Effect Of Feifukang On Pulmonary Fibrosis And Its Active Component Analysis

Background:In recent years,with China’s gradual entry into an aging society and the frequent onset of haze,the incidence of pulmonary fibrosis has increased by 11%annually,and the conservative estimate of pulmonary fibrosis patients in China is more than 1 million.At present,the pathogenesis of pulmonary fibrosis is not clear,especially idiopathic pulmonary fibrosis(IPF)has no definite and effective treatment drugs.Once diagnosed as pulmonary fibrosis,the survival time of patients is about 3-5 years.Lung transplantation is an important means to treat pulmonary fibrosis thoroughly at present,but its application is limited by its high cost and difficult donor sources.The treatment of pulmonary fibrosis is still a worldwide problem.In 2014,the FDA approved two relatively effective anti-pulmonary fibrosis drugs:pifenidone and nidanib.Although these two drugs bring hope to the patients with pulmonary fibrosis,their therapeutic effect is limited to slowing down the progress of pulmonary fibrosis,and can not significantly prolong the survival time of patients.Moreover,they also have high incidence of side effects,such as rash,gastrointestinal reactions,abnormal liver function,hypertension and so on.At present,the most commonly used therapeutic drugs in clinic are western medicine,such as glucocorticoids,immunosuppressants,antioxidants and so on.Similarly,the efficacy of these chemicals is not only controversial,but also has many adverse reactions.Our research group will develop Feifukang as a new Chinese medicine against pulmonary fibrosis,which will bring new hope to the patients with pulmonary fibrosis,and also point out new ideas for the research of other Chinese medicine compound prescriptions.Studies have found that natural Chinese medicine preparations are welcomed by more and more patients because of their dual effects of treatment and conditioning,avoiding toxic and side effects of chemical drugs.Pulmonary fibrosis belongs to the category of "pulmonary arthralgia" and "cough" in TCM.TCM syndrome differentiation belongs to the syndrome of deficiency of the root and deficiency of this sign,deficiency of the lung and the spleen qi,and in fact,phlegm,turbidity,blood stasis and toxin are mutually reinforcing.Feifukang is a pure traditional Chinese medicine preparation with complete independent property rights,which is extracted and processed by the members of this research group according to traditional Chinese medicine theory,combined with modern medical viewpoint,through repeated clinical drug screening and preparation of eight kinds of medicinal materials including Astragalus membranaceus,Codonopsis pilosula,Ophiopogon japonicus,Schisandra chinensis,Notoginseng,Fritillaria thunbergii,Anemarrhena asphodeloides and Glycyrrhiza.In the prescription,Astragalus membranaceus Qi-invigorating and Zheng-invigorating is the monarch medicine,liquorice expelling phlegm and detoxifying,and other drugs are used as assistants to prevent and treat pulmonary fibrosis by Invigorating Qi,detoxifying and expelling phlegm,removing blood stasis and dredging collaterals.Feifukang appears in the form of traditional Chinese medicine compound,and its multiple components can play a synergistic role in the process of anti-fibrosis.At present,the biggest puzzle in the study of TCM compound prescription is how to establish its target and mechanism of action.This study will study the anti-pulmonary Fibrosis Mechanism of Feifukang from the aspects of proteomics,genetic informatics and cell biology,and provide key research clues for the anti-chronic and complex diseases of Chinese medicine compound,and on this basis,provide scientific theoretical basis for the development of new anti-pulmonary fibrosis Chinese medicine.It is of great scientific significance to clarify the advantages of this Chinese medicine compound in the treatment of complex diseases and to develop Feifukang as an innovative anti-pulmonary fibrosis drug with broad clinical application prospects,which will inevitably bring good social and economic benefits.Objective1.To clarify the anti-pulmonary fibrosis effect