| Purpose:By establishing Influenza Virus-Streptococcus pneumoniae co-infection mouse model and exploring the effects of modified Xuanbai Chengqi Decoction on lung tissue and serum cytokines at different time points of co-infected pneumonia mice,combined with the general state of mice and pathological changes in lung tissue,we hope to clarify the mechanism and curative effect evaluation of modified Xuanbai Chengqi Decoction in the intervention of co-infected lung injury.Material and method:1.48 6-8-week old SPF Balb/c mice were randomly divided into normal group and model group,with 24 mice in each group.The normal group was inoculated with 0.9%normal saline,100μL per mouse;the model group was inoculated with 100μL 100LD50 influenza virus in the nasal cavity and subsequently inoculated with 100μL 1*10~6CFU Streptococcus pneumoniae 72 hours later to induce a co-infection model.8 mice from each group were killed,and blood and lung tissues were collected at the 24h,48h,and 72h time points after infection.By comparing the general status and behavioral performance of the mice,lung tissue morphologies,lung indexes of the mice,and changes in cytokine levels in the lung and serum between the normal group and the model group,we can evaluate the success of the establishment of Influenza Virus-Streptococcus pneumoniae co-infection mouse model.2.96 6-8-week-old SPF Balb/c mice were randomly divided into four groups:normal group,model group,modified Xuanbai Chengqi Decoction group,and oseltamivir+cephalosporin group,with 24 mice in each group.Except for the normal group,the other 3 groups underwent co-infection modeling,and intragastric administration of respective medication were carried out in accordance with the corresponding group was started on the second day after successful modeling,and continued for 7 days.On the 3rd,5th,and 7th day after intranasal inoculation,8 mice from each group were killed for samples.Pathological changes of lung tissue,lung and serum C-reactive protein,Interleukin-6(IL-6),Tumor necrosis factor-α(TNF-α),Interleukin-10(IL-10)expression levels and other observations were used to evaluate the efficacy of modified Xuanbai Chengqi Decoction on co-infection pneumonia and to explore its effects on cytokine levels.Results:1.Influenza Virus-Streptococcus Pneumoniae Co-infection Mouse Pneumonia Model research and evaluation.1.1 Comparing the general observation of mice in each group.During the whole observation period,the mice in the normal group were in good mental state,alert and active,shiny and smooth fur,normal food intake and excretion,smooth breathing,had no fever,sneezing,coughing,and had no significant change in body weight.In the model group,at 24 hours,the mice began to suffer from decreased mental status,decreased activity,food and water intake,more than half of the mice had shrugged and dull fur,slightly increased respiratory rate,no weight gain,and normal excretion.At 48-72 hours,the mice had worsened mental state and displayed signs of depression,physical activity is significantly reduced,slower reaction to stimulus,fur is dull and haggard,displayed shortness of breath,coughing,sneezing,and increased nasal secretions,weight loss(P<0.05),urine that was yellow and low volume,dry stool and reduced stool volume.1.2 Comparison results of lung index of mice in each group:There was no significant difference in the lung index of mice in the normal group at all time points(P>0.05).In the model group,at the 24h,there was no significant difference in the lung index compared to the normal group at 24h(P>0.05),and there was a significant increase at the 48h compared with the blank group(P<0.05),lung index continues to increase and reached its peak at 72h.1.3 Pathological analysis of lung tissue in each groupPathological and histological changes of the lung tissue of mice in each group at different time points were observed under a light microscope.At any time,point in the normal group,the bronchi,bronchioles,alveolar ducts,alveoli,alveolar sacs were intact and had distinct borders,there was also no exudation,hemorrhage,thickening of alveolar septa or lesions in the whole lung field,and occasional blue inflammatory cells aggregation was observed.In the model group,at 24h,pulmonary epithelial cell defects were seen in the lung tissue of the mice,cell infiltration and edema were observed in the bronchial wall,and expanded to the surrounding bronchial tissue.As the infection time increased,at 72h,inflammatory cells in the alveolar cavity,increased infiltration of red blood cells were observed,normal tissue structures such as alveoli,alveolar sacs,alveolar ducts,and bronchioles had disappeared,the alveolar walls are significantly thickened with connective tissue hyperplasia,and pulmonary interstitial congestion,edema,and increased inflammatory cell infiltration.Pulmonary capillary embolism and pulmonary consolidation areas can be seen in highly infected parts.1.4 Differences in serum cytokines level of each groupAt 72h,compared with the normal group,serum levels of pro-inflammatory cytokines IL-6,CRP,and TNF-αin mice of the model group were significantly increased(P<0.05,P<0.01).The expression of serum anti-inflammatory cytokine IL-10 in the model