A Study On The Mechanism Of Treating PM2.5-induced Lung Injury By Lung-Clearing And Haze-Descending Formula Based On Metabonomics And Network Pharmacology

Background:With the rapid development of economy and society,atmospheric pollution has become an urgent problem for the whole human society.People have gradually recognized the danger of air pollution to life and health as early as the urbanization and industrialization of developed countries in the last century.Currently,with the rapid development of Chinese economics and urbanization,the atmospheric pollution occurred in the different industries of developed countries emerged in China in a compressing and coexisting manner.Its characteristics are exceeding concentration of PM2.5,long duration and wide area.PM is complex.It mainly composed of mineral dust,elemental carbon,nitrogen oxides,S02,CO and organic substances.And the most harmful organic components are polycyclic aromatic hydrocarbons and derivatives,which are difficultly removed by the mucous membrane and cilium system of the upper respiratory tract,but easily deposited into the pulmonary alveoli and lower respiratory tract.When the body cannot clean the inhaled particulate matter,the local lung injury would be caused by oxidative stress,inflammatory reaction,DNA fractured damage.The particulate matter can even influence blood circulation through the lungs,and threaten the health.It is clear that PM2.5 exposure,is associated with many health effects,including the increased hospitalization and emergency rate,the aggravated respiratory symptoms,chronic respiratory and cardiovascular diseases,the decreased lung function and premature death[3].Therefore,it is increasingly important to study the monitoring,risk evaluation and health effects of PM2.5 exposure.The biological changes that occur in the body during PM2.5 exposure is discovered through metabolomics technology to measure biomarkers.It contributes to provide biological indicators for environmental risk assessment and systematically explore the impact of PM2.5 exposure on human health effects.PM2.5 is an important part of haze.China had recognized haze in traditional Chinese medicine(TCM)theory.Haze is a tangible pathogen.It is toxic,which can invade the body through the nose and mouth.The large amount of particulate matter can also adsorb toxins and deeply stay in the lungs,and obstruct qi movement,injure lung collateral.The failure in lung transforming into qi and the block of body fluids lead to internal phlegm-fluid retention.Therefore,haze combines poisonous,dry and dampness according to TCM.The pathological base of the pathogenicity of haze is exuberance of pathogenic qi with deficiency of healthy qi,the keys of pathogenesis are toxin,fire,phlegm,stasis.At the end of the disease,it may manifest as lung vessel obstruction and pulmonary lobe flaccidity.It should be treated by the differentiation of pathogenic qi and healthy qi.Eliminating pathogen and reinforcing healthy qi in stages according to the differences of the disease state and condition.After a long period of clinical practice and observation,our department has proposed that haze is a yin and turbid pathogen,which causes disease with the characteristics of "blocking,obstructing and curbing",so the prevention and treatment of haze should focus on "ventilating,relieving and transforming".Therefore,our department has created "lung-clearing and haze-descending formula" to prevent haze based on the method of ventilating lung and eliminating turbid and Liyin decoction by Qing dynasty physician Zhang Xichun.It prevents haze by clearing lung toxin.Lung-clearing and haze-descending formula is used clinically in patients who cough associated with haze with good results.Therefore,the study intends to discuss the mechanism of the lung-clearing and haze-descending formula for PM2.5 through the combination of metabolomics technology and network pharmacology platform,and guides for further research.Purpose:1.According to the metabolomics platform of high-performance liquid chromatography-high resolution mass spectrum simultaneous technique,the study analyzes the urine metabolism spectrum before and after short-term PM2.5 exposure of healthy participants,filters differential metabolites,finishes KEGG enrichment analysis,and proves the dynamics of these differential metabolites in the short-term exposure-purification cycle by time series analysis,for observing the effects of PM2.5 on human metabolism from a dynamic perspective.2.Combining the above metabolic pathways,constructing a drug-target-disease-pathway network system,making molecular docking,and discussing the mechanism of the lung-clearing and haze-descending formula for PM2.5-induced lung injury based on network pharmacology.Methods:(1)Clinical study:①Experiments on 50 healthy participants by the own before-and-after comparison method.②When the air is clean(PM2.5≤75 ug/m3 or AQI≤1 00),retaining the morning urine of participants as a baseline sample.The same clean air is required three days before sampling to avoid interference.③When the air is poor(PM2.5>115 ug/m3 or AQI>150),participants should arrive at the experimental site in the morning on an empty stomach(8:00),wear the FreeAir for 4 hours(8:00-12:00),then remove the FreeAir for another 3 hours of natural exposure(12:00-15:00),and finally wear the FreeAir for 4 hours(15:00-19:00).Cross-exposure to contaminated air and clean air is performed through the intervention of the FreeAir.To exclude dietary interference,all participants should use Abbott Allantoin(200 g per time point=900 KJ)and Watson’s distilled water as needed on the day of the trial.