Altered Intestinal Barrier Function In Food Allergy Rats

In resent years, food allergy has been recognized as a common worldwide health problem, and its incidence seems to be increasing. But so far, the mechanism of food allergy is not fully clear especially the injury mechanism of intestinal barrier function in process of food allergy is unclear. Under the influence of the mechanical and biochemical conditions, the integrity of intestinal mucosal barrier may be disrupted, so that causes TJ structures change, deletion, leading to abnormal changes in intestinal permeability. The previous study results indicated that the increase in intestinal permeability is an important incentive of food allergy, but further research in the molecular basis of intestinal barrier structural change has not been reported. So it is necessary to study the function changes of gut barrier and look for molecules of the intestinal tight junction structure associated with food allergy.ObjectiveTo investigate the changes of gut barrier when food allergy, and look for molecules of the intestinal tight junction structure associated with it, our hope is providing new theoretical foundation for prevention, diagnosis and treatment of food allergy.MethodsFood allergy models were constructed with Brown Norway (BN) rats. Rats in the experimental group were given 1mL of 1mg/mL ovalbumin (OVA) and the others in control group were given 1mL of 0.1mmol/L PBS orally everyday for 6 weeks.In modeling period, rats in each group were given lactulose (L) and mannitol (M) orally once a week, then 24h urines were collected and the urine lactulose and mannitol ratio (L/M) were detected and calculated by using high performance liquid chromatography (HPLC) to detect the intestinal permeability dynamically. At the same time, blood serums from the tail vein of rats in 1-6 weeks were obtained and antigen specific IgE, IgG were tested by ELISA dynamically. After the animals were sacrificed with ether anesthesia, 1cm of duodenum, upper jejunum, lower jejunum, upper ileum, and lower ileum were sampled from animals of each group. The samples were fixed, embedded, sectioned and stained to be observed the intestinal pathology. The ileums were also observed in electron microscopy.3 animals in each group were randomly selected and executed by clipping the ileum. The whole genome expression was analyzed by using microarray technique. After overall structure of TJ proteins changes were detected by gene chip, several key genes include ZO-1, Occludin, Claudin-2,8,9 and 15 were seleted to test mRNA levels using quantitative PCR.Results1. Model EvaluationBased on evaluation indicators of successful model,11 BN rats in the experimental group were successfully modeled, accounting for 64.7%. Serum OVA-IgE test results showed that the OVA-IgE levels were statistically distinct in different times (F=746.523, P<0.001), there were interaction between times and group (F=134.238, P<0.001), comparison of effects between the two groups also showed statistically significant difference (F=165.464, P<0.001), OVA-IgE levels between the two groups at the 6 week were statistically significant (F=686.37, P<0.001). Serum OVA-IgG test results showed that the OVA-IgG contents were statistically distinct in different times (F=30.797,.P<0.001), there were interaction between times and group (F=42.274, P<0.001), comparison of effects between the two groups also showed statistically significant difference (F=221.774, P<0.001), the OVA-IgG of the two group were statistically different in 3-6 weeks (F, respectively 38.96,93.30,155.08,454.70, P values were all less than 0.001).Observed by light microscope, the villis in control group arranged in neat rows, the structure was clear and complete, while the villis in experimental group were shown injury, loss, and inflammatory cell infiltration from duodenum to lower ileum at different degrees.2. Changes of the intestinal permeabilityUrine L/M test results showed that the intestinal permeability were statistically distinct in different times (F=1855, P<0.001), there were interaction between times and group (F=394.494, P<0.001), comparison of effects between the two groups also showed statistically significant difference (F=2127, P<0.001), the L/M of the two group were statistically different in 3-6 weeks (F, respectively 78.37,1121,937.94, 3238,3290, P values were all less than 0.001). 3. Changes of morphological structure and associated moleculars in intestinal tignt junctionObservated by transmission electron microscopic, Ileum intestinal microvillis in control group arranged in neat rows and their structures were integrity, tight junctions between cells were mutual integration. Various organelles were regularly arranged. But microvillis in the experimental group were swelling or lost, tight junction structure expansed, and all the organelles were significantly damaged. By tested with gene chip, intestinal TJ genes changed in experimental group were manily Claudins, which included Claudin-2,6,8,9,11,15,18 and 22. Claudin-9,15,18,22 increased, others decreased. The mRNA levels of Occludin and ZO-1 in experimental group compared to the control group did not change significantly.6 selected genes were analyzed by quantitative PCR, the express level of Claudin-9 increased significantly, but the ZO-1, Claudin-2, Claudin-8, Claudin-15 decreased in the allergy group. Occludin in both groups didn't change significantly.Conclusions1. The OVA-IgE and OVA-IgG both increase when food allergy, antigen-specific IgG responses occur in the early age of food allergy.2. Different inflammation degrees from the duodenum to the ileum are found in the food allergy rats.3. Intestinal permeability of sensitized rats increases visibly early in the food allergy.4. Changes of molecules in the intestinal tight junction structure when food allergy are mainly Claudins, in which Claudin-9,15,18,22 increase, but Claudin-2,6,8,11 decrease.