Establishment And Evaluation Of Mouse Model Of Exogenous Dryness

Animal model with syndrome of traditional chinese medicine (TCM) is established by making a copy of certain characteristic of mankind disease to certain animal according to theories of integer concept of TCM, determine the treatment based on differentiation of symptoms and signs, doctrine of visceral state, doctrine of etiopathogenisis and pathogenesis,et al. It is a reappearance on mankind syndrome, and it became a important pathway for investigating syndrome of TCM and pharmacology of Chinese crude drug.Dryness is one of the six climatic factors in nature, and it is the primary climatic factors in fall. When climatic change became intense, dryness could transform to a factor of pathopoiesis, called pathogenic dryness. Because it is from outside body, it was named exogenous dryness. Exogenous dryness is a important ingredient of doctrine of etiopathogenisis of TCM, but its theory and empirical research was obviously insufficient. The objective of this potic was to research means to establish and estimate the exogenous dryness animal model by summarizing the process of animal model of TCM and animal model with syndrome of TCM and the pathogenesis feature of exogenous dryness, and to provide evidence for establishing a stabile, dependable, reproductive mouse model of exogenous dryness.1. Theory research(1) Intervention factors for establishing mouse model of exogenous drynessMethods to establish animal model of TCM had distinct advantages, however, it had obvious shortcomings. According to the guidance of Professor Zhang Liutong, topic group reviewed the process of study on animal model with syndrome of TCM in the past half century, and considered that the research of animal model with syndrome of TCM should base on the following three aspects:optimizing intervention factors, establishing a unified evaluation method and selecting some characteristic indexes. Mouse model of exogenous dryness shoud be a model which consistents with pathogenesis and symptoms of exogenous dryness.The prevalence of exogenous dryness in fall was blame to intense change of fall climatic which correlates to Chinese geographical and climatic features-conversion time of wet climate→dry climate was shorter than dry climate→wet climate in fall. Mouse model of exogenous dryness, which should be established by simulating the characteristic of climate change in fall, could reflect the change law of exogenous dryness and can also avoid the effect of extreme humidity conditions to stability and repeatability of animal model.(2) Evaluation method of mouse model of exogenous drynessThere were two main characteristics of pathological changes of exogenous dryness:damaging body fluid and impacting the function of lung. Exogenous dryness can result in some symptoms,such as xeromycteria, dry pharynx, hydrodipsia, nonproductive cough.It were showed that the defensive role of WeiQi was closely related to immune response of lung, and running regularity of WeiQi was highly consistent to the immune rhythm, in addition, IgG, IgA, IgE levels in patients with deficiency of Wei Qi changed significantly. The changes of IgA, IgG, and SP-A in the bronchoalveolar lavage fluid in mouse model of exogenous dryness can discover overall the transform of immunopotency in different parts of airway, and can image the change law of WeiQi function in mouse model of exogenous dryness.The airway mucosa consists of cell layer and slime layer, and slime layer consists of gel layer and aqueous layer. The changes of airway slime layer could reflect the law of metabolism change of airway fluid correlated to inhaled air. Muc5ac mRNA and protein expression in airway to some extent may show the changes in airway gel layer and AQP5 mRNA and protein expression may indicate changes in airway aqueous layer, so the law of metabolic changes of lung fluid can be revealed by changes of Muc5ac andAQP5 mRNA and protein expression in airway.2. experiment study(1) Animals and grouping70 specific pathogen Free BALB/c mice,18±1g, male and female in half, were provided by Experimental Animal Research Center of Hubei Province. License number:SCXK (E) 2008-2005.Experimental animal, have been conventionally breeded in the laboratory for 7 days, were randomly divided into 7 groups:normal control group (C group), warm dryness model I group (W I group), warm dryness model II group (WII group), normal dryness model I group (Q I group), normal dryness model II group (Q II group), cool dryness model I group (L I group), cool