| Purpose:By observing the dynamic changes of intercellular tight junction proteins（TJs）,aquaporin 5（AQP5）and mucin 5ac（MUC5ac）,we investigated the mechanism of regulating the damage of pulmonary air-blood barrier in severe Mycoplasma pneumoniae pneumoniae（SMPP）by adding flavor to nourishing yin and clearing lung soup from the perspective of the structure and function of pulmonary air-blood barrier,and correlated with the"moistening and drying"treatment method of traditional Chinese medicine（TCM）to provide new ideas for the treatment of SMPP in TCM.Material and method:One hundred and twenty BALB/c mice were randomly divided into normal group,model group,nourishing group,aci group and aci+nourishing group. Except for the mice in the normal group,each group was anesthetized and nasal drops of MP bacterial solution at 1010CCU/ml were used to construct the SMPP model,and the drug intervention was started after the model was successfully established,and the samples were taken on the 3rd,7th,10th and 14th days.The general status of mice was observed;the dry weight and wet weight of lung tissues were weighed separately,and the dry/wet weight ratio of lung tissues was measured;the lung index of mice was calculated;the pathological morphological changes of lung tissues were observed under light microscope by HE staining,and the pathological score was calculated;the expression of tight junction proteins Occludin and ZO-1 in lung tissues was detected by immunohistochemistry;the expression of Occludin and ZO-1 in lung tissues was measured by q PCR.The gene contents of Occludin,ZO-1, AQP5 and MUC5ac in lung tissues were determined by q PCR;the protein expressions of Occludin,ZO-1 and AQP5 in mouse lung tissues were detected by Western blot;the contents of MUC5ac in mouse alveolar lavage fluid were detected by ELISA.Results:1.From the observation of general status of mice,except for the normal group,all other groups of mice showed different degrees of mental lethargy,reduced diet and activity from day 3 after MP infection,and even some mice showed vertical hair reaction and nasal secretion,among which the reaction was the lightest in the Aci+Yangyin group and the heaviest in the model group.On the 7th day,the status of the mice in the model group and all the administered groups were at the lowest level,and then gradually improved,among which the general status of the mice in the Aci+Yangyin group improved the most obviously,and the Aci group was the second.2.After MP infection,the lung indices of mice in all groups were higher than those in the normal group（P<0.05）and peaked at day 7.Compared with the model group,the lung index of mice in all groups was lower than that of mice in the model group at all time points（P< 0.05）.On day 10,the lung indices of the mice in the Archie group and the Aci+Yangyin group were lower than those in the Yangyin group（P<0.05）.On day 14,the lung indices of the mice in the Archie group were lower than those in the Yangyin group,but the difference was not statistically significant（P>0.05）,and the lung indices of the mice in the Archie+Yangyin group were lower than those in the Yangyin group（P<0.05）.Comparing the dry-to-wet ratio of lung tissues of mice in each group,we found that the dry-to-wet ratio of lung tissues of mice in each group decreased at all time points except for the normal group（P<0.05）,and reached the lowest value on day 7,and then gradually increased,but was always lower than that of the normal group（P<0.05）.The dry-to-wet ratio of lung tissues of mice in each administration group was higher than that of the model group at all times（P<0.05）.On days 7 and 10,both the Aci group and the Aci+Yangyin group were higher than the Yangyin group.There was no statistically significant difference between each administration group on day 14（P>0.05）.3.The histopathological results of lung showed that the model group mice had obvious lung pathological changes,exuded a large number of inflammatory cells,had severe capillary stasis,and the respiratory membrane was significantly thickened.The alveolar inflammatory cells in the mice in the Yangyin group exuded less than those in the model group,and the alveolar septum was widened.The mice in the Aci group had inflammatory cell infiltration and mild lung tissue stasis.Inflammatory cell infiltration was also seen in the mice of the Archie+Yangyin group.The pathological score results showed that the mice in the model group had higher pathological scores at all time points than each of the administered groups（P<0.05）,and the pathological scores in all groups peaked at day 7.Compared with the Yangyin group,the pathological scores of the aci group were lower at all time points（P>0.05）,and the pathological scores of the aci+Yangyin group were lower than those of the Yangyin group at all time points except day 3（P<0.05）,and on day 14,the pathological scores of the aci+Yangyin group were lower than those of the groups administered with Chinese medicine or western medicine alone（P<0.05）.4.The distribution of Occludin and ZO-1 in the lung tissues of SMPP mice was observed by immunohistochemistry,and it was found that Occludin and ZO-1 were mainly expressed in the alveolar epithelium and capillary endothelial cells in the normal group of mice,with strong positive staining in the cell membrane and cytoplasmic margin.In the model group,Occludin and ZO-1 were expressed in the alveolar epithelium and capillary endothelium,and the positive staining was significantly reduced.The positive staining of Occludin and ZO-1 in mice in the adoptive Yangyin group was stronger than that in the model group.Occludin and ZO-1 in the Archie group remained wavy in more alveolar wall epithelium or alveolar interstitial capillary endothelial cells.The positive staining of Occludin and ZO-1 in the Archie+Yangyin group was higher than each single administration group.5.Comparing the expression of Occludin and ZO-1 m RNA in the lung tissues of all groups of mice,we found that the expression of Occludin ZO-1 m RNA in the lung tissues of all groups of mice decreased compared with the normal group after MP infection（P<0.05）,and each administration group had different degrees of modulating effects on both,with the best effect of the combined administration.On day 3 after MP infection,the AQP5 m RNA content in the lung tissue of all groups of mice decreased to different degrees compared with the normal group,while the MUC5ac m RNA content increased to different degrees compared with the normal group,and the regulatory effect of azithromycin on AQP5 and MUC5ac was earlier than that of Yangyin Qingfei Decoction Plus,and the Chinese medicine mainly exerted its regulatory effect in the late stage of SMPP,and the combination with azithromycin The best effect was achieved by combined application with azithromycin.6.After MP infection,Occludin and ZO-1 protein expression in lung tissues of all groups of mice decreased compared with the normal group（P<0.05）,and all the administered groups were higher than the model group,which dropped to the trough value on day 7;from day 3,although the Yangyin group was higher than the model group,it was lower than the Aci group and the combination group,and the difference was statistically significant（P<0.05）.7.After MP infection,the MUC5ac level in alveolar lavage fluid was higher in all groups than in the normal group（P<0.05）,and the difference between the Yangyin group and the model group was not statistically significant at day 3（P<0.05）,and after day 7,the MUC5ac level was lower in all dosing groups than in the model group（P<0.05）.From day 10,the down-regulation advantage of the Aci+Yangyin group was obvious（P<0.05）.Conclusion:1.Yangyin Qingfei Decoction Plus Flavor has a modulating effect on the impaired lung barrier function of SMPP,which can reduce the permeability of cell membrane and restore the function of pulmonary air-blood barrier by increasing the expression of Occludin as well as ZO-1,further exploring the action targets of Chinese medicine for the treatment of SMPP.2.Yangyin Qingfei Decoction Plus Flavor can regulate water metabolism in the lung by promoting the expression of AQP5 and inhibiting the secretion of MUC5ac,thus achieving the effect of"moistening dryness",which can effectively treat SMPP. |
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