Using Transcriptomics And Metabolomics To Investigate The Mechanism And Material Basis Of The Protective Effect Of Xixin-ganjiang Herb Pair On The Lungs Of Rats With COPD

Objective:To explore the effective components,key targets and action pathway of Xixin-Ganjiang（XGHP）in the treatment of COPD rats,in order to provide a theoretical basis for clinical research and treatment.Methods:（1）The active components of XGHP were detected by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry（UPLC-MS/MS）technology,and network pharmacology method and literature reports are used for component screening and prediction of related targets,and animal experiments are used to verify the expression of related proteins.（2）40 male Sprague-Dawley（SD）rats were divided into normal group（n=10）and model group（n=30）.The COPD model was replicated by cigarette smoke+tracheal injection of LPS+cold stimulation.After successful modeling,the rats were randomly divided into COPD group,XGHP group(5.4 g·kg^-1·d^-1)and aminophylline group(0.5 g·kg^-1·d^-1),with 10 rats in each group.After treatment,selecting the lung tissues of the rats and making pathological sections.Serum levels of triglyceride（TG）,total cholesterol（TC）,high density lipoprotein cholesterol（HDL-C）and low-density lipoprotein cholesterol（LDL-C）were measured in each group.（3）High-throughput sequencing-based transcriptomics was used to analyze differential genes among lung tissues of each group;（4）Gas Chromatography-Mass Spectrometry（GC-MS）was used to analyze the differential metabolites among the lung tissues of each group.（5）Integrating the transcriptomics and metabolomics data to explore the relationships between differential genes and differential metabolites to analyze the mechanism of XGHP in the treatment of COPD rats.（6）Molecular docking of the active components of XGHP with the verified core targets,predicting the mode of action between core targets and active components based on the binding fraction between ligands and receptors,and further estimating the material basis for the role of XGHP in the treatment of COPD;（7）Western Blot technology was used to verify the expression of the core proteins.Results:（1）UPLC–MS/MS detected 412 compounds in XGHP,and 30effective compounds were obtained after screening by oral bioavailability,drug-like properties and literature reports,mainly including asarinin,sesamin,quercetin and other compounds.Network pharmacology prediction and animal experiment verification showed that XGHP could effectively increase the expression of PPARG protein in lung tissue of COPD rats and reduce the expression of PTGS2 protein;（2）Histopathological results showed that in the normal group,the alveolar cavity was intact,there were fewer inflammatory cells,and the airway mucosa was intact.In the COPD group,there were alveolar fusion,alveolar septal destruction,obvious glandular secretion,submucosal goblet cell proliferation,glandular hypertrophy,and a large number of lymphocytes and neutrophils infiltration in the pulmonary interstitium and around the airway.In the XGHP group and aminophylline group,there were less losses in the tracheal epithelium mucosa,less glandular secretion,less inflammatory cell infiltration,and less goblet cell proliferation.（3）The results of serum test showed that compared with the normal group,the levels of serum TG,TC and LDL-C in COPD rats were significantly reduced（P<0.05）,and the level of HDL-C was significantly increased（P<0.05）.Compared with the model group,the levels of serum TG,TC and LDL-C in the XGHP group were significantly increased（P<0.05）,and the level of HDL-C was significantly decreased（P<0.05）;（4）In transcriptomics analysis,by comparing the differential genes among the normal group,the XGHP group,and the COPD group,386 key genes were obtained from intersection,which were mainly enriched in oxidative phosphorylation and AMPK signaling pathway;（5）By comparing the XGHP group with the COPD group in metabolomicsanalysis,8 significantly different metabolites were found,which were mainly enriched in unsaturated fatty acid biosynthesis;（6）The transcriptomics and metabolomics data were integrated,and theresults showed that the connections between differential genes and metabolites are mainly related to the FASN and SCD genes and metabolites of oleic acid,palmitic acid and linoleic acid;（7）The molecular docking results showed that FASN,SCD and AMPK were well docked with the effective compounds of XGHP,and the docking fractions were good such as L-asarinin,sesamin,formononetin,baicalin and violacein;（8）Results in Western Blot showed that XGHP could reduce the expression of p AMPK protein in the lung tissue of COPD rats and increase the expression of FASN and SCD proteins.Conclusion:（1）XGHP can protect the lung tissue of COPD and restore the blood lipid level in rats,and the material basis of its therapeutic effect may be L-asarinin,sesamin,formononetin,baicalin,and violacein;（2）XGHP may restore the expression levels of FASN,SCD and p AMPK proteins through AMPK signaling pathway,thus enhancing the biosynthesis of unsaturated acids,restoring lipid metabolism balance and reducing inflammatory reaction;（3）XGHP may be used to treat the syndrome of COPD by increasing mitochondrial oxidative phosphorylation,fatty acid oxidation and non-trembling thermogenesis.