| Objective:Based on the results of previous literature research,clinical epidemic research and experimental research,the conditions(temperature,humidity,wind speed,light and diet)and key observation indexes for establishing the mouse model of spring dryness syndrome in Yunnan were basically determined.therefore,this experiment follows the modeling method of the previous experiment of the research group,and selects the indexes with strong correlation between observation and dryness syndrome model.For example,mouse airway Mucin5 ac,Aquaporin 5,Respiratory Mucopolysaccharide,and mouse serum IgA and IgG.To observe and compare the changes of macroscopic body surface characteristics,microstructure and molecular indexes of mice in different modeling periods of 7days,14 days and 21 days,further explain the relationship between the duration of dryness and the time of onset from multiple levels,and optimize the selection of modeling period.Yunnan spring dryness animal model was evaluated comprehensively and reasonably.Methods:After adaptive culture for 3 days,the experimental mice were weighed and divided into normal group and model group according to random grouping method.On this basis,the mice were divided into normal group and model group for 7 days,normal group and model group for 14 days,normal group and model group for 21 days.There were 15 mice in each group,a total of 90 mice were fed in animal incubator.The normal group was fed with normal climatic environment(20±2 ℃,60±2%RH),routine food and drinking water.According to the previous stage of the research group,the model group simulated the spring dry climate conditions in Yunnan and fed spicy spices.From the day of entering the incubator,the changes of macroscopic characteristics such as inspection characteristics,body weight,food intake and water intake of experimental mice were observed and recorded.On the8 th,15th and 22 nd day,the mice were killed to observe the morphological and structural changes of trachea and lung,and the concentrations of MUC5 ac,RS and AQP5 in bronchoalveolar lavage fluid and IgA,IgG in serum were detected.Results:1.Comparison of overall characteristics of miceInspection characteristics:The normal mice in the 7-day group had good mental state,bright and moist claws,shunze coat,clear urine,soft stool,few scratches and no scars;in the same cycle group,the model mice gradually showed activity excitement after feeding in the incubator for 2 days,the paw nail color was dark,the coat was dehydrated,the urine color was yellow,the stool was dry,there were often scratches and bites,and a small amount of scars could be seen in the tail.The normal mice in the 14-day group were in a good mental state,naturally active,few scratches and bites,and no scars;in the same cycle group,the model mice were obviously anxious,active and abnormal,the claw color was dark,rough and shedding,the urine color was yellow,the stool was dry and hard,and many scratches and blood stains could be seen on the back of the tail.The activity of normal mice in the 21-day group was less than that in the previous two cycles,slightly rough,occasionally scratched and bitten,and no scars were seen;in the same cycle group,the mental anxiety and abnormal activity of the model mice were less than those in the 14-day group,the nail color was dark and moist,the coat color was dull and rough,the shedding was aggravated,the urine color was yellow,the stool was dry and hard,and many scratches and blood stains could be seen on the back of the tail.Observe the amount of water consumed:From 3 days after entering the box to the end of each feeding cycle,there was a small increase or decrease in food intake per 10 g body weight of normal mice in each cycle group.After entering the box for3 days,the food intake of the model mice in the 7-day group reached the lowest value on the 5th day,and there was little change in the whole feeding cycle,the food intake decreased compared with the normal mice in the same cycle group,has the difference was not statistically significant(P>0.05).After entering the box for 3days,the food intake of the model mice in the 14-day group decreased and reached the lowest value on the 14 th day.Compared with the normal mice in the same cycle,the food intake of the model mice decreased,and there was a great difference(P<0.01).After entering the box for 3 days,the food intake of the model mice in the 21-day group decreased and decreased significantly on the 12 th day until the end of the 21 st day.Compared with the normal mice in the same cycle,the food intake in the model group decreased significantly(P<0.01).From 3 days after entering the box to the end of each feeding cycle,there was no significant increase or decrease in the amount of drinking water per 10 g body weight of normal mice in each cycle group.The amount of drinking water of the model mice in the 7-day group did not increase or decrease significantly from 3days into the box until the end of the 7-day feeding cycle,but there was no significant difference compared with the normal mice in the same cycle group(P>0.05).After entering the box for 3 days,the amount of drinking water of the model mice in the 14-day group changed little during the whole feeding cycle,and significantly increased compared with the normal mice in the same cycle group(P<0.05).The amount of drinking water of the model mice in the 21-day group gradually increased in the first 9 days from the beginning of feeding,and then decreased gradually until the end of the 21-day feeding cycle;compared with the normal mice in the same cycle group,the amount of drinking water increased in the first 14 days of the feeding cycle,and then gradually tended to normal,and there was no difference in the overall change(P>0.05).2.Comparison of organ and tissue structure in miceThe lung organ index:Compared with the normal group,the lung organ index of the normal group was 21 days group>14 days group>7 days group,and compared with the normal mice of the same cycle group,the lung organ index of model mice in each cycle group decreased,and there was significant difference between 14 and 21 days groups(P<0.01).The trachea: The results of the normal group showed that the tracheal tissue section of the 7-day group showed regular lumen morphology,complete mucosal structure,cilia arrangement,no injury and exfoliation,no inflammatory cell infiltration,no vasodilation,congestion and blood stasis;in the 14-day group,the shape of the tracheal lumen was regular,the mucosal structure was slightly defective,there was no infiltration of inflammatory cells,no vasodilation,congestion and blood stasis;in the 21-day group,the shape of tracheal lumen was relatively regular,local mucosal structure defect,a small amount of cilia exfoliated,no obvious inflammatory cell infiltration,no vascular dilatation