Protective Effect of a Cocoa-Enriched Diet on Oxidative Stress Induced by Intensive Acute Exercise in Rats.

Intensive acute exercise can induce oxidative stress, leading to muscle damage and immune function impairment. Cocoa diet could prevent this oxidative stress and its consequences on immunity. Our aim was to assess the effect of a cocoa-enriched diet on the reactive oxygen species (ROS) production by peritoneal macrophages, blood immunoglobulin (Ig) levels, leukocyte counts, and the physical performance of rats submitted to an intensive acute exercise, as well as to elucidate the involvement of cocoa fiber in such effects. For this purpose, Wistar rats were fed either a standard diet, i.e., a diet containing 10% cocoa (C10), or a diet containing 5% cocoa fiber (CF) for 25 days. Then, half of the rats of each diet ran on a treadmill until exhaustion, and 16 h later, the samples were obtained. Both C10 and CF diets significantly prevented the increase in ROS production. However, neither the cocoa diet or the cocoa fiber-enriched diet prevented the decrease in serum IgG induced by acute exercise. Therefore, although the cocoa-enriched diet was able to prevent the excessive oxidative stress induced by intensive exercise, this was not enough to avoid the immune function impairment due to exercise.

A Cocoa Diet Can Partially Attenuate the Alterations in Microbiota and Mucosal Immunity Induced by a Single Session of Intensive Exercise in Rats.

Background: Following intensive sports events, a higher rate of upper respiratory tract infections and the appearance of gastrointestinal symptomatology have been reported. We aimed to evaluate the effect of a cocoa-enriched diet on the cecal microbiota and mucosal immune system of rats submitted to high-intensity acute exercise, as well as to elucidate the involvement of cocoa fiber in such effects. Methods: Wistar rats were fed either a standard diet, a diet containing 10% cocoa providing 5% fiber and a diet containing only 5% cocoa fiber. After 25 days, half of the rats of each diet performed an exhaustion running test. Sixteen hours later, samples were obtained to assess, among others, the cecal microbiota and short chain fatty acids (SCFAs) composition, mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) lymphocyte composition, and immunoglobulin (Ig) content in salivary glands. Results: The intake of cocoa, partially due to its fiber content, improved the SCFA production, prevented some changes in PPs and in MLNs lymphocyte composition and also decreased the production of proinflammatory cytokines. Cocoa diet, contrary to cocoa fiber, did not prevent the lower salivary IgM induced by exercise. Conclusion: A cocoa dietary intake can partially attenuate the alterations in microbiota and mucosal immunity induced by a single session of intensive exercise.

Characterization of the Use of Emergency Contraception from Sentinel Pharmacies in a Region of Southern Europe.

Numerous studies have been published suggesting that emergency contraception (EC) is used repeatedly, but a lack of information regarding the profile of users makes it difficult to evaluate actual consumer habits. The aim of this study was to obtain information regarding the profile of users who obtain EC and other factors that might play a role, and to provide criteria to evaluate and improve the strategies of current contraceptive programs. This was an observational one-year study based on surveillance data on the provision of EC to women of reproductive age in 60 community pharmacies in Catalonia, Spain. In total, 941 notifications of dispensation of EC in Catalonia were received. A total of 44.2% of users said it was not the first time that they had taken the medication (repeat user). The percentage of users who used condoms was lower in repeat users compared to first-time users (56.7% vs. 64.4%, p < 0.05). A total of 25.7% of users stated that they did not use any barrier contraceptive method. The use of natural methods in repeat users was 53.8% in the subgroup who requested the medication after 48 h, significantly higher than in users who obtained the medication within the first 24 h (p < 0.05). A high percentage of repeat users with risky sexual behaviors were detected, suggesting that new measures must be implemented to provide information for this method, together with educational and preventive strategies.

Alterations in the mucosal immune system by a chronic exhausting exercise in Wistar rats.

Exhausting exercise can disturb immune and gastrointestinal functions. Nevertheless, the impact of it on mucosal-associated lymphoid tissue has not been studied in depth. Here, we aim to establish the effects of an intensive training and exhausting exercise on the mucosal immunity of rats and to approach the mechanisms involved. Rats were submitted to a high-intensity training consisting of running in a treadmill 5 days per week for 5 weeks, involving 2 weekly exhaustion tests. At the end, samples were obtained before (T), immediately after (TE) and 24 h after (TE24) an additional final exhaustion test. The training programme reduced the salivary production of immunoglobulin A, impaired the tight junction proteins' gene expression and modified the mesenteric lymph node lymphocyte composition and function, increasing the ratio between Tαß+ and B lymphocytes, reducing their proliferation capacity and enhancing their interferon-γ secretion. As a consequence of the final exhaustion test, the caecal IgA content increased, while it impaired the gut zonula occludens expression and enhanced the interleukin-2 and interferon-γ secretion. Our results indicate that intensive training for 5 weeks followed or not by an additional exhaustion disrupts the mucosal-associated lymphoid tissue and the intestinal epithelial barrier integrity in rats.

