by 1 m/min per 2 min, after which the speed was maintained at 10 m/min. The rats’ body temperatures were not measured during the running. Running was stopped when the rats were fatigued. At this time, the T${}_{\text{core}}$ of the rats was closely monitored every 5 min with a rectal thermometer, and 42 °C was considered the onset of heatstroke.

After successfully establishing the 21-day HA model, another 36 rats were randomly and double-blindly divided into 4 groups: the sham control group (CON; n = 9), exertional heat stroke group (EHS; n = 9), heat acclimation group (HA; n = 9) and heat acclimation prior to heat stroke group (HA + EHS, n = 9). The CON group was exposed to room temperature (24 $\pm$ 1 °C) freely. The 21-day heat acclimation protocol was performed directly on the HA group without temperature pill monitoring. The EHS protocol was used in the EHS group. The HA + EHS group underwent 21-day HA training and then the EHS protocol on Day 25 [33].

After the above models were established, hypothalamic tissues were collected (n = 4) under anesthesia, and blood was collected from three of the rats in each group. The rats were anesthetized under 1.5% pentobarbital sodium anesthesia (CAS:57-33-0, Sigma Aldrich, St. Louis, MO, USA) (0.3 mL/100 g) via intraperitoneal injection, the abdominal cavity was opened after disinfection, and the inferior vena cava was exposed. The blood collection needle was subsequently inserted into the blood vessel, after which the blood was collected into coagulant tubes connected to the blood collection needle. Approximately 3 mL of blood was collected per tube. Serum samples were obtained by centrifuging the blood at 3000 rpm for 15 min in a refrigerated centrifuge and then immediately stored at –80 °C. The rats were then decapitated and the brain was quickly harvested and put on ice. A vertical incision (2 mm deep) was made at the optic chiasm close to the mammillary body, enabling the removal of the hypothalamus [26]. Hypothalamic tissues from one side were placed in sterile cryotubes and immediately frozen in liquid nitrogen for Tandem mass tag (TMT)-based proteomic analysis. The hypothalamus specimen from the other side was used for Western blot (WB) analysis. Brain tissues were extracted from another 5 rats and then immersed in 4% paraformaldehyde (PFA) for fixation and subsequent hematoxylin and eosin (H&E) staining.

The brain tissues were fixed with 4% PFA, dehydrated with gradient ethanol and cleared with xylene, and subsequently embedded in paraffin (Biological Tissue Embedding Machine, Wuhan Junjie Electronics Co., Ltd., Wuhan, Hubei, China, model JB-P7). Each brain sample was then sagittally cut (4 µm) using a rotatory microtome (RM2235, Leica, Witzler, Hesse, Germany). After sectioning, the paraffin-embedded tissues were dewaxed, rehydrated, stained with hematoxylin and eosin, dehydrated, cleared and sealed in order [34]. Ten slices of brain tissue were taken from each rat. The sections were finally observed with an Olympus optical microscope (Olympus, Shanghai, China). The sections were observed at 200$\times$ magnification.

The proteomic analysis steps included protein sample pretreatment, enzymolysis, TMT labeling, reversed-phase chromatography, Liquid chromatography-mass spectrometry (LC‒MS) analysis and data analysis; for specific details, refer to the published literature [33].

A tissue grinding machine (HM-24, Huxi Industrial Co., Ltd., Shanghai, China) was used to homogenize the hypothalamus samples from all groups (n = 4) by performing three cycles of 20 seconds each at 6000 rpm. The homogenization was carried out in Radio Immunoprecipitation Assay (RIPA) Lysis buffer (R0020). The lysates were obtained from Solarbio, a biotechnology company located in Beijing, China. Phenylmethanesulfonyl fluoride (PMSF) (P0100, Solarbio, Beijing, China) was added to the lysates. After centrifugation at 12,000 rpm for 15 minutes at 4 °C, the supernatant was collected. The concentration of proteins in the collected supernatant was measured using the Pierce™ Bicinchoninic acid (BCA) Protein Assay Kit (23227, Thermo-Scientific, Waltham, MA, USA).

