Li-wen Du, Bao-qing Xu, Kai Xun, Fang-qi Zhang

1 Department of Emergency, Ningbo No. 2 Hospital, Ningbo 315000, China

2 Department of Pathology, the 900th Hospital of Joint Logistics Support Force of Chinese PLA, Fuzhou 350025, China

3 Department of Pulmonary and Critical Care Medicine, the 987th Hospital of Joint Logistics Support Force of Chinese PLA, Baoji 721000, China

KEYWORDS: Heatstroke; Glutamine; Core temperature; Intestinal apoptosis; Heat shock protein 70

High temperature is highly correlated with health risks in various populations, especially among the frail population. Heatstroke is the most hazardous heat-related illness and has a high fatality rate.[1]Heatstroke (HS) is characterized by elevated core body temperature (over 40.0 °C) and neurological disorders.[2]Therefore, a rapid diagnosis and accurate treatment are key components for a good prognosis.

Glutamine is an amino acid used in protein synthesis.[3]Amino acids are divided into essential and nonessential amino acids, and glutamine traditionally belongs to the latter class in mammals. Carbohydrates can be metabolized to glutamine via aerobic and tricarboxylic acid cycling. Additionally, glutamine is an important amino acid with many functions;[4-6]for example, it is used as fuel for fast dividing cells and can induce the expression of heat shock protein (HSP), which prevents injury-induced apoptosis and acts as a precursor to the body’s primary antioxidant glutathione (GSH).[7-9]Although glutamine is abundant in the blood, its physiological requirement may exceed its production during trauma, infection, surgery, and chemoradiotherapy. Under such conditions, glutamine becomes a conditionally essential amino acid, and glutamine supplementation is considered in therapeutic plans.

Glutamine is effective in preserving mucosal integrity and in reducing bacterial translocation resulting from increased intestinal mucosal permeability in intestinal obstructions in mice.[10]The pathogenesis of heatstroke is very complex. A significant increase in core body temperature is associated with a redistribution of blood flow, which is characterized by cutaneous vasodilation at the expense of reduced intestinal blood flow.[11]Decreased intestinal blood flow leads to ischemia of the intestinal mucosa and restricts local vascular heat exchange, which further accelerates the increase in local temperature of the intestinal tissue. Intestinal tissue ischemia and overheating may promote oxidation and stress responses, as well as damaging the stable structure of the cells, thus resulting in the dispersion of tight junctions and damage to the epithelial cells.[12,13]These physiological and pathological changes may increase the permeability of the intestinal mucosa, promote the translocation of common intestinal bacteria and endotoxins, increase the risk of systemic inflammatory response syndrome (SIRS), and eventually lead to multiple organ dysfunction syndrome (MODS) and death. As indicated above, intestinal mucosal integrity plays a key role in the pathogenesis of heatstroke, and glutamine supplementation may have a protective effect on heatstroke by reducing the intestinal mucosal permeability and plasma endotoxin concentration to improve survival.[14]

Animals

Twenty-five 12-week-old male Wistar rats (weight 305±16 g) were purchased from the Animal Experimental Center of Yangzhou University. Only male rats were used because sex is an important factor influencing the basal core body temperature. All of the rats were raised in the Animal Experimental Center of Ningbo University. They were housed in a feeding box with a temperature of 24±2 °C and humidity of 50%-65% under a 12-hour day/night cycle. Standard chow and sterile water were provided every day. All of the animal protocols were approved by the Animal Ethics Committee of Ningbo University.

Groups

After 7 d of adaptation to the feeding environment, 25 rats were randomly divided into three groups. For the control group (n=5), rats were fed standard chow for 7 d without heat exposure. For the heatstroke group (HS group,n=10), rats were fed standard chow for 7 d, after which a heatstroke experiment was performed. For the heatstroke+glutamine group (HSG group,n=10), rats were fed standard chow supplied with glutamine (0.4 g/[kg·d]) by gavage for 7 d,[15]after which a heatstroke experiment was performed.

Heatstroke experiment

An incubator (Keermei Instruments Inc., China) was preheated to maintain a temperature of 42 °C and humidity of 60%. After anesthesia, the rats in the HS group and HSG group were transferred to the incubator without food and water. The core temperatures were monitored every 20 min using a rectal thermometer inserted into the rectum. At 80 min later, the rats were taken out from the incubator and put into a feeding box maintained at a temperature of 24±2 °C and humidity of 50%-65% with chow and water. Rats in the control group underwent the same experimental procedure (except that the incubator was maintained at 24 °C).

