| Purpose:To establish the model of BALB/c mice infected with influenza virus(IV)in haze environment;and to study the regulation mechanism of Ganlu Xiaodu Micropill(GXM)on TLR3/TRIF signaling pathway of IV infected mice in haze environment from histology and molecular biology,to explore the target of anti-IV infection,and to provide experimental basis for clinical treatment.Material and method:Seventy-two SPF grade BABL/c mice were randomly divided into normal group,model group,Ganlu Xiaodu Micropill group and Oseltamivir phosphate group,with 18 mice in each group.In addition to the normal group,the other groups started to make the model on the second day.First,each group was put into the artificial climate incubator for 2 hours,and the tracheal drip was carried out with PM2.5 suspension after taking out,once every other day,three times.On the 7th day,under mild anesthesia with ether,the model group,Ganlu Xiaodu Micropill group and Oseltamivir phosphate group were inoculated with influenza A virus FM1 strain 0.1 m L per mouse in the nasal cavity.After the successful establishment of the model,the normal group,the model group were given normal saline,while the Ganlu Xiaodu Micropill group and Oseltamivir phosphate group were given intragastric administration of corresponding drugs once a day for 10 consecutive days.The mice in each group were taken3,7,10 days after the establishment of the model respectively.The pathological sections of lung tissue of mice were observed under microscope,and the degree of pathological inflammation of lung tissue of mice was scored;the lung index of mice was calculated;the gene content of TLR3 and TRIF in lung tissue of mice was determined by q PCR;the expression of IFN-? and IL-6 in serum of mice was detected by ELISA;and the expression of NF-?B and IRF3 protein in lung tissue of mice was detected by Western blot.Results:1.After co-infection of PM2.5 and IV,the pathological sections of the lung tissue of the normal group were basically normal;the infiltration of inflammatory cells and the destructionof the alveolar structure in the lung tissue of the model group were changed;a small amount of inflammatory cells were seen in the alveoli of the Ganlu Xiaodu Micropill mice,and the edema of the bronchial mucosa was significantly reduced;Small amounts of inflammatory cells were seen in the Oseltamivir phosphate group mice,and the number of inflammatory cells was significantly reduced compared to the model group.2.After co-infection of PM2.5 and IV,the lung index and pathological score of mice increased at each time point(P<0.05),and reached the peak on the 7th day.Compared with model group,the lung index and pathological score of mice in Ganlu Xiaodu Micropill group and Oseltamivir phosphate group decreased on the third day(P<0.05);the lung index and pathological score of mice in Ganlu Xiaodu Micropill group and Oseltamivir phosphate group continued to decrease on the seventh and tenth day(P<0.05);compared with Ganlu Xiaodu Micropill group,the lung index and pathological score of mice on the third day were significantly lower(P<0.05).There were significant differences in lung index and pathological score between the two groups on the 7th and 10 th day(P>0.05).3.After co-infection of PM2.5 and IV,the expression of TLR3 at each time point,peaked on the 7th day after the establishment of the model,and then decreased gradually.There were significant differences between the two groups at each time point(P<0.05).After treatment,the expression of TLR3 decreased,but the effect of Ganlu Xiaodu Micropill group was not obvious on the 3rd day(P>0.05),while that of Oseltamivir phosphate group decreased(P<0.05).On the 7th day,the Ganlu Xiaodu Micropill group began to work.TLR3 expression decreased(P<0.05)and Oseltamivir phosphate group continued to decrease(P<0.05);TLR2expression decreased significantly(P<0.05)on the 10 th day in the Ganlu Xiaodu Micropill group and the Oseltamivir phosphate group;compared with the Ganlu Xiaodu Micropill group,there was significant difference on the 3rd day in the Oseltamivir phosphate group(P<0.05);on the 7th and 10 th day,there was no significant difference between the two groups(P>0.05).4.After co-infection of PM2.5 and IV,the content of TRIF at each time point,peaked on the7 th day after the establishment of the model,and then decreased gradually.There were significant differences between each time point and the normal group(P<0.05).After treatment,the content of TRIF decreased,but the effect of Ganlu Xiaodu Micropill group wasnot obvious on the 3rd day(P>0.05),while that of Oseltamivir phosphate group decreased(P<0.05).On the 7th day,the effect of Ganlu Xiaodu Micropill group began to take effect.TRIF content decreased(P<0.05)and Oseltamivir phosphate group continued to decrease(P<0.05);on the 10 th day,the TRIF content in Ganlu Xiaodu Micropill group and Oseltamivir phosphate group decreased significantly(P<0.05);compared with Ganlu Xiaodu Micropill group,the