| Pulmonary fibrosis(PF)is a group of lung destructive diseases with different etiology and common pathological characteristics.The typical pathological characteristics of pulmonary fibrosis include alveolar epithelial cell damage,fibroblast hyperplasia,and extracellular matrix(ECM)deposition,structural deformation of lung tissue,pulmonary failure,etc.Medical research at home and abroad has shown that the causes are complex and diverse,such as inhalation of dust,toxic gases,environmental pollutants,or secondary to respiratory diseases,immune system diseases,etc.In recent years,due to the lack of effective treatment strategies,the incidence of PF is increasing every year,which seriously threatens human life and health.Therefore,exploring effective and low-toxicity therapeutic drugs is a hot research topic in the medical field.At present,Western medicine often uses glucocorticoids,immunosuppressive agents and cytotoxic drugs for clinical treatment of pulmonary fibrosis,which have many adverse reactions,are expensive,and lack long-term efficacy.As a treasure of Chinese traditional culture,traditional Chinese medicine has made good progress in the prevention and treatment of pulmonary fibrosis,whether it is Chinese medicine prescription or a single-flavored medicinal piece and Chinese herbal extract.And a large number of literature studies have confirmed that the use of traditional Chinese medicine to treat pulmonary fibrosis can better improve the symptoms of patients and delay the development of the disease.Feiwei prescription is the fifth batch of clinical experience prescriptions of Professor Zhu Qiyong,a national elder Chinese medicine practitioner,with the effect of improving pulmonary fibrosis syndrome.The prescription uses "Salvia miltiorrhiza Bunge" as the main medicine,followed by "Hirudo","Paeonia veitchii Lynch" and"Amygdalus persica L","Ophiopogon japonicus" as the third drug,and "Oroxylum indicum(L.)Kurz" and "Terminalia chebula var.chebula" as the fourth drug to nourish yin deficiency and removing stasis for treating pulmonary fibrosis.Starting from restoring the body’s dynamic balance,it stimulates the body’s autoimmunity and rehabilitation capabilities and restores normal pulmonary function.Although the clinical efficacy of Feiwei prescription is definite,the substance basis and mechanism of action of this prescription have not been clearly explained.Therefore,this study intends to use UPLC-Q-TOF/MS to analyze and identify the chemical constituents in Peiwei prescription water extract and serum after administration.In this experiment,the SD rat model of pulmonary fibrosis was used to evaluate the anti-pulmonary fibrosis activity of the prescription;the mechanism of action was elucidated from the perspective of systems biology using a metabolomics method.The research contents include 5 parts:1 Study on the extraction process of Feiwei prescriptionIn order to optimize the water extraction process of Feiwei prescription,the fingerprints of the extracts were established based on HPLC,and the peak areas of the common peaks were analyzed by principal component analysis.The L9(34)orthogonal test design was used to investigate the material liquid ratio,extraction time,and number of extractions.The total factor score was used as the evaluation index,and the optimal extraction process of Feiwei prescription was selected through the combination of range analysis and analysis of variance.The results indicate that there are 33 common peaks in the fingerprint.The results indicate that there are 33 common peaks in the fingerprint,and the cumulative variance contribution rate of 6 principal components reaches 97.72%.In this process,the optimal extraction conditions are:water is the solvent,the liquid solid ratio is 12:1,and the number of extractions is 2 times for 1 hour once.The results show that the common peaks of the fingerprint spectrum are used as the evaluation index of the compound Chinese medicine extraction,reflecting the overall extraction effect,determining a stable and feasible process,and providing a basis for the subsequent development of Feiwei prescription.2 Study on material basis of Feiwei prescriptionIn this experiment,UPLC-Q-TOF/MS was used to analyze and identify the chemical constituents in Feiwei prescription water extract and serum after administration.The analysis was performed on an ACQUITY HSS T3 reverse phase column(2.1 mm×100 mm,1.8 μm).The mobile phase consisted of 0.1%formic acid in ACN and 0.1%formic acid in water was used for gradient elution,the flow rate was 0.4 mL/min,the column temperature is 40 degrees.The mass spectrometry detection conditions were electrospray ionization source(ESI),scanning and collecting data in positive and negative ion mode.Based on accurate mass-to-charge ratios and fragmentation information provided by the secondary mass spectrometry with relevant literature and reference information,the main components include alkaloids,phenylpropanoids,flavonoids and their glycosides,sugars,organic acids,monoterpenes,diterpenes,triterpenes,etc.Among the 109 compounds,24 were derived from Ophiopogon japonicus,19 were derived from Amygdalus persica L,17 were derived from Salvia miltiorrhiza Bunge,15 were derived from Paeonia veitchii Lynch,14 were derived from Oroxylum indicum(L.)Kurz,13 were derived from Terminalia chebula var.chebula,10 were derived from Hirudo.Based on the in vitro chemical components that have been identified and combined with