Expression and mechanism of TRPV1 channel in prefrontal cortex after acute hypoxic exercise

Abstract

Objective

This study investigates the role of transient receptor potential vanilloid subtype 1 (TRPV1) in acute hypoxic exercise.

Methods

After acute hypoxia intervention, the mRNA expression levels of TRPV1 and 5-hydroxytryptamine 1 A (5-HT1A) in the prefrontal cortex of rats were detected by real-time quantitative polymerase chain reaction (RT-PCR). Meanwhile, the content of 5-hydroxytryptamine (5-HT) in this area was determined by enzyme-linked immunosorbent assay (ELISA).

Results

Hypoxic conditions significantly reduced the duration of high-load exercise performance in rats, resulting in an earlier onset of fatigue and a pronounced decline in exercise capacity (p < 0.05). Acute hypoxic exercise upregulated the expression of TRPV1, 5-HT, and 5-HT1A in the prefrontal cortex (p < 0.05), which may lead to a decrease in exercise capacity. Pharmacological blockade of TRPV1 and 5-HT1A receptors extended the duration of high-load exercise under hypoxic conditions and improved exercise capacity (p < 0.05).

Conclusion

Our findings indicate that the upregulation of TRPV1, 5-HT, and 5-HT1A is a key mechanism underlying the decline in exercise performance during acute hypoxia. Pharmacological blockade of these pathways effectively alleviates hypoxia-induced exercise fatigue, suggesting they represent promising therapeutic targets for enhancing performance under hypoxic conditions.

Significance

These findings provide a biological basis for developing nutritional strategies to counteract the initial decline in physical performance experienced by military personnel and adventurers during their ascent to high-altitude environments.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13102-025-01523-6.

Introduction

Compared to normoxic training, the duration of exercise to exhaustion is significantly shorter under hypoxic conditions, resulting in an earlier onset of fatigue [1]. This not only complicates athletes ability to sustain recommended training loads but also diminishes physical performance in military personnel at high altitudes and reduces exercise efficiency in the general population exposed to hypoxia. Regarding the early decline in exercise capacity due to hypoxia.Initial research into the early decline in exercise capacity due to hypoxia mainly focused on its effects on cardiopulmonary function and muscle oxygen utilization. However, current evidence suggests that the reduction in exercise capacity during hypoxic exercise is often due to multisystem interactions, involving peripheral factors (such as muscle fatigue), limitations in cardiovascular and respiratory functions, and functional inhibition of the central nervous system (CNS), which is often attributed to central fatigue [2]. The role of the CNS is particularly critical, as studies indicate that the decline in exercise capacity upon initial exposure to high altitudes is closely related to central fatigue [3]. The fundamental reason lies in the direct adverse effects of acute hypoxia on the brain, which impairs the brain’s ability to integrate motor information, issue motor commands, and maintain motor excitability, thereby limiting the individual’s maximum potential for executing motor tasks. During exercise in a hypoxic environment, if hypoxia persists or intensifies, it may exert a more severe inhibitory effect on the CNS by reducing cerebral oxygenation and impairing motor cortex function [4]. This dual mechanism, involving both environmental and exercise-induced hypoxia, is key to the premature onset of fatigue. Hemodynamic studies have also highlighted the crucial role of the prefrontal cortex (PFC) in regulating the exercise cessation threshold, although the precise underlying biological mechanisms remain to be elucidated [5].

While the PFC’s role is established, the molecular mechanisms that trigger this “stop” signal under hypoxia remain unclear. A key candidate in this process is the dysregulation of intracellular calcium, which is vital for governing CNS function. The calcium overload hypothesis suggests that Ca²⁺ contributes to the development of central fatigue [6]. As a member of the calcium channel family, the transient receptor potential vanilloid 1 (TRPV1) has been confirmed to be involved in cerebral ischemia-hypoxia injury [7]. Upon ligand binding, TRPV1 is activated, resulting in increased influx of calcium ions and a higher intracellular calcium concentration. This, in turn, triggers various physiological or pathological reactions, such as intracellular chemical sensing, neurogenic inflammation [8].

