Many studies have shown that intestinal microorganisms can confer healthy benefits on their host. Probiotics are involved in regulating intestinal flora, immunity, and the mucosal barrier . Moreover, some studies have suggested that an important consequence of a modified bacterial community could be a change in the expression of a range of different bacterial genes in the bowel contents, as well as in the intestinal mucosa of the host. Analogous observations with probiotics, the stimulation of cytokines, and modification of immune responses could be important in producing beneficial effects . On the other hand, the EE, which promotes “eustress” or positive psycho-social stress , not only influences brain structure and function , but also significantly inhibits tumor growth in syngeneic melanoma, colon cancer , and breast cancer models . Moreover, some studies have indicated that physical exercise influences potential preventive pathways in the colon mucosa, reducing colon cancer risk . Therefore, EE can protect the intestinal mucosal barrier of CRC patients, thereby inhibiting tumor growth to a certain extent. Therefore, the comparative study of probiotics and EE can be used to explore their protective effects on the intestinal mucosal barrier, and also to explore the effects of combined intervention on the intestinal mucosal barrier in colorectal carcinoma.
Intestinal Mucosal Mechanical Barrier
Intestinal epithelial cells and tight junctions (TJs) between intestinal epithelial cells form the structural basis of the intestinal mucosal mechanical barrier. The TJ barrier function can also be affected by changes in the distribution of specific TJ proteins and/or their expression levels. The intestinal epithelial transmembrane binding protein occludin is one of the main closely connected proteins which is transmembrane protein engaged in zonulae occludens. It has been proven to affect the permeation of ions and soluble substances and is involved in regulating the migration of cell bypass immune cells [40–42]. Therefore, the combination of occludin with the length, thickness, and muscular thickness of intestinal epithelial villi can affect the intestinal mucosal mechanical barrier to a certain extent.
In this study, the four groups of rat intestinal epithelial tissues were examined by immunohistochemical to detect occludin expression levels. The results showed that neither the EE nor probiotics had any significant effect on the secretion of occludin, nor was there any difference in the expression of occludin between the four groups. Some studies have shown that long duration and high intensity exercise will destroy the TJ of the intestinal tract. Active occludin is an important part of intestinal TJs . All three parts of the rich environment can produce physical movement . Therefore, there is no significant effect on occludin in the development of colon cancer. The regulatory effect of probiotics on the intestinal mechanical barrier of CRC is mainly produced by enhancing the gene expression of TJ proteins such as occludin . However, some studies have shown that two weeks of intervention time is too short and has no obvious protective effect on intestinal mucosal barrier . The results of our study are similar to this conclusion. Therefore, a longer intervention time should be used in further studies.
The effects of EE on intestinal mucosal morphology, whether intestinal epithelial villus length, intestinal mucosa thickness, or muscle thickness, were all better than those of the probiotics group, suggesting that a probiotics intervention for two weeks in advanced CRC has a limited effect on the intestinal mucosal mechanical barrier. Treatment with probiotics was not able to resist the damage caused by tumor’s growth and consumption to the intestinal structure of the body and did not have a synergistic effect with EE. In previous studies on the effect of probiotics on colorectal cancer, most of the subjects focused on patients with colorectal cancer after operation . In the case of tumor resection, probiotics have a protective effect on intestinal mucosal mechanical barrier. However, this study did not remove the tumors, and probiotics did not play a significant role. Therefore, we speculate that the reason may be that the regulation of probiotics can not completely resist the damage of intestinal structure caused by the growth and consumption of tumors, and can not form a more effective synergistic effect with the rich environment. Social support, cognitive stimulation and physical movement in rich environments may lead to mechanical changes of intestinal mucosa by adjusting brain-gut axis or micro-environment, which may play a more effective role in maintaining or promoting the integrity of intestinal mucosal mechanical barrier .
Therefore, in the regulation of intestinal mucosal mechanical barrier, the role of enriched environment is better than that of probiotics and the interaction between them. However, the effect of occludin on intestinal pathomorphology needs further study.
Intestinal Mucosal Immune Barrier
Cytokines have a central role in systemic changes in cancer patients . Moreover, they are the major regulators of mucosal immunity and play an important part in the intestinal immune defense. Cancer patients generally have changes in cytokine levels, which seriously affect the metabolism and immunity of the body.
There are two main types of cytokine: (1) factors that promote the inflammatory reaction, such as TNF-α, IL-1, and IL-6; and (2) suppression of inflammatory response factors, such as IL-4 and IL-10 . Our results showed that the EE can regulate cytokines in both serum and intestinal mucosal; specifically, it can adjust IL-10, IL-6, and TNF-α, with beneficial effects on the body. Probiotics also play a part in regulating serum cytokines, but their beneficial effect on the body is weaker than that of the EE. The combination of probiotics and EE can produce an interaction, sometimes with a beneficial effect on the body; however, sometimes the combined effect is weaker than that of a single factor or may even have a negative effect on the body. Overall, the results show that EE can regulate the immune function of the intestinal mucosal immune barrier, with a more marked regulatory effect in the serum and intestinal mucosa IL-10. This is similar to the results of other studies, which showed that  EE as a benign pressure can regulate the level of adiponectin by regulating the hypothalamus sympathetic nerve cell axis, thus affecting the secretion of cytokines. The effect of the single use of probiotics in immune factor regulation is weaker than that of environmental enrichment. However, the interaction of probiotics and EE on cytokines needs further study.
