Metabolic Characteristics Of Patients With Phlegm-damp Constitution And Cerebral White Matter Lesions Based On Metabolomic

Objectives:1.In order to develop new ideas for the prevention and control of White Matter Degeneration of Brain(WMLs),we’ll retrospect statistical analysis was carried out on the TCM constitution and physical examination data of patients with WMLs.The distribution characteristics and related risk factors of TCM constitution in patients with WMLs were explored to provide a certain basis for the development of WMLs disease-related prevention and treatment models.2.Based on the non-targeted metabolomics with Liquid Chromatograph Mass Spectrometer(LC-MS)of serum and urine,the potential metabolites and metabolic pathways of WMLs patients with phlegm-dampness constitution were screened.Then the prognosis of WMLs patients with phlegm-dampness constitution was explored.Metabolic characteristics to provide objective evidence for clinical prevention and treatment of WMLs.3.In order to demonstrate the internal environment mechanism of WMLs patients with phlegm-dampness constitution from multiple perspectives.Select target markers with the same biological properties as metabolic mechanisms for level determination.Confirmed that the biological mechanism plays a role in WMLs patients with phlegm-dampness constitution one more time.Methods:1.Study on the TCM Constitution and influencing factors in patients with WMLs:Using a cross-sectional study method,a total of 1133 physical examiners who had undergone brain MRI and TCM Constitution identification from Januaiy 2015 to August 2021 were selected from the physical examination department of Dongfang Hospital of Beijing University of traditional Chinese medicine.Statistical analysis was carried out on the general information,WMLs patients and WMLs phlegm-dampness constitution in TCM constitution types,WMLs-related risk factors,and physical and chemical indicators of the overall population.The statistically correlated indicators were substituted into the Logistics regression equation to obtain WMLs The distribution law and risk factors of TCM constitution.2.Study on metabolic characteristics of WMLs patients with phlegm-dampness constitution:The physical examination subjects who had undergone brain MRI and TCM constitution identification in Dongfang Hospital from Jan 2021 to Nov 2021 were selected and divided into the WMLs phlegm-damp constitution group(group A),20 subjects,and the non-WMLs phlegm-damp constitution group(group B).)16 subjects,and 19 subjects in the WMLs and physique group(group C).LC-MS technology was used to conduct non-targeted metabolomics research on serum and urine samples of subjects in each group,and SIMCA-P software was used to conduct bioinformatic analysis of metabolites in each group of subjects.Including principal component analysis(PCA)and clustering heat map drawing,using Orthogonal projections to latent structures(OPLS-DA)to calculate VIP values to identify and analyze differential metabolites;and then apply MetaboAnalyst3.0 Perform hierarchical clustering analysis on metabolites and search KEGG database for metabolic pathway enrichment analysis.The differential metabolites and differential metabolic pathways of WMLs patients with phlegm-dampness constitution were obtained,and the relevant biological mechanisms were analyzed and explained,and the metabolic characteristics were summarized.3.Corrlation between oxidative stress and chronic inflammation in WMLs patients with phlegm-dampness constitution:The physical examination subjects who had undergone brain MRI and TCM constitution identification in Dongfang Hospital from Sep 2021 to Mar 2022 were selected and divided into the WMLs phlegm-damp constitution group(group A),20 subjects,and the non-WMLs phlegm-damp constitution group(group B).)19 subjects,and 22 subjects in the WMLs and physique group(group C).Enzyme-linked immunosorbent assay(Elisa)was used to detect the levels of redox marker 3-NT and chronic inflammation marker VCAM-1,which confirmed the biological mechanism of high oxidative stress and chronic inflammation in WMLs patients with phlegm-dampness constitution.Results:1.Among the 1133 medical examiners enrolled after screening,421 were in the WMLs group and 712 in the control group.The main results of the cross-sectional study are presented below:①The distribution of TCM constitutions in patients with WMLs:Phlegm-dampness>Yang-deficiency>Yin-deficiency>Qi-deficiency>Peace and Peace>Blood-stasis>Damp-heat>Qi-stagnation>Extra-endemic In addition to the distribution characteristics of other biased constitutions,male patients have more phlegm-dampness and yin-deficiency,and female patients have more phlegm-dampness and yang-deficiency.②The statistical results of the correlation between the risk factors of WMILs and physical and chemical indicators showed:age(P=0.000),hypertension(P=0.000),carotid atherosclerosis(P=0.000),waist-to-hip ratio(P=0.001),systolic blood pressure(P=0.000),homocysteine(P=0.001),lipoprotein alpha(P=0.027),alkaline phosphatase(P=0.000),β2 microglobulin(P=0.043),serum uric acid(P=0.000)=0.000),glycosylated hemoglobin(P=0.019),and there was a statistical correlation between the two groups(P<0.05).