Influence of Hesperidin on Systemic Immunity of Rats Following an Intensive Training and Exhausting Exercise.

Intensive training and exhausting exercise can disrupt innate and acquired immunity. The flavanone hesperidin has shown immunomodulatory properties in physiological and some pathological conditions, and positive effects on exercise-induced oxidative stress. Nevertheless, it remains uncertain whether it also prevents exhausting exercise-induced immune alterations. The aim of this study was to establish the effect of oral hesperidin supplementation on the systemic immune system in rats following an intensive training and exhausting exercise. For this purpose, female Wistar rats were randomized into an intensive training group or a sedentary group. Intensive training was induced by running in a treadmill 5 days per week (including two exhausting tests) for five weeks. Throughout the training period, 200 mg/kg of hesperidin or vehicle was administered by oral gavage three times per week. At the end, blood, thymus, spleen and macrophages were collected before, immediately after and 24 h after an additional final exhaustion test. Hesperidin supplementation enhanced natural killer cell cytotoxicity and the proportion of phagocytic monocytes, attenuated the secretion of cytokines by stimulated macrophages, prevented the leukocytosis induced by exhaustion and increased the proportion of T helper cells in the thymus, blood and spleen. These results suggest that hesperidin can prevent exhausting exercise-induced immune alterations.

Alterations in the innate immune system due to exhausting exercise in intensively trained rats.

It is known that intensive physical activity alters the immune system's functionality. However, the influence of the intensity and duration of exercise needs to be studied in more depth. We aimed to establish the changes in the innate immune response induced by two programmes of intensive training in rats compared to sedentary rats. A short training programme included 2 weeks of intensive training, ending with an exhaustion test (short training with exhaustion, S-TE). A second training programme comprised 5-week training including two exhaustion tests and three trainings per week. In this case, immune status was assessed before (T), immediately after (TE) and 24 h after (TE24) an additional final exhaustion test. Biomarkers such as phagocytic activity, macrophage cytokine and reactive oxygen species (ROS) production, and natural killer (NK) cell activity were quantified. S-TE was not enough to induce changes in the assessed innate immunity biomarkers. However, the second training was accompanied by a decrease in the phagocytic activity, changes in the pattern of cytokine secretion and ROS production by macrophages and reduced NK cell proportion but increased NK cytotoxic activity. In conclusion, a 5-week intense training programme, but not a shorter training, induced alterations in the innate immune system functionality.

Protective Effect of Hesperidin on the Oxidative Stress Induced by an Exhausting Exercise in Intensively Trained Rats.

Intensive exercise can lead to oxidative stress, which can be particularly deleterious for lymphoid tissues. Hesperidin has demonstrated its antioxidant activity, but few studies focus on its influence on intensive training. The aim of this study was to assess the impact of hesperidin on the oxidant/antioxidant status of lymphoid tissues after an intensive training program. Wistar rats were trained for five weeks (five days per week), including two exhaustion tests plus three trainings per week. During this period, animals were orally administrated with 200 mg/kg of hesperidin or vehicle (three days per week). The oxidative status was determined before, immediately after and 24 h after an additional exhaustion test. The production of reactive oxygen species (ROS) by peritoneal macrophages, superoxide dismutase (SOD) and catalase activities in spleen, thymus and liver, and hepatic glutathione peroxidase activity (GPx) were assessed. Hesperidin prevented an increase in ROS production induced by the additional exhaustion test. Likewise, hesperidin avoided a decrease in SOD and catalase activities in the thymus and spleen that was found after the additional exhaustion test. The antioxidant effects of hesperidin were associated with a higher performance in the assessed training model. These results suggest that hesperidin, acting as an antioxidant, can prevent oxidative stress induced by exercise and improve exercise performance.

Role of Theobromine in Cocoa's Metabolic Properties in Healthy Rats.

