Establishment Of Dengue Animal Model With TCM Damp-heat Syndrome

Background and Objectives:Dengue virus belongs to a member of Flavivirus,it is RNA viruses conveying by mosquito and can cause dengue fever(DF),dengue haemorrhagic fever(DHF) and dengue shock syndrome(DSS).In recent years,because of global warming,the regional expansion of the distribution of mosquito-borne dengue fever epidemic was continuing an upward trend,has been distributed in more than 100 countries and regions,become the most serious pest borne infectious disease in the field of disease, death and economic losses.Dengue virus pathogenesis,vaccine or antiviral research make slow progress,a important difficult point among them lies in the experiment animal not consistent with the human body symptom,so we have no way to carry out verification on the pathopoiesia factor,vaccine or medicine directly.Traditional Chinese medicine has accumulated large amount of experience in the treatment of virus infection by the syndrome differentiation。And the studies of winess-heat animal model have made great progress,According to Chinese medicine theory,we set up a suitable model for dengue virus infected mice which employ compound factor, simulat the way natural infection,and integrat environmental climate,physical factors such as pathogenic microorganisms.Thus it is more closer to the actual clinical situation and provid a more appropriate animal model for the study of nature of dengue fever. Method:According to Chinese medicine theory,we set up a suitable model for dengue virus infected mice which employ compound factor,simulat the way natural infection,and integrat environmental climate,physical factors such as pathogenic microorganisms.1.Employing compound factors:the diet with high sugar and fat+ High-temperature warehouses + biological factor dengue virus(DEN),to reproduce l mice plague mode.2.Gan lu Xiaodu Capsule:Talc 30g,Baical Skullcap Root 20g,Virgate Wormwood Herb 20g,Grassleaf Sweetflag Rhizome 12g,Tendrilleaf Fritillary Bulb 10g,akebiae 10g,Agastache rugosa 10g,Weeping Forsythia Capsule 10g,fructus amomi rotundus 10g,Peppermint 10g,Blackberrylily Rhizome.10g。(we buy all of them in Nanfang Hospital)3.Grouping60 SPF grade BALB/c mice were devided into seven groups(each group 10 mice) randomly,the normal control group(NCG,Not giving any of the conditions),the Virus group(Inoculation of dengue virus type 2 strain GZ14(10⁴TCID₅₀) by mixed injection(intraperitoneal injection and subcutaneous multi-point injetion),0.8ml /mouse,1time/d,For 3 consecutive days),the damp-heat group(the diet with high sugar(8%honey) and fat(12%Lard) + High-temperature warehouses(temperature is 33±2℃,Relative humidity is 60%～65%),1 times / day,2hours/ time,For 3 consecutive days),the Virus and damp-heat group(the diet with high sugar and fat + High-temperature warehouses + biological factor dengue virus(DEN)),the Chinese medicine treatment group(the Virus and damp-heat group+ Gan lu Xiaodu Capsule,0.47g/20g,1 times / day,For 7 consecutive days),the Positive drug group (the Virus and damp-heat group+ Virazole,0.003g/20g,1 times / day,For 7 consecutive days).4.Indexs of observation and detection Observation of general condition Before and after the creation of modles, changes of the whole body of mice,namely,spirit,diet,activity,body temperature, urineetc,were observed contrastly.Laboratory examination PLT,TC,TG,AST,ALT,AQP2,MTL,GAS.the detection of dengue virus with Fluorescence quantitative PCR.Histological examination Executed the mice,took out of Liverand Brain tissue. Then the tissue were put into 10%formalin,imbedded byparaffin and made blade, stained with HE and observed under light microscope.5.Statistics methodAll the data were processed by statistical software(SPSS13.0).The data type was quantity data,expressed by mean±standard deviation((?)±s).The data of temperature,pit were analyzed by variance of repeated measure.The data of AST,ALT,AQP2,TC,TG,GAS and MTL were analyzed by One-Way ANOVA.Multiple comparison was carried by LSD for different groups.P＜0.05 is the significant level.Result:1.General condition of the mice The mice were tired,enjoyed to lie down, acted lazily,reacted slowly,and their hair was fluffy and withered,drank more water,some of the crissum filth appeared.2.Temperature detection The temperature of mice were frome 36.1℃to 38.1℃,there were no significant differences between the groups(F=2.814, P=0.099).3.Detection of key indicatorsAfter the DEN virus infection,the Change of PLT is similar to the reduction of varying degrees,the difference is significant(F=77.578,P=0.000).ALT and AST was generally higher than normal,the AST is more significant,which is similar to those reported abroad.After the intervention of Hot and humid,AQP2 protein expression were significantly reduced,the most representative is the Virus and damp-heat group.The level of TC is notable increase,TG is not have notable difference among every group,(F^&=1.619,P=0.185);the MTL,GAS levels of Each group is increase, explaining that interference in mice resulted in the spleen and stomach function disorder.After drug treatment,the above-mentioned index have certain degree recovering,the group is comparatively obvious with traditional Chinese medicine.4.Pathological changes in organs4.1 the liver it could see clear hepatocytes cable structure and lobules of liver in Normal liver tissue with microscope,has no liver cell degeneration and necrosis and other pathological changes,the reticular fiber structure was normal.After infecting DEN virus,we open abdominal cavity of mice,the liver tissue significantly increased in size,color is lighter.In the Light microscope,liver cells,significant swelling, osteoporosis cytoplasm,showing balloon-like change and the dengue virus typical CPE Appeare,that is,cell disruption,integration,the formation of vacuoles of varying sizes and reticular formation,loss of normal hepatic plate structure Dou-gap disappears.after the intervention of hot and humid conditions,the liver of mice microscope can see there is many inflammatory cell infiltration,focal necrosis and necrosis clastic.liver cells status has improved after drug therapy.4.2 Brain tissue normal brain tissue has a integrity Nerve cells and a larger nucleus,nucleolus clear cells,without interstitial edema.Imposed by complex factors,The majority of nerve cell nuclear pyknosis,nucleolus significant unclear,extracellular gap widened significantly,mesenchymal osteoporosis,indicating the surrounding cells and interstitial edema evident,it has improved after drug therapy.5.the detection of dengue virusWe detection dengue virus with Fluorescence quantitative PCR,it is positive except the normal control group and the damp-heat group.Among them,the virus and damp-heat group forms contents maximal,the Virus group is secondly,we estimate that it will help virus grow in damp-heat environment.The contents of virus in Chinese medicine treatment group and the Positive drug group come down,the group is the most obvious with traditional Chinese medicine. Conclusion:Animal model with the characteristics of dengue fever,dengue fever did not change the model of key indicators,while mice with a clear objective indicators of the hot and humid card changes,after treatment by Chinese herbal compound,the indicators have improved significantly.According to Chinese medicine theory,employing compound factors could establish a mouse model of infection,it provid a more appropriate animal model for the study of Chinese medicine treatment and the effects of dengue fever.