Introduction

Stress is defined as any internal or external stimulus that produces a biological response. The impact of stress varies based on the type, timing, and severity of the stimulus applied. It can result in anything from alterations in homeostasis to life-threatening consequences, and in extreme cases, death (1). The stress response is a multifaceted complex mechanism that depends on many factors such as environmental variables (ambient temperature, humidity, solar radiation and air movement) and living factors (species, breed, age, health and physiological status) (2-4).

The range of environmental temperatures in which organisms comfortably perform their regular physiological activities is termed the "thermal neutral zone" or "comfort zone". One of the primary determinants of this zone is air temperature. Disruptions occur when the ambient temperature surpasses 25°C, disrupting the equilibrium between body temperature and body heat release. This condition, known as heat stress (HS), poses risks to human and animal health, leading to substantial economic losses in public health services and livestock production (5-7).

Numerous studies report that HS causes endocrine system disorders, electrolyte imbalance, immune system disorders, and lipid and protein oxidation (8-10). Additionally, HS is associated with oxidative stress (OS), marked by an upsurge in the production of reactive oxygen species (ROS) and a decline in antioxidant capacity (11,12). OS arises as a result of an imbalance between ROS production and the antioxidant defense against them. OS, which can cause damage to biomacromolecules such as DNA, proteins and lipids, can also result in. cell dysfunction and tissue damage. OS induced by free radicals can cause many diseases such as cancer, Alzheimer’s disease and autoimmune diseases as well as leading to degenerative processes (12-14).

Tissue myeloperoxidase (MPO), a marker of neutrophil migration, plays an important role in oxidant production by neutrophils. MPO enzyme is a lysosomal enzyme secreted from leukocytes in response to active stress. In the event of inflammation, MPO is released into the extracellular environment. Therefore, measurement of MPO activity as an indicator of neutrophil secretion is a sensitive test (15). Superoxide dismutase (SOD) is an enzyme that catalyzes the dismutation of oxygen and hydrogen peroxide and is a primary protector against oxyradicals. SOD is an important antioxidant for superoxide, one of the main ROS in the cell (16). HS can stimulate systemic release of endogenous molecules and trigger local and systemic release of both pro- and anti-inflammatory cytokines such as interleukin (IL)-1 beta, IL-2, IL-6 and tumor necrosis factor (TNF)-α (17).

HS acts as the major challenge for maintaining the immune status in living organism. Acute HS may have a stimulatory effect on the immune system, however chronic HS may have an inhibitory role on the capacity of the immune system to maintain homeostasis (18). In addition, the negative impact of HS is predicted to worsen with the global warming (19).

This study aims to elucidate the effects of elevated temperature stress, specifically at 30°C and 35°C, on OS and inflammation biomarkers across different tissues. The selected parameters for this study are geared towards assessing inflammation and OS. TNF-α, IL-2, and IL-6 were chosen to probe inflammatory pathways, with MPO activity reflecting pro-oxidant processes, and SOD representing the enzymatic component of antioxidant activities.

Methods

In this study, 18 male Sprague Dawley rats from Atatürk University Experimental Animal Research Center were used. The Atatürk University local ethic committee for animal experiments approved the study, with protocol number: 2019-16/238.

Animals and Experimental Design

Eighteen Sprague Dawley male rats weighing between 250-300 g were randomly divided into three groups with six rats in each group. The groups were determined as Thermo-neutral (TN), 30 °C HS1 and 35 °C HS2.

Thermo-neutral (TN) group: The rats in this group were housed at room temperature at 22-24 °C for 24 hours/day and were fed with a normal diet and standard conditions were applied.

Temperature stress (HS1) group: The rats in this group were exposed to a temperature of 30 °C for 8 hours (20) a day (9:00 to 17:00/whole study) in wire cages in temperature-controlled rooms for fourteen days.

Temperature stress (HS2) group: The rats in this group were exposed to a temperature of 35 °C for 8 hours a day (from 9:00 to 17:00/whole study) in wire cages in temperature-controlled rooms for fourteen days.

At the end of the study, our experimental animals in the groups were stopped from being fed, as they were free to drink water from 24:00 the day before they were sacrificed by cervical dislocation method. Brain, duodenum, liver, thyroid gland and testis tissues of rats were taken for biochemical and histopathological analysis and stored at -80.