In addition to calcium dysregulation, another well-established pathway to central fatigue involves the serotonergic system. Research indicates that an increase in brain serotonin (5-hydroxytryptamine, 5-HT) levels during prolonged exercise can impair central nervous system function, leading to a decline in exercise performance [9]. 5-HT is regulated by various factors, including its synthesis rate-limiting enzyme, tryptophan hydroxylase (TPH), and the 5-hydroxytryptamine receptor 1 A (5-HT1A) [10]. The 5-HT1A receptor is one of the most important subtypes involved in serotonergic function, and subjects engaging in strenuous exercise tend to experience faster fatigue when taking 5-HT1A receptor agonists [11, 12]. Critically, emerging evidence suggests a potential crosstalk between these two pathways. Research indicates that TRPV1 function can be modulated by serotonergic signaling, where the release of 5-HT contributes to the sensitization of TRPV1 channels, a mechanism implicated in heightened sensory responses. These neurotransmitters, in turn, activate downstream nerves, facilitating nerve impulse transmission and contributing to increased visceral sensitivity and hyperalgesia [13]. Based on these findings, this study aims to investigate the roles of TRPV1, 5-HT1A, and 5-HT in the prefrontal cortex in issuing movement-stop instructions during acute hypoxia, and to explore whether the relationship between TRPV1 and 5-HT plays a crucial role in the biological mechanism of central fatigue. To achieve the above objectives, this study first investigated the expression of TRPV1, 5-HT, and 5-HT1A following acute hypoxic exercise and analyzed the role of the prefrontal cortex in exercise cessation signaling. Second, by separately injecting TRPV1 and 5-HT1A inhibitors, we explored the functional role of TRPV1 and its associated mechanisms during acute hypoxic exercise.

Related works

Experimental animals and groups

Forty-eight male Wistar rats (body weight: 280–300 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. All procedures were approved by the Institutional Animal Care and Use Committee (Approval No. 2020176). Four rats per cage were placed in standard cages, with the room temperature maintained at 20–24 °C, a relative humidity of 40–60%, and a 12-hour light/12-hour dark cycle implemented. Animals were fed standard feed according to national standards, and the water and food intake were not controlled [14, 15]. The rats were assigned to 6 groups(n = 8): O(normoxic rest), OE(normoxic exercise exhaustion), H(hypoxic rest), HE(hypoxic exercise exhaustion), HE1(hypoxic exercise exhaustion + TRPV1 antagonist AMG9810), and HE5 (hypoxic exercise exhaustion + 5-HT1A antagonist WAY100635) [16]. Antagonists were administered intraperitoneally 30 min pre-exercise. Strict monitoring for humane endpoints (≥ 20% weight loss, lethargy, dyspnea) was implemented.Animals meeting any of these criteria were euthanized according to the approved protocol.

Rats adaptive treadmill training program

Treadmill adaptation training was conducted for the rats before the formal intervention began. The adaptation protocol involved 5 days of training, 15 min per day, at a constant speed of 10 m/min. After completing the adaptation, the rats were given a one-day rest period, after which the subsequent experimental procedures were initiated.

Rat incremental load exercise program

The rat exercise protocol was adapted from Leandro’s model for assessing maximal oxygen uptake in Wistar rats. In this study, we followed Leandro’s protocol for maximal oxygen uptake testing in rats [17]. The rats were subjected to an incremental exercise protocol on a treadmill with a 10° incline, starting at a speed of 5 m/min for 4 min and then increasing the speed by 5 m/min every 3 min until they could no longer maintain their pace or reached the maximum treadmill speed of 50 m/min. The maximum speed reached was 50 m/min [17]. Exhaustion was determined when the rats were unable to continue running on the treadmill under electrical stimulation conditions.