Protein samples (20 µg) were subjected to electrophoresis on 10% sodium dodecyl sulfate (SDS)‒polyacrylamide gels and subsequently transferred to a Polyvinylidene fluoride (PVDF) membrane with a pore size of 0.22 µm (PR05505, Immobilion-P, Boston, MA, USA). After being blocked with a 5% nonfat milk solution for 2 hours, the membrane was subsequently exposed to the following antibodies: ASS1 (16210-1-AP, rabbit, 1:1000; Proteintech, Wuhan, Hubei, China), HMGB2 (14597-1-AP, rabbit, 1:1000; Proteintech), vimentin (10366-1-AP, rabbit, 1:1000; Proteintech) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, mouse, 1:1000; Abcam, Cambridge, UK). Overnight at the following 4 ℃, the membrane underwent incubation in a secondary antibody, which was a conjugate of horse-radish peroxidase (HRP), and specifically targeted rabbit and mouse IgG using goat anti-rabbit and goat anti-mouse IgG (1:10,000, ZB-2301, ZB-2305, ZSGB Biotechnology Co., Ltd., Beijing, China), respectively, in a concentration ratio of 1:10,000, at room temperature for 1 hour and rinsed with triethanolamine buffered saline solution with Tween-20 (TBST) three times. Protein bands were visualized using an enhanced chemiluminescence (ECL) Western Blotting Detection kit (ab133406, Abcam, UK), and X-ray imaging was used visualize the bands. The software ImageJ 5.0 (National Institutes of Health, Bethesda, MD, USA) was used to compute the grayscale values of the bands.

Enzyme-linked immunosorbent assay (ELISA) kits were used to measure the serum levels of IL-4, IL-1, IL-8, IL-1$\beta$, IL-10, and IFN-$\gamma$ (Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s instructions (https://www.mlbio.cn/) (n = 3). Three replicate wells were used, and the experiment was repeated three times. The absorbance was measured at 450 nm with an optical absorption enzyme marker (Molecular Devices, SpectraMax 190, Sunnyvale, CA, USA). The concentrations of IL-4, IL-1, IL-8, IL-1$\beta$, IL-10, and IFN-$\gamma$ were calculated from standard curves and are presented in pg/mL.

All of the data are presented as the mean $\pm$ standard error (SE). Statistical analysis was performed using SPSS Statistics Software Version 23.0 (SPSS, Inc., Chicago, IL, USA), while GraphPad Prism Software Version 9.0 (GraphPad Software, La Jolla, CA, USA) was utilized for data visualization. Two-way repeated-measures Analysis of Variance (ANOVA) with the Bonferroni correction was used to compare T${}_{\text{core}}$ and body weight. Multiple comparisons were used to compare two groups at the same point in time. Differences in WB, ELISA protein quantification and cell counts after H&E staining were evaluated via one-way ANOVA. The Shapiro-Wilk test was used to determine the normal distribution of the data. Levene’s test was used to determine the homogeneity of variance. A significance level of p 0.05 was considered acceptable.

The sample size was decided according to sample size calculation, previous researches and practical conditions. In 21-day HA regimen, G.Power was used to calculate the sample size, and t tests were used; the effect sizes were 0.5, $\alpha$ = 0.05, and 1-$\beta$ = 0.8, and the total sample size was 128. However, this number is too high for animal experiments. According to the principle of reduction in the Replacement, Reduction and Refinement Principle (3Rs) of animal experiments and with reference to previous studies [35, 36, 37], we ultimately divided 10 rats into groups; the temperature data of 5 rats in each group were published, and the expression levels of heat shock proteins in various organs after the completion of heat acclimation training were also published [33]. Unpublished data on rat body temperature and body weight are presented here.

