Metabolomics And Metabolic Flux Analysis Reveals The Alterations Of Pulmonary Metabolism In IPF Mice

PartⅠNon-targeted Metabolomics Database to Explore the Changes of Physiological State Rhythm of LungBackground and objectivesSince its discovery in the early 20th century,circadian rhythms have garnered widespread attention.Under physiological conditions,factors such as light exposure,diet,and sleep can induce 24-hour circadian oscillations in the body through the regulation of circadian gene transcription,translation,and hormone secretion.In addition to these factors,rhythmic changes in lung tissue are closely associated with respiratory frequency,airway resistance,and end-tidal carbon dioxide levels.Various respiratory diseases such as acute lung injury,chronic obstructive pulmonary disease,pulmonary arterial hypertension,pulmonary fibrosis,and lung cancer exhibit varying degrees of rhythmic changes,leading to impaired immune regulation and exacerbation of disease progression.Metabolomics,being the omics closest to the phenotype,can rapidly reflect changes in the body.A deeper understanding of the rhythmic patterns of lung tissue at the metabolic level holds significant importance for discovering new biomarkers and potential therapeutic targets.However,the complexity of metabolite structures,incomplete metabolite databases,and the expensive cost of metabolite standards pose challenges to the accurate identification of metabolites in non-targeted metabolomic analysis,highlighting the urgent need for solutions in this area.To delve deeper into the rhythmic changes in lung tissue at the metabolic level,this section of the research work first integrated electrospray ionization（ESI）and atmospheric pressure chemical ionization（APCI）for the simultaneous detection of isotopically labeled metabolites in a yeast system using liquid chromatography-tandem mass spectrometry（LC-MS）.This allowed for the simultaneous detection of polar and non-polar metabolites and established an analysis database containing approximately 1300 small molecule metabolites.Subsequently,based on this database and integrated detection process,metabolic changes in lung tissue under physiological conditions were revealed from the perspectives of lipid changes,amino acid changes,and changes in energy metabolism pathways,elucidating the rhythmic oscillations of metabolic processes in lung tissue.MethodsThis study initially employed ¹³C and ¹⁵N isotope-labeled media to culture yeast,which was harvested during the logarithmic growth phase for LC-MS analysis.To enhance database expansion,we utilized two ion sources,ESI and APCI,for LC-MS analysis.For improved detection,various voltages and extraction agents were applied to detect changes in metabolite peak intensities,further optimizing detection conditions.The detected data were subjected to statistical analysis using software such as MS-convert,EL-Maven,Metaboanalyst 5.0,and R language to determine metabolite chemical formulas.Subsequently,the Human Metabolome Database（HMDB:http://www.hmdb.ca/）and Pub Chem database（http://pubchem.ncbi.nlm.nih.gov/）were utilized for comparison and MS2 detection to determine the final metabolite names,thus establishing a non-targeted metabolomics database.This established database was applied to study the rhythmic changes in metabolism in mouse lung tissues under physiological conditions.Results1.Yeast cultured in media enriched with ¹³C and ¹⁵N isotopes were subjected to ESI/APCI-LC-MS detection,resulting in the characterization of 798 metabolites through database matching and MS2 detection.Among these,214 metabolites were exclusively detected using APCI,with amino acids comprising the highest proportion at approximately38.4%,followed by lipids at approximately 33.5%.Conversely,approximately 452metabolites were exclusively detected using ESI,with water-soluble lipids representing the majority at approximately 52.8%,followed by carbohydrates at approximately 15.3%.Combining this database with our existing collection of standard compounds,we obtained relative retention times and MS2 spectrum identification information for over 1300metabolites.2.Analysis of lung tissue from mice under physiological conditions matched and identified a total of 1003 metabolites from the database of around 1300 metabolites.Among these,143 metabolites and their metabolic pathways exhibited rhythmic oscillations over time,primarily including glycerophospholipids,arginine,proline,phenylalanine,tyrosine,tryptophan,and glycine-serine-threonine related metabolites and pathways.After categorizing the metabolites,it was observed that peak levels of lipid,nucleotide,and peptide metabolites occurred during the daytime,while amino acid and carbohydrate metabolite levels peaked during the nighttime.These results strongly indicate a close relationship between changes in lung metabolites and circadian rhythm regulation.ConclusionBy integrating both ESI and APCI ionization sources,simultaneous detection of polar and non-polar metabolites was achieved,leading to the establishment of a comprehensive metabolite database.Based on the developed detection methods and database,the rhythmic changes in lung tissue at the metabolic level under physiological conditions were revealed.This provides an effective research foundation for further investigating the rhythmic alterations during the formation of pulmonary fibrosis.PartⅡStudy on the