relevant literature,using MetaboLynx XSTM data processing software to infer the mass spectral information,48 blood-inducing components were characterized,including 35 prototype components and 13 metabolic components.The research results provide a basis for controlling the drug quality,material basis and treatment mechanism of Feiwei prescription3 Study on the quality evaluation of Feiwei prescriptionIn order to conduct a preliminary quality evaluation study of Feiwei liquid extract,this experiment uses TLC qualitative method and HPLC quantitative method.The TLC method was used to identify the seven Chinese herbal medicine in the liquid extract.Finally,a qualitative analysis method for 5 Chinese herbal medicine such as Salvia miltiorrhiza Bunge,Paeonia veitchii Lynch,Amygdalus persica L,Ophiopogon japonicus,and Hirudo with good reproducibility and specificity was established.The methodological results show that the HPLC method for the determination of amygdalin,paeoniflorin,and salvianolic acid B in Feiwei liquid extract has been established,which has good linearity,precision,stability,repeatability,and recovery.According to the determination results of 3 batches samples,temporary prescribe that the content of amygdalin per g of Feiwei liquid extract shall not be less than 0.810 mg/g,the content of paeoniflorin per g of Feiwei liquid extract shall not be less than 0.195 mg/g,and salvianolic acid B per g of Feiwei liquid extract shall not be less than 0.363 mg/g.The 3-month accelerated stability results show that the RSD of the 33 common peak areas in the fingerprint of Feiwei liquid extract are all less than 5.0%,indicating that it has good stability.The results showed that the quality of Feiwei liquid extract was stable and controllable,which provided a reliable guarantee for further research on the efficacy and mechanism of anti-pulmonary fibrosis.4 Study on anti-pulmonary fibrosis in Feiwei prescriptionIn order to verify the efficacy of Feiwei prescription,this experiment investigated the anti-pulmonary fibrosis activity.54 SD rats were randomly divided into 6 groups,which were the normal group,model group,prednisone group and high-dose FWP group,mid-dose FWP group and low-dose FWP group,with 9 rats in each group.A model of pulmonary fibrosis was made by dripping bleomycin into the trachea of rats.After 8 hours of modeling,the rats were continuously administered with gastrointestinal administration for 28 days.On the 7,14,and 28 days after modeling,the general conditions of the animals were observed,the lung coefficients were measured,and the lung tissues were stained with HE and Masson.Pathological changes.Serum transforming growth factor β1(TGF-β1),rat interleukin-17(IL-17),and rat interleukin-6(IL-6)were measured in serum.The results showed that compared with the model group,high-dose FWP group,mid-dose FWP group and low-dose FWP group could improve general conditions such as mental state,hair,and diet in rats,reduce the increase in lung coefficient caused by pulmonary fibrosis,the scope of fibrotic lesions,the degree of the disease and the levels of TGF-β1,IL-17,and IL-6 in the serum(p<0.05).The above confirms the anti-pulmonary fibrosis activity of Feiwei prescription.5 Study on serum metabolomics mechanism of Feiwei prescription anti-pulmonary fibrosisBasis on the pharmacodynamic research,we explore the mechanism of Feiwei prescription’s anti-pulmonary fibrosis through the metabolomics.UPLC-Q-TOF/MS technology and MetaboAnalyst 4.0 platform were used to analyze the metabolic profile of serum samples from each group of rats in pharmacodynamic experiments,combined with principal component analysis(PCA),orthogonal partial least squares discrimination analysis(OPLS-DA)and other multivariate statistical analysis methods to identify biomarkers which related to pulmonary fibrosis.Comparing the normal group with the model group,find out 17 potential biomarkers in the serum,and 21 related metabolic pathways were constructed with the help of MetPA.Finally,the experiment pointed out 5 main metabolic pathways:including Ubiquinone and other terpenoid-quinone biosynthesis,Inositol phosphate metabolism,Tyrosine metabolism,Arginine biosynthesis,Arginine and proline metabolism(impact>0.05).Compared with the serum of pulmonary fibrosis model rats,11 metabolites were up-regulated and 6 metabolites were down-regulated in the administration group,indicating that each of the administration groups could make these,17 potential biomarkers withdraw to the normal group.Based on the analysis of the experimental results,it is speculated that Feiwei prescription may exert anti-pulmonary fibrosis effects through multiple targets and multiple pathways.For all these reasons,this study optimized the Feiwei prescription extraction process,determined the most suitable extraction process parameters;characterized the transition components of the extract in serum,clarified the material basis;established qualitative and quantitative methods to ensure stable and controllable quality.To evaluate the activity further confirmed the efficacy of anti-pulmonary fibrosis.To identify biomarkers and major metabolic pathways,elucidate the mechanism of metabolomics.In order to develop Feiwei prescription into a modern Chinese medicine preparation with a clear material basis,stable technology,controllable quality,precise curative effect,and clear mechanism of action,which provides basis. |
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