Study On The Effect And Mechanism Of Radix Astragali And Radix Angelicae Sinensis In The Treatment Of Idiopathic Pulmonary Fibrosis Based On Evidence-based Medicine And Bioinformatics

Objective:At present,the treatment options for idiopathic pulmonary fibrosis(IPF)are limited,and the purpose of the treatment is still to delay the progress of the disease,improve the quality of life and prolong survival.Traditional Chinese medicine(TCM),including Danggui Buxue decoction(DGBXD)plays a certain role in the treatment of IPF,having less side effects and being easy to accept for patients.Therefore,based on the experimental research and clinical research on Radix Astragali(RA)and Radix Angelicae Sinensis(RAS)treatment of IPF,the comprehensive exploration of RA and RAS treatment of IPF from multiple perspectives is needed.We will explore the modern scientific connotation of the curative effect and action mechanism of RA and RAS treating IPF from a comprehensive perspective based on evidence-based medicine and bioinformatics,make the further basic research from surface to point more scientific connotation,and provide a deeper scientific basis for the future experimental research and clinical research.Methods:1.Randomized controlled trials(RCTs)on RA and RAS treating IPF were retrieved through the main Chinese and English database.RCTs meeting the criteria were included for systematic review according to the inclusion criteria and exclusion criteria.The quality of included studies was assessed using the Jadad rating scale and the risk of bias in the included studies was reference to the Cochrane Reviewer’s Handbook for guidelines.We extracted the main outcomes of included RCTs and a meta-analysis was conducted using the Cochrane Collaboration’s RevMan5.3 software.2.The active compounds and active genes of RA and RAS were retrieved using Traditional Chinese Medicine Systems Pharmacology Database(TCMSP)and UniProt database.IPF-related target genes were obtained through GeneCards database.Intersecting the active genes of RA and RAS and IPF-related target genes,the target genes acting on IPF were obtained.A medicine-compound-target-disease network was constructed using the Cytoscape software to screen for key compounds.A protein-protein interaction(PPI)network of target genes acting on IPF was constructed to screen for key target proteins.Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed using the DAVID database and the KEGG pathway database.3.We retrieved microarray datasets containing differentially expression genes(DEGs)of IPF tissue samples compared to normal tissue samples in the Gene Expression Omnibus(GEO).The active compounds and active target genes of RA and RAS were determined from TCMSP.The DEGs were intersected with the active target genes of RA and RAS,and the medicine-compound-gene network and the PPI network were established using the Cytoscape software.GO functional enrichment and KEGG pathway enrichment analyses were performed using RGUI software and its packages.Then a gene-pathway network for RA and RAS treating IPF was constructed.Molecular docking simulations of the target protein receptors and their corresponding compounds were performed using AutoDockTools software and AutoDock Vina software.4.The mouse model of bleomycin-induced IPF was established,which was divided into control group,model group,DGBXD treatment group and prednisone acetate positive treatment group.Pathologies among the groups were observed by hematoxylin-eosin(HE)staining,and the degrees of lung fibrosis were assessed by Masson staining.The amount of hydroxyproline in the lung tissue of each group was examined to quantify the extent of lung fibrosis.Inflammation-specific NF-κB(nuclear factor-κB)p65 and collagen type I in lung tissue were detected by immunohistochemical staining.Immunofluorescence staining was performed to detect the lung tissue leukocyte surface marker CD45,and macrophage surface marker F4/80.5.The GEO database was used to obtain datasets,which were subsequently used to identify differentially expressed miRNAs(DE-miRNAs)and differentially expressed mRNAs(DE-mRNAs).Utilizing miRNet,the predicted target genes of identified DE-miRNAs were estimated,and then the target genes of DE-miRNAs in IPF were examined thoroughly.The Enrichr database was used for the purpose of conducting GO functional enrichment and KEGG pathway enrichment.TCMSP were employed to obtain the target genes of RA and RAS as well as active compounds.A putative miRNA-mRNA regulation network of RA and RAS acting on IPF was developed by intersecting the target genes of RA and RAS with the DE-miRNA target genes in IPF.A bleomycin-induced mouse model was established and used to perform histopathology as well as real-time quantitative polymerase chain reaction(qRT-PCR)analyses of some miRNA-mRNA pairs.Results:1.Seventeen eligible RCTs were identified and made a systematic review and meta-analysis.The quality of the included studies was assessed using the Jadad rating scale and the risk of bias in the included studies was assessed with reference to the Cochrane Reviewer’s Handbook for guidelines.The main outcome indices included in the RCTs