Tissue drawing

Anesthesia was induced via intraperitoneal injection of a 3% pentobarbital sodium solution.

(1.5-2.0 ml/kg body weight). After confirmation of deep anesthesia, the animals were euthanized by.

cervical dislocation. Brain tissue was then promptly harvested.

Lab environment

Normoxic-group rats were trained in the standard housing(21%O₂). Hypoxia-group rats exercised within sealed chambers inside the facility, where a simulated hypoxia generator maintained an oxygen concentration of 12.7%, equivalent to 4,000 m. Compared with humans and other advanced mammals, rodents such as rats are more tolerant to hypoxic environments and have a shorter acclimation period. Considering that athletes usually train at an altitude of 2,500 to 3,500 m, and rodents have stronger oxygen tolerance and require a lower oxygen concentration during training, the target al.titude for rats is 3,500 to 4,500 m. Studies suggest that an oxygen level corresponding to 4,000 m (12.7% O₂) is a reasonable intervention dose, effectively stimulating the rats under hypoxia while allowing control of training intensity. Thus, 4,000 m was set as the intervention dose for the hypoxia group in this study [18].

ELISA

This study employed the ELISA (Sangon Biotechnology, China) to measure the concentration of 5-HT in the prefrontal cortex of rats. Before performing ELISA on tissue samples, it is essential to extract proteins and ensure uniform protein concentrations across all samples to prevent inaccuracies arising from variations in tissue sample sizes. The specific procedure is as follows: Pre-chilled Phosphate Buffered Saline (PBS) was mixed with the protease inhibitor cocktail at a ratio of 50:1. 250 µL of this mixed buffer was added to each sample. Subsequently, the samples underwent tissue homogenization followed by centrifugation at 12,000 rpm for 10 min. After centrifugation, the supernatant was carefully collected, and its protein concentration was determined using the BCA method. The protein concentration determination was carried out strictly according to the instructions provided with the BCA Protein Quantification Kit: Protein standard solutions and sample lysis buffer were prepared on ice. A standard curve was generated using the known protein content and corresponding absorbance values of the standards. Based on the measurement results, an appropriate amount of PBS buffer was added to each sample to adjust all sample protein concentrations uniformly to 400 µg/mL. During this process, the sample with the lowest protein concentration could be used as a reference standard. After completing these sample preparation steps, the subsequent ELISA experiment was conducted strictly according to the instructions provided with the ELISA kit.

Real-time PCR

The RT-PCR method was used to determine the messenger ribonucleic acid (mRNA) expression levels of TRPV1 and 5-HT1A in the rat prefrontal cortex. The primers for amplifying the corresponding gene transcripts were designed based on relevant literature and confirmed by reference to the National Center for Biotechnology Information (NCBI) database. Gene expression was normalized to GAPDH as the endogenous control. The primers were then synthesized by Shanghai Sangon Biotech Co., Ltd. Ribonucleic acid (RNA) was extracted using the Trizol method. Using rat RNA as the template, SYBR Green one-step quantitative detection was performed using the ABI 7500 Fast Thermal Cycler. Different template loads of varying concentrations (120, 60, 30, 15, 0 ng/well) were set up to amplify the corresponding genes, obtaining amplification curves and melting curves. After diluting the RNA of each sample to the same concentration (5 ng/µL), the PCR reaction was carried out according to the table below, with a total volume of 20 µL per well. The experiment was set up with duplicate wells. The primers exhibited good amplification specificity and linearity, making them suitable for detecting gene expression in the samples.