Blood collection and measurement

Three hours after the heatstroke experiment, all of the rats were anesthetized via intraperitoneal injection of 2% pentobarbital sodium (40 mg/kg). Blood samples were obtained from the femoral vein and collected into anticoagulant tubes for routine blood examination and into pro-coagulation tubes for serologic analysis. Routine blood examination was performed by using a hematology analyzer (Sysmex Corporation, Japan). Aspartate transferase (AST), alanine aminotransferase (ALT), serum creatinine (SCr), and creatine kinase (CK) levels were detected using an automated biochemical analyzer (ADVIA, Siemens, Germany). Additionally, prothrombin time (PT) and activated partial thromboplastin time (APTT) were detected by an automated blood coagulation analyzer (Werfen, Spain). Diamine oxidase (DAO) levels were determined via colorimetry using an ELISA kit (Xingke Biotechnology Inc., China). Furthermore, the levels of IL-6 and IL-8 were measured by ELISA kits (Chundu Biotechnology Inc., China).

Histological analysis of the small intestine tissue

Small intestinal tissues located 3 cm away from the stomachus pyloricus were harvested, fixed in 10% neutral formalin, dehydrated in ethanol and embedded in paraffin. Four-micrometer-thick sections were stained with hematoxylin and eosin (HE) for histological analysis.

Terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL) staining

Servicebio’s TUNEL staining kit (Servicebio®, China) was used for TUNEL staining. In brief, the sections were rehydrated with xylene, gradient alcohol and water. After permeabilization of the specimen, the sections were incubated with TUNEL solution. Afterwards, diaminobenzidine (DAB) solution was added. The sections were counterstained with hematoxylin, and dark brown signals indicated positive staining. Three different fields of view were photographed under an optical microscope (DM500, Leica, Germany), and optical density values were quantitatively analyzed by Image-Pro Plus 6.0 software.

Immunohistochemistry (IHC)

IHC was used to detect the expression levels and distribution of heat shock protein 70 (HSP70). In brief, paraffin sections were baked, deparaffinized and rehydrated. Antigen retrieval was performed using citrate buffer (pH=6.0). Endogenous peroxidases were inactivated with hydrogen peroxide solution (3%). After blocking with 1% bovine serum albumin (BSA) for 15 min, the sections were incubated with anti-HSP70 antibody (Cell Signaling Technology, USA) at 4 °C overnight. Following incubation with horseradish peroxidase (HRP)-labeled secondary antibody for 1 h, DAB solution was added. Three different fields of view were photographed under an optical microscope (DM500, Leica, Germany), and optical density values were quantitatively analyzed using Image-Pro Plus 6.0 software.

Western blotting

Small intestinal tissues (100 mg) from each group were lysed on ice by adding radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitor cocktail and quantified by using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). Equal amounts of protein samples (20 μg) were separated by using 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene difl uoride (PVDF) membranes. The membranes were blocked in 5% skim milk for 1 h and then incubated with the primary antibodies anti-HSP70 and β-actin (Cell Signaling Technology, USA) overnight at 4 °C. The membranes were subsequently incubated with HRP-conjugated secondary antibody (Solarbio, China) for 2 h at room temperature. The protein bands were detected using enhanced-electrochemiluminescence (ECL) solution (Solarbio, China) with a UVP gel imager. The expression of HSP70 was quantified using ImageJ software.

Statistical analysis

All of the statistical analyses were performed via GraphPad Prism 5. Data are presented as the means±standard deviation. Multiple comparisons were analyzed by using one-way analysis of variance, and the Bonferroni test was conducted for the post hoc comparisons. A two-tailedP-value ＜0.05 was considered to be statistically significant.

Glutamine supplementation attenuated the increase in core body temperature during heat exposure

The weights of the rats before the heatstroke experiment were 313.50±23.70 g (control group), 300.60±13.22 g (HS group), and 305.60±14.40 g (HSG group), which showed no statistically significant difference among the three groups. The core temperature of the rats before the heatstroke experiment was 33.44±0.18 °C. After 80 min of heat exposure, the core temperature of the HS group was 40.65±0.31 °C (supplementary Figure 1), which was higher than the criterion of heatstroke, thus indicating that heatstroke was successfully induced. To ensure the survival rate of the rats, the heatstroke experiment was stopped. The core temperature of the HSG group was 39.45±0.14 °C, which was significantly lower than that of the HS group (P＜0.001), thus indicating that glutamine supplementation attenuated the increase in core body temperature during heat exposure.

Glutamine supplementation restored the increased white blood cells and coagulation indicators that were induced by heatstroke to normal levels

Routine blood examination showed that there was a statistically significant difference in the numbers of white blood cells, neutrophils, and lymphocytes among the groups (Figure 1A). Specifically, the numbers of white blood cells, neutrophils, and lymphocytes in the HS group were significantly higher than those in the control group (P＜0.001), thus showing that heat exposure caused an increase in white blood cells, neutrophils (P＜0.001), and lymphocytes (P＜0.001) in the blood. Conversely, the numbers of white blood cells, neutrophils, and lymphocytes in the HSG group were significantly lower than those in the HS group, thus showing that glutamine supplementation prevented the increase in white blood cells, neutrophils, and lymphocytes that was caused by heat exposure.