difference was significant on the 3rd day(P<0.05);on the 7th and 10 th day,there was no significant difference between the two groups(P>0.05).5.After co-infection of PM2.5 and IV,the expression of NF-?B protein at each time point,peaked on the 3rd day after model establishment,and then decreased gradually.There were significant differences between the normal group and each time point(P<0.05).After treatment,the expression of NF-?B protein decreased,but the effect of Ganlu Xiaodu Micropill group was not obvious on the 3rd day(P>0.05),while that of Oseltamivir phosphate group decreased on the 7th day(P<0.05);and Ganlu Xiaodu Micropill on the 7th day(P<0.05).The expression of NF-?B protein decreased in Ganlu Xiaodu Micropill group(P<0.05)and persistently decreased in Oseltamivir phosphate group(P<0.05);the expression of NF-?B protein decreased significantly in Ganlu Xiaodu Micropill group and Oseltamivir phosphate group on the 10 th day(P<0.05);compared with Ganlu Xiaodu Micropill group,the difference was significant on the 3rd day(P<0.05);on the 7th and 10 th day,there was no significant difference between the two groups(P>0.05).6.After co-infection of PM2.5 and IV,the expression of IRF3 protein at each time point,peaked on the 3rd day after model establishment,and then decreased gradually.There were significant differences between the two groups at each time point(P<0.05).After treatment,the expression of IRF3 protein decreased,but the effect of Ganlu Xiaodu Micropill group was not obvious on the 3rd day(P>0.05),while that of Oseltamivir phosphate group was decreased(P<0.05);on the 7th day,the Ganlu Xiaodu Micropill group was opened(P<0.05).Initially,the expression of IRF3 protein decreased(P<0.05),while that of Oseltamivir phosphate group continued to decrease(P<0.05);on the 10 th day,the expression of IRF3 protein in Ganlu Xiaodu Micropill and Oseltamivir phosphate group decreased significantly(P<0.05);compared with Ganlu Xiaodu Micropill group,the difference was significant on the3 rd day(P<0.05);on the 7th and 10 th day,there was no significant difference between thetwo groups(P>0.05).7.After co-infection of PM2.5 and IV,the content of IL-6 at each time point,peaked on the3 rd day after model establishment,and then decreased gradually.There were significant differences between the normal group and each time point(P<0.05).After treatment,the content of IL-6 decreased,but the effect of Ganlu Xiaodu Micropill group was not obvious on the 3rd day(P>0.05),while that of Oseltamivir phosphate group decreased on the 7th day(P<0.05);and Ganlu Xiaodu Micropill on the 7th day(P<0.05).The the content of IL-6decreased in Ganlu Xiaodu Micropill group(P<0.05)and persistently decreased in Oseltamivir phosphate group(P<0.05);the the content of IL-6 decreased significantly in Ganlu Xiaodu Micropill group and Oseltamivir phosphate group on the 10 th day(P<0.05);compared with Ganlu Xiaodu Micropill group,the difference was significant on the 3rd day(P<0.05);on the 7th and 10 th day,there was no significant difference between the two groups(P>0.05).8.After co-infection of PM2.5 and IV,the content of IFN-? at each time point,peaked on the7 th day after model establishment,and then decreased gradually,but it was still higher than that in normal group.Compared with normal group,there were significant differences at all time points(P<0.05);after treatment,the content of IFN-? increased,and the content of IFN-?in Ganlu Xiaodu Micropill group and Oseltamivir phosphate group increased at all time points(P<0.05);compared with Ganlu Xiaodu Micropill group,the difference was significant on the3 rd day(P<0.05);on the 7th and 10 th day,there was no significant difference between the two groups(P>0.05).Conclusion:1.In this study,the mouse model of inflammation was successfully established by intratracheal drip combined with IV nasal drip.It has been proved that this method is simple and reliable,and can be widely used in repeated infusion of respiratory system in mice,which is conducive to the further study of lung diseases and other inflammation mechanisms.2.The expression trend of TLR3 in lung tissue of IV infected mice in haze environment was similar to that of TRIF,NF-?B.It was suggested that IV infection might participate in inflammatory response by relying on TLR3/TRIF signaling pathway.3.Ganlu Xiaodu Micropill can control the pulmonary inflammation after IV infection in haze environment,down-regulate the expression of TLR3,TRIF,NF-?B and IRF3 in the lung tissue of IV infected mice under haze environment,down-regulate the expression of IL-6 and up-regulate the expression of IFN-? in serum;and it can regulate the immune function of mice infected with IV in haze environment.4.It was found that the anti-IV infection mechanism of Ganlu Xiaodu Micropill in haze environment may be related to blocking the TLR3/TRIF signaling pathway and reducing the release of pro-inflammatory factors. |
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