| Background:Pulmonary interstitial fibrosis(PF)is a kind of chronic and progressive pulmonary interstitial injury disease,and it is also the final outcome of a variety of interstitial lung diseases(ILD).PF-ILD is one of the key issues in clinical research.The incidence of the disease is increasing year by year,the mortality is high,which seriously affects patients’ quality of life,and there is still a lack of specific drugs in clinics.According to the classification of western medicine,PF can be divided into idiopathic pulmonary fibrosis(IPF)and secondary pulmonary fibrosis(SPF),while Traditional Chinese Medicine(TCM)belongs to the category of "Fei Wei".TCM has rich experience in the treatment of lung diseases.Professor Zhang Shunan,a mentor,after summing up his clinical experience for many years,believes that different types of PF have the same pathogenesis of deficiency of both lung and kidney,deficiency of qi and blood stasis.Under the idea of treating different diseases with the same treatment,tonifying the lung and kidney,replenishing qi and activating blood circulation as the treatment method,the formulation of Feiwei granules(FGs)prescription,clinical treatment of all kinds of PF has a clear effect.Clinical studies have confirmed that FGs can improve the clinical symptoms,improve the quality of life and delay the decline of lung function in patients with PF;it can inhibit the process of epithelial-interstitial transformation(EMT)and regulate the level of autophagy in vivo and in vitro.Current studies have shown that the phosphatidylinositol 3kinase/serine/threonine kinase/mammalian rapamycin target protein(PI3K/AKT/mTOR)signal pathway is the key signal pathway to regulate the level of autophagy and apoptosis,and autophagy deficiency and abnormal apoptosis are important pathogenesis of PF.This study summarized the clinical syndrome characteristics and mentor prescription of PF patients based on data mining technology,and discussed the pharmacodynamic mechanism of FGs based on the process of PI3K/AKT/mTOR signal pathway targeting regulation of autophagy and apoptosis,autophagy and apoptosis regulation of EMT.Objective:Clinical study:by collecting the four diagnostic data of patients with PF and the prescription information of Professor Zhang Shunan,to explore the distribution characteristics of clinical syndromes of patients with PF and the medication rule of Professor Zhang Shunan in the treatment of PF.so as to provide ideas for the clinical prescription.Experimental study:through the intervention of FGs on the mouse model of pulmonary fibrosis induced by bleomycin,to explore the effects of FGs on the survival rate,the degree of PF and collagen deposition,the level of EMT,autophagy and apoptosis and PI3K/AKT/mTOR pathway in mice with PF,and to clarify the efficacy and mechanism of FGs.Methods:1 Clinical studyThrough retrospective study,the general data,four diagnosis data,and prescription information of patients with PF who were treated for the first time in the Department of Traditional Chinese Medicine for Pulmonary Diseases of China-Japan Friendship Hospital were collected and inputted into Excel 2010 software.SPSS Modeler 18.0 and SPSS Statistics 20.0 software were used for association rule analysis,frequency statistics,visual processing,and cluster analysis,using a variety of data mining.To explore the distribution characteristics of clinical syndromes in patients with PF and the characteristics of prescription medication of Professor Zhang Shunan in the treatment of PF.2 Experimental studyThe mouse model of PF was established by bleomycin by one-time intratracheal instillation.The mice were randomly divided into four groups:blank group(Sham group),model group(Model group),rapamycin group(Rapamycin group),and Feiwei granules group(FGs group).All mice except the blank group were given 2 mg/kg dose of bleomycin intratracheally on day 0,while the blank group received the same amount of normal saline on the 1st to 21st day,rapamycin group received intraperitoneal injection of 2 mg/kg dose every other day,FGs group was given intragastric administration of 7.8 g/kg Feiwei granules every day,and other mice were injected intraperitoneally and intragastrically with the same amount of saline.In the first group,after continuous intervention for 21 days,the survival rate of each group was counted and survival analysis was carried out,while in the second group,the lung coefficient and lung appearance were recorded on the 7th,14th,and 21st day respectively,and the pathological morphology of lung tissue was observed by HE staining and Masson staining.The content of hydroxyproline(HYP)in lung tissue was detected by the Elisa method.Real-time quantitative polymerase chain reaction(qPCR),immunofluorescence staining,and Western Blot were used to detect the expression of EMT transcription factor Snail,epithelial and stromal markers αsmooth muscle actin(α-SMA),E-cadherin(E-cad)and vimentin(Vim)in lung tissue on the 21st day.The relative expression levels of autophagy-related proteins Beclin-1,SQSTM1/p62,LC3-II/LC3-I and apoptosis inhibitory Bcl-2 in lung tissue were detected by qPCR and Western Blot,autophagosome was observed by transmission electron microscope,apoptosis was detected by Tunel staining(fluorescence),and the phosphorylation level of PI3K,AKT,mTOR and p70(S6K)protein downstream of the pathway were detected by qPCR and Western Blot methods.Results:1 Clinical study results1.1 Distribution of Clinical Syndromes in patients with Pulmonary FibrosisA total of 262 patients with PF were included,including 59 cases of IPF,160 cases of secondary pulmonary fibrosis(SPF),and 43 cases of PF excluding secondary factors.SPF is mainly secondary to autoimmune diseases.The gender composition was 153 males and 109 females,slightly more males than females.The most age composition were 61-70 years old,mainly distributed in the 51-80 years old,mainly middle-aged and elderly.High-frequency symptoms include shortness of breath,wheezing,cough,suffocation,fatigue,white sputum,spontaneous sweating,clubbing fingers,dry pharynx,loose stool,loss of appetite,and so on.The tongue is dark,light red,purple dark,red,light-dark,accompanied by tooth marks,and the tongue coating is thin,white,thick and greasy,white and less fluid,and the pulse is characterized by string,sinking,fine,deficiency,fast and so on.The clustering results of the four