Stress is caused by a variety of stressors, that is, harmful stimuli or challenges that lead to various physiological, behavioral, emotional and cognitive alterations known as stress responses. It is an adaptive response mediated by the stress system, which includes the central nervous system and peripheral components in various physiological and pathological states. The primary components of the stress system are corticotropin-releasing hormone (CRH), also known as corticotropin-releasing factor, locus coeruleus-norepinephrine-autonomic systems and their peripheral effectors, the hypothalamus-pituitary-adrenal (HPA) axis and the limbs of the autonomic system (1–3). Stress responses are highly organized and regulated, and act to reduce the adverse impact of a stressor. By contrast with the view that the stress response restores the stability of an organism's internal environment (homeostasis), it is now hypothesized that stressors cause well-organized responses with their own homeostasis, to promote adaptive coping (1). These adaptive changes are usually time-limited, which may improve an individual's survivability. However, when stress is severe or chronic, or there is a functional defect of inadequate response to stress, the organization and regulation of stress responses may be disrupted, resulting in various endocrine, metabolic, autoimmune and psychiatric disorders (1,2). Therefore, to maintain a healthy state, it is crucial to limit the extent of harmful external stimuli, reduce the adverse effects of severe or chronic stress and enhance the body's resistance to stressors.

Following acute exposure to stressors, including fatigue, heat shock, skin burn, cooling, frostbite, immobilization and swimming under load, the amplitude and synchronization of the CRH and arginine-vasopressin pulsations in the hypophyseal portal system markedly increase, causing adrenocorticotropic hormone (ACTH) and cortisol secretion levels to increase (4). CRH serves a crucial function in the activation of the stress response. A circumscribed group of parvocellular neurosecretory neurons in the paraventricular nuclei (PVN) of the hypothalamus is the principal source of CRH for delivery to the portal capillary zone of the median eminence, although CRH is broadly expressed throughout the central nervous system (5,6). Glucocorticoids, as the final effectors of the HPA axis, help to maintain homeostasis and the body's response to stress, resulting in behavioral and peripheral changes that improve the ability to adjust to external challenges.

Adaptogens augment resistance to stress and increase concentration, performance and fatigue endurance (7–10). In the 1960s, on the basis of the results of numerous pharmacological and clinical studies, three plant species, namely Eleutherococcus senticosus, Rhodiola rosea (R. rosea) and Schisandra chinensis (S. chinensis), were incorporated into official medical practice in the Union of Soviet Socialist Republics as adaptogens, and were considered to exert stimulatory, restorative and anti-stress effects (10). These plants may be used by healthy individuals as tonics during states of fatigue and stress, and in sports medicine for preventing and treating injuries. In addition, they have been employed in occupational medicine for protection against adverse environmental factors, including extreme conditions such as high noise levels and low temperature, and in clinical medical practice for treating acute hepatic poisoning, ischemia due to oxygen deprivation and for accelerating recovery following surgery.

As medicinal plants, R. rosea and S. chinensis appear to possess the characteristics of adaptogens (10–13), which were described by Brekhman (14) as follows: i) Produces a non-specific response in an organism, for example, an increase in power of resistance against multiple stressors, including physical, chemical or biological agents; ii) has a normalizing influence on physiology, irrespective of the direction of change from physiological norms caused by the stressor; and iii) is incapable of influencing normal body functions more than required to gain non-specific resistance. By definition, adaptogens reinforce the non-specific resistance against stressors, increase the capacity to withstand stress, and thus protect against disease caused by excessive stress. Plant adaptogens do not exert unwanted effects on normal physiological functions (15). By contrast, stimulants such as caffeine and nicotine that increase alertness and concentration by enhancing the activity of the sympathetic nervous system, may possess addictive, tolerance and abuse potential and their long-term use may impair mental function and result in psychiatric disorders (15).

Previous studies have shown that ADAPT-232 forte, which comprises E. senticosus, S. chinensis and R. rosea extracts, prolongs the period until exhaustion by ~7-fold and repeated administration of this adaptogen may increase basal levels of heat shock protein 72 (Hsp72) in the serum of mice (16). A number of other stress-related factors have been extensively studied in the process of stress, including neuropeptide-Y (NPY) (17,18), Hsp70, activating protein-1 (AP-1) and cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) (19). In addition, the rapid induction of immediate early genes such as c-Fos, Fos-related antigen (Fra)-1, Fra-2 and c-Jun may alter gene transcription in hormone-secreting cells in the hypothalamus, pituitary gland and adrenal gland, which may result in changes in behavior, morphology, and the survival or degeneration of neurons in response to pathophysiological stimuli (19). Previous studies have shown that c-Fos in the hypothalamus is a reliable marker for activated cells and central nervous system circuits that respond to stress challenges (20–25). Furthermore, Fra-2 mRNA expression has been detected in the hippocampus (26), and the suprachiasmatic nucleus and PVN in the hypothalamus during stress. These stress-related factors have been demonstrated to be crucially involved in the mediation of the stress process.

