The Effect Of D-4F,an Apolipoprotein A-I Mimetic,on Pulmonary Fibrosis And Related Mechanism

Background:Idiopathic pulmonary fibrosis(IPF)is a progressive fibrotic interstitial pneumonia and is one of the most common interstitial lung diseases.However,the specific pathogenesis of IPF remains unclear.There is no specific drug at present.Pirfenidone and Nintedanib are FDA approved for the treatment of IPF,but there are adverse reactions such as upper gastrointestinal symptoms,photosensitivity,rash,anorexia and hepatotoxicity.As the epidemic of COVID-19 began in 2020,pulmonary abnormalities,including pulmonary fibrosis,have been found in lung tissue from patients who have died from COVID-19.This requires us to find new potential therapeutic targets for IPF.Apolipoprotein A1(Apo-A1)is the highest apolipoprotein in HDL.Apo-A1 decreased in the lung tissues of patients with IPF and it could alleviate pulmonary fibrosis in animal models of IPF,suggesting that Apo-A1 is a potential therapeutic target of IPF.However,because of the ethical and purification issues,Apo-A1 therapy cannot be used in patients with IPF.D-4F is a polypeptide containing 18 D-amino acids,which is a mimic of Apo-Al and has shown good effects in the treatment of cardiovascular diseases.Recently,studies have shown that D-4F inhibits TGF-?1-mediated epithelialmesenchymal transition in alveolar epithelial cells.D-4f also inhibits TGF-?1 transcription and translation in macrophages.These results suggest that D-4F has a good potential of anti-pulmonary fibrosis in vitro.Transcriptomics and lipidomics have been widely used in IPF research.IPF is a result of multiple etiologies,involving alveolar epithelial cells,fibroblasts,immune cells and other cells.Transcriptomics can be used to systematically analyze the pathophysiological changes of IPF,finding molecular markers and new mechanisms.Both RNA-seq technology and single-cell RNA-seq technology have been used to study IPF,to provide potential targets for the individualized treatment of IPF.Lipidomics is a omics developed from metabonomics.Through the results of lipidomics,we can have a better understanding of the lipid metabolism changes in pulmonary fibrosis,especially in type 2 alveolar epithelial cells.In this study,transcriptomics and lipidomics were used to reveal D-4F's intervention on gene and lipid changes in pulmonary fibrosis in animals,which is helpful to explain the mechanism of D-4F.Objectives:1.The anti-fibrosis effect of D-4F in mice with pulmonary fibrosis model.2.Transcriptomic methods were used to analyze the gene changes in the lung tissues of D-4F interfered mouse model of pulmonary fibrosis and identify the differential genes and pathways related to pulmonary fibrosis.3.Lipidomics method was used to analyze the lipid changes in the lung tissues of D4F interfered mouse model of pulmonary fibrosis and identify the different lipids and pathways related to pulmonary fibrosis.4.Bioinformatics methods were used to comprehensively analyze transcriptome and lipidomics data to find out the pathway and possible targets of D-4F.Method:A.the effects of D-4F,an apolipoprotein A-I mimetic,on the mouse model of IPFa)Animal model and grouping:42 male C57/BL6 mice aged 7 weeks were selected.All mice were divided into 3 groups with 14 mice in each group,including blank group,model group and intervention group.b)Bleomycin-induced pulmonary fibrosis model:exposed trachea after anesthesia in mice.The model group and the intervention group were given 60?l,2mg/kg bleomycin solution,and the blank group was given the same amount of normal saline.The intervention group was intraperitoneally injected with 3mg/kg D-4F every day from the second day.Meanwhile,the blank group and model group were given the same amount of normal saline for 28 days.28 days later,all the mice were anesthetized and sacrificed.Serum,lung histopathology,lung transcriptome and lipidomics samples were collected.Cholesterol,high density lipoprotein,low density lipoprotein and triglyceride of each group were measured by biochemical analyzer.Serum TGF-?1 levels of each group were detected by Elisa.Lung histopathological samples were stained by HE staining,Masson staining and aSMA staining.B.Transcriptome analysis of D-4F,a mimic of Apo-A1,in a mouse model of IPFa)Frozen lung tissue samples were collected,and n=3 for each group after intragroup mixing.Total RNA was extracted,cDNA library was constructed and sequenced.The genes obtained by sequencing were determined according to the differential multiple>2 or<0.5,and P<0.05.The differential genes were analyzed by geneontology and KEGG analysis.b)The same differential genes of blank group and model group,model group and intervention group were analyzed together to obtain KEGG pathway.C.Lipidomics analysis of D-4F,a mimic of Apo-A1,in a mouse model of IPF.a)The frozen lung tissue was taken.The tissue was broken by shock and then dissolved with isopropanol-methanol solution(volume ratio 1:1).Positive ion and negative ion modes were used for separation by ULTRA performance liquid chromatography.Software LipidSearch 4.1.30 was used to conduct qualitative and quantitative analysis on the data obtained by positive and negative ion modes.Differential lipids were screened according to VIP>1 and P value<0.05.b)KEGG pathway was obtained by co-analysis of common differential lipids in blank group and model group,model group and intervention group.D.Combined transcriptomic and lipidomic analysis reveal the mechanism of D-4F,an apolipoprotein A-I mimetic,in the mouse model of IPF.The results of lipidomics and transcriptomics were analyzed by using proteinprotein interaction network to find the target of D-4F intervention in bleomycininduced pulmonary fibrosis model.The target was verified by qRT-PCR.Results:A.the effects of D-4F,an apolipoprotein A-I mimetic,on the mouse model of IPFa)After 28 days of modeling,5 mice in the model group died,2 mice