Statistical analyses

All experimental data are expressed as mean ± standard deviation (SD), rounded to two decimal places. Statistical processing and analysis were carried out using SPSS version 23.0. Prior to analysis, the normality of data distribution was assessed using the Shapiro-Wilk test, and homogeneity of variance was evaluated using Levene’s test. The following statistical procedures were employed based on the research objectives: 1) A two-factor analysis of variance (ANOVA) was conducted to assess the main and interactive effects of exercise (rest and fatigue) and oxygen environment (normoxia and hypoxia) on the expression levels of TRPV1, 5-HT, and 5-HT1A receptors. In the event of a significant interaction, simple effects analysis was performed to decompose the interaction. When the interaction was non-significant, the main effects were interpreted directly.2) A one-way ANOVAs were performed to compare exercise exhaustion time among the OE, HE, HE1, and HE5 groups, and the mRNA expression of TRPV1,5-HT1A, and 5-HT among the HE, HE1, and HE5 groups. Significant ANOVA results were subjected to post-hoc pairwise comparisons using the Bonferroni method to control for Type I error.The significance level of the test was set as P < 0.05, and the very noteworthy significance level was set as P < 0.01.

Research results

Rat exhaustion time

Compared to the OE group, the HE group exhibited a significantly shorter exercise time (P < 0.01). This finding further indicates that the hypoxic conditions contributed to a decrease in exercise duration and a decline in the exercise capacity of the rats, confirming the successful establishment of the hypoxic exercise fatigue model in this study. Administration of AMG9810 significantly prolonged the time to exhaustion in the HE1 group (P < 0.01), and similarly, WAY100635 treatment extended the exercise duration to exhaustion in the HE5 group (P < 0.01). These findings indicate that both AMG9810 and WAY100635 are effective in delaying exercise exhaustion(Table 1).

Table 1.

Rat exhaustion exercise timetable

Group	Time(min)
OE	114.17 ± 33.58
HE	49.29 ± 8.06**
HE1	76.17 ± 30.68##
HE5	76.17 ± 19.52##

*Indicates comparison with OE, p < 0.05

**Indicates comparison with OE, p < 0.01

#Indicates comparison with HE, p < 0.01

##Indicates comparison with HE, p < 0.01

Expression of TRPV1 mRNA in rat prefrontal cortex

In the normoxic environment, the expression of TRPV1 mRNA does not show a statistically significant difference in the OE group compared to the O group(P > 0.05). In hypoxic conditions, the expression of TRPV1 was significantly upregulated in the HE group compared to the H group(P < 0.05). When comparing across oxygen environments, the expression of TRPV1 mRNA is not significantly different in the OE group compared to the O group(P > 0.05), and it was also not significantly different in the HE group compared to the OE group(P > 0.05)(Table 2; Fig. 1).

Table 2.

Relative expression levels of TRPV1mRNA、5-HT1AmRNA and 5-HT in the rat prefrontal cortex

	O	OE	H	HE
TRPV1	1.09 ± 0.11	1.26 ± 0.25	1.05 ± 0.84	1.39 ± 0.22*
5-HT1A	0.99 ± 0.08	1.88 ± 0.04**	2.32 ± 0.07##	2.91 ± 0.04**##
5-HT	1.03 ± 0.03	1.36 ± 0.13**	1.28 ± 0.13##	1.68 ± 0.15**##

Significant difference: Comparison within the group:* Indicates comparisons with the resting group of equal oxygen concentration, p<0.05,** Indicates comparisons with the resting group of equal oxygen concentration, p<0.01

Comparison between groups:#Indicates comparisons with normoxic peers, p<0.05,## Indicates comparisons with normoxic peers, p<0.0.1

Fig. 1.

TRPV1 PCR amplification and dissociation curves. A Amplification curve; (B) Dissociation curve

After intraperitoneal injection of TRPV1 blocker, the expression of TRPV1 in the prefrontal cortex of rats in the HE1 group was notably decreased. These results suggest that AMG9810 effectively downregulates the expression of TRPV1 in the prefrontal cortex. After intraperitoneal injection of 5-HT1A blocker in rats, the expression of TRPV1mRNA in the prefrontal cortex of the HE5 was not significantly different from that of the HE group (P > 0.05) (Table 3; Fig. 1).

Table 3.

