Establishment Of Dengue Virus Infection Mouse Model With TCM Damp-heat Syndrome And Study The Effect Of Hot-Clear And Dehygrosis Medicine On It

Background and Object: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 is continuing an upward trend, has became major public health problem which threats human life and health. Regrettably,dengue virus pathogenesis, vaccine or antiviral research make slow progress, an 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.In recent years, "disease and syndrome combination" model has become a new trend in the development of animal models.Considering "disease" (cause or pathogeny) of Western medicine and "syndromes" of Traditional Chinese Medicine (TCM) to prepare animal models has a strong objectivity,high credibility and much more closer to clinical characteristics In this way, our research trys to build an animal model combining dengue disease with syndrome. Remarkably,there is no "dengue fever" in TCM. According to development and symptoms characteristics, it can divide to "hot and humid epidemic" and "summer dry epidemic". The disease more commonly occur in LingNan which it's wet and heat. Thus, most people think it's caused by extrnal infection of hot and humid and internal injury of spleen and stomach,and close with damp-heat.On this basis, our research simulate the environment of LingNan where is wet and hot with high-temperature cabin. According to the damp-heat method of TCM make models (fatty, sugary feed and infection factor DV), aim to establish dengue virus infection mouse model with TCM damp-heat syndrome. At present BALB/C mice are used in most study, but in recent years, C57BL/6mice are successfully used to build dengue hemorrhagic fever and liver injury model by Chen Xuan-jing's study. Therefore,this experiment studys with both BALB/C and C57BL/6 mice, meanwhile it also adopts different attack way (single DV2 attack, simulated ADE attack) and heat exposure mode (hot injury, hot acclimatization), to discuss the effect of compound factors with different rat, attack way and heat exposure mode in order to seek the ideal mice modle and reveal the pathogenic mechanism of. dengue viruses.Basical study of application of TCM damp-heat model includes development of damp-heat animal model and mechanism study of removing heat and dampness. Later traditional Chinese medicine are used to therapy to prove the reliability of the model. Removing heat and dampness method is adopted, comparing with hot disease and toxin removing method (Qingwen Baidu drink). ribavirin (western medicine) and a placebo. To explore therapeutic effect and its possible mechanism,futher research on its molecular mechanism of targets (the relationship between damp-heat and aquaporins) and establish foundations to clarify the mechanism of removing heat and dampness method.Methods:1,Dengue virus cultivation and establishment of DV2 fluorescence quantitative polymerase chain reaction (PCR) standard curve:DV1 and DV2 were reproduced using animal and cell culture methods.Plasmids that contains DV2 (NGC) cDNA fragment of restructuring genome were diluted 10 times, after Real-time PCR reaction obtained the corresponding amplification and standard curve.2,To establish Damp-heat model of dengue fever:BALB/C and C57BL/6 mice were divid-ed into short-term heat acclimation and heat exposure, while divided into single dengue virus 2 infections (referred to as DV2) and ADE effects (the ADE) model of two differe-nt virus challenge respectively; So, in accordance with the principles of permutations and combinations, there were 8 model. Finally, there were 14 group adding the blank control group, DF2 control group, damp-heat group included 14 group. Then through the observation of mice body temperature, symptoms, signs, and platelet (PLT), liver function (ALT, AST), blood lipids (TG, CHOL) and liver pathology, to evaluate the results of model establishment and filter out the best damp-heat syndrome of dengue fever in animal.3,To compare and investigate the mechanism of the intervention effects of different drugs on mice of Damp-heat model of dengue fever:Mice selected by the second experiment were divided into 5 groups randomly:normal group, model group, Artemisia Scutellar-ia Decoction treatment group, treatment group Qingwenbaiduyin drink, Ribavirin treatment group, saline treated group. Then to observe body temperature, symptoms, signs, platelet (PLT), liver function A (LT, AST), blood lipids (TG, CHOL) and liver pathology, and to detect brain aquaporin 4 (AQP4), renal water channel protein 2 (AQP2), stomach aquaporin 4 (AQP4). Finally, to compare the therapeutic effects of different drug treatments and explore therapeutic mechanism of removing heat and dampness method.Results:1,Virus cultivation and establishment of the standard curve for DV2:suckling mouse were infected DV2 (NGC strains), after 7 days began to sicken, characterized by refus milk,far from the group, slow-moving, outlining, side, double lower limbs paralysis or twitching and other symptoms. C6/36 cells were cultured 5～7 days after inoculation DV,90% of the cells appear CPE phenomenon, performanced by cell aggregates, cell falls off or appear empty bubble. According to amplification curve, a corresponding standard curve equation were obtained:Y=-3.576 logX+52.23. Slope for 3.576, E for 0.94, all these are instances of good amplification efficiency. Correlation coefficient:R2=1.0>0.99, explain the initial concentration of numerical template with multiple Ct value has good linear correlation.2,General Performance:After a virus attack, mice indicated fatigue condition, lower mperament, less movement, tired lying, slow respond to stimuli, hair standing on flow, less shiny, less diet and water intake, more smelly urine and soft feces. Into the ware-, house mice manifested irritability, less activities, less diet and respond to stimuli, more water intake and tired lying. After put into the warehouse several times. The difference of BALB/c and C57BL/6 mice wasn't significant. Haoqin- qingdan decoction had paid a certain role in improving the symptoms of model group.3,Detection of the main indexes:After virus attacks, PLT slowed down to a certain extent similarly. ALT and AST increased to varying degrees, especially AST; TG, CHOL increased in mice given high fat diet fed. But liver function and blood lipids had no significant increase in heat acclimation group. So Haoqin decoction could decrease ALT, AST and TG, CHOL, while Qingwenbaidu decoction could upgrade PLT.4,After established modle with complex method, the expression of AQP4 in brain tissue and AQP2 in gastric mucosa increased, but renal AQP2 re- duced. Haoqin decoction could reverse those expressions to the nomal levels, and the differences were statistical-ly significant, compared with other groups. Through correlation analysis three AQP above, the in- dependent variables of renal AQP2 had a significant correlation with the dependent variable of gastric AQP4 and The independent variables of renal AQP2 had no correlation with the dependent variable of gastric AQP4.5,Liver pathology:There are varying pathological changes in specimens of each groups. Liver cells had edema from mild to sever. Individual cells had eosinophilic changes; Liver cells had punctate, small focal necrosis, and necrosis area was infiltrate by inflammatory cells; There was no steatosis and fibrosis. Microscopy shows slight swelling of liver cells in group with damp-heat intervention; The state of liver cells had im- proved after the treatment of Haoqinqingdan decoction.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 with symptoms, fever, liver damage and blood lipid, the model group of BALB/c+DV2+thermal indicated damp-heat characterristic of dengue fever, so it's the best method. Haoqin decoction had therapeutic effects on mice with damp-heat syndrome of dengue fever, while have improved symptoms, decreased blood lipids and liver pathological damage, and regulated the water channel protein adjustment disorders.