Antagonism Effects And Signal Transduction Path Of Hao Qin Qing Dan Decotion On Damp-heat Syndrome Of Pneumonia Disease Infected By Influenza Virus

Research backgroud:Damp-heat syndrome of seasonal febrile disease was common clinical disease disease caused by exogenous damp-heat factor and interal damage of the spleen and stomach..The body in hot and humid evironemt for long time,and with bad eating habit could decrease the immunity,at the time the viruses or bacteria invade,damp-heat syndrome would happen.Damp-heat syndrome could made the body on a series of physiological and pathological changes.Influenza viruses are sub-segments of ssRNA viruses.Influenza viruses charactered by variant and infectious.Over the years,the prevalence of influenza virus has be threat to human health.Variation of influenza virus exist,and quick dissemination of such nature, easily give rise to a pandemic;Guangdong province,charactered by high temperature and rainy,profit to bacterial and viral propagation.It was important significance to study damp-heat syndrome in Guangdong province.Nowadys,people prevention of influenza viruses mainly through vaccine and antiviral drugs.However,vaccine production is hard to play the role of variation of the virus.Anti-viral agents must be use an early stage if you want to achieve the desired result,.Chinese medicine in the treatment of such diseases has unique advantages.In the clinical,Hao Qin Qing Dan decotion in the treatment of heat caused by influenza virus has a significant effect; However,its role and mechanism was not clear.Therefore,this experiment was sign to explore the role of Hao Qin Qing Dan decotion of antagonism effects,and look for the possible signal pathway.The experiment is significance to the ground research.Objective:To establish the animal model of damp-heat syndrome of pneumonia infected by influenza virus according to clinical symptom.To treat the model by Hao Qin Qing Dan decoction.To observe the effect of Hao Qin Qing Dan decoction.To explore the possible signaling pathway,to evaluate the experiment.Methods:The experiments consisted of two parts.The first part:Establish the animal model of damp-heat syndrome of pneumonia infected influenza virus.Mice were randomly divide into three groups:control group,virus group(infecting by influenza virus),model group(richly fatty and sweet diet + Humid heat environment + infecting by influenza virus).From the 1d to 9d,the control group and the viruse group fed with normal diet;the model group fed with high fat diet(normal diet, honey,lard 20:2:3 ratio of preparation;preparated by the Guangdong Laboratory Animal Medicine Center).At the 10d,the mice in model group and virus group infected by influenza virus,the control group give equivalent sterile liquid LB medium.After 24h,put the model group on the high-temperature environment: temperature(32±0.5 )℃,humidity(55～60)%for 3h;once every two days,and totally the model group was heat treat 3 times.Measure the body weight of mice in each group at the 1d,7d,10d,14d of 9:00 AM,and detect their anus temperature.At the 5d,8d,10d,the level of blood lipid in serum of mice were detected.RT-PCR were used to detect the quantity of virus in mice lung tissue.To observe the pathological changes of mice in each group.The anus temperature,blood-fat and the levels of lung pathological changes of mice in each group were compared.The second part:Hao Qin Qing Dan decotion to treat on Damp-heat syndrome of pneumonia disease infected by influenza virus and to explore its signal transduction mechanism.Mice were randomly divide into five groups:control group,virus group(infecting by influenza virus),model group(richly fatty and sweet diet + Humid heat environment + infecting by influenza virus),Virazole group(mouse of model group was treated by virazole),Hao Qin Qing Dan decoction group(mouse of model group was treated by Decoction of Hao Qin Qing Dan).To establish the mice modle reference to the first part of the experiment.After the virus infect,the medicine was give to the medicine groups,twice a day.When the model established,weighting the mice body weight and lung weight,calculate the lung indexes.The level of TLR2-mRNA and NF-κB-mRNA expressions of peritoneal macrophages in each group of mice were quantitated by reverse transcription-polymerase chain reaction(RT-PCR).The level of IL-4 and IFN-γ,in mouse serum were detected by ELISA,to calculate the Th1/Th2(IFN-γ/IL-4).Results:The first part:Establish the animal model of damp-heat syndrome of pneumonia infected influenza virus.1.General state In normal temperature,the model group mice weight were quickly increased,their fur are smooth,vivid and healthy.Access to high-temperature environment,there is a restless first period of activity,increased water intake, shortness of breath,and then enter a quiet state,scattered in the supine,but the breath is still rapid.Return to room temperature,mice in model group become to normal condition in about 1h.24h after the virus infection,the intake of virus group mice were significant decrease,which symptoms was not obvious in model group.48h later, the mice in model model show the symptoms of the above.96h later,the mice in virus group claw gentian violet is dark purple,sparse body hair,shortness breath.The mice in model group is smiliar to the virus group,but lighter than the virus group.2.The physiological change of mice①Mice in each group changes in body weight:at the begining,control group,virus group,model group data separately were (12.50±0.64)g,(12.62±0.61)g,(12.67±0.69)g;at the 7d times,control group, virus group,model group data separately were(16.65±0.67)g,(17.88±0.91)g, (18.80±0.7)g;at the 7d times,control group,virus group,model group data separately are(18.8±0.75)g,(18.75±1.24)g,(22.7±1.26)g;model group significantly heavier than the normal weight group(P＜0.05).