Cocoa is rich in polyphenols and methylxanthines, and it has been reported that its consumption, among other properties, has beneficial effects on metabolism. This study aimed to investigate the role of theobromine in cocoa's metabolic properties in healthy rats. In addition to morphometric measurements, biochemical markers of lipids and glucose metabolism and gene expression of molecules related to immune cells in adipose and hepatic tissues were assessed after 7 or 18 days of diet. Additionally, a metabolomic analysis was carried out at day 7. This study revealed the presence of six discriminant metabolites in plasma due to the diets. Moreover, the results showed that theobromine is the main responsible factor for cocoa's effects on body weight gain as well as on lipid and glucose metabolism. The effects on body weight and lipids appeared as early as after 7 days of diet, whereas those affecting glucose metabolism required a longer intervention.

Hesperidin Effects on Gut Microbiota and Gut-Associated Lymphoid Tissue in Healthy Rats.

Hesperidin, found in citrus fruits, has shown a wide range of biological properties. Nonetheless, a more in-depth investigation is required on the effects on the immune system, and in particular, on the gut-associated lymphoid tissue, together with its relationship with the gut microbiota. Therefore, we aimed to establish the influence of oral hesperidin administration on the intestinal lymphoid tissue and on the gut microbiota composition in healthy animals. Lewis rats were orally administrated 100 or 200 mg/kg hesperidin three times per week for four weeks. Microbiota composition and IgA-coated bacteria were determined in caecal content. Mesenteric lymph node lymphocyte (MLNL) composition and functionality were assessed. IgA, cytokines, and gene expression in the small intestine were quantified. Hesperidin administration resulted in a higher number of bacteria and IgA-coated bacteria, with changes in microbiota composition such as higher Lactobacillus proportion. Hesperidin was also able to increase the small intestine IgA content. These changes in the small intestine were accompanied by a decrease in interferon-γ and monocyte chemotactic protein-1 concentration. In addition, hesperidin increased the relative proportion of TCRαß+ lymphocytes in MLNL. These results show the immunomodulatory actions of hesperidin on the gut-associated lymphoid tissue and reinforce its role as a prebiotic.

Changes in Lymphocyte Composition and Functionality After Intensive Training and Exhausting Exercise in Rats.

Exhausting exercise can have a deleterious effect on the immune system. Nevertheless, the impact of exercise intensity on lymphocyte composition and functionality remains uncertain. The aim of this study was to establish the influence of intensive training on lymphoid tissues (blood, thymus, and spleen) in Wistar rats. Two intensive training programs were performed: a short program, running twice a day for 2 weeks and ending with a final exhaustion test (S-TE group), and a longer program, including two exhaustion tests plus three runs per week for 5 weeks. After this last training program, samples were obtained 24 h after a regular training session (T group), immediately after an additional exhaustion test (TE group) and 24 h later (TE24 group). The composition of lymphocytes in the blood, thymus, and spleen, the function of spleen cells and serum immunoglobulins were determined. In the blood, only the TE group modified lymphocyte proportions. Mature thymocytes' proportions decreased in tissues obtained just after exhaustion. There was a lower percentage of spleen NK and NKT cells after the longer training program. In these rats, the T group showed a reduced lymphoproliferative activity, but it was enhanced immediately after the final exhaustion. Cytokine secretion was modified after the longer training (T group), which decreased IFN-γ and IL-10 secretion but increased that of IL-6. Higher serum IgG concentrations after the longer training program were detected. In conclusion, the intensive training for 5 weeks changed the lymphocyte distribution among primary and secondary lymphoid tissues and modified their function.

Theobromine Is Responsible for the Effects of Cocoa on the Antibody Immune Status of Rats.

Background: A 10% cocoa-enriched diet influences immune system functionality including the prevention of the antibody response and the induction of lower immunoglobulin (Ig) concentrations. However, neither cocoa polyphenols nor cocoa fiber can totally explain these immunoregulatory properties. Objectives: This study aimed to establish the influence of cocoa theobromine in systemic and intestinal Ig concentrations and to determine the effect of cocoa or theobromine feeding on lymphoid tissue lymphocyte composition. Methods: Three-week-old female Lewis rats were fed either a standard diet (AIN-93M; RF group), a 10% cocoa diet (CC group), or a 0.25% theobromine diet (the same amount provided by the cocoa diet; TB group) in 2 separate experiments that lasted 19 (experiment 1) or 8 (experiment 2) d. Serum IgG, IgM, IgA, and intestinal secretory IgA (sIgA) concentrations were determined. In addition, at the end of experiment 2, thymus, mesenteric lymph node (MLN), and spleen lymphocyte populations were analyzed. Results: Both CC and TB groups in experiments 1 and 2 showed similar serum IgG, IgM, and IgA and intestinal sIgA concentrations, which were lower than those in the RF group (46-98% lower in experiment 1 and 23-91% lower in experiment 2; P < 0.05). In addition, in experiment 2, the cocoa and theobromine diets similarly changed the thymocyte composition by increasing CD4-CD8- (+133%) and CD4+CD8- (+53%) proportions (P < 0.01), changed the MLN composition by decreasing the percentage of T-helper (Th) lymphocytes (-3%) (P = 0.015), and changed the spleen composition by increasing the proportion of Th lymphocytes (+9%) (P < 0.001) after 1 wk of diet treatment. Conclusions: The theobromine in cocoa plays an immunoregulatory role that is responsible for cocoa's influence on both systemic and intestinal antibody concentrations and also for modifying lymphoid tissue lymphocyte composition in young healthy Lewis rats. The majority of these changes are observed after a single week of being fed a diet containing 0.25% theobromine.