In a previous study, we established an acute stress model in which adult rats were subjected to water-floating and high-intensity exercise, with the aim of simulating psychological and physical stress, respectively (27). The aim of the present study was to investigate the effects of S. chinensis and R. rosea in rats subjected to acute compound stress, and to determine the underlying mechanisms by evaluating the serum levels of ACTH, CORT and IL-1β and the mRNA expression levels of CRH, NPY, c-Fos and Fra-2 in the whole hypothalamus of the rats exposed to S. chinensis and R. rosea.

In the present study, a 3-h water-floating experiment including a feline predator stress element, and a 2-h treadmill exercise were conducted as psychological and physical stressors, respectively. Prior to the initiation of the experiment, all rats underwent 6 days of training to enable them to adapt to the long-term and intense stimulations on day 7. Following the experiment, serum levels of CORT and ACTH were determined, in addition to the CRH mRNA expression levels in the hypothalamus. CRH mRNA expression was significantly increased following exposure to prolonged compound stress; however, no significant differences in ACTH and CORT levels were observed. Furthermore, ACTH levels appeared to decrease slightly following the compound stress. It has been reported that the magnitude and duration of increases in serum ACTH concentration may be associated with the duration and intensity of the ACTH-releasing stimulus, and that elevated levels of ACTH persist throughout a 2-h period of ether stress (>700 pg/ml) (29). By contrast, ACTH levels have been shown to reduce markedly under prolonged mobilization stress, with a reduction to almost twice the basal levels after 6 h, from >600 pg/ml to <50 pg/ml; these levels persisted for the remainder of the stress period (30). Previous results suggest that the elevation in plasma CORT levels in response to stress is biphasic, whereas plasma ACTH levels exhibit only a single peak that rapidly declines to a sustained plateau level (31). Plasma levels of CORT, but not those of ACTH, have been found to remain elevated after 180 min restraint (32). In addition, it has been reported that running training attenuates the ACTH response induced by physical or psychological stress (33,34). In the present study, serum ACTH levels were evaluated after a total of 5 h exposure to psychological and physical stress. On the basis of the results of previous studies, it is not unexpected that the ACTH levels in the present study were not markedly increased, and decreased to a certain extent in the stress control group (<300 pg/ml on average) compared with the quiet control. In addition, a previous study has shown that blood concentrations of adrenal glucocorticoids increase to peak levels at 15–30 min and subsequently decline gradually to pre-stress levels at 60–90 min (35). This increase appears to be attenuated by repeated exposure to stress (36), which is consistent with the present result showing insignificant differences in CORT levels following the water-floating and treadmill exercise experiments. Therefore, this insignificance may be explained by the neural adaption caused by the previous repeated stress training during the first 6 days, indicating a possible mechanism of chronic stress characterized by reduced sensitivity of the HPA response to similar stressors, such as posttraumatic stress disorder (37–40).

Notably, the present results demonstrated that S. chinensis and R. rosea significantly decreased CRH mRNA expression levels (P<0.05) and the levels of serum CORT (P<0.05) in the rats exposed to water-floating and exhaustion experiments. CRH and CORT are pleiotropic, and exert effects via ubiquitously distributed intracellular receptors (41,42). S. chinensis increased the mildly stress-reduced ACTH levels, and R. rosea showed a similar, but less marked effect. These results suggest that S. chinensis and R. rosea are able to reduce the changes in CORT and ACTH levels caused by prolonged and intense compound stress, thus balancing the HPA axis and maintaining homeostasis in rats.

In the present study, the effects of S. chinensis and R. rosea on the immune system were investigated by evaluating the serum levels of IL-1β, which is a typical stress-associated cytokine and the most extensively studied inflammatory cytokine. The immune inflammatory response is considered to be closely associated with the stress system. Activation of the HPA axis produces marked inhibitory effects on the immune inflammatory response, as virtually all the components of the immune response are inhibited by cortisol (43). In turn, cytokines including TNF-α, IL-1β and IL-6 stimulate the HPA axis independently, or in combination with one another (44). IL-1 is a critical mediator of adaptive stress responses, in addition to stress-associated neuropathology and psychopathology (45). A recent study demonstrated that the activation of the HPA axis by IL-1β is dependent on IL-1 type 1 receptors in non-hematopoietic cells, such as brain endothelial cells, but not in perivascular macrophages (46). Furthermore, a 15-min swim stress has been shown to provoke a significant reduction in lipopolysaccharide (LPS)-induced IL-1β production at 120 and 240 min following LPS challenge (<200 pg/ml), compared with the control (>600 pg/ml) (47). In the present study, serum IL-1β levels in the rats were mildly, but not significantly, suppressed by compound stress. Treatment with S. chinensis and R. rosea appeared to increase the stress-reduced IL-1β levels. However, their effect on peripheral IL-1β levels was also mild and requires further investigation.