The number of animals chosen for hypothalamus tissue collection and blood withdrawal were also determined by calculation, reported researches and practical limitations. G.Power was used to calculate the sample size, and univariate analysis of variance was used to analyze the data. The results were as follows: effect size = 0.4, $\alpha$ = 0.05, 1-$\beta$ = 0.8, and total sample size = 76. However, our experiment is limited by practical conditions. In fact, in addition to those from the hypothalamus, we also collected the cortex, hippocampus, and kidneys from each group of rats for proteomics. There were 64 protein-sequenced samples. To minimize the cost and not exceed the project budget, we chose to add one additional rat to the minimum sample size of three, that is, four rats in each group, for proteomic analysis. In a previous study [38], rats were classified into five groups, and three liver samples from each group were randomly selected for TMT-labeled quantitative proteomics. Therefore, four hypothalamic samples from each group used for proteomics was acceptable. In addition, when blood was collected from rats, the blood exhibited a hypercoagulable state after exertional heatstroke, which was also consistent with the pathological coagulation involved in heatstroke [39]. Blood collection was indeed difficult, so only three rats were collected from each group in the EHS and HA + EHS groups, and a sample size of three in each group was also used in the control and HA groups for ease of description.

The results of this section demonstrate the feasibility of our proposed heat acclimation scheme. The changes in body weight and T${}_{\text{core}}$ during the 21-day heat-exercise exposure shown in Fig. 1 were observed in some of the rats during the process of establishing the HA model, and the rest of the changes can be found in our published article [31]. The experimental scheme of establishing the HA model is shown in Fig. 1A. All of the rats (n = 5) gained weight during the HA modeling period. After 14 and 21 days of heat-exercise exposure, the increase in weight in the HA group was slower than that in the CON group (69.20 $\pm$ 3.18 vs. 103.90 $\pm$ 4.10 g (p = 0.003) and 101.16 $\pm$ 1.87 vs. 159.20 $\pm$ 5.70 g (p = 0.000), respectively) (Fig. 1B). The T${}_{\text{core}}$ of the HA group rats during the training periods on Days 1, 7, 14 and 21 are presented in Fig. 1C. The rats’ core temperatures increased after they were placed in the preheated chamber, peaked at the end of the running, and then gradually decreased. The T${}_{\text{core}}$ tended to increase more slowly and decline more quickly as the training progressed. Following the 21-day HA training, acclimatized and control rats (n = 5) were exposed to an ambient temperature of 39.5 °C for 30 minutes and then moved to room temperature (~24 °C). The results of the acquired heat tolerance test are shown in Fig. 1D. During the 20–60 min period, T${}_{\text{core}}$ was significantly higher in the control group than in the HA group (t = 20, 25, 45 min, p = 0.001; t = 30 min, p = 0.002; t = 35, 40, 50, 55 min, p = 0.000; t = 60 min, p = 0.005). The increase in T${}_{\text{core}}$during the 10–20 min in the HA group was evidently lower than that in the CON group (0.063 $\pm$ 0.008 vs. 0.096 $\pm$ 0.007) ℃/min (p = 0.007). During the 30–40 min period, T${}_{\text{core}}$ recovered faster in the HA group (0.176 $\pm$ 0.015 vs. 0.095 $\pm$ 0.007) than in the control group ℃/min (p = 0.001). In addition, the core body temperature of the rats also changed after achieving HA. Fig. 1E shows significant differences in the basal T${}_{\text{core}}$ between the HA and control groups after 21 days of exercise-heat stress. The basal T${}_{\text{core}}$ of HA rats was evidently lower than that of unacclimated rats (36. 89 $\pm$ 0.023 vs. 37.28 $\pm$ 0.022) ℃ (p = 0.042). These results are consistent with previous findings in an HA rat model [26, 31].

Fig. 1.