Cocoa Diet and Antibody Immune Response in Preclinical Studies.

The ability of cocoa to interact with the immune system in vitro and in vivo has been described. In the latter context, a cocoa-enriched diet in healthy rats was able to modify the immune system's functionality. This fact could be observed in the composition and functionality of lymphoid tissues, such as the thymus, spleen, and lymph nodes. Consequently, immune effector mechanisms, such as antibody synthesis, were modified. A cocoa-enriched diet in young rats was able to attenuate the serum levels of immunoglobulin (Ig) G, IgM, and IgA and also the intestinal IgM and IgA secretion. Moreover, in immunized rats, the intake of cocoa decreased specific IgG1, IgG2a, IgG2c, and IgM concentrations in serum. This immune-regulator potential was then tested in disease models in which antibodies play a pathogenic role. A cocoa-enriched diet was able to partially prevent the synthesis of autoantibodies in a model of autoimmune arthritis in rats and was also able to protect against IgE and T helper 2-related antibody synthesis in two rat models of allergy. Likewise, a cocoa-enriched diet prevented an oral sensitization process in young rats. In this review, we will focus on the influence of cocoa on the acquired branch of the immune function. Therefore, we will focus on how a cocoa diet influences lymphocyte function both in the systemic and intestinal immune system. Likewise, its potential role in preventing some antibody-induced immune diseases is also included. Although further studies must characterize the particular cocoa components responsible for such effects and nutritional studies in humans need to be carried out, cocoa has potential as a nutraceutical agent in some hypersensitivity status.

Influence of Hesperidin on the Systemic and Intestinal Rat Immune Response.

Polyphenols, widely found in edible plants, influence the immune system. Nevertheless, the immunomodulatory properties of hesperidin, the predominant flavanone in oranges, have not been deeply studied. To establish the effect of hesperidin on in vivo immune response, two different conditions of immune system stimulations in Lewis rats were applied. In the first experimental design, rats were intraperitoneally immunized with ovalbumin (OVA) plus Bordetella pertussis toxin and alum as the adjuvants, and orally given 100 or 200 mg/kg hesperidin. In the second experimental design, rats were orally sensitized with OVA together with cholera toxin and fed a diet containing 0.5% hesperidin. In the first approach, hesperidin administration changed mesenteric lymph node lymphocyte (MLNL) composition, increasing the TCRαß+ cell percentage and decreasing that of B lymphocytes. Furthermore, hesperidin enhanced the interferon (IFN)-γ production in stimulated MLNL. In the second approach, hesperidin intake modified the lymphocyte composition in the intestinal epithelium (TCRγδ+ cells) and the lamina propria (TCRγδ+, CD45RA+, natural killer, natural killer T, TCRαß+CD4+, and TCRαß+CD8+ cells). Nevertheless, hesperidin did not modify the level of serum anti-OVA antibodies in either study. In conclusion, hesperidin does possess immunoregulatory properties in the intestinal immune response, but this effect is not able to influence the synthesis of specific antibodies.

Effect of cocoa's theobromine on intestinal microbiota of rats.

SCOPE: To establish the role of cocoa theobromine on gut microbiota composition and fermentation products after cocoa consumption in rats. METHODS AND RESULTS: Lewis rats were fed either a standard diet (RF diet), a diet containing 10% cocoa (CC diet) or a diet including 0.25% theobromine (TB diet) for 15 days. Gut microbiota (fluorescence in situ hybridization coupled to flow cytometry and metagenomics analysis), SCFA and IgA-coated bacteria were analyzed in fecal samples. CC and TB diets induced lower counts of E. coli whereas TB diet led to lower counts of Bifidobacterium spp., Streptococcus spp. and Clostridium histolyticum-C. perfingens group compared to RF diet. Metagenomics analysis also revealed a different microbiota pattern among the studied groups. The SCFA content was higher after both CC and TB diets, which was mainly due to enhanced butyric acid production. Furthermore, both diets decreased the proportion of IgA-coated bacteria. CONCLUSION: Cocoa's theobromine plays a relevant role in some effects related to cocoa intake, such as the lower proportion of IgA-coated bacteria. Moreover, theobromine modifies gut microbiota although other cocoa compounds could also act on intestinal bacteria, attenuating or enhancing the theobromine effects.