Relative expression levels of TRPV1mRNA and 5-HT1A mRNA in rat prefrontal cortex after intraperitoneal injection blocker

	HE	HE1	HE5
TRPV1	1.39 ± 0.22	1.00 ± 0.26#	1.35 ± 0.15
5-HT1A	2.91 ± 0.04	2.39 ± 0.08##	2.26 ± 0.27##
5-HT	1.68 ± 0.15	1.30 ± 0.10##	1.56 ± 0.12

Significant difference: #Indicates compared to HE, p<0.05, ##Indicates compared to HE, p<0.01

Results of 5-HT concentration in rat prefrontal cortex

In the normoxic environment, the expression of 5-HT was significantly upregulated in the OE group compared to the O group (P < 0.01). In hypoxic conditions, the expression of 5-HT was significantly upregulated in the HE group compared to the H group (P < 0.01). When comparing across oxygen environments, the expression of 5-HT was significantly upregulated in the H group compared to the O group (P < 0.01), and it was also significantly upregulated in the HE group compared to the OE group (P < 0.01)(Table 2).

After intraperitoneal injection of TRPV1 blocker, the expression of 5-HT in the prefrontal cortex of rats in the HE1 group was notably decreased. These results suggest that AMG9810 effectively downregulates the expression of 5-HT in the prefrontal cortex. After intraperitoneal injection of 5-HT1A blocker in rats, the expression of 5-HT in the prefrontal cortex of the HE5 was not significantly different from that of the HE group (P > 0.05) (Table 3).

Expression of 5-HT1A mRNA in rat prefrontal cortex

In the normoxic environment, the expression of 5-HT1A mRNA was significantly upregulated in the OE group compared to the O group(P < 0.01). In hypoxic conditions, the expression of 5-HT1A mRNA was significantly upregulated in the HE group compared to the H group(P < 0.01). When comparing across oxygen environments, the expression of 5-HT1A mRNA was significantly upregulated in the H group compared to the O group(P < 0.01), and it was also significantly upregulated in the HE group compared to the OE group(P < 0.01) (Table 2; Fig. 2).

Fig. 2.

5-HT1A PCR amplification and dissociation curves. A Amplification curve; (B) Dissociation curve

After intraperitoneal injection of 5-HT1A blocker, the expression of 5-HT1AmRNA in the prefrontal cortex of rats in the HE5 group was dramatically declined compared with the HE. This suggest that WAY100635 effectively downregulates the expression of 5-HT1A mRNA in the prefrontal cortex (P < 0.01). After intraperitoneal injection of TRPV1 blocker, the expression of 5-HT1A mRNA in the prefrontal cortex of the rats in the HE1 group was dramatically declined compared with the HE(P < 0.01). This suggests that the activation of TRPV1 may affect the activation of 5-HT1A(Table 3; Fig. 2).

Discussion

Research indicates that as elevation increases in plateau environments, the efficiency of the human body decreases. At an altitude of 4,500 m, the body’s work capacity is only 60% of that at lower altitudes. In areas above 5,500 m, the maximum labor capacity is just 30% of that in the plains [19]. The decline in athletic performance in high-altitude environments can be attributed to hypoxic conditions, which reduce athletes’ ability to train effectively. The main reason is that low oxygen levels lead to a decrease in both the oxygen concentration and blood flow to the prefrontal cortex of the brain [20]. The capacity to maintain high minute ventilation in the face of reduced arterial oxygen hemoglobin saturation affects endurance exercise performance in hypoxic conditions. Additionally, hypoxic environments induce greater neuronal excitation in localized brain regions compared to normoxic environments, leading to increased oxygen consumption. Consequently, these brain regions require more nutrients to replenish the consumed oxygen, resulting in an earlier decline in exercise capacity in hypoxic environments [4].

