Effect of chronic stress and chewing on serum interleukin-6 and interferon-gamma levels.

INTRODUCTION

Walter Cannon first described stress and the concept of homeostasis in 1915, then Hans Selye investigated “the physiological response to stress” (1936) defining it as an alarm reaction. In those days, stress had a more static value, because the permanent dynamism of physiological processes and adaptation within a specific context were not well understood(1,2). A stressor agent has a frequency and duration; and becomes chronic if it is extended over time(3,4). Research on the biology of chronic stress involves various families of cytokines, among which IL-6 and interferons (IFN) stand out, and have been most frequently studied as markers of the interface between psychology and biology(5,7). This is because these cytokines are associated with psychosocial stimuli, influencing behavior and psychological states; causing changes in the biological processes within the nervous system that are manifested with biopsychological reactions such as anxiety.(5-8)

Due to the immune alterations it produces, stress increases the expression of certain cytokines augmenting susceptibility to infections, cancer and cognitive alterations, it can also increase allergic or autoimmune conditions due to the imbalance it causes in circulating cytokines(9-11). Himmerich et al.(11), observed increased levels of IL-6 and a decreased production of INF-? in rodents exposed to stress by immobilization, compared to rats that were not stressed. They concluded that the increase in IL-6 would is a sign of a pathophysiological pathway that begins with acute stress, and continues with chronic stress, developing into depression, cognitive alterations, autoimmune disorders, allergies and cancer. Furthermore, the decrease in INF-? would explain the greater susceptibility to infection observed in periods of life associated with sustained levels of stress. On the other hand, it has been found that IL-6, and other members of this family of cytokines, would have not only pro-inflammatory but also anti-inflammatory functions(12). Pagliarone et al.(13) reported on finding inhibition in the production of IFN-? in mice exposed to stress. Li et al.(14) It was then related to the increase in cortisol due to stressful stimuli, discovering that an increase in cortisol decreases serum levels of IFN-? and allows the appearance of autoimmune and infectious diseases. In addition, it has been reported that the increase in circulating catecholamines, due to stress, also inhibits the release of IFN-?.(7,11)

In general, chewing is known as the first process within the digestive function; however, it is now understood that the masticatory function would also have regulatory functions on the nervous and immune system(15-17). In humans, nail, teeth clenching, and biting objects are considered stress responses; thus, it is known that chewing or biting attenuates stress-induced diseases such as: gastric ulcers, cognitive problems, and psychological disorders(15). This information can be found on several research in rodents, in which the masticatory function of the animals was stimulated. This action decreased effects of stress on the hypothalamus-pituitary-adrenal axis and on the reactive activity of the autonomic nervous system, diminishing the secretion and consequently the plasma levels of corticosteroids(15,18). In addition, it has been reported that stress induced in rodents without masticatory stimulation increased serum levels of IL-6. On the other hand, an adequate chewing stimulus decreased the levels of this cytokine, as well as IL-1?

and IL-1?.(19)

Due to the importance that stress, and cytokines have on physiology and pathophysiology, highlighting their effect on the nervous system and human behavior, and the role that chewing has on the serum expression of cytokines during stress, are subjects that lack studies and are still unclear clear. This research has addressed these variables, with the hypothesis that chewing reduces the effects of chronic stress on serum concentrations of IL-6 and INF-? in mice belonging to Balb/cc strain.

METHODOLOGY

Animals

The experimental study was approved by the Vice President for Research of the Universidad Nacional Mayor de San Marcos (UNMSM), and 64 8-week-old Balb/c male albino mice (National Institute of Health of the city of Lima, Peru) weighing between 25 and 30 g were used. After acquisition, the animals were acclimated for a week in the animal facility of the School of Medicine at UNMSM, where they were fed ad libitum with the same grain diet, (common grains for mice) that they received since weaning. The mice were exposed to alternating light and dark cycles of 12 hours with controlled humidity and temperature, according to the Guide for the Care and Use of Laboratory

Animals.(20)

Procedures

After a week of adaptation in the vivarium, the mice were randomly distributed into 4 equal groups, applying the following:

NE group: Normal chewing + stress due to immobilization. The same grain diet and stress due to immobilization was applied.

N Group: Normal chewing without exposure to stress, continued with the same grain diet and no stress due to immobilization was applied.

DE Group: Inferior chewing + stress due to immobilization. Mice were fed with powdered grains (powder diet) to induce masticatory deficiency and stress due to immobilization was applied.

D Group: Inferior chewing without being exposed to stress. They were fed with pulverized grains (powdered diet) to induce masticatory deficiency and no immobilization stress was applied.

Paradigm for stress induction by immobilization: After a week of setting, the NE and DE groups were induced to stress by immobilization. The rodent was placed in a transparent polystyrene restraint box to keep it immobile, according to the design of Lam-Figueroa et al(21). This procedure was performed only once a day between 08:00 and 12:00 for 60 minutes throughout the research.

Paradigm to vary the type of chewing: All animals received the same grain diet (conventional for mice) since weaning. After the week of setting, groups N and NE continued with the same diet, while groups D and DE switched to a powdered diet, with powdered grains. This design allows modifying the type of chewing by varying diet consistency.(22)

IL-6 and IFN-? quantification: To analyze cytokines, eight mice from each of the 4 experimental groups were sacrificed by cervical dislocation(23) at the end of the fourth week of treatment. The same was done with the other half of the sample at the end of the eighth week of treatment. Blood from the mice was collected by cardiac puncture in Minicollect® Z serum tubes using a 1 ml 25Gx5/8 sterile Segurimaxx® brand syringe, for analysis purposes. The sample was incubated at 37°C for 30 minutes and centrifuged at 10,000 rpm for 10 minutes at room temperature. Serum was stored at -80°C until analysis. For the quantification of cytokines, sandwich ELISA kits for IL-6 and IFN-? (Invitrogen®, 88706488 and 88831488, correspondingly) were used and the procedure was carried out according to the manufacturing instructions. The concentration of cytokines in the serum samples of the treated mice in the 4 experimental groups was calculated using 2-order polynomial regression by ELISA reader (Sinowa® ER500) at D.O450 and D.O630.