③Further substituting all the influencing factors of WMLs correlation into multivariate logistic regression analysis,the regression equation was obtained:Y=-6.918+0.057age+0.511 waist-hip ratio+0.027homocysteine+0.49β2 microglobulin-0.026peace quality+0.014 phlegm-dampness quality,indicating age(OR 1.059,95%CI 1.037～1.081),waist-to-hip ratio(OR1.667,95%CI 1.089～2.552),homocysteine(OR 1.028,95%CI 1.003～1.054),β2 microglobulin(OR 1.632,95%CI 1.215～2.191),peaceful quality(OR 0.974,95%CI 0.965～0.983),phlegm-dampness(OR 1.667,95%CI 1.089～2.552)yes Among the influencing factors of WMLs,peace and quality are protective factors,and the rest are risk factors.2.WMLs phlegm-dampness constitution group compared with non-WMLs phlegm-dampness constitution group(group A and group B):Plots of PCA and OPLS-DA scores showing significant differences between the two groups with or without WMLs.32 potential serum biomarkers related to WMLs(20 metabolites were up-regulated and 12 metabolites were down-regulated)were screened in serum samples by the combination of positive and negative ions.11 metabolites(Bilirubin,Biliverdin,Fructose Lysine,Glycosine,L-Ornithine,N-Acetyl Ornithine,L-Indolecin,Lysophosphatidylethanolamine,1-Palmitoylglycerol Choline,1-Linoleic acid glycerophosphocholine,1-stearoylglycerol choline).They were enriched in statistically significant porphyrin and chlorophyll metabolic pathways,phosphotransferase system(PTS)pathway,arginine biosynthesis pathway and glycerophospholipid metabolism pathway.Urine samples were screened for 19 potential urinary biomarkers associated with WMLs(5 metabolites were up-regulated,14 metabolites were down-regulated).2 metabolites(p-cymene,4-pyridoxine)were enriched on the xylene degradation pathway with statistical significance.Energy metabolism,glucose metabolism,arginine metabolism,glycerophospholipid metabolism,and xylene metabolism were altered in patients with WMLs,and the physiological functions of differential metabolites and pathways in serum and urine suggested that metabolic changes were related to oxidative stress.It is speculated that these metabolites and pathways lead to the occurrence of WMLs through oxidative stress processes.WMLs phlegm-damp constitution group compared with WMLs peaceful constitution group(group A and group C).Plots of PCA and OPLS-DA scores showing clear differences between the two constitutions.26 potential serum biomarkers related to phlegm-dampness constitution(18 metabolites were up-regulated and 8 metabolites were down-regulated)were screened in serum samples by the combination of positive and negative ions.12 metabolites(D-mannose,L-cystine,L-Proline,lysine,7-methylguanine,o-palmitoylcarnitine,octanoylcamitine,decanoylcarnitine,caproylcamitine,butyryl carnitine,acetylcholine,3-methyl Crotonyl glycine).Enriched to statistically significant ABC transporter pathway,amino acid metabolism pathway,fatty acid metabolism pathway and cAMP signaling pathway.Urine specimens screened out 26 potential urinary biomarkers related to phlegm-dampness constitution(6 metabolites were up-regulated,20 metabolites were down-regulated).4 metabolites(methylmalonic acid,1-methylguanine,uric acid,and arachidonic acid)were enriched in the statistically significant purine-pyrimidine metabolic pathway and arachidonic acid metabolic pathway,respectively.Compared with patients with phlegm-damp constitution,lipid metabolism,amino acid metabolism and energy metabolism have changed.Physiological functions of differential metabolites and pathways suggest that metabolic changes in patients with phlegm-dampness constitution are related to chronic inflammation and oxidative stress.They are intrinsic mechanisms in patients with phlegm-dampness constitution.3.We detected the levels of serum oxidative stress marker 3-nitrotyrosine(3-NT)and chronic inflammation marker vascular cell adhesion molecule-1(VCAM-1).Compared with the non-WMLs phlegm-damp constitution group,the level of 3-NT(P=0.005)increased in the WMLs phlegm-damp constitution group.The levels of 3-NT(P=0.007)and VCAM-1(P=0.003)increased in the WMLs phlegm-dampness constitution group compared with the WMLs peaceful constitution group.It is confirmed that the WMLs patients with phlegm-dampness constitution present the characteristics of oxidative stress and chronic inflammation,and the chronic inflammatory response may mainly come from the factors of phlegm-dampness constitution.Conclusions:1.WMLs is a chronic disease related to TCM constitution,and phlegm-damp constitution is the most common type of constitution.Logistics regression analysis showed that age,waist-to-hip ratio,homocysteine,β2-microglobulin and phlegm-damp constitution were risk factors for WMLs.It should be considered to introduce the above five items into the early screening of WMLs,and use traditional Chinese medicine and other methods to correct the controllable factors as soon as possible,so as to achieve the purpose of early prevention and treatment of WMLs.2.Compared with the non-WMLs phlegm-damp constitution group,etabolism of energy,sugar,amino acids,and lipids have occurred in the body had changed.The WMLs phlegm-damp constitution group had a significant oxidative stress response.We can pay attention to the changes in potential biological markers and differential pathways,then help people with phlegm-dampness constitution identify WMLs in early identification.Compared with the patients in the WMLs normal constitution group,the WMLs phlegm-damp constitution group showed obvious inflammation and oxidative stress and the metabolism of lipids,amino acids and energy occurred in the body.We can pay attention to Potential biological markers and differential pathways which can be used as objective indicators of phlegm-dampness constitution.Then providing ideas for the regulation and prevention of WMLs.Metabolomics research has deepened the understanding of the pathogenesis of WMLs and the characteristics of the internal environment of patients with phlegm-dampness constitution,and provided objective evidence at the micro level for clinical prevention and treatment of WMLs.3.Enzyme-linked immunosorbent assay once again proved that WMLs patients with phlegm-dampness constitution have internal environment characteristics of oxidative stress and chronic inflammation at the immune level,and the chronic inflammatory response may mainly come from factors of phlegm-dampness constitution.It is suggested that in the prevention of WMLs in patients with phlegm-dampness constitution,not only attention should be paid to the control of risk factors related to WMLs and cerebrovascular disease,but also to correct the biased phlegm-dampness constitution.This study opens up new ideas for the prevention and treatment of WMLs.