| With the acceleration of our living pace, and with the increase of pressures from various aspects, the psychological stress has become a significant factor that threatens our health. Some certain stress has been acknowledged as related factors of common chronic diseases of digestive system, such as FGID, IBD, GERD and PU et al. Both clinical and laboratorial experiments have confirmed that acute and chronic psychological stress could cause detectable pathophysiological change of gastrointestinal tract. However, since the exact pathogenesis of these diseases is still unknown, we are still in dark about how to prevent and treat them effectively.As the pathogenesis and progression of diseases that are caused by psychological stress varies among different people, the researches about the treatments are quite restrained. Presently most of the researches in this field depend on animal models. And in order to find out the potential relationships between daily life stress and certain diseases, researchers developed several non-invasive physiological-stress-provoked animal models, among which the cold-restrain stress (CRS) model is a well-developed and easy-to-apply method. This model could immediately change the permeability of intestinal mucosa, without any surgical operations or drugs, and could be repeated easily. Therefore, many researchers have put a lot of emphasis on it nowadays.Enteric epithelium ceils layer is the frontier defensive barrier of the body. It could protect organs against harmful microorganisms and potentially pathogenic bacteria in the intestinal tracts. The breakdown of intestinal mucosa barrier function could lead to the invasion of bacteria, toxins and other macromolecule antigens into the body, and the exposure of mucosal immune system to the massive antigens from food and bacteria penetrated through intestinal epithelium will stimulate the immune system, which will cause inflammation intestinal diseases. Experiments have confirmed that acute constraint stress could loosen the tight junctions of epithelium cells, and increase the permeability of intestinal mucosa, which damages the barrier function and then does more damage to other systems in the body. Therefore, it is essential to protect the intestinal barrier function for preventing and treating stress-induced inflammation intestinal diseases, which is also the key point of this research.Recently with the development of microecology, the protective effects of microecology on intestinal barrier function and overall immune function have been noticed. Prebiotics, especially prebiotics oligosaccharide could help balance the bacteria in intestinal tracts as micro-ecological regulator. And it could improve the permeability of intestinal mucosa, and enhance immune function, then help maintain the intestinal barrier function. In various oligosaccharides, galacto-oligosacchardes(GOS) is considered of great potentials. Many researches have been applied to treat severe clinical diseases, but the exact mechanisms are still not known.This research focuses on the protective effect of GOS on CRS rat mucosal mechanical barrier function and its mechanism. The research was carried out from three parts:1, Establishment of model of cold-restrain stress in ratsRats were fasting for 24 hours before experiments, but free to drink water. Then the rats were anesthetized with ethylether and fixed into fixation box to restrain their movements; then they were put into 4℃refrigerator and restrained for 3 hours.After successful model construction, the HE staining results showed that the pathological damage to intestinal mucosa is obvious: mucosa was thinner and shrinked, there was necrosis in the superficial layer, and the epithelium cells shedded, on the top of villus propria lamina there was obvious haematoma, and the distance between intestinal epithelium cells and propria lamina was increased.There were no rat died, and this method was suitable for this research because the pathological change in the intestinal mucosa was satisfying, and it could be repeated simply.2, Effect of prebiotics on CRS rat intestinal barrier permeability and its mechanic barrier change2.1 Effect of prebiotics on CRS rats intestinal barrier permeabilityThe plasma DAO level in CRS groups was significantly higher than that of control group, which prompted that there was necrosis and shed of the intestinal epithelium cells in CRS rats, therefore DAO entered plasma. The level in C group (basal diet + CRS) was higher than that in D group (GOS+CRS), which suggested that GOS could reduce the damages to intestinal mucosa and decrease the level of plasma DAO, despite that it could not completely reverse the situation of impaired intestinal mucosa in CRS.2.2 Effect ofprebiotics on small intestinal mechanic barrier change of CRS ratTo investigate the role of epithelial cell apoptosis in intestinal barrier function in CRS rats, the apoptosis index (AI) of intestinal mucosa epithelial cells was analyzed with terminal deoxynucleotidyl-transferase mediated dUTP--biotin nick and labeling (TUNEL) method. The AI in CRS model groups was significantly higher than that in control group, which indicated an obvious increase in CRS rats. The results in D group (GOS+CRS) was lower than that in C group (basal diet + CRS), which suggested that prebiotics, especially GOS was quite helpful in keeping intestinal mechanic barrier by decreasing the occurrence of epithelial cell apoptosis.2.3 The effective mechanism of prebiotics on integral membrane protein occludin localizing at tight junction of intestinal epithelial cells in CRS ratThe expression of occludin protein in intestinal epithelial tight junction was analyzed by immunohistochemistry method. The results showed that the occludin protein level in CRS groups was significantly lower than that in control group, and that in C group (basal diet+CRS) was significantly lower than that in D group (GOS+CRS). There was no significant difference between A and B group. The occludin mRNA level was detected by real-time fluorescent quantitative PCR. The occludin mRNA level in CRS groups was significantly lower than that in control group, with that in C group lower than that in D group, and there was no significant difference between A and B group. The results suggested that prebiotics could increase the expression of occludin protein and mRNA level, which could be one main cause for its effect in enhancing tight junction and maintaining intestinal barrier function, and the up-regulation for occludin protein might be at transcriptional level. |
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