diagnostic information showed that the clinical syndromes were lung qi deficiency,yin deficiency and lung dryness,spleen and stomach qi deficiency and phlegm dampness,kidney yang deficiency and blood stasis as common syndromes,and lung,spleen,and kidney deficiency and phlegm dampness and blood stasis as syndrome characteristics.1.2 Professor Zhang Shunan’s regular use of drugs in the treatment of Pulmonary FibrosisA total of 262 prescriptions for PF were collected,with a total frequency of 3637.There were 18 herbs with frequency≥60,including Panax notoginseng powder,fried perilla seed,licorice,Ophiopogon,peach kernel,Radix Astragali,Schisandra vinegar,and so on.The drug taste is mainly warm,flat,sweet,bitter,and pungent,which is mainly attributed to the lung meridian and spleen meridian.A total of 20 association rules were obtained by high-frequency drug association rule analysis,including 6 two-point drug combinations and 14 multi-point drug combinations;cluster analysis showed 5 kinds of drug combinations,and further obtained 10 drug pairs and 3 drug combinations.Association rules and cluster analysis showed that the main efficacy of drug combinations was warm lung and reduce fluid,nourishing Yin and promoting Jin,tonifying Qi and activating Blood Circulation,relieving cough and relieving Asthma,invigorating spleen and resolving phlegm.2 Experimental study2.1 Survival analysisAfter 21 days of modeling,the survival rate of mice in each group was observed.there was no death in the blank group,and the survival rates of mice in the model group,rapamycin group,and FGs group were 41.67%,58.3 3%,and 66.67%,respectively.The survival rate in the FGs group was higher than that in the model group,and there was no significant difference between the two groups(p>0.05).2.2 Body weight,lung coefficient,lung histomorphology and HYP content in lung tissue of miceAfter modeling,the body weight of mice in the blank group increased slowly,and the body weight of mice in the other three groups decreased significantly(p<0.05).The lung coefficient of the blank group was significantly lower than that of the other three groups on the 7th,14th,and 21 st day,and the lung coefficient of the FGs group was lower than that of the model group(p<0.05).HE and Masson staining of lung tissue showed that the structure of lung tissue in the blank group was normal and there was no collagen deposition.In the model group,there was large consolidation of lung tissue,obvious thickening of the alveolar septum,inflammatory exudation and fibroblast formation,and a large amount of blue collagen deposition.With time,the number of inflammatory cells decreased,fibroblasts increased and collagen deposition increased.The pathological changes in the FGs group were slighter than those in the model group.Ashcroft score showed that the fibrosis score of the FGs group was lower than that of the model group(p<0.05).Elisa test showed that the content of HYP in the model group was significantly higher than that in the blank group,and increased continuously with time,and the content of HYP in the FGs group was lower than that in the model group(p<0.05).2.3 EMT level in lung tissue of miceThe results of Western Blot and immunofluorescence staining showed that E-cad in the model group was lower than that in the blank group,while α-SMA and Vim in the model group were higher than those in the blank group(p<0.05).After intervention with FGs,E-cad increased and α-SMA and Vim decreased compared with the model group(p<0.05).qPCR showed that EMT transcription factor Snail was significantly increased in the model group(p<0.05),and significantly lower in the FGs group than in the model group(p<0.05).2.4 Levels of autophagy and apoptosis in lung tissue of miceWestern Blot detection showed that the autophagy-related proteins Beclin-1 and LC3BII/LC3B-I in the model group were lower than those in the blank group,while SQSTM1/p62 was increased in the model group(p<0.05).Beclin-1 and LC3B-II/LC3B-I in the FGs group were significantly higher than those in the model group,and SQSTM1/p62 was significantly lower than that in the model group(p<0.05).It was observed by transmission electron microscope that the autophagosome decreased significantly in the model group and increased in the FGs group compared with the model group.qPCR detection showed that the apoptosis inhibitory gene Bcl-2 in the model group was significantly higher than that in the model group(p<0.05),while that in the FGs group was significantly lower than that in the model group(p<0.05)and the apoptosis index(AI)in the model group was significantly higher than that in the blank group(p<0.05),and the apoptosis index in the FGs group was significantly lower than that in the model group(p<0.05).2.5 The expression level of PI3K/AKT/mTOR pathway in lung tissue of miceWestern Blot detection showed that the phosphorylation levels of PI3K,AKT，and mTOR in the model group were significantly higher than those in the blank group(p<0.05),while those in the FGs group were significantly lower than those in the model group(p<0.05).The levels of p70(S6K)downstream of PI3K/AKT/mTOR signal pathway in the model group were significantly higher than those in the blank group(p<0.05),while those in the FGs group were significantly lower than those in the model group(p<0.05).Conclusion:1 Clinical studyThe clinical syndromes of patients with PF are mainly characterized by the deficiency of lung,spleen and kidney,phlegm-dampness and blood stasis.From the point of view of diseasesyndrome-combination,Professor Zhang Shunan pays attention to tonifying qi,blood,yin and yang,reconciling cold and heat,especially tonifying the deficiency of lung,spleen and kidney,and removing pathological products such as phlegm,dampness and blood stasis at the same time.Flexible use of blood circulation method to tonify but not stagnant;and antitussive,asthma and phlegm-relieving drugs to improve clinical symptoms.2 Experimental studyFeiwei granules can increase the median survival time,improve the survival state,and reduce the destruction of lung tissue structure and collagen deposition in mice with PF.Its pharmacodynamic mechanism is through inhibiting the excessive activation of the PI3K/AKT/mTOR signal pathway,thus increasing autophagy activity and inhibiting abnormal apoptosis,and then inhibiting the process of EMT. |
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