Metabolites Of Local Microenvironment In Acupoint Sensitization Of Knee Osteoarthritis Based On Metabonomics

Objective:Knee osteoarthritis(KOA),a common disease in acupoint sensitization research,was selected as the research object,and the local temperature and mechanical pain threshold of acupoint were selected as the important indicators of acupoint sensitivity,and the local microenvironment metabolomics of acupoint was used as the starting point to carry out the research.To construct a small molecule metabolic map of the sensitized acupoint microenvironment in patients with knee osteoarthritis.Screening the differential small molecule metabolites in the sensitized acupoint microenvironment of KOA patients.In order to explore the substance basis of acupoint sensitization from the perspective of metabonomics,the important signal substance groups of acupoint sensitization microenvironment of KOA patients were explored.The relationship between acupoint sensitization and acupuncture effect was discussed from the perspective of small molecular metabolites.Method:(1)Screening of sensitized acupoints in patients with knee osteoarthritis:KOA patients(KOA group,referred to as group K)were selected as the research object,healthy volunteers(Control group,referred to as group C)were selected as the Control,acupoints ST36,ST35,ST34,SP9,SP10,EX-LE2,EX-LE4,LR,GB34 were selected as the screening range.Fotric 200 infrared thermal imager was used for infrared thermal imaging of the above acupoints of the two groups of subjects.Fotric Analyz IR image analysis software was used to analyze and extract the local temperature of acupoints,and the temperature difference between the two groups was used to calculate the local temperature change rate of acupoints.Then Life Science pain meter was used to detect the mechanical pain threshold of the above acupoints,and the change rate of local mechanical pain threshold of the acupoints was calculated by the difference of the mechanical pain threshold of the two groups.Sensitized acupoints were screened by local temperature change rate and mechanical pain threshold change rate of acupoints,and the correlation between local mechanical pain threshold change rate and temperature change rate of acupoints was compared and analyzed.(2)Metabolic study on the microenvironment of acupoint sensitization in patients with knee osteoarthritis:1)Untargeted metabolomics studiesBased on the screening results of sensitized acupoints in patients with knee osteoarthritis in the early stage,human tissue microdialysis probe,human tissue microdialysis buffer and human tissue microdialysis instrument were used to collect local tissue dialysis fluid from sensitized acupoints in group K and the same acupoints in group C at a rate of 0.3ul/min.High performance liquid tandem mass spectrometry(LC-MS/MS)was used to screen the differently-expressed small molecule metabolites in microdialysate of acupoint local tissues of PATIENTS with KOA.The QE HF-X mass spectrometry tandem Vanquish UHPLC system was used.The Accucore HILIC column was selected as the column.ESI+ and ESI-modes were used for mass spectrometry ionization,and data were compared and identified by Mz Cloud database.With threshold VIP >1.0,difference multiple FC >1.2 or FC <0.833 and P value<0.05 was the standard for screening differential metabolites.Bioinformatics methods were used to analyze the KEGG metabolic pathway of differentially expressed metabolomics,so as to explore the important signaling substances in the microenvironment of acupoint sensitization in KOA patients and analyze their biological functions.2)ATP and Histamine mass spectrometry verification of local microenvironment of sensitized acupointsValidation of ATP and histamine targeted metabolomics in the local microenvironment of sensitized acupoints.Qualitative mass spectrometry was performed in multiple reaction monitoring(MRM)mode using Agilent 6460 triple quadrupole mass spectrometer equipped with AJS-ESI ion source.Agilent Mass Hunter Work Station Software(B.08.00,Agilent Technologies)was used to complete all Mass spectrometry data collection and quantitative analysis of target compounds.(3)The relationship between acupoint sensitization and acupuncture effect based on metabonomics:KOA patients were treated with electroacupuncture acupoint sensitization 5times a week for 4 consecutive weeks.After the treatment,the mechanical pain threshold and infrared thermal imaging temperature of local acupoints were measured again.Furthermore,microdialysis samples of local tissue fluid at the acupoint were taken again,and liquid tandem mass spectrometry(LC-MS)was used to analyze the hydrophilic metabolites and lipidomic metabolites of three groups: group K,group C and group AK(KOA patients after acupuncture).The hydrophic metabolites were separated by a Waters ACQUITY UPLC tandem Xevo G2-XS QTOF mass spectrometry system.Reversed-phase chromatography(RP)mode was selected for chromatographic separation.ESI+ and ESI-modes were used for mass spectrometry ionization.The Shimadzu Nexera UHPLC X2 LC-30 AD chromatographic series SCIEX QTRAP 6500+ mass spectrometry system was used for lipidomics.The chromatographic separation mode was combined with reverse phase chromatography(RP)and hydrophilic interaction chromatography(HILIC).ESI+ and ESI-mass spectrometry ionization modes were used for determination,and multiple response monitoring(MRM)mode was used for data collection.Lipid Search software(V4)was used for lipid annotation and calibration,and the differences of lipid metabolites between groups were compared by statistical analysis.Results:(1)Screening results of sensitization points in patients with knee osteoarthritis:A total of 22 KOA patients(group K)and 18 healthy volunteers(group C)were included.Patients in group K showed decreased mechanical pain threshold and temperature sensitivity at relevant acupoints compared with patients in group C.The decrease of mechanical pain threshold was obvious in ST34,GB34,ST36,ST35 and SP9.The infrared temperature of acupoints increased obviously EX-LE4,ST34,ST35,EX-LE2 and ST36.Combined with the correlation analysis of mechanical pain threshold,infrared temperature changes of acupoints and Pearson coefficient,Liang Qiu was identified as the sensitized acupoint for the next step of this study.(2)Metabolomics results of sensitized acupoint microenvironment in patients with knee osteoarthritis:1)Untargeted metabolomics studies482 kinds of small molecular differential metabolites were screened,38 of which were significantly different,30 under cationic mode and 8 under anionic mode.Among them,33 were up-regulated and 5 were down-regulated.According to the comparison of M-z Cloud database,8 different metabolites were completely consistent,namely,acetyl L-carnitine,L-Pyroglutamic acid,Proline,L(-)-carnitine,N-phenylacetyl glutamine,Indole-3-acetic acid,Propyl carnitine and isoquinoline.All the above 8 small molecule metabolites were significantly increased at the local liang Mound point of KOA.KEGG enrichment pathway found that Glutathione metabolic signaling pathway may be closely related to KOA nociceptive sensitization.2)Targeted metabolomics studiesThe results of targeted metabolomics of dialysis fluid in local tissues of KOA patients and healthy volunteers at sensitized acupoints showed that ATP was not detected,while histamine was 25.12 ± 0.956nmol/ L in KOA group and 24.07 ±1.724nmol/L in control group,P > 0.05,showing no statistical significance.However,the sensitization points of KOA patients showed an increasing trend.(3)Study on the relationship between acupoint sensitization and acupuncture effect based on metabonomicsA total of 23 samples of local tissue dialysate in group C(N=6),group K(N=9)and group AK(N=8)were collected for hydrophilic metabolite and lipid omics analysis:1)Acupuncture at local acupoints of knee joint can reduce WOMAC functional score in patients with mild KOA,and the mechanical pain threshold of local acupoints has an increasing trend after acupuncture.2)Hydrophilic metabolite analysis was performed on 23 samples: PCA analysis of the total samples showed that there were overlaps among the data of the three groups,but the overlaps between the C and AK groups were more obvious,and K showed a relative separation trend,suggesting that there were relatively different metabolites between the K group and the other two groups.A total of 4525 kinds of small molecule differential metabolites were identified by metabonomics,among which 123 were statistically significant,including 24 negative ions and 99 positive ions.The 123 metabolites were used to analyze the metabolic pathways,and it was found that the pathways related to glycerophospholipid metabolism and sphingolipid metabolism were closely related to the changes of local acupoint pain sensitivity.In addition,24 metabolites were found to revert after acupuncture.3)A total of 453 lipids were detected in lipid group analysis,among which 57 lipids including ceramide,sphingosine,glycerol and fatty acid showed statistical differences between group K and group C.Six lipids in AK group had significant callback compared with K group,which were Cer(D18:0/20:0),DAG(20:4/16:0),DAG(18:1/18:3),DAG(18:1/18:2),TAG54:8-FA20:4,TAG60:3-FA24:0.Conclusion:(1)There were mechanical pain sensitization in local acupoints of knee joint in KOA patients;ST34,GB34,ST36 are significant acupoints of KOA mechanical pain threshold sensitization.(2)A variety of small molecule metabolites are involved in the changes of local microenvironment after acupoint sensitization in KOA patients.Acetyl L-carnitine,Proline,Cer(D18:0/20:0),DAG(20:4/16:0,18:1/18:3,18:1/18:2)and other small molecule differential metabolites may be the key metabolites involved in acupoint sensitization of KOA patients.(3)The effect of electroacupuncture on KOA may be related to the changes of small molecule metabolites in local microenvironment of sensitized acupoints.