Analysis and interpretation of information: The analysis and interpretation of the data was performed using the Graphpad Prisma version 5.0 software for Windows. Descriptive statistics were first performed, where the data were grouped according to their means and standard deviation. Inferential statistics were then applied, where it was determined that the variables followed a normal distribution according to the Shapiro-Wilk test and had homoscedasticity according to Levene’s test A variance analysis was performed using Tukey’s test. A significance of 0.05 was considered.

RESULTS

The four experimental groups of Balb/c mice were evaluated at the end of the 4th and 8th week of treatment. Table 1 shows the means ± standard deviation found in the serum for IL-6 and IFN-?.

In the control carried out at the end of the 4th week of treatment, it was found that the mice in the DE group increased the production of IL-6 compared to the other groups (p<0.05), while the NE treatment did not produce significant results regarding its N control. In relation to IFN-?, mice from groups DE and NE increased their production compared to D and N (p<0.0001) and (p<0.01) correspondingly, as shown in Figure 1A and 1B.

In the control carried out at the end of week 8 of treatment, an increase in the production of IL-6 was observed in the mice of the NE group compared to the other groups (p<0.0001), while the DE treatment did not show a significant difference compared to D. IFN-? increased significantly in the DE treatment compared to the other groups (p<0.0001); on the other hand, the NE treatment did not produce significant results regarding N control, as shown in Figura 2A y 2B.

DISCUSSION

The aim of this study was to determine the effect of chewing and chronic stress over serum levels of IL-6 and INF-?. Four groups of 16 mice each were used. Two groups were exposed to stress, one of them, additionally had poor chewing (DE), while the other had normal chewing (NE). In the other 2 groups, only chewing was modified, one group with normal chewing (N) and the other with poor chewing (D). To quantify the cytokines using the ELISA test, half of the members of each experimental group were sacrificed at the fourth week, and the other half at the eighth week of starting the experiment.

From the mice exposed to stress, the ones within DE group, had increased IL-6 and in IFN-? action at the end of week 4 of, as well as the IFN-? at the end of week 8. Additionally, at the end of the 4th week, an increase in IFN-? was found in the NE group compared to treatments N and D; Also, in this group there was a significant increase in IL-6 at the end of the 8th week compared to the other groups evaluated. The results obtained coincide with those of Voorhees et al.(24), and Himmerich et al.(11), who established that prolonged stress, induced by movement restriction in mice, increases IL-6. Cheng et al.(25) showed that mice twice stressed by electrical shocks in the legs accelerated the increase in IL-6 levels in comparison to mice stressed only once with this method. It has also been shown that mice exposed to acute stress induced by radiation significantly increased their levels of IL-6 and IFN-?(26). Other researchers have found that the induction of chronic stress in rodents leads to depressive behavior and an increase in the cytokines IL-6 and TNF-?(25-29). In addition, it has been reported that the impact of stressful aggressive encounters increases the plasma levels of IL-6 in mice(29). All the referenced studies coincide with what was found in this study regarding the increase in IL 6 in the groups exposed to stress (NE and DE). It is relevant to mention that in the study by Rong et al.(26), acute stress was evaluated, whereas in the present study chronic stress was used. However, what is observed is that stress, regardless of its duration, increased the serum expression of IL-6.

The results of this study, show that chronic stress and poor chewing increase the production of IL-6 and IFN-? at the end of the fourth and eighth week of treatment, as a difference to animals with normal chewing and with no stress (group N). Chronic stress activates the hypothalamic-pituitary-adrenal axis and the sympathetic-adrenal-medullary axis, which secrete glucocorticoids and catecholamines, which increase the secretion of proinflammatory cytokines(30). Wang et al.(31), showed that mice induced to stress by immobility significantly increased the production of IL-6, which is related to the hyperactivity of the axis. Assaf et al.(32), showed differences in the production of IFN-? in people under academic stress; thus, in the initial and the middle stage of the academic semester, a significant increase in IFN-? was observed, while at the end of the semester IFN-? decreased. The researchers concluded that the cytokines decrease at the end of the semester, was related to the increase in cortisol at that stage, as it was a period of greater stress for the students. However, in our study, the groups exposed to chronic stress (NE and ED) had high serum levels of IFN-? both in the fourth and eighth week of the study. This finding could suggest a different behavior in rodents compared to humans regarding the concentration of cytokines. Given the important role that this molecule plays in human physiology and pathophysiology, further research on IFN-? is needed. This is also needed in the case of cytokines behavior assessed in this study, under conditions in which they have not yet been evaluated, such as: chewing and acute stress or chewing and intermittent stress. This is due to the important role that the masticatory function seems to play in stress modulation, and that unfortunately, especially in Latin America, even public policies do not prioritize maintenance and timely rehabilitation, which would not only affect a digestive function but, above all, its modulating role of various functions in the nervous and immune systems.

In conclusion, chronic stress related to poor chewing increased IL-6 and IFN-? levels in Balb/c mice. A protective role of adequate chewing can be understood by not increasing the level of IL-6 to the same extent as in the NE group, when compared to the ED at the end of week 4 of evaluation. However, this protective role is no longer observed at the end of week 8, which allows us to understand that stress exceeds, over time, the beneficial effects that chewing would have.