in the intervention group died,and no mice in the blank group died.Survival curve analysis showed no statistical difference between the intervention group and the model group.The weight of mice in the model group and the intervention group was lower than that in the blank group,and the weight of mice in the model group was lower than that in the intervention group,which was statistically differentb)The serum cholesterol of model group was significantly higher than that of blank group,but there was no difference between model group and intervention group.The serum HDL-C of model group was significantly higher than that of blank group,but there was no difference between model group and intervention group.The serum LDL-C of model group was significantly higher than that of blank group,and the serum LDL-C of intervention group was significantly lower than that of model group.There was no difference in serum triglyceride of blank group,model group and intervention group.The level of serum TGF-?1 in model group was significantly higher than that in blank group and intervention groupc)HE staining showed that the lung tissue structure of the model group was severely deformed,and most of the lung tissue was replaced by fibrous tissue.Masson's staining of the same field shows numerous red muscle fibers and blue collagenous fibers in the consolidated lung tissue.The ?-SMA staining showed a large amount of lung tissue consolidation,with hyperchromatic particles in the lung tissue.HE staining in the intervention group showed lung tissue damage with fibrous bands and clumps,but focal thickened alveoli could also be seen in some areas.Masson staining of the same field shows fewer red muscle fibers and blue collagen fibers.In ?-SMA staining,there are small clumps of fibrous tissue,some areas showing focal thickened alveoli,with few deeply stained particles.The blank group showed no abnormality.Ashcroft score and mean optical density statistics showed that the degree of fibrosis in the model group was significantly higher than that in the intervention group and the blank group.?B.Transcriptome analysis of D-4F,a mimic of Apo-A 1,in a mouse model of IPFa)A total of 2179 differentially expressed genes were detected in the blank group and the model group,including 904 up-regulated genes and 1273 down-regulated genes.A total of 216 differentially expressed genes were detected in the model group and the intervention group,including 78 up-regulated genes and 138 down-regulated genes.b)After the blank group,model group and intervention group were analyzed together,99 differentially expressed genes appeared in both transcriptome results.KEGG analysis was performed on the 99 differentially expressed genes,and 13 pathways with statistical differences were obtained:cytokine-cytokine receptor interaction,influenza A,rheumatoid arthritis,measles,MAPK signaling pathways,cytosolic DNA-sensing pathway,transcriptional misregulation in cancer,chemokine signaling pathway,taurine and hypotaurine metabolism,hematopoietic cell lineage,Ras signaling pathway,Toll-like receptor signaling pathway and glycosaminoglycan biosynthesis-keratan sulfate.C.Lipidomics analysis of D-4F,a mimic of Apo-A1,in a mouse model of IPFa)There were 1100 differential lipids in the blank group and the model group,among which 740 were down-regulated and 360 were up-regulated.There were 92 lipid differences between the model group and the intervention group,including 57 down-regulated lipid ions and 35 up-regulated lipid ionsb)When the blank,model,and intervention groups were analyzed together,35 different lipids appeared in the two lipidomics analysis.KEGG analysis of the 35 differentially alter lipid revealed five statistically significant lipid metabolic pathways:glycerophospholipid metabolism,glycosylphosphatidylinositol(GPI)anchor biosynthesis,autophagy-other,ferroptosis and autophagy-animalc)In order to search for biomarkers of D-4F alleviating pulmonary fibrosis,we performed ROC curve analysis on 35 differentials alter lipids in blank group,model group and intervention group.We obtained the sensitivity,specificity and AUC of each group.Depending on sensitivity>0.8,specificity>0.8 and AUC>0.8,we identified 16 lipid molecules that could be used to differentiate the effects of D-4F,including DG(56:4)+H,DGDG(33:3)-H,GM3(d34:1)-H,LPC(20:2)+HCOO,LPS(24:0)+H,PC(17:4/22:1)+H,PC(20:1/22:6)+HCOO,PC(22:5/22:6)+HCOO,PE(16:0p/20:5)+H,PE(16:0p/22:6)-H,PE(18:2p/18:2)+H,phSM(d32:1)+H,SQDG(38:9)+HCOO,TG(15:2/18:2/21:6)+H,TG(18:0e/18:2/22:2)+NH4 and TG(18:0p/18:2/22:1)+NH4?D.Combined transcriptomic and lipidomic analysis reveal the mechanism of D-4F,an apolipoprotein A-I mimetic,in the mouse model of IPF.a)We comprehensively analyzed the common differential genes and common differential lipids in blank,model,and intervention groups.In the KEGG analysis of lipidomics,most of the different lipids were involved in the glycerophospholipid metabolism.So we made the protein-protein interaction network diagram of glycerophospholipid metabolism.The differential genes in the protein-protein interaction network diagram is pla2g4c.b)We used qRT-PCR to verify the pla2g4c gene.We found that the expression of pla2g4c in the model group was increased,which was significantly different from that in the blank group and the intervention groupConclusion1.this study proved that D-4F can alleviate bleomycin-induced pulmonary fibrosis in mice by reducing the level of peripheral blood TGF-?1.The anti-fibrosis effect of D-4F was confirmed.D-4f can be used as a potential therapy to treat IPF in the future.2.In this study,transcriptome and lipidomics methods were used to analyze the mechanism of D-4F in alleviating pulmonary fibrosis from the levels of RNA and lipid molecules.The roles of glycerophospholipid metabolism and pla2g4c gene in D-4F intervention were revealed,which put a foundation for the mechanism of D4F in alleviating pulmonary fibrosis.