of Feifukang.2.To explore the targeting gene and pathway of Feifukang against pulmonary fibrosis.3.To clarify the active ingredients of Feifukang and its pharmacokinetic characteristics.Methods1.In vitro evaluation of anti-pulmonary fibrosis effect of Feifukang.TGF-beta 1 stimulates L929 cells to establish pulmonary fibrosis cell model in vitro.The effect of Feifukang on pulmonary fibrosis cell model was analyzed by real-time proliferation/migration.Immunofluorescence staining showed the expression of alpha-SMA in the cells of each group.Western blot was used to detect the expression levels of collagen type Ⅰ,collagen type Ⅲ and alpha-SMA in the cells of each group.2.In vivo evaluation of anti-pulmonary fibrosis effect of Feifukang.C57BL/6 mice were randomly divided into normal control group,pulmonary fibrosis model group and Feifukang treatment group.The forced vital capacity(FVC)of mice in each group was analyzed by animal lung function instrument,and the difference of FVC of mice in each group was evaluated.Lung histopathological sections of mice were stained with HE and Mason.Hydroxyproline(Hyp)in lung homogenate was determined.Western blot was used to detect the expression of alpha-SMA and Collagen I to evaluate the anti-pulmonary fibrosis effect of Feifukang.3.Network Pharmacology Searches for the Effective Components of Feifukang and the Signaling Pathway Possible Participation.TCMID database and Herb BioMap database were used to collect and extract the active chemical constituents of various drugs in Feifukang.Drug components were imported into TCMSP database,and the main bioactive components were screened by ADME system TCMSP database model.Through the network pharmacology method,three levels of network were established:compound drug network to find the effective active ingredients of Feifukang;compound target network to find the corresponding action targets of the effective active ingredients of Feifukang;target-path network to reflect the possible action targets and corresponding target signaling pathways of Feifukang against pulmonary fibrosis.4.RNA sequence further reveals the anti-pulmonary Fibrosis Mechanism of Feifukang.Lung tissues of mice in each experimental group were collected,total RNA was extracted and purified,and RNA sequence was performed.The differentially expressed mRNAs were evaluated among the three groups.Through GO and KEGG analysis,differentially expressed genes were further collated to find the involved signaling pathways.Real-time quantitative PCR(qRT-PCR)reconfirmed differentially expressed genes.Western blot was used to detect the differentially expressed proteins corresponding to the mRNA,and to verify the JAK-STAT signaling pathway of Feifukang in anti-fibrosis.Western blot analysis of p-STAT3,p-JAK1 and p-smad3 showed that protein phosphorylation was involved in JAK-STAT signaling pathway.Using the sequencing data of IncRNA,the co-expression of STAT3 and JAK1 was analyzed in order to provide a basis for future multi-target research.5.U-HPLC-MS/MS Analysis of Main Active Components and Pharmacokinetic Characteristics of Feifukang.Ultra-high performance liquid chromatography tandem mass spectrometry(U-HPLC-MS/MS)was used to determine the main active ingredients of Feifukang by comparing the retention time of the reference substance,the first-order mass spectrometry data and the second-order mass spectrometry data.Blood was collected from the infraorbital vein of the pulmonary fibrosis model mice before Feifukang was administered intragastrically.Blood was collected at 0.08,0.17,0.33,0.5,0.75,1,1.5,2,3,4,6,8,12 and 24 hours after treatment.The concentration of each active component of Feifukang was determined by U-HPLC-MS/MS at different time points to obtain its pharmacokinetic characteristics.Result1.Feifukang can inhibit the activation of myofibroblasts,inhibit the proliferation and migration of L929 cells induced by TGF-beta 1,and reduce the expression of Collagen Ⅰ and Collagen Ⅲ.2.The FVC of pulmonary fibrosis model group was significantly lower than that of pulmonary fibrosis model group,while the FVC of mice in Feifukang treatment group was slower than that of pulmonary