②The changes of anus temperature:in the first day,control group,virus group,model group data separately were(34.7±0.3)℃,(34.9±0.4)℃,(34.6±0.5)℃;in 7d times,control group,virus group,model group data separately were(35.6±0.4)℃,(35.±0.4)℃,(36.7±0.3)℃; in 14d times,normal group,virus group,model group data separately were(35.8±0.2 )℃,(35.8±0.2)℃,(36.8±0.3)℃;the mice in model group anus temperature higher than the normal group and the virus group,there is statistical significance (P＜0.05).③Changes in blood lipids(TG):The data between model group and the control group were(6.74±1.03)mmol / L and(5.74±1.79)mmol / L;in 5d times;in 8d times,model group and control group data separately were(6.74±1.03) mmol/L, (5.74±1.79) mmol/L;in 10d times,model group and control group data separately were(6.74±1.03) mmol/L,(5.74±1.79) mmol/L;there is statistical between them (P＜0.01).3.The lung exponential and lung pathological changes of each group:①The lung exponential:control group,virus group,model group data separately were (0.79±0.11)%,(1.93±0.38)%,(1.41±0.26)%,there was statistical difference between them(P＜0.01).②The lung pathological changes of model group:Compared to the normal,the mice in virus group and model group were change.The second part:Hao Qin Qing Dan decotion to treat on Damp-heat syndrome of pneumonia disease infected by influenza virus and its signal transduction mechanism.1.Drugs effect on mice lung index in each group:control group,virus group, model group,virazole group and Hao qin Qing dan decoction group were(0.79±0.11)%,(1.93±0.38)%,(1.41±0.26)%,(1.10±0.26)%and(1.02±0.16)%;The mice of virazole group and Hao Qin Qng Dan decoction group lung index were decreased(P＜0.05).2.Each group of mice lung tissue expression of NF-κB picture analysis and the average optical density:①Mice infected with influenza virus,the lung tissue of the NF-KB activation;The Activate NF-KB(p65) expression in the nucleus,showed brown particles.②The mice NF-κB average value of optical density data separately were 0.208±0.005,0.321±0.033,0.305±0.029,0.254±0.041 and 0.247±0.041; the expression in Virazole group and Hao Qin Qing Dan decoction group are decreased(P＜0.05)3.TLR2 mRNA expression The results showed that the control group,virus group,model group,virazole group and Hao Qin Qing Dan Decoction group TLR2-mRNA expression were separately:0.145±0.017,0.991±0.149,0.903±0.124,0.257±0.03 and 0.413±0.031;Compared to the model group,Haoqin Qingdan decoction group and virazole group were decreased(P＜0.01).4 NF-κB mRNA expression Control group,virus group,model group,virazole group and Hao Qin Qing Dan decoction group NF-κB-mRNA expression were separately:0.075±0.148,0.379±0.019,0.291±0.012,0.169±0.026 1 and 0.175±0.033;the expression in virazole group and Hao Qin Qing Dan decoction group are decreased(P＜0.01).5.Cytokine levels in serum①The change of IFN-γ:the level of IFN-γin mice serum of control group,virus group,model group,virazole group and Hao Qin Qing Dan decoction group data separately were:(7434.06±323.27)pg / ml,(8679.77±198.70)pg / ml,(8068.78±113.8) pg / ml,(7454.66±301.30) pg / ml and(7484.56±229.85) pg / ml;the IFN-γlevel in serum of Hao Qin Qing Dan decoction group and virazole group were decreased(P＜0.05).②Each group of mice IL-4 contents were(3701.74±256.00) pg / ml,(3569.64±161.35) pg / ml,(3530.88±334.63 ) pg / ml,(3481.84±282.25) pg / ml and(3618.00±262.16 )pg / ml;no significant difference.③Th1/Th2 type cells in a state of equilibrium(means IFN-γ/IL-4) were: 2.02±0.19,2.38±0.10,2.36±0.14,2.22±0.17 and 2.07±0.15;and model group Hao Qin Qing Dan decoction group and virazole group were decreased.Conclusion:1.In this experiment,the first time use influenza virus as a biological factor, with "hot and humid environmental + fat food + biological infection factor" to replicate damp-heat syndrome of pneumonia disease.The incidence of the conditions, signs of model are consistent with the symptoms of Damp-heat.The production process of model is simple and it also in advantages of good reproducibility,the animal model is very useful.This study expand the research of the content of the previous model for its innovative;in addition,model as a prerequisite for future research.2.Hao Qin Qing Dan decoction can decreased the mice lung index of model group,it can reduce the immune damage and protect the lung tissue.The effective of Hao Qin Qing Dan decoction on Damp-heat syndrome of pneumonia disease infected by influenza virus were confirmed in vivo experiment.3.When influenza virus invade the body,TLR2 is activated;Hao Qin Qing Dan decoction can decreased the expression of TLR2 mRNA.Maybe it is the way to decreased the body's immune response4.The expression of NF-κB increased in the mice with damp-heat syndrome of pneumonia disease infection by influenza virus.Hao Qin Qing Dan decoction can reduce the expression of NF-κB.The change of NF-κB mRNA expressions of peritoneal macrophages in each group were consistent with expression TLR2 mRNA.5.Damp-heat syndrome of pneumonia disease infection by influenza virus IFN-γin serum is significantly increased,and IL-4 did not significantly increase,Th1/Th2 shift to Th1,the dominant of cell-mediated immunity lead to lung tissue injury, aggravated the disease.Hao Qin Qing Dan decoction can reduce the level of IFN-γin serum,so that the ratio of Th1/Th2 would return to balance.6.Hao Qin Qing Dan decoction is an effective drugs to the damp-heat syndrome of pneumonia disease infection by influenza virus in mice.The effective mechanism maybe through decrease the express of TLR2 mRNA and NF-κB mRNA in cell surface to regulate the activation of cytokine secretion,reduce the body content of IFN-γto improve the imbalance in the immune status,and ultimately protect the lung tissue to reduce the role of immune injury.