Body weight and core temperature (T${}_{\text{𝐜𝐨𝐫𝐞}}$) change during the establishment of heat acclimation (HA). (A) Illustration detailing the experimental paradigm to develop the 21-day HA rat model. AT, ambient temperature; RH, relative humidity. (B) The body weight of the HA rats was significantly lower than that of the control rats after 14 days (*p = 0.003) and 21 days (*p = 0.000) of heat exercise training (n = 5). (C) Changes in T${}_{\text{core}}$ during training on Days 1, 7, 14 and 21 are shown. *p = 0.000 on Day 21 vs. Day 1; #p = 0.000 on Day 21 vs. Day 7; &p = 0.003 on Day 21 vs. Day 14 (n = 5). (D) Impact of heat acclimation on T${}_{\text{core}}$ responding to acute heat stress (n = 5). *p = 0.001, t = 20, 25, 45 min; *p = 0.002, t = 30 min; *p = 0.000, t = 35, 40, 50, 55 min; *p = 0.005, t = 60 min, HA vs. Control (n = 5). (E) Basal T${}_{\text{core}}$ of the HA and control rats. *p 0.05 HA vs. Control (n = 5).

H&E staining was used to visualize the hypothalamic microstructure in each group. Compared with the hypothalamic neurons in the control group (Fig. 2A), those in the EHS group exhibited swelling, loosening of the cytoplasm, widespread vacuolation and nuclear pyknosis (Fig. 2B). In the HA group, nuclear pyknosis and cell swelling were inconspicuous (Fig. 2C). Nuclei exhibiting pyknosis and cellular edema were significantly less severe in the HA + EHS group (Fig. 2D) than in the EHS group. In addition, the number of cells in hypothalamic tissue decreased significantly after EHS (p = 0.000; Fig. 2E), while the number of cells in the HA + EHS group was significantly higher than that in the EHS group (p = 0.006; Fig. 2E).

Fig. 2.

Structural alterations in hypothalamus. Heat acclimation (HA) can improve structural abnormalities in the hypothalamus in rats after exertional heat stroke (EHS). Representative images of hypothalamic hematoxylin and eosin (H&E) staining in the control group (A), exertional heat stroke (EHS) group (B), heat acclimation (HA) group (C) and HA + EHS group (D). Arrows indicate cell swelling and cellular vacuolation. (E) The total number of hypothalamic cells in the control, EHS, HA and HA + EHS groups. *p = 0.000 on EHS vs. Control; *p = 0.006 on EHS vs. HA + EHS; *p = 0.001 on HA vs. Control (n = 5).

The differentially expressed proteins (DEPs) in the hypothalamus from rats in the EHS and HA + EHS groups were analyzed using TMT-based proteomic analysis. The threshold fold change (FC) values were above 1.2 or below 0.8, respectively, with a statistically significant p value $\le$ 0.05. Compared with those in the EHS group, 153 upregulated proteins and 161 downregulated proteins were observed in the HA + EHS group. The differences between the groups are listed in Supplementary Table 1 and visualized in volcano plots (Fig. 3A). Volcano plots revealed significant differences in protein expression between the HA + EHS and EHS groups. The blue points on the left represent the underexpressed proteins, and the overexpressed proteins are indicated by red points on the right. Hierarchical clustering analysis of protein expression was further performed to determine the protein expression profiles (Fig. 3B). Notably, among these DEPs, proinflammatory proteins such as argininosuccinate synthetase (ASS1; gene name: Ass1), high mobility group protein B2 (HMGB2; gene name: Hmgb2), metalloproteinase inhibitor 2 (TIMP2; gene name: Timp2), programmed cell death protein 10 (PCDP10; gene name: Pdcd10), S100 calcium-binding protein A13 (S100A13; gene name: S100a13), and vimentin (VIM; gene name: Vim) [40, 41, 42] were expressed at lower levels in the HA + EHS group.. These findings suggested that EHS could disturb hypothalamus function by altering the abundance of certain proteins that regulate the inflammatory response and that HA could reverse some of the changes caused by EHS, thereby exerting a protective effect on organ function.

Fig. 3.

Identification of the differentially regulated proteins. (A) Volcano plots delineated by using the fold changes (FCs) and p values between the heat acclimation followed by exertional heat stroke (HA + EHS) group and the EHS group are presented. Proteins with significantly higher levels are dotted in red, and those with lower levels are dotted in blue. The differentially expressed proteins of interest are labeled in the diagram. (B) Heatmaps showing the hierarchical clustering of differentially expressed proteins between HA + EHS and EHS (red indicates upregulated proteins, and blue represents downregulated proteins). Each group contained four individuals (fold change $>$1.2 or 0.8; p 0.05). ASS1, Argininosuccinate synthetase; HMGB2, High mobility group protein B2; VIM, Vimentin; Dbn1, Recombinant Drebrin 1; Fn1, Fibronectin-1; Hpx, Hemopexin.