fibrosis model group.3.Compared with the pulmonary fibrosis model group,the pulmonary fibrosis degree of Feifukang treatment group was lighter and the deposition of collagen was less.It could reduce the content of hydroxyproline(HYP)and the expression of actin alpha(alpha-SMA)and type I collagen(Collagen I)in lung tissue.4.Through the compound drug network(c-m network)in network pharmacology,90 bioactive compounds were analyzed in eight prescription drugs of Feifukang.Among them,M05 anthocyanin,M07 nicotinic acid,M66 pilose isoflavone and M76 mangiferin are replicated in three herbal components,and have high oral bioavailability(OB),which may be the main active compounds of Feifukang.5.Through the compound target network(c-t network)and target-path network(T-P network)in network pharmacology,90 bioactive compounds of Feifukang were displayed,and 129 potential target molecules were corresponded through 914 relationships.The average degrees of compounds and molecular targets were 10.16 and 7.20,respectively.Among these potential target molecules,83 molecules can target 26 signal pathways related to pulmonary fibrosis,including JAK-STAT3 signaling pathway.The degree of molecular target and signal pathway are 3.35 and 10.30,respectively.6.JAK-STAT signaling pathway is involved in the anti-pulmonary Fibrosis Mechanism of Feifukang.RNA-Sequence was used to enrich the function of differential genes in lung tissue of mice.It was found that there were many differentially expressed genes related to JAK-STAT signaling pathway in Feifukang group.The representative JAK1,STAT3 and ADAM 17 genes were selected for qRT-PCR and Western blotting analysis.The results showed that JAK1,STAT3 and ADAM 17 expressed more in the model of pulmonary fibrosis at the gene level and the corresponding protein level,while the expression of JAK1,STAT3 and ADAM 17 decreased significantly in the treatment group of Feifukang.7.Protein phosphorylation may be involved in JAK-STAT signaling pathway expression.Western blot was used to detect the expression of SMAD3,p-STAT3 and p-JAK1 phosphorylated proteins.The expression of phosphorylated protein decreased in Feifukang treatment group.8.Based on RNA sequencing data,9 lncRNAs were found to co-express with STAT3 and 10 lncRNAs were co-expressed with JAK1.Among them,lncRNA-MSTRG7464.1 and lncRNA-NONMMUG052541.1 had the highest degree of co-expression with JAK 1 and STAT3,and the values of co-expression were 2.13 and 1.9,respectively.9.The main active ingredients of Feifukang were determined by ultra-high performance liquid chromatography-tandem mass spectrometry(U-HPLC-MS/MS).The regression equations of mangiferin,pilose isoflavone glucoside,mangiferin,pilose isoflavone and stigma anthocyanin were Y=7.185× 10-4X+8.95× 10-5(r=0.9992),Y=9.82×10-2X-6.354×10-2(r=0.9975),Y=4.014×10-2X+6.446×10-2(r=0.9926),Y=6.854×10-2X+5.510×10-2(r=0.9943),Y=7.140×10-2X+2.316×10-2(r5=0.9978)10.The retention time of the above five compounds in blood was determined by U-HPLC-MS/MS,respectively,2.39 min for mangiferin,2.56 min for pilose isoflavone glucoside,4.12 min for glycyrrhizin,5.02 min for mangiferin,6.77 min for pilose isoflavone and 4.12 min for mangiferin.Mulberry isoflavones,mangiferin and mangiferin had higher serum concentrations(greater than 5.0 ng/ml).Compared with normal rats,the metabolic parameters of pilose isoflavone,mangiferin and mangiferin were different in pulmonary fibrosis rats.Among them,there were significant differences between fibrotic rats and normal rats in pili isoflavone and stigma anthocyanin T1/2.Conclusions1.Animal experiments in vivo and in vitro prove that Feifukang has a good anti-pulmonary fibrosis effect.2.Feifukang has the effect of anti-pulmonary fibrosis through multiple genes and pathways.JAK-STAT signal transduction pathway is one of the mechanisms of anti-pulmonary fibrosis of Feifukang.3.Protein phosphorylation and some IncRNA may be involved in the expression of JAK-STAT signal transduction pathway.4.Mulberry isoflavone,Mulberry isoflavone glucoside,Mangiferin,Mangiferin and Mangiferin are the main active ingredients of Feifukang.Feifukang showed satisfactory pharmacokinetic performance.