and hyperemia and blood stasis.The results of the model group showed that the trachea tissue section of the 7-day group could be seen under the microscope,the local injury and exfoliation of the mucous membrane,no obvious inflammatory cell infiltration,slight dilatation of blood vessels,no obvious hyperemia and blood stasis were found in the 7-day group;in the 14-day group,the local structure of tracheal mucosa was defective and thinned,with a small amount of cilia shedding,inflammatory cell infiltration,slightly thickening and hyperemia of blood vessel wall;in the 21-day group,the trachea was slightly irregular,with obvious defect of mucosal structure,infiltration of inflammatory cells,thickening of blood vessel wall and obvious hyperemia.The lung tissue: The results of the normal group showed that the lung tissue sections of the mice in the 7-day group showed clear bronchial structure,intact mucosal structure,no inflammatory cell infiltration,intact alveolar wall and a small amount of hyperemia;in the 14-day group,a small number of exfoliated cells were found in the bronchial lumen,no inflammatory cell infiltration,no obvious edema and hyperemia;in the 21-day group,the bronchial structure was clear,the shape was slightly irregular,there were a small number of exfoliated cells in the mucous layer,no inflammatory cell infiltration and slight hyperemia.The results of the model group showed that the lung tissue sections of the mice in the 7-day group showed irregular bronchial morphology,exfoliation of a few epithelial cells,mild hyperemia of the alveolar wall,infiltration,exudation and bleeding of a small amount of cells;in the 14-day group,the bronchial lumen was irregular,the mucosal defect cells were exfoliated,the inflammatory cells infiltrated and exudated,the alveolar wall was obviously congested,and a small amount of bleeding and congestion were seen in the 14-day group;in the 21-day group,the bronchial lumen was irregular,the wall of the bronchus was hyperplastic and thickened with obvious hyperemia,and a large number of pulmonary vesicles fused.3.Comparison of the concentrations of Mucin5 ac,mucopolysaccharide RS and Aquaporin 5 in mouse airwayConcentration of MUC5 ac in airway :Compared with the normal mice in the same cycle group,the decrease of MUC5 ac concentration in the model mice in the7-day group was not statistically significant(P>0.05);the concentration of MUC5 ac in model mice decreased in 14 days group,and the difference was statistically significant(P<0.05);there was no significant decrease in the concentration of MUC5 ac in the model mice in the 21-day group(P>0.05).Concentration of mucopolysaccharide RS :Compared with the normal mice in the same cycle group,the concentration of mucopolysaccharide RS in the 7-day group was significantly lower than that in the same cycle group(P<0.01);the concentration of mucopolysaccharide RS in the 14-day group was significantly lower than that in the model mice(P<0.05);there was no significant difference in the concentration of mucopolysaccharide RS in 21-day group(P>0.05).Concentration of Aquaporin 5:Compared with the normal mice in the same cycle group,the concentration of Aquaporin 5 in the 7-day group was slightly higher than that in the same cycle group(P>0.05);the concentration of Aquaporin 5in 14-day group was significantly lower than that in model mice(P<0.05);the concentration of Aquaporin 5 in the 21-day group was lower than that in the control group,and there was no significant difference between the two groups(P>0.05).4.Comparison of serum IgA and IgG concentrations in miceThe concentration of IgA in serum :Compared with the normal mice in the same cycle group,the serum IgA concentration of the model mice in the 7-day group was significantly lower than the same cycle group(P<0.05);the serum IgA concentration of the model mice in the 14-day group decreased significantly,and the difference was statistically significant(P<0.01);the serum IgA concentration of the model mice in the 21-day group was not significantly higher than that in the control group(P>0.05).The concentration of IgG in serum :Compared with the normal mice in the same cycle group,the serum IgG concentration of the model mice in the 7-day group was significantly higher than that in the same cycle group(P<0.01);the concentration of serum IgG in the 14-day group was significantly higher than that in the model group(P<0.01);the concentration of serum IgG in the 21-day group was significantly higher than that in the model group(P<0.01).Conclusion:(1)Under the intervention of Yunnan spring dryness environment,the model mice showed different degrees of symptoms and signs of dryness syndrome,and their organ tissue structure and physiological function were damaged to varying degrees,and the 14-day cycle was an important stage for dryness pathogen to damage body fluid and dryness syndrome.(2)Under the intervention of Yunnan spring dryness environment,the body fluid metabolism of model mice was damaged in varying degrees.Based on the overall inspection characteristics of mice and the changes of organ and tissue structure,it was concluded that the 14-day cycle was the most severe stage of body fluid dryness.With the extension of feeding time to 21 days,the adaptability of mice to Yunnan spring dryness environment was enhanced,the body fluid was redistributed,and the dryness syndrome was alleviated to a certain extent.(3)Because the concentration changes of MUC5 ac,RS and AQP5 indicate the profit and loss of body fluid in the body,IgG and IgA in serum are related to the function of defending evil and protecting surface.Based on a comprehensive analysis of the concentration changes of body fluid and immune indexes in mice,it is considered that dryness pathogen invades muscle surface,suppresses Wei qi and damages lung,stomach and body fluid,which makes mice show internal and external signs of body fluid damage,that is,"dryness" syndrome,especially in the14-day cycle group.However,because the external dryness syndrome is light and shallow,the vital qi is not deficiency,and with the extension of feeding time to 21 days,the mice make adaptive changes to the environment,and the body fluid redistribution at the same time,the ability to resist evil can also be restored or even enhanced,which is consistent with the pathogenesis characteristics of external dryness syndrome.To sum up,based on the intervention of Yunnan spring dryness environment,the overall characteristics,tissue and organ structure,body fluid index and immune index of each cycle model mice showed abnormal changes in varying degrees,especially the modeling cycle of 1-14 days.This experiment puts forward the conclusion that the 14-day cycle is the best modeling cycle of Yunnan spring dryness mouse model. |
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