rhythm and metabolic changes of lung tissue in IPF miceBackground and objectivesIdiopathic pulmonary fibrosis（IPF）is a chronic progressive disease characterized by diffuse damage to alveolar epithelial cells,and effective therapeutic drugs are still lacking due to its unclear pathogenesis.It has been reported that the expression regulation of rhythm-related genes can alter the normal biological rhythm of the lungs and is closely related to the development of IPF.For instance,in tissues with pulmonary fibrosis lesions,the rhythm-regulating factor Nuclear factor erythroid2-derived 2-like 2（Nrf2）can accelerate the synthesis of glutathione by regulating the transcription and translation of antioxidant proteins,thereby enhancing antioxidant activity and playing an anti-fibrotic role.In addition,genes encoding inflammatory molecules such as chemokine ligands and tumor necrosis factor ligands exhibit diurnal rhythm oscillations in lung expression,directly or indirectly participating in pulmonary immune regulation processes.Therefore,exploring and revealing rhythmic changes in IPF lung tissue is of great significance for finding new therapeutic approaches and better preventing and controlling the disease.This study elucidates the metabolic changes in the lungs of IPF mice under conditions of circadian rhythm disruption.By employing metabolic flux analysis（MFA）,we delve deeper into the metabolic changes associated with IPF,revealing a metabolic reshaping process in which more amino acids are consumed for oxidative energy supply during the occurrence and progression of IPF.We propose that targeting the inhibition of amino acid oxidation could serve as a potential therapeutic target or adjunctive treatment for IPF.MethodsTo establish an IPF mouse model,C57BL/6J mice were intratracheally instilled with bleomycin,while the Control group received an equal volume of phosphate-buffered saline（PBS）.At the third week post-bleomycin instillation,mice underwent blood routine tests,lung function assessments,and histological evaluation of fibrotic lesions using Hematoxylin and Eosin（H&E）staining.For studying circadian changes,mice were kept under suitable temperature and humidity conditions.Zeitgeber times（ZT）were set with ZT0 as the lights-on time（9:00 am）and ZT12 as the lights-off time（9:00 pm）.Tissues（blood,lung,liver,etc.）from both the IPF and control groups were collected every 4 hours（ZT0,ZT4,ZT8,ZT12,ZT16,ZT20）.Transcriptome sequencing was performed on lung tissues,and metabolomics analysis was conducted on lung and liver tissues.Data obtained were subjected to joint statistical analysis using software such as MS-convert,EL-Maven,Metaboanalyst 5.0,and R language to reveal the metabolic rhythmic changes in lung tissue during fibrosis.Furthermore,eye orbital injection of ¹⁵NH₄Cl was performed,and lung and liver tissues were collected at 2 minutes,15 minutes,and 30 minutes post-injection.Changes in metabolite labeling were used to calculate the flux changes of urea cycle-related metabolites in lung and liver tissues.Quantitative studies on amino acid metabolic pathways were conducted using in vivo isotope tracing technology.Both IPF and Control group mice underwent jugular vein catheterization.After the incision healed well,a continuous infusion of ¹³C,¹⁵N-labeled leucine was administered for 48 hours,followed by a fasting period.Subsequently,²H-labeled leucine was injected after a 4-hour fasting period,and protein degradation rates in lung tissue were calculated by multiplying the ratio of ¹³C-leucine to ²H-leucine by the infusion rate.Mice with well-healed jugular vein catheterization in both IPF and Control groups received a continuous infusion of ¹³C,¹⁵N-labeled leucine for 2.5 hours and 4 hours while fasting.Blood,lung,and liver tissues were collected for metabolomics analysis.Protein synthesis rates were calculated based on the measured labeling fraction,tissue dry weight,protein content,and the percentage of branched-chain amino acids in proteins.We get the result according to the formula about protein synthesis rate=^{tissue dry weight×protein content×Leu percentage×slope}.The direct Leu molecular weight×Leu serum enrichment contribution of amino acids to the TCA cycle function in lung tissue was calculated based on the enrichment of TCA cycle intermediates（succinate,fumarate,andα-KG）and¹³C,¹⁵N-labeled leucine in the blood.Results1.The IPF mouse model was induced by intratracheal instillation of bleomycin,with weight monitoring indicating a significant decrease in body weight compared to the PBS group.In the IPF group,hydroxyproline levels were significantly elevated.Histopathological examination of lung tissue sections stained with H&E revealed disruption of lung tissue architecture,interstitial proliferation,and collagen deposition,indicating successful modeling of IPF.2.Transcriptomic analysis of mouse lung tissue showed significantly increased expression of genes associated with fibrosis formation and disrupted oscillation of circadian genes in IPF.Lipid-related metabolic analysis data indicated lipid accumulation and loss of original rhythmic oscillation patterns.Lyso PA and PE lost significant rhythmic oscillations in the pathological state,while PC and PG showed significant daytime elevation in IPF.Changes in energy metabolism in IPF mouse lung tissue indicated a significant increase in TCA intermediates malate,succinate,and fumarate