Gut Microbiota in a Rat Oral Sensitization Model: Effect of a Cocoa-Enriched Diet.

Increasing evidence is emerging suggesting a relation between dietary compounds, microbiota, and the susceptibility to allergic diseases, particularly food allergy. Cocoa, a source of antioxidant polyphenols, has shown effects on gut microbiota and the ability to promote tolerance in an oral sensitization model. Taking these facts into consideration, the aim of the present study was to establish the influence of an oral sensitization model, both alone and together with a cocoa-enriched diet, on gut microbiota. Lewis rats were orally sensitized and fed with either a standard or 10% cocoa diet. Faecal microbiota was analysed through metagenomics study. Intestinal IgA concentration was also determined. Oral sensitization produced few changes in intestinal microbiota, but in those rats fed a cocoa diet significant modifications appeared. Decreased bacteria from the Firmicutes and Proteobacteria phyla and a higher percentage of bacteria belonging to the Tenericutes and Cyanobacteria phyla were observed. In conclusion, a cocoa diet is able to modify the microbiota bacterial pattern in orally sensitized animals. As cocoa inhibits the synthesis of specific antibodies and also intestinal IgA, those changes in microbiota pattern, particularly those of the Proteobacteria phylum, might be partially responsible for the tolerogenic effect of cocoa.

Effect of a cocoa diet on the small intestine and gut-associated lymphoid tissue composition in an oral sensitization model in rats.

Previous studies have attributed to the cocoa powder the capacity to attenuate the immune response in a rat oral sensitization model. To gain a better understanding of cocoa-induced mechanisms at small intestinal level, 3-week-old female Lewis rats were fed either a standard diet or a diet containing 10% cocoa for 4 weeks with or without concomitant oral sensitization with ovalbumin (OVA). Thereafter, we evaluated the lymphocyte composition of the Peyer's patches (PPL), small intestine epithelium (IEL) and lamina propria (LPL). Likewise, gene expression of several immune molecules was quantified in the small intestine. Moreover, histological samples were used to evaluate the proportion of goblet cells, IgA+ cells and granzyme+cells as well. In cocoa-fed animals, we identified a five-time reduction in the percentage of IgA+ cells in intestinal tissue together with a decreased proportion of TLR4+ IEL. Analyzing the lymphocyte composition, almost a double proportion of TCRγδ+cells and an increase of NK cell percentage in PPL and IEL were found. In addition, a rise in CD25+, CD103+ and CD62L- cell proportions was observed in CD4+ PPL from cocoa-fed animals, along with a decrease in gene expression of CD11b, CD11c and IL-10. These results suggest that changes in PPL and IEL composition and in the gene expression induced by the cocoa diet could be involved, among other mechanisms, on its tolerogenic effect.

Cocoa Diet Prevents Antibody Synthesis and Modifies Lymph Node Composition and Functionality in a Rat Oral Sensitization Model.

Cocoa powder, a rich source of polyphenols, has shown immunomodulatory properties in both the intestinal and systemic immune compartments of rats. The aim of the current study was to establish the effect of a cocoa diet in a rat oral sensitization model and also to gain insight into the mesenteric lymph nodes (MLN) activities induced by this diet. To achieve this, three-week-old Lewis rats were fed either a standard diet or a diet with 10% cocoa and were orally sensitized with ovalbumin (OVA) and with cholera toxin as a mucosal adjuvant. Specific antibodies were quantified, and lymphocyte composition, gene expression, and cytokine release were established in MLN. The development of anti-OVA antibodies was almost totally prevented in cocoa-fed rats. In addition, this diet increased the proportion of TCRγδ+ and CD103+CD8+ cells and decreased the proportion of CD62L+CD4+ and CD62L+CD8+ cells in MLN, whereas it upregulated the gene expression of OX40L, CD11c, and IL-1ß and downregulated the gene expression of IL-17α. In conclusion, the cocoa diet induced tolerance in an oral sensitization model accompanied by changes in MLN that could contribute to this effect, suggesting its potential implication in the prevention of food allergies.