The Study In Mechanism Of Using The Liver-dredging And Spleen Fortifying Method To Treat Abnormalities In Brain-gut Axis In IBS-D Rat Model

ObjectiveTo improve IBS-D rat model and its evaluation methodology; To discuss the regulation of Chang ji’an decoction, a reprehensive formula of the liver-dredging and spleen fortifying method, on CD3+、CD4+、CD8+in IBS-D rats. To analyze the2dimensional gel electrophoresis maps of hypothalamus and colon proteins between the Chang ji’an capsule-administration group and model group, and the mass spectrometry was used to distinguish and analyze the differential expressed proteins in order to further clarify the regulatory mechanism of liver-dredging and spleen fortifying method on abnormalities of brain-gut axis pathway in IBS-D rats.Methods(1) Newborn rats were separated from maternal rats from the second day after birth to the fourteenth day, with3hours per day. From the thirtieth day on, senna leaf water decoction was administered to newborn rats in1ml/100g by gavage and the front shoulder, the front upper limbs and chest was fixed by paper bags in order to restrain the rats scratching their face and head in one hour after administration in order to prepare the IBS-D rat model. In control group, the rats were administered with saline by gavage with no restraining measurements.(2) The content of CD3+、CD4+、CD8+in serum of IBS-D rats administered with Chang ji’an decoction was assayed with flow cytometery.(3) The differential expressed proteins in hypothalamus and colon between IBS-D model group and Chang ji’an capsules-administration group were separated by two-dimensional gel electrophoresis and MALDI TOF/TOF time-of-flight mass spectrometry was used to identify the above proteins. Next, Gene Ontology was undertaken to classify the identified proteins, in order to analyze the key protein involved in the pathological mechanism of IBS-D rats by using the liver-dredging and spleen fortifying method.(4) The expression of PRDX1in hypothalamus and expression of LGALS9were tested by Western Bloting technique.Results(1) IBS-D rat model was successfully established through the method of separating newborn rats from maternal rats, administering senna leaf water decoction to newborn rats by gavage and fixing the activity of newborn rats. The model can create such symptoms as high-sensitivity of internal organs, bowel dysfunction, diarrhea and depression, etc. In addition, the model has no abnormal intestinal lesion in histology and can well stimulate the pathological features of chronic functional bowel disorders.(2) There is no statistical significance in CD3+contents among model group, pinaverium bromide group and Chang ji’an capsules group. Compared with control group, the content of CD4+and CD8+in model group increased significantly and the ratio of CD4+/CDg+decreased significantly too. The content of CD4+and the ratio of CD4+/CD8+in Chang ji’an capsules group increased and the content of CD8+decreased compared with model group.(3) The proteomic approach was used in the present study in order to find out the differentially expressed proteins in hypothalamus and colon between IBS-D rat model and Chang ji’an capsules group, and the results indicated that nine types of proteins in colon by using the biomass spectrometry technique were found, they are LONP1、 LTA4H、 UAP1、 ALDH2、 SET2、HADHSC、LGALS9、 KRT19、RPS12, among which eight decreased and one increased compared with the model group. Meanwhile, ten types of proteins in hypothalamus were found, and they are TPI1、 PAFAH1B3、 PRDX1、 NDUFB10、 AK1、 S100B、 DSTNL1、 PPIA、 COX6A1、 HBB-B1respectively, among which five increased and the other five decreased compared with the model group and drug group.ConclusionIBS-D rat model was successfully established through the method of separating newborn rats from maternal rats,administering senna leaf water decoction to newborn rats by gavage and fixing the activity of newborn rats. The mechanism of using the liver-dredging and spleen fortifying method to treat abnormalities in brain-gut axis may be associated with the increase of CD4+content and the ratio of CD4+/CD8+, and the decrease of CD8+content. The mechanism may be also associated with the decrease of PRDX1expression in hypothalamus and the decrease of LGALS9protein expression in the colon.