~1HNMR-based Metabonomic Study Of COPD Induced By Environmental Smoke Exposure In Rats

Chronic obstructive pulmonary disease(COPD)is a chronic respiratory disease,which is associated with abnormal inflammatory reactions of harmful gases or harmful particles in the lungs.The treatment effect of COPD is not optimistic,showing an upward trend year by year.At present,harmful gases are considered to be the most important risk factors for inducing COPD,especially cigarette smoke,but the underlying pathogenesis has not yet been fully elucidated.It may involve oxidative stress,protease/antiprotease imbalance,immune imbalance,and signaling pathways such as Nrf-2 and NF-?Bp65.Cigarette smoke can damage airway epithelial cells and airways.Harmful components in cigarette smoke,such as nicotine and tar,are irritating,toxic and potentially carcinogenic,and can damage various tissues and organs of the body.One third of these chemicals come directly from tobacco.Taking tobacco endogenous compounds as the starting point to study the toxicological effects of smoke,which is of great significance to reveal the pathogenic effects of smoke.Metabonomics is a systematic science to study the changes of endogenous metabolites after disturbance.Compared with genomics and proteomics,metabonomics can more intuitively study the dynamic changes of organisms after disturbance by external stimuli.Metabonomics can be used to reveal the mechanism of external factors acting on organisms.Its application in COPD has become a hot spot.The COPD rat model has been successfully constructed by smoke exposure in our laboratory.Toxicological effects of different genotypes of tobacco were found to be different by investigating inflammation injury,oxidation/antioxidant imbalance and apoptosis in COPD rats.In order to reveal the relationship between endogenous compounds of different genotypes of tobacco and their smoke toxicity.In this study,metabolomic techniques were used to analyze endogenous differential metabolites of tobacco and body fluids of COPD rats. Immunohistochemistry was used to detect the expression of NF-?Bp65 and Nrf2 in lung and extrapulmonary tissues.Furthermore,the toxicological effects of tobacco smoke of different genotypes on rats are analyzed,which is of great significance to elucidate the toxic effects of tobacco smoke and to reveal the pathogenesis of diseases.Methods: Three genotypes of tobacco(NDY-1,NDY-2 and NDY-3)were cultured in MS medium.When tobacco reaches its seedling stage,it is placed on ice for sampling.After lyophilization,take 200 mg sample for hydrogen nuclear magnetic detection.48 healthy male SD rats of clean grade were randomly divided into control group(CK group)and three model groups(NDY-1,NDY-2,NDY-3).The COPD model was constructed by exposure to smoke.Urine and plasma samples were collected at 30 and 60 days for nuclear magnetic resonance(NMR)detection.Livers,spleens,lungs and kidneys were taken to detect the expression of nuclear transcription protein NF-?B p65 and Nrf2 by immunohistochemistry.Result:1.After 60 days of tobacco culture in MS medium,pretreatment of tobacco samples for nuclear magnetic resonance detection.Eighteen compounds were identified by ~1H-NMR.Leucine,alanine,proline,glutamic acid,gamma-aminobutyric acid,choline,beta-glucose,alpha-glucose,aspartic acid,sucrose and glycine were the differential metabolites.2.Metabolites in plasma and urine samples were analyzed after exposure to smoke.Compared with the healthy control group,the differential metabolites in plasma were: ?-glucose,?-glucose,trimethylamine oxide,valine,alanine,succinic acid,O-acetylated glycoprotein,creatinine,betaine,lactose,glycine and choline.Glycine is contained in the model group.Valine and creatinine are unique to the NDY-1 group,and choline is specific to the NDY-3 group.the differential metabolites in urine were: citric acid,creatine,horse urate,ketoglutaric acid,allantoin,dimethylamine,succinic acid,alanine,pyruvic acid,taurine,dimethylglycine,betaine,thioglycolate and acetic acid.Citric acid,horse urate,allantoin and dimethylamine are contained in the model group.In addition,citric acid,creatine,ketoglutarate,and alanine are common differential metabolisms exposed to smoke for 30 days.Taurine is a common differential metabolite that occurs when smoke is exposed for 60 days.It involves glycine,serine and threonine metabolism,tricarboxylic acid cycle,valine,leucine and isoleucine biosynthesis,taurine and hypotaurine metabolism,glycolysis or gluconeogenesis,pyruvate Metabolism,glyoxylic acid and dicarboxylic acid metabolic pathways.3.Compared with the control group,the expression levels of Nrf 2 in lungs,livers,spleens and kidneys and NF-?Bp65 in livers,spleens of model groups were significantly increased after exposure to smoke(p<0.01);when exposed to smoke for 30 days,the expression levels of NF-?Bp65 in lungs of NDY-1 and NDY-2 groups had no significant difference,and at 60 days of smoke exposure,the expression levels of NF-?B p65 in kidneys increased significantly(p<0.01).The expression of NF-?Bp65 in kidneys was significant difference in NDY-3 group(p<0.01)after smoke exposure for 30 days.Conclusion: The contents of choline and glycine in NDY-1 were higher.The contents of sucrose,proline,glutamine and alanine in NDY-2 were higher.The contents of ?-glucose,and ?-glucose in NDY-3 were higher.Smoke exposure can affect the metabolism of plasma and urine in rats.Compared with healthy control group,the changes of metabolites in different model groups are different,which may be due to the different chemical components of tobacco smoke caused by endogenous substances,resulting in different biological effects.In addition,glycine in plasma and hippurate,dimethylamine and allantoin in urine can be used as potential biomarkers of COPD.The expressions of Nrf2 and NF-?B p65 in lungs and extrapulmonary tissues of rats increased significantly after exposure to smoke,suggesting that cigarette smoke can stimulate the rats and may produce inflammatory reaction and oxidative stress.