were extracted,and the meta-analysis was performed using the RevMan5.3 software.Meta analysis showed that the total effective rate and TCM syndrome effective rate were statistically significantly higher in the experimental group than the control group;the force vital capacity(FVC),FVC%predicted(%pred),total lung capacity(TLC)%pred,diffusion capacity of the lung for carbon monoxide(DLCO)and DLCO%pred were statistically significantly higher in the experimental group than the control group;the six minute walking distance(6MWD)was statistically significantly higher in the experimental group than the control group;the Borg scale questionnaire scores were statistically significantly lower in the experimental group than the control group;the incidence of adverse reactions was statistically significantly lower in the experimental group than the control group;cough,wheezing,short of breath,fatigue,thirst,coated tongue and pulse manifestation syndrome scores of TCM were statistically significantly lower in experimental group than the control group.2.The 22 active compounds and 100 active target genes of RA and RAS were obtained using TCMSP and UniProt database.The 2232 IPF-related target genes were obtained from GeneCards database.Sixty-nine overlapping genes were obtained by intersecting 100 compound target genes with 2231 IPF target genes,corresponding to 17 effective compounds.The "medicine-compound-target-disease" network was constructed using Cytoscape software,and the key compounds were quercetin,kaempferol,isorhamnetin,7-Omethylisomucronulatol,formononetin,beta-sitosterol,stigmasterol,3,9-di-O-methylnissolin and calycosin.The PPI network of intersection genes was constructed using the STRING website,and the key target proteins were IL6,VEGFA,MAPK8,CASP3,EGFR,MYC,ESR1,CCND1,FOS and AR.The GO functional enrichment analysis revealed that target genes for RA and RAS treating IPF were mainly enriched in DNA-binding transcription activator activity/RNA polymerase Ⅱ-specific,nuclear receptor activity,transcription factor activity/direct ligand regulated sequence-specific DNA binding,steroid hormone receptor activity,protein heterodimerization activity and so on.KEGG pathway enrichment showed that the target genes of RA and RAS treating IPF mainly enriched in Kaposi sarcoma-associated herpesvirus(KSHV)infection,hepatitis B,human cytomegalovirus(HCMV)infection,advanced glycation end product(AGE)-receptor for AGE(RAGE)signaling pathway in diabetic complications,proteoglycans in cancer and so on.3.Microarray datasets GSE2052,GSE21369,GSE24206,and GSE53845 of the GEO database containing DEGs of IPF tissue samples compared to normal tissue samples were retrieved and 1566 DEGs were identified.While 22 active compounds and 196 active target genes were identified from the TCMSP platform.After intersecting DEGs with active target genes,40 intersection genes were considered as target genes of RA and RAS acting on IPF.The "medicine-compound-gene" network and PPI network identified key compounds as quercetin,kaempferol,stigmasterol,7-O-methylisomucronulatol,formononetin,and beta-sitosterol;key targets as NTRK1,MYC,CUL3,FN1,TP53,VCAM1,MCM2,ITGA4,ESR1,APP,CDK2,EGFR,CUL7,COPS5,CUL1.We performed GO functional enrichment and KEGG pathway enrichment analysis of target genes acting on IPF and constructed a"gene-pathway" network to sort out major pathways as well as potential hotspot pathways.Finally,the potential interactions between key compounds and their target proteins were initially verified by simulated molecular docking.4.A bleomycin-induced IPF mouse model was established.HE staining results showed that the model group could show pulmonary congestion,different degrees of emphysema and inflammatory cell infiltration;pulmonary congestion and emphysema were lower on day 14,and inflammatory cell infiltration on day 21 was significantly lower in treatment group than in the model group.The model group showed impaired alveolar structure and pulmonary interstitial hyperplasia on day 14,and significant alveolar dilation on day 21;the treatment group had retained alveolar structure on day 14,pulmonary interstitial hyperplasia was inhibited,and alveolar dilation on day 21 were slightly compared with the model group.Masson staining revealed collagen fiber deposition in lung tissue on day 14 and heavily stained blue in the lung stroma on day 21;mild pulmonary fibrosis was observed on days 14 and 21 in the treatment group.The detection of hydroxyproline in the lung tissue was significantly higher in the model group compared to the control group,and was significantly lower in both the treatment and positive treatment groups compared to the model group.Immunohistochemical staining showed that NF-κB p65 expression increased in bleomycin-induced IPF mice compared with controls,especially in the IPF model group compared with treatment and positive treatment groups;the model group was higher in the nucleus.Bleomycin-induced IPF showed significantly increased expression of collagen type I compared to controls;RA and RAS inhibited the expression of collagen type I and reduced collagen deposition in lung tissue.Immunofluorescence staining