The results of the coagulation tests showed that the PT and APTT of the HS group were significantly longer than those of the control group (Figure 1B,P＜0.001), thus indicating that heat exposure may affect endogenous coagulation systems. The APTT of the HSG group was significantly shorter than that of the HS group (P＜0.001), which indicated that glutamine supplementation had a protective effect on endogenous coagulation systems in heatstroke rats.

Glutamine supplementation restored the increased blood biochemical indicators and infl ammatory cytokines that were induced by heatstroke to normal levels

Based on clinical experience and a literature review, AST, ALT, SCr, and CK were selected for examination among the blood biochemical indicators. Three hours after the heatstroke experiment, the above-mentioned indicators were quantitatively detected. The results showed that the serum levels of AST and ALT in the HS group were significantly higher than those in the control group; however, the serum levels of SCr and CK in the HS group were not significantly different from those in the control group (Figure 1C), thus suggesting that heat exposure can cause an increase in AST and ALT levels. However, no statistically significant difference in the four indicators was found between the HS group and the HSG group.

Compared with the control group, the serum levels of DAO in the HS group were significantly increased (Figure 1D), thus suggesting that intestinal mucosal permeability was increased and that the intestinal mucosal integrity was damaged. In contrast, the serum levels of DAO in the HSG group were significantly decreased compared with those in the HS group, which suggested that glutamine supplementation had a protective effect on intestinal mucosal integrity. Moreover, the serum levels of IL-6 and IL-8 in the HS group were significantly higher than those in the control group (Figure 1D), which indicated that heat exposure had a stimulative effect on inflammatory factors. Additionally, the serum level of IL-8 in the HSG group was significantly lower than that in the HS group, thus indicating that glutamine supplementation may alleviate the stimulative effect of heat exposure on infl ammatory factors.

G lutamine supplementation a lleviated i ntestinal apoptosis induced by heatstroke

Under a light microscope, the intestinal mucosa was injured, and the structure of tight junctions was damaged in the HS group; however, the structure of intestinal mucosal epithelial cells was stable in the HSG group (Figure 2A), thus suggesting that g lutamine supplementation had a protective effect by sustaining intestinal integrity. Additionally, TUNEL staining showed that TUNEL-positive cells were increased in the HS group compared with the control group, whereas glutamine supplementation reduced TUNEL-positive cells compared with the HS group (Figure 2B). The above-mentioned results confirmed that glutamine supplementation contributed to the alleviation of apoptosis of small intestinal epithelial cells induced by heatstroke.

Figure 1. Glutamine supplementation restored the increased indicators induced by heatstroke to normal levels. A: numbers of white blood cells, neutrophils and lymphocytes in the blood; B: PT and APPT; C: concentrations of AST, ALT, SCr and CK in the serum; D: concentrations of DAO, IL-6 and IL-8 in the serum in the control, HS and HSG groups. **P＜0.01, ***P＜0.001. HS: heatstroke; HSG: heatstroke+glutamine; PT: prothrombin time; APTT: activated partial thromboplastin time; AST: aspartate transferase; ALT: alanine aminotransferase; SCr: serum creatinine; CK: creatine kinase; DAO: diamine oxidase.

Gl utamine supplementation up-regulated HSP70 expression

The IHC results showed that the expression of HSP70 in the HS group was significantly increased compared with that in the control group, and the expression of HSP70 in the HSG group was higher than that in the HS group (Figure 3A). The protein level verified via Western blotting also matched the IHC results (Figure 3B). These results demonstrated that glutamine supplementation significantly up-regulated the expression of HSP70 in heatstroke rats.

Figure 2. Glutamine supplementation alleviated intestinal apoptosis induced by heatstroke. A: representative pathological images of the small intestine from the control, HS and HSG groups stained with hematoxylin-eosin (HE) staining at magnifications of ×100 and ×400; B: TUNEL staining at a magnification of ×400, and the densitometric values statistically quantified via Image-Pro Plus (IPP) 6.0. ***P＜0.001. HS: heatstroke; HSG: heatstroke+glutamine.

Figure 3. Glutamine supplementation upregulated the expression of HSP70. A: representative images of the HSP70 expression detected by IHC staining at magnifications of ×100 and ×400, and the densitometric values were statistically quantified via Image-Pro Plus (IPP) 6.0; B: Western blotting detection of HSP70 protein expression and statistical quantification of optical density values via ImageJ. ***P＜0.001. HS: heatstroke; HSG: heatstroke+glutamine; IHC: immunohistochemistry.