group was significantly reduced(P<0.05).The results suggested that the up-regulation of pro-inflammatory factors and the down-regulation of anti-inflammatory factors were significantly related to lung tissue damage.2.Effects of modified Xuanbai Chengqi Decoction on cytokines levels in influenza Virus-Streptococcus pneumoniae co-infected mouse pneumonia model.2.1 Comparing the general observation of mice in each groupThe mice in the normal group were alert and active,with smooth skin fur,normal food intake,no fever,sneezing,etc;the mice in the model group were listless,with significantly reduced activity,had fever,cough,shortness of breath,poor diet,and arched and haggard fur,dry stool,weight loss.After oral administration of modified Xuanbai Chengqi Decoction,on 3d,the general state of the mice was significantly improved compared with the model group,mental state improved,the activity level and food intake were increased,and sneezing and coughing symptoms were significantly reduced,fur gradually regained its luster,and the stool gradually returned to normal and soft stool.On 5d,the general state of the mice recovered to that of the normal group.The general state of the mice in the oseltamivir+cephalosporin group was similar to the normal group at 3d,and remained until the end of the experiment.2.2 Comparing the lung index of mice in each groupThe result showed that compared with the normal group,the lung index of the model group increased significantly with time(P<0.05),and there was a continuous increasing trend;Compared with the model group,the lung index of Xuanbai Chengqi Decoction and oseltamivir+cephalosporin group mice started to decrease on 5d,with a significant difference(P<0.05),but there was no significant difference between the two groups(P>0.05),suggesting both modified Xuanbai Chengqi Decoction and oseltamivir+cephalosporin can inhibit lung tissue damage.2.3 Pathological analysis of lung tissue after co-infection in each groupThe alveolar tissue of mice in the normal group had a clear shape throughout the experiment,no exudation,hemorrhage,inflammatory cells,thickening of the alveolar septum were observed;the lung tissue structure of the mice in the model group disappeared significantly with the development of the experiment,and the structure of the alveolar cavity disappear,alveolar wall was widened and thickened,alveolar cavity and pulmonary interstitium filled with inflammatory cells and bloody exudate,with areas of pulmonary consolidation.Compared with the model group,after the administration of modified Xuanbai Chengqi Decoction,the alveolar structure of the mice basically recovered after 5 days,and the alveolar were filled,hyperemia,infiltration of inflammatory cells,alveolar septal rupture and alveolar wall thickening were all reduced,degree of lung injury was significantly reduced after treatment.The lung histopathological manifestations of mice in Xuanbai Chengqi decoction group and oseltamivir+cephalosporin group were similar after 5 days.2.4 Changes of lung and serum cytokines in each groupCompared with the normal group,the levels of IL-6 and TNF-αin the serum and lung tissue of the mice in the model group increased(P<0.05),and they showed a surge state after 7d and5d respectively;the serum CRP level of the mice in the model group was at 3d was significantly higher than that of the normal group(P<0.05),but it was down-regulated at 5d and 7d,but the level of CRP in the lung tissue was continuously higher than that of the normal group(P<0.05);The serum and lung tissue levels of IL-10 were lower than that of the normal group,and gradually decreased with the progress of time.Compared with the model group:the serum and lung tissue IL-6,TNF-αand CRP levels of the mice in the Xuanbai Chengqi Decoction group were significantly reduced(P<0.05);while the level of serum and lung tissue IL-10 in the Xuanbai Chengqi Decoction group increased significantly after 5 days(P<0.05).The serum and lung tissue cytokines level of mice in the Xuanbai Chengqi decoction group and the oseltamivir+cephalosporin group showed similar trends.Conclusion:1.By observing the results of the study,it was verified that an influenza virus-Streptococcus pneumoniae coinfection mouse pneumonia model can be established by firstly inoculating with Influenza virus,followed by Streptococcus pneumoniae inoculation 72 hours later.2.Compared with the model group,the modified Xuanbai Chengqi Decoction can effectively improve the general state and lung damage of co-infected mice and has a significant regulatory effect on cytokines.The curative effect of modified Xuanbai Chengqi Decoction is comparable to that of oseltamivir and cephalosporin combination treatment.3.Through comparative analysis of the pathological changes of lung tissue in mice and the changes in the serum and lung tissue levels of cytokines,IL-6,CRP,TNF-α,and IL-10,it shows that modified Xuanbai Chengqi Decoction can decrease the expression of pro-inflammatory cytokines and increase the expression of anti-inflammatory cytokines,thereby regulate the immune microenvironment,and play a therapeutic role in the lung tissue damage caused by Influenza Virus-Streptococcus pneumoniae co-infection. |
PDF Full Text Request