Mechanism Of Ren Shen Ping Fei Decoction On Idiopathic Pulmonary Fibrosis By Bioinformatic Analysis And Network Pharmacology

Object:Idiopathic pulmonary fibrosis(IPF)is a chronic and fetal lung disease,affecting majorly aged population.To date,its pathogenesis is still not clear and pharmaceutical choices are limited.In this study,transcriptomic data analysis was applied to explore the underlying mechanism of IPF while patterns in different groups were compared;Network pharmacological strategy was applied to elucidate the regulatory network of Ren Shen Ping Fei San(RSPFS)on IPF;Molecular docking and in vitro experiment were conducted to study how RSPFS would intervene in IPF.Method:1.RNA sequencing data of explant lung tissue from GSE150910,GEO database(https://www.ncbi.nlm.nih.gov/geo/)was selected for analysis.Gene set enrichment analysis(GSEA),Reactome pathway database and differential gene expression analysis were applied to discover meaningful genes and pathways.2.Weighted gene co-expression network analysis(WGCNA)was used to find gene modules related to different clinical traits,Reactome pathway database was used to find significant pathways,differential gene expression analysis was used to find significant genes in different traits.3.Published articles,TCMSP,SwissTargetPrediction,pharmapper were used to predict the therapeutic targets of RSPFS,Bisogenet was used to build protein protein interaction(PPI)network of the targets of RSPFS and IPF,Reactome pathway database was used to discover and compare significant pathways among groups.4.TGF-? signaling pathway was one of the significant results generated by the above studies and.Representative ingredients of RSPFS namely Ginsenoyne D,Ginsenoside 20-Glc-Rf,Notoginsenoside R1,Ginsenoside Rg1,6-Acetylacetonide,Caffeic acid,Timosaponin A1,Timosaponin B ?,Resveratrol,Rosmarinic acid,Xuelianlactone,Tectorigenin,sarsasapogenin,isomeranzin,glycyrol and Liquiritigenin were chosen as ligand,15 proteins in TGF-? signaling pathway namely HDAC1?XPO1?HS90B?HS90A?A4?1433G?FYN?SRC?EGFR?LRRK2?ESR1?TGFB1?SMAD2 and SMAD3,as well as COL1A1 were selected as protein targets.AutoDock Vina software was used to perform the molecular docking analysis.5.In vitro experiment was conducted to explore the effect of RSPFS and its subgroups(RSPFS group,BuYi group,QingFei group and HuaTan group)on phenotype shift in human embryonic lung fibroblast(MRC-5)induced by TGF-?1 stimulation.PCR and Western Blotting were used to test the mRNA and protein expression change of SMAD2/3,?-SMA,COL1A1 and NOX4Result:1.9 statistically significant gene sets were revealed by GSEA analysis,namely Hallmark Epithelial-Mesenchymal Transition gene set,HALLMARK_Kras downregulated gene set,HALLMARK_Glycolysis gene set,HALLMARK_angiogenesis gene set,HALLMARK_G2/M checkpoints gene set,HALLMARK_coagulation gene set,HALLMARK_Myc targets V2 gene set,HALLMARK_E2F targets gene set and HALLMARK_apical junction gene set,as well as the respective 485 leading edge genes.Reactome enrichment reveals extracellular matix,cellular senescence,cell cycle,coagulation,DNA repair defect,apical junction,carbohydrates metabolites and programmed death defect,etc.The most significant signaling pathways were Ras downregulated pathways such as PIK3/AKT,MAPK6/MAPK4,Rho GTPase,RTK,TGF-?,NOTCH,Integrin,TP53 and estrogen receptor signaling pathways.Interlukin 4 and 13 family were most significant among senescence associated secreted phenotype(SASP)factors.Differential gene expression(DEG)analysis showed senescence associated genes such as CCND2?CDKN2A?TERT?GDF15 and E2F8 were significantly upregulated in IPF patients.2.WGCNA and Reactome analysis revealed that the most relavant results were extracellular matrix,cell stress,immune system,cell cycle,coagulation,apical junction,muscle contraction,metabolism of carbohydrates,proteins and lipids,the results were overlapped with GSEA study.Gene modules related to senior group(over 65 y.o.)and middle group(age between 55-65 y.o.)were discovered,as well as 1076 and 422 genes within the modules.Both were significant enriched in extracellular matrix and immune system.In middle age group,irregular cell cyle,innate immune system and adaptive immune system were more significantly enriched.In senior group,cell senescence,unfoled protein reaction and endoplasmic reticulum stress were more significantly enriched.DEG