| Objective:The combination of microdialysis(MD),which is an in vivo sampling technique,and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS)was applied to the metabolomic study of jugular blood(JB)and synovial fluid(SF)of adjuvant arthritis(AA)rats.The established metabonomics platforms aimed to decipher the metabolic changes of AA rats and investigate the therapeutic effect of geniposide(a natural anti-inflammatory compound extracted from Gardenia jasminoides Ellis).Methods:First,the rat model of rheumatoid arthritis(RA)was established by using freund's complete adjuvant,which was verified by the second paw swelling and arthritis index.After the model was successfully established,the rats were randomly divided into the control group,AA model group,GE group and methotrexate(MTX)(positive drug)group.Pathological sections were used to detect the pathological conditions of synovial tissue in each group of rats.Serum levels of IL-6,IL-10,TNF-?,and RF in each group of rats were determined by ELISA.Second,JB and SF dialysate of rats in the GE group,control group,and AA model group were collected by using MD sampling method.Then,the UPLC-Q-TOF/MS~Eanalysis method of endogenous metabolites in the two biological samples was established to obtain the metabolic profile and the original metabonomic data.Third,combined with Progenesis QI software to perform multivariate statistical analysis on metabonomic data,differential metabolites between the GE group,control group,and AA model group were initially characterized by online databases.Moreover,these potential biomarkers were further identified by the MS/MS fragments in the high energy model.To further reveal the effect of GE on metabolic changes in AA model rats,differential metabolites of AA group and GE group and its corresponding metabolic pathways were compared and characterized.Last,to further explore the reasons for the metabolic changes in different groups,western blotting was conducted to verify the target protein of interest using the synovial tissues.Results:1.Compared with normal rats,the secondary paw swelling degree and AI levels of AA rats were significantly increased.Histological examination showed that synovial tissues of AA rats were accompanied by the synovium hyperplasia,pannus formation and infiltration of inflammatory cells.ELISA results showed that serum levels of TNF-?,IL-6,and RF in AA rats were significantly higher than that in normal rats,while IL-10 levels were significantly lower.After GE treatment,the development of AA-induced arthritis was ameliorated,which was demonstrated by the alleviated paw swelling,the decreased level of AI,the reversal of synovial histopathological changes and the secretion balance of TNF-?,IL-6,RF,and IL-10.2.MD probes were successfully implanted into the jugular vein and articular cavity of AA rats.JB and SF samples collected from each rat as well as the prepared quality control(QC)samples were analyzed by UPLC-Q-TOF/MS.The results of instrument precision showed that the relative standard deviation(RSD)of retention time is less than 1.70%and the RSD of the peak area is less than 4.20%.The results of sample stability showed that the RSD of retention time is less than 0.88%and the RSD of the peak area is less than 5.70%.3.JB metabonomics study showed that 22 differential metabolites induced by AA were screened and identified,among which 9 metabolites were detected by negative ion mode and 13 metabolites by positive ion mode.The 22 different metabolites were mainly involved in three metabolic pathways,namely,arginine and proline metabolism,histidine metabolism and glycerophospholipids metabolism.GE had altered regulation of 8 above differential metabolites,including PG(18:2(9Z,12Z)/18:0),DG(18:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)/0:0),PG(18:1(11Z)/18:1(11Z)),SM(d18:0/14:1(9Z)(OH)),L-Arginine,N-Acetyl-L-glutamic acid,4a-Carbinolamine tetrahydrobiopterin and L-Histidine.4.SF metabonomics study demonstrated that 20 differential metabolites induced by AA were screened and identified,among which 9 metabolites were detected by negative ion mode and 11 metabolites by positive ion mode.The 21 different metabolites were mainly involved in two metabolic pathways,including glycerophospholipid metabolism and sphingolipid metabolism.GE had altered regulation of 13 above differential metabolites,namely,palmitoylethanolamide(PEA),Phenylacetic acid,Cer(d18:0/22:0),PE(16:0/20:2(11Z,14Z),Glycogen,N4-Acetylaminobutanal,PC(18:1(11Z)/16:1(9Z)),3'-AMP,ADP,(R)-3-Hydroxy-Octadecanoic acid,PE(22:5(7Z,10Z,13Z,16Z,19Z)/16:0),PG(18:1(9Z)/18:1(11Z))and PA(16:0/18:1(11Z)).Western blotting results showed that the reason for the adjusted level of PEA in GE-treated rats was attributed to the down-regulated expression of N-acylethanolamine-hydrolyzing acid amidase(NAAA)in synovial tissues.Conclusion:Based on the metabonomic platform combining MD with UPLC-Q-TOF/MS,42 biomarkers induced by AA were screened and identified,which were closely associated with lipid metabolism and amino acid metabolism.GE had altered regulation of 21 biomarkers of AA,involving arginine and proline metabolism,histidine metabolism,glycerophospholipid metabolism,and sphingolipid metabolism.The present study provided a new perspective to explore the anti-inflammatory effect of GE. |
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