Analysis of TRPV1 results

The research results show that compared with H, the expression of TRPV1 in HE significantly increased, while there was no such increase in OE and O. This hypoxia-specific upregulation indicates that hypoxic stress initiates TRPV1’s participation in the fatigue pathway. Further finding on the use of TRPV1 inhibitors (AMG9810) to enhance exercise capacity under hypoxia also supports the above view. Studies have shows that TRPV1 has a broader range of functions in the central nervous system, mediating multiple pathways including glial and neuronal responses, as well as cytokine release [21, 22]. The activation of TRPV1 may lead to the opening of calcium channel, calcium influx, and subsequent an increase in intracellular calcium concentration through ligand binding. This process can trigger a series of physiological or pathological events, such as regulating neurogenic inflammation and neurotransmitter release [8, 23]. In addition, the activation of TRPV1 can induce cell death in astrocytoma [24]. Previous studies have shown that the activation of TRPV1 channels stimulates sensory neurons and induces calcium accumulation in microglia. This increases the mitochondrial calcium load, leading to damage to the mitochondrial membrane potential and mitochondrial rupture [25].

Further study after the administration of a TRPV1 antagonist prolonged the exercise duration in rats. Therefore, strategic attenuation of TRPV1 may contribute to prolonged exercise endurance in hypoxic conditions. Inhibiting TRPV1 has shown potential benefits in enhancing exercise-induced cognitive functions and improving behavioral performance. For example, Razavinasab et al. demonstrated that inhibition of TRPV1 receptors can alter cellular distribution in the substantia nigra and effectively mitigate declines in motor and cognitive function induced by 6-hydroxydopamine in a rat model of Parkinson’s disease [26]. In the context of levodopa-related complications, studies have demonstrated that suppressing TRPV1 receptor activity can effectively prevent the development of motor disorders [27]. Furthermore, studies on mice with traumatic brain injury have shown that inhibiting TRPV1 receptors through intraperitoneal injection of an antagonist can reduce neuronal apoptosis in the brain tissue following the injury, significantly improving behavioral performance [28].Overall, our experimental results provide preliminary evidence that the activation of the TRPV1 channel may be one of the reasons for the decline in exercise capacity under hypoxic conditions.

Analysis of 5-HT results

The research results show that a significant increase in 5-HT levels in the prefrontal cortex immediately after exhaustive exercise under normoxic conditions. This increase is even more pronounced following hypoxic exercise. Comparing the normoxic resting state to the hypoxic resting state, there is a notable augmentation in 5-HT expression in the prefrontal cortex. This increase is particularly significant when comparing exhaustive exercises under normoxic and hypoxic conditions. This phenomenon may be related to the role of monoaminergic neurotransmitters in mediating ischemic neuronal injury during cerebral ischemia and hypoxia. Ischemic stroke triggers the release of monoaminergic neurotransmitters, leading to impairments in reuptake and degradation, resulting in an excessive accumulation of neurotransmitters in the extracellular space and even abnormal reuptake into surrounding neurons, causing acute neural damage. The excessive discharge of monoamines may act as potent agonists that open calcium channels, causing an influx of intracellular calcium, activation of phospholipases, and subsequent neuronal death [29]. The results suggest that the changes in the level of 5-HT in the prefrontal cortex during acute hypoxic exercise may lead to a premature decline in exercise capacity under hypoxia.

Central fatigue is thought to involve neurotransmitters including 5-HT, NE, and DA, with substantial evidence indicating that 5-HT plays a crucial role in its development [29]. Studies consistently demonstrate that elevated 5-HT concentrations promote fatigue, whereas reducing 5-HT levels extends exercise duration and attenuates fatigue. Prolonged exercise increases cerebral 5-HT concentrations, potentially impairing central nervous system function by disrupting central-peripheral signaling and compromising motor control [30, 31]. Gomez et al. [32] reported that in untrained rats subjected to a 120-minute run, extracellular levels of the neurotransmitter 5-HT and its metabolite, 5-HIAA, increased concurrently in the hippocampus and cortex. Both compounds rose significantly after 90 min of exercise and peaked within the first 30 min of recovery. These findings align with evidence that prolonged exercise elevates cerebral 5-HT, which inhibits motor neuron rhythmic activity and firing, ultimately inducing central fatigue [9–11]. Current research has found that increased prefrontal cortex 5-HT expression is associated with fatigue occurrence during normoxic and hypoxic exhaustive exercise, with post-exercise concentrations significantly exceeding controls. Thus, hypoxia-induced alterations in prefrontal cortical 5-HT levels may contribute to the early decline in exercise capacity under hypoxic conditions.