Subsequently, Gene Ontology (GO) functional annotation clustering and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted on the hypothalamus of the HA + EHS vs. EHS groups to further explore the biological functions of the DEPs. The results of the GO analysis of hypothalamic proteins are shown as follows. Biological process (BP) analysis revealed that the differentially expressed proteins in the HA + EHS and EHS groups were involved in nervous system development, regulation of cell homeostasis, and Cyclic adenosine monophosphate (cAMP) biosynthetic process. The enriched molecular functions (MF) were structural molecule activity, calcium-dependent protein binding, and the receptor for advanced glycation end products (RAGE) receptor binding. The enriched cellular component (CC) terms included cell junctions, cell projections and plasma membrane-bounded cell projections (Fig. 4A; Supplementary Table 2). Notably, the proinflammatory differential proteins are showed in Fig. 5A. ASS1 and HMGB2 mentioned above are involved in BP (Fig. 5B) related to the regulation of the response to cytokine stimuli and inflammatory reactions, and they also participate in MF (Fig. 5C) associated with RAGE receptor binding, cell adhesion molecule binding and integrin binding, which mediate cytokine activities and the immune response process. The CC terms were synapses, cell junctions, and neural projections (Fig. 5D). KEGG pathway analysis revealed that the hypothalamic DEPs in HA + EHS vs. EHS rats were associated with spliceosome pathways (Fig. 4B; Supplementary Table 3). Protein‒protein interaction network analysis (PPI network analysis) (Supplementary Fig. 1) revealed the predicted functional associations between these different proteins and inflammation.

Fig. 4.

Bioinformatics analyses of the differentially expressed proteins. (A) Gene Ontology (GO) enrichment analysis of biological process (BP), cell component (CC), and molecular function (MF) terms between heat acclimation followed by exertional heat stroke (HA + EHS) group and EHS group are shown. (B) Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways identified in the HA + EHS vs. Control (CON) comparison are shown.

Fig. 5.

Heatmaps and Gene Ontology (GO) enrichment analysis between exertional heat stroke (EHS) and heat acclimation (HA) + EHS revealed the key differential proteins involved in inflammation. (A) Heatmap of the HA + EHS group vs. the EHS group shows the relative abundances of the differentially expressed proteins involved in inflammation. The downregulated proteins included metalloproteinase inhibitor 2 (TIMP2), programmed cell death protein 10 (PCDP10), S100 calcium-binding protein A13 (S100A13), argininosuccinate synthetase (ASS1), high mobility group protein B2 (HMGB2) and vimentin (VIM). (B) GO enrichment analysis showed that the BPs enriched in these differentially expressed proteins were associated with the response and regulation of cytokines such as interferon-gamma and tumor necrosis factor. (C) The results of the molecular function (MF) analysis are shown. The enriched MFs in the differentially expressed proteins were associated with cell adhesion molecular binding, the receptor for advanced glycation end products (RAGE) binding and integrin binding related to immune inflammation. (D) The cell component (CC) enrichment results are shown. The CCs associated with these differentially expressed proteins were synapses, cell junctions, and neural projections.

Thus, controlling inflammatory conditions could be a key strategy employed by HA to mitigate the hypothalamic dysfunction induced by EHS. ASS1, HMGB2 and VIM may be candidate proteins involved in the mechanism underlying the protective effect of HA against hypothalamic injury after EHS.