Heatstroke is one of the most serious heatrelated diseases and has a high mortality rate.[16]The pathophysiological mechanism of heatstroke is complex, which increases the difficulty of treatment; however, heatstroke is a preventable disease. In this study, we examined the protective effect of glutamine on the intestinal mucosal barrier to understand the pathogenesis of heatstroke. Before heat exposure, we supplied glutamine by gavage as a prevention method for heatstroke. The results showed that after heat exposure, the core temperature of the HS group was higher than the criterion of heatstroke, and the core temperature of the HSG group was lower than that of the HS group, thus indicating that glutamine supplementation before heat exposure inhibited the increase in core temperature during heat exposure.

The serum levels of AST and ALT in the HS group were significantly higher than those in the control group, which showed that liver function was abnormal. Heat exposure may cause intestinal bacterial translocation, after which endotoxin enters the liver through the portal vein and eventually leads to liver dysfunction.[17]The serum levels of SCr and CK in the HS group were not significantly different from those in the control group, which showed that rhabdomyolysis and renal dysfunction had not yet occurred. Heat exposure can cause rhabdomyolysis and release large molecules (such as creatine kinase and myoglobin), which damage the kidneys and cause renal dysfunction.[18]The results of the coagulation tests showed that heat exposure may affect endogenous coagulation systems. It is generally believed that the change in APTT mainly reflects abnormalities in the endogenous coagulation system. Early activation of endogenous coagulation and excessive consumption of endogenous coagulation substances are related to disseminated intravascular coagulation that occurs at later stages.[19,20]DAO is an intracellular enzyme in the intestinal mucosa of mammals, and its specificity is very high because it is rarely found in other tissues. When the intestinal mucosa is injured, DAO is released into the blood. Therefore, it is positively correlated with the presence of intestinal bacteria and endotoxin and is often used to evaluate intestinal function.[21]The serum levels of DAO in the HS group were significantly increased, thus demonstrating that the intestinal mucosa was damaged by heat exposure. Endotoxemia triggers an immune response within half an hour, and the immune response peaks within 2 h.[22]The serum levels of IL-6 and IL-8 in the HS group were significantly higher than those in the control group, thus indicating that heat exposure had a stimulative effect on infl ammatory factors. Moreover, the serum level of IL-8 in the HSG group was significantly lower than that in the HS group, which indicated that glutamine supplementation may alleviate the stimulative effect of heat exposure on infl ammatory factors.

Histological changes in the small intestine showed that heat exposure directly led to damaged villi in the small intestine, which were partly unconnected from the lamina propria, with visible fracture deformities, visible resorptive cells and goblet cells, a vague structure of the central chylous duct and sparse intestinal villi. Moreover, intestinal mucosal barrier function was impaired, and the tight connection structure was destroyed. Glutamine pretreatment stabilized the barrier and function of intestinal mucosal epithelial cells in rats after heat exposure. HSP family proteins bind to damaged proteins caused by hea t stress to inhibit apoptosis, and HSP70 is the most stress-responsive protein among this family.[23]HSP70 increased in response to heatstroke to protect the cells from proteotoxic damage. A previous animal study revealed that the overexpression of HSP70 can inhibit the apoptosis of intestinal cells and reduce mortality in rats.[24]Glutamine is important in inducing the expression of HSP to enhance cell survival.[25]Furthermore, the glutamine-induced up-regulation of HSP70 protected the integrity of the intestinal mucosa from heat stress and prevented intestinal mucosal injury.[26]

When considering the survival rate and animal ethics factors, the time of heat exposure was restricted to 80 min. A longer duration of heat exposure may provide more accurate physiological data of more serious heatstroke. Furthermore, the time of recovery was defined as 3 h, and a longer time of recovery may provide more complete physiological data of heatstroke. Biomarkers such as serum troponin[27]and urinary HSP72 to urinary creatinine ratio,[28]as well as the effects of drugs like myosin light-chain kinase (MLCK) inhibitors on HSP expression after heat exposure, require more research. In addition, the inhibition of epithelial MLCK may be an effective treatment for heatstroke.[29]

Our current study shows that glutamine supplementation before heat exposure may inhibit the increase in core temperature during heat exposure, restore the increased white blood cells, coagulation indicators, blood biochemical indicators and infl ammatory cytokines induced by heatstroke to normal levels, and alleviate intestinal apoptosis by upregulating the expression of HSP70.

Funding:This study was supported by the Research Foundation of Hwa Mei Hospital, University of Chinese Academy of Sciences, China (2020HMKY22); Zhejiang Medicine and Health Science and Technology Project (2021KY1015); Ningbo Key Support Medical Discipline (2022-F16).

Ethical approval:All of the animal protocols were approved by the Animal Ethics Committee of Ningbo University.

Confl icts of interest:None.

Contributors:LWD and BQX contributed equally to this work. All authors read and approved the final version.

The supplementary file in this paper is available at http://wjem.com.cn.