dryness model II group (L II group), for modeling in "temperature-relative humidity-the wind" conditions. Mice in C, W I, Q I, L I group were managed by experimental requirements on the 7th morning and in WII, QII, LII group were managed on the 13th morning. Breed had been prohibited for 4 hours before mice were managed. (2) indicatrix detection1) general state of health in mice, such as demeanor, activity, color and luster of fur, dietary and physical properties of excrement, were observed.2) Lung tissue structure were observed with hematoxylin eosin (HE) staining method.3) IgG, IgA,, and SP-A protein content in bronchoalveolar lavage fluid(BALF) were detected by Quantitative ELISA kit.4) Expression of Muc5ac mRNA and AQP5 mRNA in airway were detected by real time quantitative fluorescence PCR.5) Muc5ac Protein expression in airway of were measured by immunohistochemical staining.6) AQP5 protein expression in airway were measured by Western blotting.(3) statistical analysisAll data were analyzed by SPSS 15.0 statistical software. Experimental data were showed with mean±standard deviation. t test was used to compared between the two groups, and repeated measures for ANOVA were performed in order to compare the differences between the various groups. A P< 0.05 level was used to indicate statistically significant difference.3. ResultsIntervention factors for establishing mouse model of exogenous dryness in this topic not only embodied the process of climate change in fall, but also reflected the characteristics of climate change in fall. This process reflected the pathopoiesis characteristics of exogenous dryness.Exogenous dryness damaged the function of WeiQi and fluid in airway. Changes of IgA, IgG and SP-A content in BALF in mouse model of exogenous dryness indicated diversify of WeiQi function. Compared to changes of IgG content, changes trend of IgA, SP-A content in BALF in warm dryness model, normal dryness model and cool dryness model showed consistently, although there were some differences between groups. Degression of AQP5 mRNA and protein expression in mouse model of exogenous dryness demonstrated that fluid content in airway was lower and fuild transport level decreased. Increase of Muc5ac mRNA and protein expression may be a protective response to prevent further airway fuild loss. However, thickening of gel layer, showed by increase of Muc5ac mRNA and protein expression, and thinning of aqueous layer, displayed by decrease of AQP5 mRNA and protein expression, could reduce airway mucociliary transit rate and descend airway defense capability. Symptoms, such as fur becoming dry and dall, quantiry of water intake raising, excrement showing slightly dry, exhibit the performance of exogenous dryness too.The cough occurring was because of stimulation of airway mucus and enhancement of airway responsiveness. Increase of Muc5ac mRNA and protein expression and decrease of AQP5 mRNA and protein expression not only could lead to increase of airway responsiveness, but also resulted in decline of airway mucociliary transport rate. These change made it become possible that airway mucus transform into sputum, and prompted sputum was viscous too.In summary, mouse model of exogenous dryness not only exhibited pathopoiesis characteristics of exogenous dryness, but also displayed basic symptoms and physical sign of exogenous dryness, so it could work as a means for researching etiopathogenesis of exogenous dryness.4. ConclusionThis topic established successfully mouse model of exogenous dryness, the model displayed pathogenesis, etiopathogenesis of exogenous dryness and symptom of generating sputum and cough.(1) Intervention factors for establishing mouse model of exogenous dryness Intervention factors simulating climate characteristic of fall were as follows:early fall (warm dryness):temperature 22±2℃, relative humidity 33±2%; mid-fall (normal dry):temperature 15±2℃, relative humidity 33±2%; later autumn (cool dryness):temperature 8±2℃, relative humidity 33±2%; wind speed is 2.5m/s in artificial environment.(2) evaluation of mouse model of exogenous dryness1) It was showed that fur became dry and dall, quantiry of water intake raised, excrement displayed slightly dry in mouse model.2) It was showed that mucosal folds increased, goblet cells proliferated, and airway mucus thickness had a decreasing trend.3) It was showed that IgA content increased and SP-A content decreased in BALF.4) It was showed that AQP5 mRNA and protein expression decreased and Muc5ac mRNA and protein expression increased.