Study On The Mechanism Of Qingfei Huatan Decoction Against MUC5AC In High Secretion Of COPD Mucus

Abjective: to study the anti-COPD mechanism of Qingfei Huatan decoction and to confirm that Qingfei Huatan decoction has a close relationship with the high secretion of mucus in the treatment of recurrent COPD.Thus the mechanism of anti-mucus hypersecretion was revealed in order to provide new methods and strategies for the treatment and prevention of COPD and lighten the burden of patients.Methods: In the part of animal experiment,the model of COPD rats was established by smoke inhalation and LPS lung irrigation.After the model was made,the rats were treated with Qingfei Huatan decoction with different doses(low,middle and high).In order to explore the therapeutic effect of Qingfei Huatan decoction on COPD rats,a positive drug(ambroxol hydrochloride)was set up in order to explore the therapeutic effect of Qingfei Huatan decoction.The methods include:1 to study the effect of Qingfei Huatan decoction on pulmonary function of COPD rats by measuring the four pulmonary function indexes of PIF,PEF,EF50%,MV with lung respiratory apparatus.The results were as follows:(1)the effect of Qingfei Huatan decoction on the pulmonary function of COPD rats was studied.(2)to explore the effect of Qingfei Huatan decoction on the gross appearance of lung in COPD rats by observing the changes of the gross appearance volume and color of the intact lungs in each group.(3)to explore the pathological changes of lung tissue of COPD model with Qingfei Huatan decoction by HE staining;(4)the pathological sections of lung tissue of rats in each group were observed by AB-PAS staining method to explore the effect of Qingfei Huatan decoction on the proliferation of goblet cells in lung tissue of COPD model.(5)The ultrastructureof lung tissue cells was observed by transmission electron microscope in order to explore the effect of Qingfei Huatan decoction on the ultrastructure of lung tissue cells in COPD rats.(6)to explore the effect of Qingfei Huatan decoction on the expression of MUC5 AC in lung tissue of COPD rats by using RT-PCR method and immunohistochemical method to study the expression of MUC5 AC mRNA and protein in lung tissue of COPD rats.In the part of cell experiment,the optimal concentration of LPS-induced inflammation of NCI-H292 cells was screened by MTT method,and the optimal time of LPS-induced inflammation of NCI-H292 cells was selected by immunocytochemical method.Based on the concentration and time obtained from the above screening,the expression of mRNA and protein in MUC5 AC was determined by RT-PCR technique and immunocytochemistry after treatment with Qingfei Huatan decoction.To explore the effect of serum containing Qingfei Huatan decoction on NCI-H292 cells stimulated by LPS.The expression of EGFR signal in NCI-H292 cells stimulated by LPS was measured by Weston-blot method,and the expression of EGFR in the cells was compared after treated with Qingfei Huatan decoction,which was treated with Qingfei Huatan decoction.To explore the signal pathway of regulating the expression of MUC5 AC in the treatment of COPD with Qingfei Huatan decoction.Results: in the animal experiment part;1.Compared with the blank control group,the pulmonary respiratory function of the model animal decreased obviously,and the large number of lung function of the model group decreased obviously,while the lung function of the rats treated with Qingfeihuatan decoction improved obviously.The PIF,PEF,EF50%,MV of the rats in the control group was significantly lower than that in the control group.2.Compared with the blank control group,the gross appearance of the lung in the model group was characterized by emphysema,inflammation and cellular fibrosis.For example,the volume of lung tissue in the model group increased obviously,and the lung tissue showed fold and collapse,which reflected the obvious pathological changes in the model group.All groups of Qingfei Huatan decoction improved the appearance of lung lesions.3.HE staining,a large amount of alveolar fusion,fibrosis and severe inflammation were found in the lung tissue of the model group.However,Qingfei Huatan decoction improved obviously in each group,and the alveolar fusion decreased obviously in each administration group.It is suggested that the Ming and Qing Dynasty Lung Huatan decoction has obvious therapeutic effect on the pathological changes of COPD rats.4.Compared with the normal control group,the goblet cells in the lung tissue of the model group were obviously proliferated by AB-PAS staining,and a large amount of mucus was produced to block the airway,while the Qingfei Huatan decoction was low and middle,and the lung tissue of the model group was lower than that of the normal control group.The degree of goblet cell proliferation was not significant in high-dose groups.5.the ultrastructural changes of lung tissue cells in each group were observed by transmission electron microscope(TEM).The results showed that Qingfei Huatan decoction could improve the ultrastructural damage of lung cells in COPD rats.6.Real-time fluorescence quantitative(RT-PCR)method was used to measure the expression of MUC5 AC mRNA in lung tissue of rats in each group.The results showed that the expression of MUC5 AC gene in lung tissue of model group was significantly up-regulated compared with that of normal control group.The expression of MUC5 AC in the treatment group of Qingfei Huatan decoction decreased obviously,indicating that Qingfei Huatan decoction could regulate the expression of MUC5 AC in the treatment of COPD.7.The expression of MUC5 AC protein in lung tissue of COPD rats was observed by immunohistochemical method.Compared with the normal control group,the expression of MUC5 AC protein in model group was significantly increased,while the expression of MUC5 AC protein in each group of Qingfei Huatan decoction increased with the increase of dosage.The expression of MUC5 AC protein decreased obviously.In the cell experiment section: 8.The stimulation concentration of NCI-H292 cells induced by LPS was screened by MTT method.Combined with the experimental results and literature resources,the stimulation concentration was determined to be 10 ug / ml.(9)immunocytochemical method was used to screen the stimulation time of NCI-H292 cells induced by RT-PCR LPS.The optimal stimulation time was 24 hours combined with the experimental results and literature resources.The expression of MUC5 AC protein and mRNA in NCl-H292 cells stimulated by LPS was detected by immunocytochemistry and RT-PCR method.Compared with the blank control group,the expression of MUC5 AC protein and mRNA in the cells stimulated by LPS was significantly increased,while the expression of MUC5 AC protein and mRNA in the cells of each group added with serum containing Qingfei Huatan decoction was significantly lower than that in the model group.It is proved that Qingfei Huatan decoction is achieved by inhibiting the expression of MUC5 AC in the treatment of COPD.(10)The expression of EGFR signal in NCl-H292 cells stimulated by LPS was measured by Weston-blot method,and the expression of EGFR in the cells was compared after treated with Qingfei Huatan decoction,which was treated with Qingfei Huatan decoction.The results showed that the expression of EGFR in the cells induced by LPS was up-regulated,and the expression of EGFR after blocking the EGFR signaling pathway by EGFR blocker was significantly lower than that of the normal control group,while the expression of EGFR in each intervention group of Qingfei Huatan decoction was down-regulated.It is suggested that the inhibition of cytoplasmic MUC5 AC expression by feihuatan decoction in Ming and Qing dynasties may be achieved by inhibiting EGFR signaling pathway.Conclusion: Qingfei Huatan decoction can inhibit the expression of MUC5 AC to treat the mucus hypersecretion of COPD,and its mechanism may be related to the inhibition of EGFR signaling pathway.