| Rheumatoid arthritis(RA)is a chronic,inflammatory,systemic autoimmune disease.The disease is widespread in the world,and the long course of disease and persistent attacks,being one of the main causes of the loss of labor force and disability in the Chinese population.The common drugs for clinical treatment of RA are non-steroidal,glucocorticoids and other anti-inflammatory drugs.But they also have varying degrees of side effects,and need require long-term use.Therefore,finding and developing effective medicines with low toxicity and side effects from traditional Chinese medicine has become a research hotspot.However,traditional Chinese medicine has the characteristics of multi-component and multi-target,which brings difficulties to the study of traditional Chinese medicine.The use of metabolomics to study the dynamic changes of the overall metabolites of biological systems provides new ideas for the study of the overall mechanism of action of traditional Chinese medicine.Our presious spectrum-effect study analgesic and anti-inflammatory of RCE have showed that the anti-inflammatory substances of RCE are a combination of multiple components.Therefore,this study used metabonomics research methods to explore the anti-inflammatory mechanism of Rubia cordifolia L..ObjectiveIn our study,the UPLC-QTOF/MS method based metabolomics study was performed to obtain the compounds in adjuvant-induced arthritis(AIA)rats.Multivariate statistical analysis was used to analyze the AIA rat serum mass spectrometry data to screen potential biomarkers and metabolic pathways associated with RA.The identification of potential biomarkers,metabolic pathways and physiological and clinical biochemical indicators will be helpful to explore the anti-inflammatory activity and underlying mechanisms of RCE in the treatment of RA.Methods1.Evaluation of anti-inflammatory activity of RCE in AIA ratsIn our study,the model of adjuvant-induced arthritis(AIA)in rats was duplicated to evaluate the anti-inflammation activity with the degree of paw swelling and the levels of lipid peroxidation and inflammatory factors in tissues,pathological and clinical biochemical indicators.2.Metabolomics analysis of the serum and urine in AIA rats after treatment with RCEThe UPLC-QTOF/MS metabolic fingerprints of serum and urine from AIA rats were established with sample pretreatment,optimizing analysis conditions,and methodological investigations.The raw data of rat serum and urine were preprocessed,with peak alignment,peak identification and normalization,and which were analyzed by PCA and OPLS-DA of SIMCA-P14.0.Score plot directly showed the distinction between different groups.First of all,the metabolism of serum and urine was compared between Con group and AIA group.Secondly,the metabolism of serum and urine at different time points and of rats at different time points were compared.After the drug intervention,the metabolism of serum and urine was observed in AIA rats,which given the trend of metabolites in AIA rats.VIP value(VIP>1)and t-test test(P<0.05)were used to found out potential biomarkers.According to the molecular weight and the ion fragment fragmentation rule of the secondary mass spectrometry compound,the structure was identified by online database retrieval such as METLIN and HMDB.Potential biomarkers and metabolic pathways were founded out by searching in databases such as KEGG Pathway,HMDB,and Metabo Analyst.In our study,we evaluated the potential anti-inflammatory of RCE for treatment of RA,and explored its potential anti-inflammatory mechanisms.Results1.Evaluation of anti-inflammatory activity of RCE in AIA ratsAfter administration,the degree of paw swelling in Dex group was significantly decreased(P<0.01).Because of its adverse reaction,the weight gain of rats was the slowest in the test.The paw swelling of rats in RCE middle and high dose groups was significantly decreased than that in AIA group(P<0.05,P<0.01),and they have better mental status.The indexes of spleen and liver in AIA group were significantly decreased(P<0.01,P<0.05).RCE could significantly increase the liver and spleen visceral index(P<0.01,P<0.001).RCE can significantly improve the AIA rat liver,spleen and joint tissue pathological damage,and the RCE high dose group has the best effect.The levels of TNF-α,PGE2 and NO of rat paw tissues in all administration groups were significantly decreased(P<0.01,P<0.01,P<0.01).RCE significantly down-regulated the expression of IL-1β,PGE2 and P65(P<0.01,P<0.001,P<0.05),and upregulated the expression of Bax protein(P<0.001).The levels of MDA and MPO of rat liver tissue in all administration groups were decreased significantly,RCE high dose group significantly increased the level of SOD(P<0.01),significantly reduced the level of NO(P<0.01).The expression of Foxp3 protein was significantly decreased in each RCE group(P<0.01,P<0.001).2.Metabolomics analysis of serum and urine in AIA rats after treatment with RCEThe optimal serum and urine preparation and UPLC-QTOF/MS analysis conditions were established.In this study,SIMCA-P14.0 software was used to perform multivariate statistical analysis on the mass spectrum data of serum and urine in AIA rats.With the increase of administration time,each administration group gradually separated from AIA group and approached to Con group.On the 21 st day of administration,RCE high,middle and low dose groups were clearly separated from AIA group,indicating that RCE can reverse the metabolic disorders of serum and urine in AIA rats.A total of 10 potential biomarkers were identified in serum,mainly related to six metabolic pathways: fatty acid metabolism,glycerophospholipid metabolism,tryptophan metabolism,terpenoid backbone biosynthesis,glucuronidation metabolism and biosynthesis of unsaturated fatty acids.A total of 7 potential biomarkers were identified in urine,mainly related to 5 metabolic pathways: phenylalanine metabolism,pyrimidine metabolism,arginine and proline metabolism,oxidative injury and tryptophan metabolism.The results of metabolic pathway analysis showed that,metabolic pathways such as terpenoid backbone biosynthesis,glycerophospholipid metabolism,tryptophan metabolism and pyrimidine metabolism had the largest impact values and were severely disturbed after inflammation.They may be considered as potential metabolic pathways for RA,suggesting that RCE may regulate the four metabolic pathways to produce therapeutic effects.ConclusionIn this study,we established the metabolic fingerprints of serum and urine in AIA rats.A total of 10 potential biomarkers were identified in serum and urine,mainly related to six metabolic pathways: fatty acid metabolism,glycerophospholipid metabolism,tryptophan metabolism,terpenoid backbone biosynthesisand,glucuronidation metabolism and biosynthesis of unsaturated fatty acids;a total of 7 potential biomarkers were identified in urine,mainly related to 5 metabolic pathways: phenylalanine metabolism,pyrimidine metabolism,arginine and proline metabolism,oxidative injury and tryptophan metabolism.The results of metabolic pathway analysis showed that,metabolic pathways such as terpenoid backbone biosynthesis,glycerophospholipid metabolism,tryptophan metabolism and pyrimidine metabolism had the largest impact values and were severely disturbed after inflammation,which may be considered as potential metabolic pathways for RA.The results suggested that RCE may regulate the four metabolic pathways to produce therapeutic effects. |
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