The Study Of Key Metabolic Pathway In Acupoint-embedding Therapy Of Phlegm-dampness Overweight/Obese People Based On Transcriptomics And Metabolomics

Objective1.To explore the correlation between overweight/obesity and traditional Chinese medicine(tcm)constitution,blood pressure(BP),biochemical indexes and abdominal color doppler ultrasound(CDU);2.To assess the clinical efficacy of acupoint-embedding treatment for overweight/obese people with phlegm and dampness by body composition analysis data such as body weight,BMI,waist-hip ratio,InBody score,total body water percentage,body protein percentage,body inorganic salt percentage,body fat percentage,and extracellular water ratio.3.To search the possible signaling pathways from the changes of gene expression by transcriptomics techniques after thread-embedding therapy.4.To search the possible signaling pathways from the changes of metabolite expression by metabonomics techniques after thread-embedding therapy.5.To explore the possible signal pathways by transcriptome and metabolome cross-analysis and to provide a new idea for the treatment of overweight and obesity in the future.Methods1.Retrospective study:A total of 2329 physical examination patients from the Physical examination Center of the Third Affiliated Hospital of Guangzhou University of Chinese Medicine from 2020 to 2021 were collected to analyze the distribution characteristics of phlegm-moisture constitution in different body mass index BMI stratification,and analyze the physical examination data of 744 physical examiners with body mass index≥24 and TCM constitution identification in the physical examination items.The data included gender,age,height,weight,blood pressure,BMI,physical type,liver function,four items of blood lipid,abdominal color ultrasound(liver,bile,spleen and pancreas),and blood glucose.The collected data were statistically analyzed using SPSS 25.0.2.Clinical observation:Fifty patients with overweight/obese of phlegm-dampness constituion were selected in Health Construction Center in the Third affiliated hospital of Guangzhou University of TCM from June 2022 to November 2022.were selected.Body composition analysis,traditional Chinese medicine constitution identification score were performed,while whole blood(placed in PAXgene tube)and serum samples were taken before and after acupoint-embedding.12 times(about three months)treatment was performed.The data were analyzed by SPSS 25.0 which could evaluate the changes in weight,BMI,waist-hip ratio,phlegm-dampness ratio,physical score,InBody score,total body water ratio,body protein ratio,body inorganic salt ratio,body fat percentage,and extracellular water ratio in overweight/obese patients.3.Transcriptomics:A total of 10 whole blood samples collected in part(1)were selected and divided into pre-treatment and post-treatment groups,and transcriptome sequencing was performed using an Illumina NovaSeq 6000 sequencer with Q value<0.05 and FC2(fold change),while any set of FPKM>1(Fragments Per Kilobase of exon model per Million mapped reads)was used as a screening condition for differentially expressed genes,which were subjected to GO(gene ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis.4.Metabolomics:A total of 10 serum samples matched with part(2)were selected and divided into pre-treatment and post-treatment groups for metabolomic analysis based on liquid chromatography-mass spectrometry(LC-MS/MS)combined quadrupole Orbitrap mass spectrometer(Q Exactive Orbitrap)for metabolomic analysis,with a Variable Importance for the Projection(VIP)>1 and p value<0.05 as the screening condition for differential metabolites,followed by pathway analysis.5.Joint multi-omics analysis:Integration of transcriptomics and metabolomics data for correlation analysis and co-expression network construction for differential expression based and differential metabolites.Results1.Retrospective study(1)The proportion of phlegm-dampness patients was larger in the group with higher BMI,and the proportion of phlegm-dampness patients with BMI≥)24 increased significantly,with statistical significance.BMI was positively correlated with Alt(Alanine transaminase),triglyceride,systolic and diastolic blood pressure,and BMI was positively correlated with AST(L-Aspartic Acid enzyme),total cholesterol,there was no correlation between blood glucose(fasting).2.The clinical effectiveness of acupoint-embedding(1)After acupoint-embedding treatment,the BMI and weight were significantly reduced(P<0.05),the waist-hip ratio was significantly reduced(P<0.01).(2)After acupoint-embedding treatment,the phlegm-dampness index was significantly reduced(P<0.0001),with significant statistical differences.(3)There was no significant statistical difference in InBody score of overweight and obese people after acupoint embedding treatment.The change trends of each components of human components were different:①the basic metabolic rate of the subjects after acupoint embedding treatment was decreased without statistically different;②after acupoint embedding treatment,the body fat rate of the subjects was significantly reduced(P<0.05);③after acupoint embedding treatment,the total water,protein and extracellular water ratio of the subjects were increased;④after acupoint embedding treatment,the inorganic salt ratio in the subjects was increased(P<0.05).3.Transcriptomics After screening of 80 differentially expressed genes,including 56 up-regulated genes and 24 down-regulated genes,the main enriched signal pathways are human papillomavirus infection,bacterial dysentery,platelet activation and hematopoietic system and ECM receptor interaction signal pathway such as immune system,endocrine system,amino acid metabolism process.4.Metabolomics.The positive ion mode mainly caused the following metabolites changes:phenylalanine,tyrosine,tryptophan metabolism and chlorophyll or steroid hormones.Linoleic acid,unsaturated fatty acid,bile acid and purine metabolites in negtive ion mode are the key metabolic substances involved in acupoint embedding treatment.The results suggest that there may be a close relationship with the metabolic pathway of amino acids.5.Joint multi-omics analysis.In addition to the biological normal metabolic pathways such as protein digestion and absorption,cancer carbon metabolism,bile secretion,tiyptophan metabolism,biosynthesis of unsaturated fat,sphingolipid signaling pathway,sphingolipid metabolism,metabolism of pyrimidine metabolism,purine metabolism,glutathione metabolism,primary bile acid biosynthesis,mineral absorption,and aminoacyl-tRNA biosynthesis,as well as biological functions such as prolactin signaling pathway,necrosis,and melanogenesis.The changes in body signaling pathways caused by group analysis of overweight patients also involve Parkinson’s disease,dopaminergic synapses,insulin resistance,cocaine addiction,amphetamine addiction,and alcohol dependence.ConclusionThose who are phlegm-dampness constitution have a higher risk of overweight/obesity than other constitutions,and ALT(Alanine transaminase)in liver function,triglyceride in blood lipids,and blood pressure are risk factors for overweight obesity.There was a significant decrease in BMI,waist-to-hip ratio and the score of phlegm-dampness index after acupoint-embedding therapy in overweight/obese patients.Acupoint-embedding intervention in phlegm-dampness overweight patients mainly involves amino acid metabolism,lipid metabolism and other physiological processes,as well as the signaling pathways of small molecule compounds,involving the immune system,endocrine system and hematopoietic function.It is also related to Parkinson’s disease,dopaminergic synapses,insulin resistance,cocaine addiction,amphetamine addiction,alcoholism metabolic pathways,which provides new ideas for subsequent research.The insulin resistance signaling pathways of sphingolipid-ACKR2 and biliverdin-LDB2 will be the key pathways worth further exploring.