SIgA is the most secreted immunoglobulin in the body and also an important part of the intestinal immune barrier. SIgA is resistant to proteolysis in the gut and does not activate the alexine and inflammatory reaction. SIgA is an ideal protective agent for intestinal mucosa and has an important role in determining the composition of the intestinal mucosal immune barrier. Therefore, SIgA can be used to evaluate the function of the intestinal mucosal immune barrier [40, 50, 51]. The results of this study showed that the EE and probiotics had no obvious effect on SIgA secretion. Upon further comparison, there were no significant differences between the different groups. Therefore, we cannot conclude that EE and probiotics play a part in the secretion of intestinal immunoglobulins. This may be because the intervention time was too short. A longer intervention time should be used in future studies to investigate the effects of EE and probiotics on SIgA.
The EE can directly or indirectly regulate the hypothalamic-pituitary-adrenal and hypotha-lamic- sympathoneural–adipocyte(HSA) axes, by utilizing the humoral system, neuroendocrine system, and immune system; regulating the gene expression of the hypothalamus; and promoting the proliferation of splenic lymphocytes and the mitosis of T cells. In this way, it can regulate serum and intestinal mucosal cytokine levels to protect the intestinal mucosal immune barrier . Probiotics can directly affect the intestinal tract, strengthening the activity of macrophages and other non-specific defense functions, thus protecting the intestinal mucosal immune barrier. However, their effect was weaker than that of EE on the body fluid-endocrine system. However, the production of SIgA is the result of a synergistic effect of B cells, T cells, and cytokines of the local microenvironment . The regulation mechanism of external factors needs further study.
Intestinal Mucosal Biological Barrier
Generally, BT refers to the translocation of intestinal bacteria from the intestinal lumen to the mesentery or other organs. Under normal conditions, intestinal BT does not occur easily, owing to tight intestinal junctions. However, BT increases during bacterial pathogenesis in the intestinal tract or during periods of stress, when the mucosal epithelium is damaged. Therefore, BT can be used to evaluate the permeability of the intestinal mucosal barrier . In this study, we found that there was no combined effect of EE and probiotics on BT; however, analysis of the individual factors showed that environmental enrichment and probiotics could both affect BT. Upon further analysis, the BT rate in the EE group was found to be lower than that of group P, indicating that the protection by probiotics of the intestinal mucosa biological barrier in CRC is limited compared with the protection by EE. The effect of EE on the intestinal mucosa biological barrier is thus more beneficial. Probiotics can decrease intestinal dysbacteriosis and the BT rate, and enhance the effect of resistance to pathogens, thereby protecting the intestinal barrier, inhibiting tumor growth and reducing intestinal complications. However, in this study, we found that the role of probiotics was not as important as that of the EE in CRC. A possible reason is that stress can reduce intestinal bacteria, such as lactic acid bacteria, while eustress can increase intestinal bacteria, prevent dysbacteriosis, and promote TJs of the intestinal mucosa, thereby reducing the incidence of BT . EE, as a kind of eustress stimulation , also helps to maintain the biological barrier function of the intestinal mucosa.
CRF is the main mediator for the central nervous system to participate in stress response. Under stress conditions such as diseases, CRF can be overexpressed to regulate gastrointestinal motility, secretion and sensation through the HPA axis. Ghrelin is an endogenous brain-gut peptide composed of 28 amino acids discovered by Japanese scientist Kojima in 1999.The binding of Ghrelin with its receptor can produce a wide range of biological effects, such as stimulating the secretion of growth hormone, regulating food intake and energy metabolism, regulating immune function, protecting gastrointestinal mucosa, regulating gastrointestinal motility, promoting gastric acid secretion, controlling the proliferation of gastrointestinal cancer cells and improving gastrointestinal dysfunction. Therefore, both corticotropin-releasing factor and Ghrelin can be used as important indicators for evaluating brain-gut axis function.
In the regulation of CRF, EE and probiotics could cross-react. Both EE and probiotics were beneficial to the secretion of CRF, but the effect of EE was greater than that probiotics, and the interaction of EE and probiotics was similar to the effect of probiotics alone.
Regarding the secretion of ghrelin, the intervention condition only influenced the secretion of hypothalamic ghrelin and had no effect on the secretion of intestinal mucosa ghrelin. There was no effect of the interaction on hypothalamic ghrelin secretion, but EE and probiotic separately can both effect the secretion of hypothalamic ghrelin. The effect of EE was greater than that of probiotics. Through two-two comparision, there was no significant effect on hypothalamic ghrelin secretion when using probiotics alone. When probiotics were combined with EE, the effect became more marked.
Therefore, in the study of brain-gut peptides, the role of EE is greater than that of probiotics. Studies have shown that both inside and outside body pressure and cognitive disorders affect the brain-gut axis, damaging the intestinal mucosal barrier with detrimental effects on its function. The cognitive training, social support, and physical exercise involved in the EE help to relieve body pressure and cognitive impairment [58, 59], thus protecting the intestinal mucosal barrier. In addition, some studies have shown that probiotics also modulate brain activity [22, 60], although our results suggest that this effect is less beneficial compared with environmental enrichment.
In summary, in the study of the intestinal mucosal barrier and brain-gut peptides, the effect of EE was greater than that of probiotics. However, the combined effect was not better than that of EE alone. In future studies, we intend to investigate the role of the environment and probiotics in determining SIgA levels, body weight, and the intestinal mucosal mechanical barrier by extending the intervention time.
There were no significant differences in body weight between different groups or within groups. The reason for this may be the short intervention time. In addition, we did not measure tumor weight. This was because the tumors in the rat were mostly multiple after the rats were dissected, which made it difficult to measure the weight of tumors. Therefore, to understand the effects of this intervention on body weight, further studies are needed with longer duration and deeper observation.