To validate the above differential proinflammatory proteins, we performed Western blotting. Compared with those in the CON group, the expression of ASS1 (p = 0.024), HMGB2 (p = 0.009) and VIM (p = 0.009) in the EHS group was significantly greater, and the protein levels of ASS1 and HMGB2 in the HA group were not significantly different, while the expression of VIM in the HA group was significantly greater (p = 0.000). Compared with those in the EHS group, the relative expression levels of ASS1 (p = 0.047), HMGB2 (p = 0.000) and VIM (p = 0.009) were decreased in the HA + EHS group (0.71-, 0.67-, and 0.64-fold, respectively; Fig. 6). Supplementary Figs. 2,3,4,5 present the full-length blots/gels of HMGB2, ASS1, VIM and GAPDH.

Fig. 6.

Western blotting identified candidate proteins related to inflammatory responses. (A) Blots of argininosuccinate synthetase (ASS1), high mobility group protein B2 (HMGB2), vimentin (VIM) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown. Full-length blots/gels of ASS1, HMGB2, Vim and GAPDH are presented in the Supplementary Figs. 2,3,4,5. (B) Increases in ASS1, HMGB2 and VIM expression were detected in the exertional heat stroke (EHS) group compared to the control group. There was evident upregulation of VIM and no changes in ASS1 or HMGB2 in the heat acclimation (HA) group compared to the control group. Similarly, the expression of ASS1, HMGB2 and VIM was significantly lower in the heat acclimation followed by exertional heat stroke (HA + EHS) group than in the EHS group. p values were calculated by one-way analysis of variance (ANOVA); ns, not significant; *, p value; data are based on one representative experiment from at least 3 independent experiments (mean $\pm$ SE). SE, standard error. p 0.05.

Proinflammatory cytokines such as IL-1 and IL-8 are involved in the inflammatory response promoted by ASS1, HMGB2 and VIM [43, 44, 45]. Thus, we examined the expression of IL-1$\beta$, IL-1, IL-8, IL-4, IL-10 and IFN-$\gamma$ in the serum samples obtained from the rats in each group. IL-1, IL-1$\beta$ and IL-8 expression levels were greater in the EHS and HA samples than in the control samples (IL-1: p = 0.000 EHS vs. CON, p = 0.017 HA vs. CON; IL-1$\beta$: p = 0.000 EHS vs. CON and HA vs. CON; IL-8: p = 0.000 EHS vs. CON and HA vs. CON), with the EHS sample exhibiting a more pronounced increase. However, there the total cytokine level in the HA + EHS group was lower than that in the EHS group (p = 0.000) (Fig. 7A–C). The expression levels of IL-4, IL-10, and INF-$\gamma$ were lower in the EHS and HA groups than in the control group (p = 0.000), with the EHS group showing a more substantial decline. Although there was no change in INF-$\gamma$, the IL-4 and IL-10 levels in the HA + EHS group were noticeably greater than those in the EHS group (p = 0.017 and p = 0.001, respectively) (Fig. 7D–F). These data suggested that HA could improve the hypothalamic proinflammatory reaction provoked by heatstroke by regulating the activities of ASS1, HMGB2, VIM, and related cytokines.

Fig. 7.

The ability of heat acclimation (HA) to regulate the inflammatory response in exertional heat stroke (EHS) was validated by measuring interleukin (IL)-1, IL-1$\beta$, IL-8, IL-4, IL-10 and interferon-gamma (IFN-$\gamma$) levels via Enzyme-linked Immunosorbent Assay (ELISA). (A–C) The expression levels of IL-1$\beta$, IL-1 and IL-8 in the EHS and HA samples were greater than those in the control samples, and the cytokine levels in the EHS samples were more obviously elevated. However, the levels of these cytokines in the heat acclimation followed by exertional heat stroke (HA + EHS) group were evidently lower than those in the EHS group. (D–F) IL-4, IL-10 and INF-$\gamma$ levels in the EHS and HA groups were reduced compared with those in the control group, and cytokine levels in the EHS group presented a more visible decrease. IL-4 and IL-10 levels in the HA + EHS group were evidently greater than those in the EHS group, although there was no change in INF-$\gamma$. p values were calculated by one-way analysis of variance (ANOVA); ns, not significant; *, p value indicates statistical significance; the data show one representative experiment from at least 3 independent experiments with triplicate biological replicates in each (mean $\pm$ SE).