showed that the infiltration of leukocytes and macrophages increased significantly in the model group,and the treatment of RA and RAS and prednisone acetate could reduce the accumulation of inflammatory cells in the lung tissue.5.DE-miRNAs were screened in IPF tissues compared to normal tissues from the GEO database miRNA dataset,and downstream target genes of DE-miRNAs were predicted by the miRNet platform.The DE-mRNAs between the IPF tissues and the normal tissues were also obtained by analyzing the mRNA dataset in the GEO database.Then,target genes of DE-miRNAs comprising 49 downmodulated and 53 upmodulated target genes were further screened to perform GO functional enrichment and KEGG pathway enrichment analyses.Subsequently,a sum of 196 target genes of RA and RAS was obtained from TCMSP,being 6 downregulated target genes identified as well as 6 upregulated target genes of RA and RAS acting on IPF.A promising miRNA-mRNA regulatory network of RA and RAS acting on IPF was developed,including upregulated miRNA and downregulated gene regulatory networks miR-654-3p-PKIA/ADRB1,miR-493-5p-FOS/OLR1,miR-410-3p/miR-495-3p-VEGFA and miR-493p-3p-MAP2;downregulated miRNA and upregulated gene regulatory network miR-338-3p-MMP2/HIF1A/MMP9 and miR-203a-3p-TOP2A/MMP1/TP63.Finally,the relevant miRNA-mRNA axis was verified by a bleomycin-induced IPF mice model and by qRT-PCR.The results showed that the mir-493 expression was remarkably elevated in the IPF model group in contrast with the control group and considerably attenuated in the DGBXD treatment group in contrast with the model group;the mRNA expression of Olrl was shown to have a significant decrease in the IPF model group in contrast with the control group and remarkably elevated in the DGBXD treatment group in contrast with the model group.The mir-338 expression was significantly attenuated in the IPF model group as opposed to the control group and significantly elevated in the DGBXD treatment group in contrast with the model group;the mRNA expression of Hifla was shown to have a substantial increase in the IPF model group in contrast with the control group and substantially attenuated in the DGBXD treatment group as opposed to the model group.Conclusions:This study looks for evidence-based evidence on the effectiveness and safety of IPF patients,reveals that it is safe and effective for IPF patients and can improve pulmonary function and exercise tolerance of these patients,which make more modern scientific connotation for clinical treatment of IPF,provide reference for further RCT and real world research,and provide clinical efficacy for further research on basic experimental mechanism.This study analyzed the mechanism of action of RA and RAS on treating IPF through the multi-level network of "compound-target-pathway-disease",and clarified the mechanism of action on the multi-component,multiple target and multi-pathway of IPF.At the same time,we further combined with network pharmacology and system bioinformatics method.Through microarray dataset analysis of IPF samples and normal samples between DEGs,the IPF target genes,target proteins and pathway relationship were accurately revealed,which established the "gene-pathway" network.Through simulated molecular docking method,preliminary molecular docking verification was completed,which further provided value for IPF treatment.This study clarified the mechanism of multi-component,multi-target and multi-pathway of IPF,established the "gene-pathway"network,obtained the important pathway for the treatment of IPF,and further explored.On the overall research,NF-κB-related and miRNA-related pathways were studied from surface to point.In terms of NF-κB-related pathways,the mechanism of involvement in anti-inflammatory effect of DGBXD to alleviate IPF by interfering in NF-κB and macrophage activation,providing modern scientific implications for further studies.In terms of miRNA-related pathways,it revealed the potential mechanism of miRNA-mRNA regulatory axis in IPF pathogenesis,established the miRNA-mRNA regulatory network of RA and RAS to improve IPF,and conducted experimental verification of some miRNA-mRNA axis to further study the mechanism through experiments.In conclusion,this project sought for the evidence-based medical evidence of the effectiveness and safety of using RA and RAS in treating IPF patients from the perspective of evidence-based medicine,and explored the scientific connotation of treating IPF from a clinical perspective.Combined with the overall perspective of systematic bioinformatics method,network pharmacology method and molecular docking method,we clarified the role of multi-component,multi-target and multi-pathway in the treatment of IPF,and scientifically explore the important pathways of RA and RAS in the treatment of IPF.Based on the basis of the overall study,the NF-κB-related pathway and miRNA-related pathway were explored from face-to-point.This project has carried out a series of research on the treatment of IPF by RA and RAS,which makes it more modern scientific connotation,more in line with the internationalization of TCM research,and better scientific development and promotion.