Analysis of 5-HT1A results

The serotonin (5-HT) receptor family encompasses seven distinct subfamilies (ranging from 5-HT1 to 5-HT7), each with multiple subtypes. The 5-HT1A receptor, a critical subtype, plays a pivotal role in the serotonin system, controlling mood, motor function, thermoregulation, and behavior. Studies indicate that 5-HT1A receptors are widely distributed on the soma and dendrites of serotonin neurons, acting as autoregulatory receptors for these neurons. The neurotransmission mediated by 5-HT1A receptors operates through an autoregulatory feedback loop. When activated by 5-HT or its agonists, these autoreceptors modulate the activity of serotonin neurons through a negative feedback mechanism, reducing the firing frequency of neurons, the release of neurotransmitters, and the activation of protein kinases [32]. The research results show that a rapid increase in 5-HT1A expression in the prefrontal cortex following strenuous exercise under normoxic and hypoxic conditions. Compared to the normoxic rest group, the hypoxic resting group showed a significant increase in 5-HT1A protein expression in the prefrontal cortex; similarly, compared to the normoxic exercise group, the hypoxic exercise group showed a significant in 5-HT1A protein levels in the prefrontal cortex. Kim et al.‘s study [29]found that strenuous exercise can enhance the expression levels of 5-HT1A receptors and serotonin transporters (5-HTT); conversely, treatment with colostrum serum can mitigate the expression of 5-HT1A receptors in the dorsal raphe region following intense exercise. Perrier et al.‘s study [33] revealed that the influx of 5-HT into the axon initial segment and subsequent activation of 5-HT1A receptors can inhibit the action potentials of motor neurons, thereby preventing muscle over-contraction during prolonged activity. Consistent with our findings, the increased expression of 5-HT1A may further suppress neural excitability, leading to an early decline in exercise performance.

Research suggests that the activation of 5-HT1A receptors plays a pivotal role in inducing exercise-induced fatigue [34]. Participants who engage in strenuous physical activity and are administered 5-HT1A receptor agonists experience a heightened sense of fatigue [12]. A study on rats exposed to chronic stress showed that 5-HT1A receptor agonists effectively reduced pathological aggressive behaviors in rats under chronic stress [35]. Our study observed that a decrease in endurance among rats subjected to simulated high-altitude exercise at 4000 m. Interestingly, administering a 5-HT1A antagonist to rats led to a significant increase in their exercise duration. Overall, Our research results indicate that the increase in 5-HT1A expression may further inhibit neural excitability, leading to an early decline in motor performance. Strategic regulation of the 5-HT1A signal may help improve the endurance performance of rats during acute hypoxic exercise.

Furthermore, this study also found that in the acute hypoxia exercise model, injecting a specific antagonist targeting TRPV1, AMG9810, into the abdominal cavity of rats could significantly down-regulate the expression of 5-HT1A mRNA and 5-HT in the prefrontal cortex of the rats. This suggests that the activation of the TRPV1 channel has a positive regulatory effect on the expression of 5-HT1A and 5-HT1A during the pathophysiological process of hypoxia-induced central fatigue. This finding provides a key mechanism explanation for the behavioral results observed in this study at the molecular level. We speculate that the regulatory mechanism of TRPV1 may involve the following pathways: the activation of TRPV1 may promote the release of 5-HT, and after binding to 5-HT1A receptors, it may trigger a positive feedback regulatory loop, thereby up-regulating its own expression. Although this study has revealed the regulatory relationship between TRPV1 and 5-HT1A at the transcriptional level, it still has limitations. Future studies using immunofluorescence double-labeling and other co-localization techniques to clarify the co-expression of TRPV1 and 5-HT1A in specific neuronal subtypes will help to elucidate the precise interaction mechanism between the two at the cellular level. These studies will jointly construct a more complete biological mechanism model of acute hypoxia-induced central fatigue.