In addition to stress-induced changes in the HPA axis and cytokine expression following stimulation with various stressors, a number of ‘molecular chaperones’ appear to be crucially involved in the regulation of the neuroendocrine system and immune response (16). Stress-induced CRH expression causes ACTH release from the pituitary gland, and ACTH stimulates the release of adrenal hormones and NPY to mobilize energy and help the body to cope with stress, and in turn NPY activates the release of CRH (18,48). Adaptogens are known to activate the expression of NPY in brain cells (49). Therefore, NPY production by brain cells has been suggested as a characteristic marker of the adaptogenic activity of plant extracts (50). Numerous studies have reported that acute and repeated physical stress may significantly increase the protein and mRNA expression levels of NPY in the amygdala (17,51,52). In the present study, NPY expression in the hypothalamus following exposure to stress was investigated. The results showed that NPY mRNA expression levels in the hypothalamus were relatively low, with no statistically significant differences among the four groups. These results suggest that NPY expression in the hypothalamus does not have such a strong association with stress as does NPY expression in the amygdala. Further experiments are required to investigate the association between adaptogens and the production of NPY (protein and mRNA) in the amygdala after stress.

Numerous studies have identified the central pathways mediating the stress response by mapping neuronal activation using c-Fos as a dependable indicator (36,53). Weinberg et al demonstrated that the kinetics of the c-Fos response to acute stimuli is transient (32). In their study, c-Fos gene expression in the prefrontal cortex (PFC), lateral septum (LS) and PVN peaked at 15 min of restraint, moderately declined at 60 min of restraint and had reduced by 180 min of continuous restraint; PVN c-Fos expression at 180 min of restraint remained increased compared with the expression levels in control rats; restraint for 60 min produced a significant increase in Fra-2 mRNA expression in the PFC, LS and PVN, and by 180 min of restraint, Fra-2 expression returned to near-basal levels in all brain regions examined.

A previous study has demonstrated that stress induces a marked elevation in the expression levels of the AP-1 complex in the rat hypothalamus, pituitary gland, adrenal gland and gastric mucosa, suggesting that AP-1 binding may be a sensitive tool for monitoring the severity of inputs of extracellular stress signals (54). The protein Fra-2, which is a Fos-family member, competes with Fos protein for participation in the AP-1 transcription factor complexes. Each protein contributes different transactivation consequences to an AP-1 complex, leading to changes in the downstream expression of genes containing AP-1 elements in their promoter region (24,55). The present results demonstrated that c-Fos and Fra-2 gene expression levels were significantly increased by stress, as demonstrated by the c-Fos and Fra-2 mRNA expression levels remaining markedly elevated after the 5-h stress. In a previous study of seizure activity, Beer et al (26) observed the rapid induction of Fra mRNA by kainic acid and persisting levels after 6 h, with prolonged Fra-2 protein expression for 7 days in the hippocampus. It has been reported that Fra-2 protein levels in the adrenal medulla are significantly elevated after 2 h of a single or repeated immobilization stress, and are particularly pronounced at the termination of the immobilization, demonstrating the crucial transcriptional role of Fra-2 gene expression in the regulation of catecholamine biosynthetic enzymes in the stress response of the adrenal medulla (56).

In the present study, following persisting stimuli, Fra-2 mRNA expression was significantly induced in the hypothalamus after 5 h of physical and psychological stress. Furthermore, following treatment with S. chinensis or R. rosea, the compound stress-induced increase in c-Fos mRNA expression was attenuated, and was comparable with the levels in the quiet control rats. By contrast, Fra-2 mRNA expression remained unchanged at high levels following the administration of S. chinensis or R. rosea, and the effect of these herbs on the protein product of the Fra-2 gene and Fos protein expression in the hypothalamus requires further investigation.

The stress-associated markers investigated in the present study are associated with each other, participating in the complex process of the stress response. For example, a synthetic analogue of an ACTH (4–10) fragment, known as Semax, has been shown to reduce stress-induced Fos protein expression in the PVN and medial septum of emotionally stressed rats (57). This may explain the observation in the present study that ACTH levels were reduced and c-Fos levels increased in the stress control group compared with the quiet control group. As previously mentioned, IL-1β is able to independently stimulate HPA activity, leading to stress, potentially by enhancing the expression of c-Fos and CRH within the parvocellular division of the PVN (58).

In conclusion, the present study demonstrated that CRH, c-Fos and Fra-2 levels were significantly elevated after 5 h of prolonged compound stress. S. chinensis and R. rosea appear to exert anti-stress effects on stressed rats by balancing the HPA axis, and may reduce the expression of c-Fos in the hypothalamus. The present study had some limitations. As laboratory conditions were limited, alterations in the hypothalamic expression levels of proteins could not be investigated. The present study was also limited in that the whole hypothalamus was isolated, rather than one specific area; therefore, further studies are required to fully characterize the stress-protective effects of S. chinensis and R. rosea and to elucidate the mechanisms involved. These may include the analysis of Fos protein expression and its association with CRH production in hypothalamic neurons following treatment with S. chinensis or R. rosea and the potentially associated intracellular signaling pathways. The relatively moderate side effects and marked anti-stress effects of adaptogens indicate that they may provide an improved therapeutic option for the treatment of acute compound stress in the future.