Hypersensitivity In ICR Mice Triggered By Fenneropenaeus Chinensis Muscle Proteins

Sensitization of edible proteins from shrimp and other aquatic products has significantly potential threatens to the consumers’health and safety. However, the sensitization depends mainly on the allergen types and amount in foods. For people who easily got allergy, the intake of shrimp or other foods which contain a variety of allergens may cause systemic hypersensitivity, leading to intestinal mucosal allergic syndrome and changes of some physiological indexes in the body. Therefore, in the present work, the muscle protein of the shrimp (F. chinensis) was extracted for intraperitoneally injection to the ICR mice, and finally allergic model of ICR mice with the shrimp extracts was constructed. Moreover, the indirect ELISA was used to detect the levels of specific IgE antibodies in sera of mice so as to validate if the model has been successfully established. In addition, hematoxylin and eosin (H&E)/Toluidine blue dye and immunohistochemistry were used to analyze intestinal mucosal immune responses in mice caused by the shrimp extracts. Finally, the related organ weight ratios, antioxidase activities and allergy indicator enzymes were tested to clarify the relationship of allergic process and changes of some physiological indicators in the body. The study aims to provide a theoretical basis for the allergy caused by intake of the shrimp (F chinensis) and related immune regulation in the body, as well as practical reference value for the clinical evaluation of potential allergenicity in the shrimp (F. chinensis). The results are concluded as follows:1. Intraperitoneally injection of shrimp (F. chinensis) muscle protein extracts was used to establish the allergic model of ICR mice with the shrimp extracts. SDS-PAGE electrophoresis and image analysis software AlphaView SA3.4.0were applied to initially speculate the allergen types in the shrimp muscle protein extracts. After mice allergic model was constructed, the indirect ELISA was used to test the levels changes of specific IgE antibodies in sera of mice during four stages of the challenging period. The OD490nm values of different sensitized groups were mostly2.1times higher than that of the control, which indicated that the model has been successfully established and could be used for further experiment.2. Several stained methods and immunohistochemistry were demonstrated to analyze intestinal mucosal immune responses in mice caused by the shrimp (F. chinensis) extracts. Epithelium and mast cells were observed by light microscopy. Associated T lymphocytes (CD3+、 CD4+、CD8+T lymphocytes) and IgA+plasma cells were detected by the method of immunohistochemistry. The results showed that the shrimp protein sensitized mice had severe intestinal inflammation and epithelial damage, as well as intestinal mast cell degranulation and significant reduction in number of the intact mast cells. Besides, the individuals of the sensitized group had higher positive rate of these T lymphocytes and IgA+plasma cells within the gut compared to the control. Therefore, we speculate that shrimp muscle proteins can cause IgE-and T-lymphocyte dependent hypersensitivity in ICR mice, which is accompanied by sustained B cell differentiation to the IgA+plasma cells.3. Changes of the immune-related organ weight ratios and enzymes caused by the shrimp (F. chinensis) muscle protein were also detected in the further study. Antioxidase (SOD, POD, CAT, GSH-Px, GST) and NOS/iNOS activities were tested by enzyme activity kits. The concentration of PLA2was tested by double-antibody sandwich ELISA. The mRNA expression of Ca2+-ATPase was tested by quantitative RT-PCR. The results showed that he shrimp protein sensitized mice had liver and kidney atrophied and spleen enlarged. With the increase of exposure dose, the atrophic/swelling degree became more severe. Besides, the activities of these antioxidases were significantly increased, including the activities of NOS/iNOS and the concentration of PLA2, which are two of the three indicator enzymes for food allergy. The mRNA expression of another indicator enzyme (Ca2+-ATPase) significantly reduced in the liver, kidney and spleen of the shrimp protein sensitized mice compared to the control. Therefore, some physiological indicators, such as the weight ratios of organs (liver, kidney, and spleen), the activities of antioxidant enzymes, NOS/iNOS activity, PLA2concentration and the mRNA expression of Ca2+-ATPase, might be associated with the allergic process in the body. It could be possible that the hypersensitivity is stimulated by the interations of these physiological indicators with other immune cells and cytokines.