(2)Methods of metabolomics studies:①Data acquisition and processing:Full-scan detecting samples by ultra-liquid chromatography analysis platform and gathering raw data and pre-processing data.②Qualitative analyzing metabolites by the Human Metabolome Database(HMDB)and METLIN database.③Filtrating differential metabolites by multivariate statistical analysis and single variable analysis combined with experimental design,and enrichment analyzing metabolic pathway by KEGG.(3)Methods of network pharmacology studies:①Analyzing the active elements and corresponding targets of lung-clearing and haze-descending formula by TCMSP platform,and gathering the disease targets of PM2.5-induced lung injury,getting the intersection targets from the intersection between drug targets and disease targets.②Constructing an active element-target network by intersection targets through Cytoscape platform.Drawing PPI network through STRING platform,and making GO enrichment analysis and KEGG pathway analysis by DAVID database.③According to the results of metabolomics studies,common metabolic pathways from two studies were filtered.Collecting active elements,targets and shared metabolic pathways from network pharmacology studies for constructing a drug-target-disease-pathway network system.④Predicting active elements and potential targets of lung-clearing and haze-descending formula and making molecular docking verification for the further research.Results:(1)Clinical study:According to the inclusion and exclusion criteria,a total of 50 non-smoking healthy participants were included in this study.Finally,42 participants(22 females and 20 males)completed this trial,8 subjects dropped out due to incomplete sample collection,and all participants had no significant discomfort during the trial.(2)Results of metabolomics studies:①The urine metabolism spectrum before and after short-term-exposure of 42 healthy participants at 5 time points confirmed by the untargeted LC-MS.It identifies that 31 differential metabolites associated with PM2.5 exposure are closely related to oxidative stress,inflammatory reaction,and genetic epigenetics.Among these differential metabolites,sphinganine,phytosphingosine,monoisobutyl phthalic acid,phthalic acid,etiocholanolone,16-dehydroprogesterone,7-methylguanine,1-methyladenosine could be used as indexes for monitoring human metabolic disturbances by outdoor transient air pollution,and biomarkers for the diagnosis of PM-related diseases.②31 metabolic pathways confirmed by KEGG enrichment analysis,five metabolic pathways were more significant(P<0.05)among of them:sphingolipid signaling pathway,sphingolipid metabolic pathway,morphine addiction pathway,alcoholism pathway,and purine metabolic pathway.(3)Result of network pharmacology studies:①159 active elements of lung-clearing and haze-descending formula and 2606 targets of active elements confirmed by TCMSP platform.2288 disease targets of PM2.5-induced lung injury confirmed by GeneCards and other databases,and got the intersection targets from the intersection between drug targets and disease targets.②Constructed an active element-target network by intersection targets throughCytoscape platform.Drawn PPI network through STRING platform,and made GO enrichment analysis and KEGG pathway analysis by DAVID database.③According to the results of metabolomics studies,13 metabolic pathways fromtwo studies were filtered.Through the above studies,active elements,targets,and shared metabolic pathways were collected for constructing a drug-target-disease-pathway network system.④It was predicted that the active elements of lung-clearing and haze-descendingformula are quercetin and beta-sitosterol,and the targets of potential function are MAPK1 and JUN.Through review of literature,beta-sitosterol could inhibit the production of CAS1 and the activation of MAPK signaling pathway by inhibiting the activation of inflammation body NLRP3 in epidermic cell and macrophage,with the result that the significant reduction of inflammatory factors,such as TNF-α,IL-1β,IL-6,IL-8,for anti-inflammatory.To sum up,it was concluded that the anti-inflammatory mechanism of the lung-clearing and haze-descending formula to prevent PM2.5-induced lung injury is closely related to the activation of beta-sitosterol to inhibit MAPK signaling pathway.Molecular docking verification suggested that the combination of elements and targets is reasonable,and lung-clearing and haze-descending formula was predicted by the effects of the above elements and targets in preventing and treating PM2.5-induced lung injury.Conclusion:(1)The results of untargeted metabolomics show that the short-term PM2.5 exposure could bring about changes in humans,and the changes are closely related to oxidative stress,inflammatory responses,and genetic epigenetics.(2)After analyzing results through untargeted metabolomics and constructing"drug-element-target-disease-signal" network,the study shows the anti-inflammatory mechanism of the lung-clearing and haze-descending formula to prevent PM2.5-induced lung injury may be closely related to the activation of beta-sitosterol to inhibit MAPK signaling pathway.