at ZT12,with a significant phase shift in the redox index GSH/GSSG.Amino acid metabolism also showed rhythmic metabolic disruption,with no significant change in essential amino acids but significant accumulation of amino acids related to the urea cycle in IPF mouse lung tissue at ZT12.Eye orbital injection of ¹⁵NH₄Cl revealed accelerated urea cycle flux in lung tissue.3.The application of in vivo stable isotope tracing metabolic flux analysis technique quantitatively studied the amino acid metabolic pathway in IPF mice.It was discovered that in IPF mice,the rate of protein synthesis in lung tissue significantly increased,while it decreased in the liver.There was no significant change observed in the rate of protein degradation in both lung and liver tissues;however,the rate of amino acid oxidation accelerated in lung tissue.These results suggest a shift from fatty acid oxidation to amino acid oxidation for energy supply in lung tissue after fibrosis formation.Conclusion1.Using multi-omics approaches including metabolomics and transcriptomics,we investigated the changes in rhythmicity of lung tissue in wild-type and bleomycin-induced IPF mice.Several metabolic pathways associated with lipid metabolism and amino acids showed significant alterations in the lung rhythmicity of IPF mice.Accumulation of urea cycle metabolites and accelerated flux of ¹⁵N-labeled urea cycle-related metabolites were observed in the IPF lung.2.Employing stable isotope tracing technology,we quantitatively studied protein synthesis,amino acid oxidation,and protein degradation pathways in the amino acid metabolism pathway.We found increased rates of protein synthesis and amino acid oxidation in lung tissue of IPF mice,suggesting enhanced urea cycle activity for ammonia clearance and a shift from fatty acid oxidation to amino acid oxidation for energy generation in lung tissue to maintain energy homeostasis.Targeting amino acid metabolism may provide insight into delaying fibrosis progression.PartⅢ Exploring the effects of protein restriction diet on IPF miceBackground and objectivesIncreasing evidence suggests that disturbances in energy metabolism in lung tissue drive pathological changes in the respiratory system.Amino acids involved in collagen synthesis,including glycine,serine,proline,and arginine,are significantly elevated in IPF lung tissue,indicating the importance of amino acid metabolism in IPF.Dietary and nutritional therapy is a method of treating diseases or improving health conditions by adjusting diet and nutrient intake.Numerous animal studies have demonstrated that sustained low-sugar diets,intermittent fasting,and ketogenic diets can reduce tumor incidence and delay tumor progression through various mechanisms.However,there has been limited research on dietary therapy in the treatment of IPF.In order to investigate the effects of amino acid intake on the occurrence and development of IPF,this study subjected IPF mice to a protein-restricted diet,aiming to explore the possibility of anti-fibrotic therapy by adjusting diet,reducing amino acid intake,and lowering oxidative energy rates.MethodsEight-week-old C57BL/6J mice were divided into two groups:one group received a protein-restricted diet（LP）containing 5%protein content,and the other group received a standard normal diet（NP）containing 20%protein content.After a brief two-day adaptation period,bleomycin was intratracheally instilled to induce IPF mouse models.The mice were weighed daily,and on the 21st day,lung tissues were collected for analysis.Blood routine tests and lung function tests were performed on the mice,and H&E staining and collagen III immunohistochemistry staining were conducted to evaluate collagen deposition in IPF mouse lungs.Additionally,fresh lung and liver tissues were collected,and the weight of lung tissues was assessed after drying at 65°C for 24 hours to evaluate fibrosis.Metabolomics analysis was performed on the collected lung tissues.The obtained data underwent statistical analysis using software such as MS-convert,EL-Maven,Metaboanalyst 5.0,and R Studio.Through correlation analysis between metabolites and lung function,metabolites with strong correlations were screened to determine if they could serve as potential therapeutic targets for further exploration of mechanisms and evaluation of efficacy.ResultsThrough providing protein-restricted diet to IPF mice,although weight monitoring of protein-restricted diet IPF mice（LI group）showed a significant decrease in body weight,lung function in LI group improved compared to the control diet IPF mice（CI group）.Immunohistochemical staining for collagen III deposition indicated a reduction in LI group lung tissue compared to an increase in CI group.Additionally,certain metabolites showed a strong correlation with lung function,such as serine,α-KG,and S1P,exhibiting opposite trends between the LI and CI groups.This suggests that protein restriction diet may impact lung function and collagen deposition in IPF mice,closely correlated with specific metabolic changes.ConclusionBy providing a low protein diet to bleomycin-induced IPF mice,it was observed that the lung function and collagen deposition in IPF mice receiving the low protein diet improved compared to those on a standard normal diet.Adjusting protein intake through dietary modification to reduce collagen deposition may have a supportive role in anti-fibrotic therapy.