Conclusion

This study indicates that acute hypoxic exercise leads to an upregulation of TRPV1 and 5-HT/5-HT1A expression in the rat prefrontal cortex, which is likely closely related to the decline in exercise capacity under hypoxic conditions. By separately applying the TRPV1 antagonist AMG9810 and the 5-HT1A antagonist WAY100635 for intervention, we found that inhibiting these two pathways not only downregulated their respective expression levels in the prefrontal cortex but, more importantly, significantly prolonged the animals’ incremental load exercise time under hypoxic conditions. These results suggest that the TRPV1 and 5-HT1A receptor pathways in the prefrontal cortex jointly participate in the process of hypoxia-induced decline in exercise endurance, and their upregulation may be one of the important mechanisms leading to limited exercise capacity. Therefore, targeting these two receptor pathways may offer new strategies for improving exercise performance under hypoxic conditions.

Prospects and limitations

The research provides preliminary evidence indicating that the decline in exercise capacity during acute hypoxia in rats is associated with the increased expression of TRPV1, 5-HT and 5-HT1A in the prefrontal cortex. This study highlights the key regulatory roles of TRPV1 and 5-HT1A in the decline of exercise capacity caused by acute hypoxia, suggesting that appropriate down-regulation of these molecules may prolong the exercise time of rats. This discovery has laid the foundation for future research on nutritional intervention. 1)The research only preliminarily demonstrated the potential role of blockers in enhancing hypoxic exercise capacity. Further experimental research is needed to explore and verify these findings. 2) Additionally, the animal model adopted in this experiment is designed to study the related mechanisms of hypoxia failure and may not fully reflect the situation of hypoxia failure in real life. The enhanced performance of AMG9810 (TRPV1 antagonist) and WAY100635 (5-HT-1-hydroxyl antagonist) supports the participation of these two pathways in fatigue attacks. However, we admit that the off-target effects of these inhibitors cannot be completely ruled out. Supplementary methods (for example, tissue-specific knockout models) are needed to confirm target specificity. 3) The independent efficacy of TRPV1 and 5-HT 1 antagonists suggests that fatigue pathways are parallel, but potential crosstalk (such as TRPV1 activation regulating 5-HT release) still requires further research to clarify.

Abbreviations

TRPV1

Transient receptor potential vanilloid subtype 1

5-HT1A

5-hydroxytryptamine1A

5-HT

5-hydroxytryptamine

Authors’ contributions

All authors contributed to the study. Jing Ma and Xing Huang conceived and designed research. Material preparation, data collection and analysis were performed by Jing Ma, Xing Huang, Lijing Gong, Yizhu Tang, Chi Xu. The first draft of the manuscript was written by Jing Ma, Xing Huang, Lijing Gong, Yizhu Tang, Chi Xu. All authors read and approved the final manuscript.

Funding

This work was supported by the Science and Technology Innovation Project of the General Administration of Sport of China (25KJCX004), Beijing Higher Education Teaching Reform Project(202310029005), and Mengniu-Capital University of Physical Education and Sports Collaborative Innovation Laboratory for Sports Nutrition.

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information.

Declarations

Ethics approval and consent to participate

This study is reported following the ARRIVE guidelines, was conducted following the Basel Declaration, and was approved by the Ethics Committee of Sports Science Experiment of Beijing Sport University (approval number: 2020176).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.