Cocoa and cocoa fibre intake modulate reactive oxygen species and immunoglobulin production in rats submitted to acute running exercise

Acute high-intensity exercise can impair the immune system, and lead to oxidative stress. Cocoa intake might help in protecting against oxidative damage and impaired immune functioning. The aim of this study was to establish the effect of cocoa and cocoa fibre on the oxidative status and the immunoglobulin (Ig) production of rats following a bout of acute exercise on a treadmill. The production of reactive oxygen species (ROS) by macrophages and the concentration of serum and mucosal Ig was assessed 16 h after the running session. Exercise increased ROS production and decreased the serum IgG concentration and the salivary gland IgM content. A cocoa fibre-enriched diet prevented the increased ROS production and the reduction in salivary IgM induced by exercise, although it decreased the IgA content in serum and the salivary glands. Overall, cocoa, by means of its fibre content, can partially prevent the alterations in ROS and Ig production induced by a single session of intensive running exercise.


Introduction
Although regular bouts of moderate-intensity exercise provide several health benefits, acute high-intensity exercise can impair the immune system, particularly the mucosal immune system [1], and lead to oxidative stress [2]. Due to its content of polyphenols and other bioactive compounds, cocoa intake might help protecting against the oxidative damage and the impaired immune function [3]. Moreover, cocoa fibre, by influencing the intestinal immune system [4], could also play a protective role.

Animals
Four-week-old female Wistar rats (N = 48) were purchased from Envigo (Huntingdon, United Kingdom) and maintained in the animal facilities of the Faculty of Pharmacy and Food Science at the University of Barcelona under controlled temperature and humidity conditions, in a 12/12h light/dark cycle. Animals were housed in polycarbonate cages (4 rats per cage) and were given ad libitum access to food and water. All animal procedures were approved by the Ethical Committee for animal Experimentation of the University of Barcelona and the Catalonia Government (CEEA/UB ref. 517/18).

Acute Exercise Protocol and Diets
Exercise was performed on a treadmill for rodents with electric shock stimulation (Exer3/6, Columbus, OH, USA). At the beginning of the study, rats were adapted to the treadmill for 1 week by increasing time and speed. Then, all rats performed a pre-selection exhaustion test with an initial speed of 18 m/min with increasing increments of 3 m/min every 2 min until exhaustion, which was defined as the time that rats remained at the end of the treadmill despite the shock stimulation. From the physical performance in the pre-selection exhaustion, rats were randomly distributed into three dietary groups (n = 8/each): reference (REF), 10% cocoa (C10) and 5% cocoa fibre (CF). The reference group (REF) was fed with a standard AIN-93M diet (Envigo); the cocoa group (C10) received a special chow containing 10% cocoa (Idilia Foods S.L., Barcelona, Spain) and the 5% cocoa fibre group (CF) was fed with a diet containing the same fibre proportion as C10 diet but with a lower amount of polyphenols (Table 1), as in previous studies [4].

Sample Collection
In order to assess the oxidative stress and the Ig production, runner rats were euthanized 16 h after running the exhaustion test (ET). Likewise, sedentary rats were euthanized randomly over the same days. Animals were anaesthetized by intramuscular injection of ketamine (Merial Laboratories S.A., Barcelona, Spain) and xylazine (Bayer A.G., Leverkusen, Germany) (90 mg × Kg −1 and 10 mg × Kg −1 , respectively) and exsanguinated. Blood, peritoneal macrophages, submaxillary salivary glands (SMG), caecal content (CC) and the small intestine were collected.
The proximal part of the small intestine was used to obtain gut wash (GW), as previously described [6]. Briefly, the intestine was washed with cold PBS (pH 7.2) in order to remove faecal content, opened lengthwise, cut into small pieces, weighed, incubated for 10 min in a shaker at 37 °C (55 shakings × min −1 ) and centrifuged (538 g, 4 °C, 10 min). Then, supernatants were collected and stored at −20 °C until IgA quantification.

Peritoneal Macrophage Isolation and ROS Production
Peritoneal macrophages were obtained as described previously in our laboratory [7]. Briefly, cells were collected after a 2 min massage with a 40 mL sterile PBS injected in the peritoneal cavity, centrifuged and resuspended with cold Roswell Park Memorial Institute (RPMI) medium without phenol red and supplemented with 1% heat-inactivated fetal bovine serum, 100 IU/mL streptomycinpenicillin, 2 mM L-glutamine and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich, Madrid Spain). After cell counting using a Spincell hematology analyzer (MonLab Laboratories, Barcelona, Spain), marophages were plated (10 4 cells/well) on a 96-well black plate (Sigma-Aldrich) and incubated overnight in order to assess the ROS production [7]. Then, cells were washed and incubated for 30 min with 20 µM of reduced 2′,7′-dichlorofluorescein diacetate probe (H2DCF-DA, Invitrogen, Paisley, UK) in order to oxidise the H2DCF-DA to a fluorescent compound (2′,7′-dichlorofluorescein) by means of the macrophage-derived ROS. The obtained fluorescence was measured at 0 and 120 min by the fluorimeter Modulus Microplate Multimode Reader (Turner BioSystems, CA, USA). ROS data are expressed as the percentage of increase between 0 and 120 min.

Immunoglobulin Quantification
The concentrations of IgG, IgM and IgA in serum, SMG or GW were quantified by a sandwich ELISA (Bethyl Laboratories Inc., Montgomery, TX, USA), as previously described [8]. Absorbance was measured on a microplate photometer (Labsystem Multiskan, Helsinki, Finland) and data were interpolated according to the respective standard curve by Ascent v.2.6 software (Thermo Fisher Scientific, Barcelona, Spain). In order to normalize the Ig content in SMG, total protein concentration was measured using the Pierce-660 nm Protein Assay Reagent (Thermo Fisher Scientific).

ROS Production by Peritoneal Macrophages
The production of ROS from peritoneal macrophages obtained from sedentary and runner rats, fed either with the REF diet, CF diet and C10 diet, was quantified ( Figure 2). The ROS production was increased in the acutely exercised rats from the REF group (p < 0.05 vs. REF-SED group), which was prevented by the cocoa intake, especially by means of its fibre content (p < 0.05, CF-RUN group vs. REF-RUN group).

Immunoglobulin Production
The serum contents of IgG, IgM and IgA 16 h after performing the running exhaustion test were quantified (Figure 3).  In summary, acute exercise increased ROS production and decreased the serum IgG concentration and the salivary gland IgM content. The cocoa fibre-enriched diet prevented both the higher production of ROS and the reduction in salivary IgM induced by exercise, although it decreased the IgA content in serum and salivary glands. The cocoa diet also altered the Ig profiles, evidencing a higher impact in runner animals than in the sedentary ones.

Conclusions
Overall, cocoa, by means of its fibre content, can partially prevent the alterations in ROS, and Ig production induced by a single session of intensive running exercise.