analysis showed 203 and 39 DEGs in senior and middle age groups.Correlated co-expression differential expressed genes were foundCDH2?SFRP1?SERPINE2?CXCL12?SFRP4 and GEM;GREM1?VCAM1?PTHLH and RGS4;SLC7A5 and HIF1A;UGT2B17 and SLC12A3.3.608 protein targets of RSPFS were predicted,PPI network of leading genes from GSEA and RSPFS's targets was built and 152 hub proteins were found.Reactome analysis revealed the regulatory mechanisms were focused on post translational protein modification,cell cycle,DNA repair,TP53 and FOXO mediated transcribed regulation,oxidative stress and oncogene stress induced senescence,SASP,intrinsic apoptosis pathway,chaperone mediated authphagy,cytokins and innate immune system.Estrogen receptor mediated signaling pathway,Ras downregulated and TGF-?,tyrosine kinase receptor,NOTCH,WNT and integrin signaling pathways.83 and 90 hub proteins were found by building PPI network between RSPFS and gene modules related to senior or middle age group.Reactome analysis revealed that the most significant mechanisms are about post-translational protein modification,cell cycle,TP53 transcribed regulation,DNA repair,chaperone mediated autophagy,oxidative stress induced cell senescence,SASP and immune system.In senior group,the metalloprotease ubiquitination,SKP2-mediated p27/p21 degradation and adaptive immune reaction was significantly enriched while FOXO and E2F6 mediated transcript regulation,TLR 3 cascades in innate immune system were enriched in middle aged group.4.Molecular docking analysis showed most scores were less than-5 kcal mol-1,suggesting that ingredients of RSPFS were nicely docked with components direct or indirect in TGF-? signaling pathway.5.In vitro study showed that compared to the module group,RSPFS and all its subgroups decreased the mRNA and protein expression level of ?-SMA and COL1A1,suggesting that RSPFS and all its subgroups could regulate transcription and post-transcription activity of ?-SMA and COL1A1.RSPFS and Qingfei group reduced the mRNA and protein expression level of p-SMAD2,suggesting that RSPFS and Qingfei group could regulate transcription and post-transcription activity of p-SMAD2 while Buyi and Huatan group could not.RSPFS and Qingfei group dropped the the mRNA and protein expression level of p-SMAD3 while Buyi group only dropped the mRNA level,suggesting that RSPFS and Qingfei group could regulate transcription and post-transcription activity of p-SMAD3,Buyi group could affect the transcription activity,Huatan could affect neither.RSPFS and all its subgroups decreased the mRNA and protein expression level of NOX4,suggesting that RSPFS and all its subgroups could regulate transcription and post-transcription activity of NOX4.Conclusion1.Epithelial mesenchymal transition,coagulation,angiogenesis,glycolysis were pathogenesis found in this study,senescence associated genes such as p16,TERT?CCND2 and GDF15 were regulated in IPF patients suggested oncogene(Kras,Myc)associated cell senescence.My study confirmed the irregular pathogenesis and molecular signatures proved by previous studies,as well as some novel discoveries,such as cell cycle irregular was appeared in 55-65 y.o.IPF patiens while cell senescence was reached in population over 65.Innate and adaptive immune system reaction were more significant in 55-65 y.o.population,endoplasmic reticulum stress was more pronounced in poluation over 65.2.The regulation mechanism of RSPFS on IPF is mainly about post translational protein modification,cell cycle,DNA repair,gene transcription,oxidative stress and oncogene induced cell senescence,apoptosis,autophagy,SASP and immune system.Ras downregulated sinalling,TP 53 mediated transcription,TGF-??tyrosine kinase receptor,NOTCH,WNT and integrin signaling pathways were pronounced.3.RSPFS ingredients were nicely docked with proteins associated with TGF-? signaling pathways,paving the way for further experiment.By downregulating the rised expression level of?-SMA COL1A1 and NOX4,RSPFS and all its subgroups could reverse fibroblast phenotype shift,suppress extracellular matrix deposition and oxidative stress.RSPFS and QingFei group could cut off TGF? signaling pathway by inhibiting the phosphating of SMAD2/3,In general,RSPFS could mitigate the mesenchymal transition,ECM deposition and oxidative stress induced by TGF ?1 through inhibiting the phosphating of SMAD2/3 in TGF-? signaling pathway.