Analysis of Oxidative Stress (MPO) and Antioxidant Enzymes (SOD) in Various Tissues

Biochemical kits were used for measuring: Myeloperoxidase; Rat MPO ELISA kit, (Sunred biological technology; Shangai, China) and Superoxide dismutaz; Rat SOD ELISA kit, (Sunred biological technology; Shangai, China)

Determination of Inflammatory Markers (TNF-α, IL-2, IL-6) in Brain, Duodenum, Heart, Liver, Thyroid and Testis Homogenate of Adult Male Rat

Serum cytokines; IL-2, IL-6 and TNF-α, were measured using a commercial available kits specific for rats (ELISA kits, Sunred biological technology; Shangai, China). Levels of IL-2 and TNF-α in serum samples were measured according to manufacturer’s instructions.

Statistical Analysis

Descriptive statistics of quantitative variables. in the groups were expressed as mean and standard error. The Shapiro-Wilk test was used to confirm the normality of the distributions of quantitative variables. The homogeneity of variances, which was a prerequisite for parametric tests, was checked with Levene’s test. Differences between groups within the same parameter were checked with one-way ANOVA and Kruskal-Wallis test. Tukey HSD and Dunnett's post-hoc tests were used to determine the groups caused.the significant difference. Groups with significant differences within the same parameter were indicated with different superscript letters. P<0.05 was considered significant in all statistical calculations. SPSS (version 20.0, SPSS Inc, Chicago) package program was used for statistical analysis of the data.

Results

In this study, 18 male Sprague Dawley rats were used. The experimental animals were divided into three groups. The groups were exposed to a comfort temperature, 30 °C, and 35 °C, respectively. At the end of the experimental procedure, inflammatory markers (IL-2, IL-6, and TNF-α) in the brain, duodenum, heart, liver, thyroid, and testis tissues; MPO as an oxidative stress marker; and SOD levels indicating antioxidant activity were determined. (Tables 1, 2, 3 and Figure 1).

Biochemical Results

Brain

As shown in Table 1 and Figure 1, heat exposure in the brain tissue resulted in a higher IL-6 level and SOD activity, and a lower TNF-α concentration in the HS2 group compared to the control group (p<0.05, Table 1 and Figure 1).

Duodenum

Compared to the control group, HS1 group duodenal IL-2 level was higher in rats exposed to heat, and HS2 group duodenal TNF-α concentration was higher compared to the control group as well (p<0.05, Table 1 and Figure 1).

Heart

As shown in Table 2 and Figure 1, IL-6 and MPO levels of the heart tissue of the HS2 group exposed to 35 °C temperature were higher than the control and HS1 groups (p<0.05).

Liver

Heat exposure in the liver resulted in decreased IL-2 levels compared to the control group (p<0.05). Hepatic IL-6 level increased in the HS2 group compared to the HS1 group (p<0.05, Table 2 and Figure 1).

Thyroid

As shown in Table 3 and Figure 1, no statistically significant change was observed in thyroid tissue IL-2, IL-6, TNF-α, MPO and SOD values at 30 °C and 35 °C exposure (p>0.05).

Testis

Exposure to heat stress in testicular tissue resulted in an elevation of IL-2 levels in the HS1 group compared to controls (p<0.05). Testicular concentrations of TNF-α were increased in both HS1 and HS2 groups relative to the control group (p<0.05), and SOD activity in testicular tissue also showed a marked increase compared to controls (p<0.05).

Discussion

HS is increasingly significant due to global warming (21,22). Exposure of the body to heat can cause different physio/pathological conditions. Behavioral and autonomic temperature-defense negative feedback mechanisms are used to maintain normal body temperature. Physiologically, successful activation of defense mechanisms results. in temperature tolerance. With repeated, chronic heat exposure, tolerance intensifies, leading to heat acclimation. Heat-related diseases develop when the pathological effects of heat load are not prevented. Syndromes range from less severe such as heat syncope to severe forms such as fatal heatstroke (23) .

There are number of internal environment changes and systemic effects caused by HS in the organism as determined in the literature. However, our study determined the effects of high-temperature stress exposures, beyond the comfort temperature, on the oxidant system (MPO, SOD) and inflammation markers (IL-2, IL-6, TNF-α levels) in various tissues, supplementing existing literature (Tables 1, 2, 3 and Figure 1).

HS changes the physiology of all systems in the body with impaired redox homeostasis (24). MPO, secreted from activated polymorphonuclear leukocytes, increases OS by promoting free oxygen radical production.Therefore, excessive MPO production can damage tissues and organs (25). It was determined in our study that the heart tissue MPO level of the HS2 group was higher than that of the control group and HS1 group (p<0.05, Table 2 and Figure 1). This result shows that exposure to 35 °C HS increases the oxidant level associated with heart tissue leukocyte activation. In a study conducted by Horowitz et al. (26), the tissue damage caused by HS was associated with increased free radical production in animals exposed to high ambient temperature (42 or 43 °C). In another study, it was reported that chickens exposed to 36 °C for 8 hours a day showed a rapid increase in ROS production in mitochondria of duodenal, jejunal and ileal epithelial cells (27). An in vitro study with intestinal epithelial cells showed that exposure to 42 °C for 60 minutes increased ROS concentration and mitochondrial dysfunction and early apoptotic rates (28).

Free radicals produced continuously in the cell are destroyed by the antioxidant defense systems that are produced during normal metabolism in the body. Antioxidants basically prevent or delay cell damage by scavenging free radicals in the cell. Antioxidants can either be produced naturally in the body or obtained from external foods (29). SOD is the most important antioxidant defense system against ROS and superoxide anion radicals (30). When Table 1 and Table 3 were examined, it was seen that 35 °C temperature exposure increased the SOD activity of brain and testis tissues (p<0.05). The increase in the activity of SOD, an antioxidant enzyme, in brain and testicular tissues may be an antioxidant defense response to increased reactive oxygen intermediates.

Cytokines are intracellular peptides that act as immune system mediators. In both human and animal models, the levels of pro-inflammatory and anti-inflammatory cytokines increase in heat stroke (17). The inflammatory reaction associated with heatstroke includes activation of the humoral and cellular components of the immune system, as well as alterations in the production and/or suppression of numerous cytokines (31-33). The findings of this study suggest that HS is associated with changes in proinflammatory cytokine levels. When the inflammatory responses of the tissues are examined; duodenum TNF-α concentration, heart and brain IL-6 levels increased, while brain TNF-α concentration and liver IL-2 levels decreased (p< 0.05). Ex vivo and in vitro studies characterizing the direct effects of heat have shown that it activates numerous pathways related to the physiological or cellular/biochemical axis (17,34). In a study by Helwig and Leon (35) using a mouse model of heat stroke, it was reported that the IL1 cytokine family played a role in the development of organ damage (spleen and liver). Another study showed that exposure to heat directly affected intestinal permeability and function The inflammatory response in animals exposed to mild to severe heat has the potential to impair nutrient absorption, gut health, and immunological status of the affected organism (34,36).

The potent inflammatory response to heatstroke increases after the end of HS and is intricately involved in both the damaging processes and the repair mechanisms activated during the healing phase. In survivors, the magnitude of this response decreases as time progresses, allowing a return to normal homeostasis. A stronger inflammatory response is often associated with poor prognosis and even death, indicating an imbalance in the immune system and causing a dysregulated inflammatory response (17). Consequently, HS can cause organ dysfunction associated with the accumulation of oxidants and the release of proinflammatory mediators. Future research should aim to elucidate the role of the immune inflammatory response in heat-stressed organisms. Nutritional and therapeutic interventions can prevent damage to organs due to HS and increase thermal tolerance to heat by reducing the accumulation of oxidants, but additional data are needed to support this theory.

Study Limitations

It is important to acknowledge the limitations of the current study. Because only male rats used in the experiments, these findings might not apply to female rats. Further studies that include different genders, ages, ete. are necessary. Future research should consider this difference.

Conclusion

As a result, exposure to high temperatures (30 °C or 35 °C) outside the room temperature of 24 °C, which is known as the comfort temperature, causes changes in the redox status of rats, in oxidant and antioxidant levels, and also in the levels of cytokines, known as immune system mediators. This indicates that exposure to high temperatures may lead to homeostasis changes in living organisms, resulting in deterioration of tissue and organ functions.

Ethics

Ethics Committee Approval: The approval to our study was granted by Atatürk University Local Ethical Committee for Animal Experiments with the decision number 7704040475-641.04-E.1900352247 (dated: December 31, 2019 and numbered: 2019/238).

Informed Consent: This study does not apply because it involves animal subjects.

Peer-review: Externally peer reviewed.

Authorship Contributions

Concept: E.S., H.U., Design: E.S., H.U., Data Collection or Processing: E.S., H.U., Analysis or Interpretation: E.S., H.U., Literature Search: E.S., H.U., Writing: E.S., H.U.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.