| Objective:RA is a systemic autoimmune disease characterized by aggressive arthritis.In this study,the AA rat model was established to simulate RA disease,and metabolomics method was used to study the abnormal metabolism of AA rats.Together with other pharmacodynamics experiments,it is to reveal the therapeutic effect and mechanism of TGPH on rheumatoid arthritis,and to provide experimental basis for clinical application of TGPH.Methods:1.The adjuvant arthritis rat model was established by injecting 0.1ml Complete Freund's adjuvant?CFA?of 10mg/m L through the right plantar region.The model was successfully established in 17day after injection.TGPH was formulated into solutions of 0.058g·L-1,0.116g·L-1,0.232 g·L-1,subsequently,oral medication for 30 days.Rats'paw swelling coefficient,body weight coefficient,thymus coefficient and adrenal coefficient were detected.2.ELISA was used to detect the content of NO,CRP,TNF-?,IL-1?,IL-17 of serum of rat model.And the content of NF-?B of synovium of knee joint was detected in the same way.3.The pathological changes of rat ankle synovium were observed by HE staining4.By methods of LC-MS combined with Principal component analysis?PCA?,Partial least squares discriminant analysis?PLS-DA?,metabolomics database was used to analyze the metabolic changes in serum of rats in normal group,AA model group and drug groups.It was possible to find the differential metabolites in serum of AA rats by analyzing the metabolic changes of rats in normal group and AA model group.Through analyzing the metabolic changes of rats in AA model group and TGPH groups,the therapeutic effect of TGPH on AA rats was observed in search for differential metabolites that can be used to evaluate the efficacy of TGPH.Results1.In drug treatment for 10 d,20 d,30 d,the model group compared with normal group,rats paw swelling coefficient increased significantly?P<0.01?;TGPH groups compared with the model group,rats paw swelling coefficient decreased?P<0.05?.In drug treatment for 10 d,20 d,the model group compared with normal group,the body weight coefficient of rats decreased significantly?P<0.01?;TGPH groups compared with the model group,the body weight coefficient of rats was an upward trend?P>0.05?.In drug treatment for 30 d,the model group compared with normal group and TGPH groups compared with the model group,there were no significant changes in thymus coefficient and adrenal coefficient?P>0.05?.In drug treatment for30 d,the model group compared with normal group,the spleen coefficient of rats increased significantly?P<0.01?;TGPH groups compared with the model group,there was a decreasing trend of spleen coefficient?P>0.05?.2.In drug treatment for 30 d,compared with normal group,the content of NO,CRP,IL-1beta,TNF-a,IL-17,NF-kappa B in rats of model group increased significantly?P<0.01?;compared with model group,the levels of NO,CRP,IL-1,TNF-a,IL-17,NF-kappa B in rats of TGPH groups decreased in different degree?p<0.05?.3.In drug treatment for 30 d,compared with normal group,the synovial tissue of the model group showed the proliferation of synovial cells,macrophages,fibrous tissue hyperplasia,and the infiltration of a large number of inflammatory cell infiltration;Compared with model group,TGPH groups showed reduction in the proliferation of synovial cells,macrophages,fibroblasts and inflammatory cells infiltration in different degrees.4.Metabonomics study showed that metabolic expression in serum of rats has changed significantly in the normal group,AA model group and the drug groups.Firstly,compared with metabolites in the serum of the normal group rats,27 different metabolites were identified in the serum of AA model group.These differential metabolites included Urea,Tryptophan,Pyroglutamic acid,Proline,Phosphocholine,Indoleacrylic acid,Indole,Histidine,Glutamin,Arginine,Succinic acid semialdehyde,Serine,Purine,Oleic Acid,Malic acid,Linolenic Acid,Glycine,3-Formylpyruvic acid,Creatine,5?S?-HpETE,PS?14:0/12:0?,PS?21:0/0:0?,PS?20:0/0:0?,PS?19:0/0:0?,PG?17:0/0:0?,PG?12:0/12:0?,PC?19:3?10Z,13Z,16Z?/0:0?.The metabolic pathways involved including Arginine and Proline metabolism,Glycine,Serine and Threonine metabolism,Alanine,Aspartic acid and Glutamic acid metabolism,Phenylalanine,Tyrosine and Tryptophan biosynthesis,Tryptophan metabolism,Purine metabolism,Glutathione metabolism.Secondly,through the PCA score plot analysis,TGPH had a therapeutic effect on AA rats.Compared with metabolites in the serum of AA model group rats,the 15 metabolites were differentially returned in the serum of the TGPH groups rats by tracking the 27 differential metabolites in serum of AA rats.Callback metabolites included urea,tryptophan,proline,choline phosphate,indole acrylic acid,indole,histidine,glutamine,arginine,serine,malic acid,creatine,linolenic acid,PS?14:0/12:0?,PS?21:0/0:0?.Conclusions:The TGPH showed therapeutic effect on AA rats.The therapeutic effect may be related to the anti-inflammatory effect of the drug through the influence of inflammatory factors in the body and the joint synovial NF-kappa B content.Metabolomics results showed that there were 27 different metabolites in AA serum,which significantly affected amino acid metabolism,purine metabolism and glutathione metabolism.15 of the 27 differential metabolites were returned in the serum of the TGPH group rats.Tryptophan,Indole,Arginine,Proline,Serine,and Glutamine can be used as potential biomarkers for clinical RA disease.The results showed that the therapeutic effect of TGPH on RA disease was associated with inflammatory response,NF-?B signaling pathway,TCA cycle,tryptophan metabolism,arginine and proline metabolism,histidine And serine and glycine metabolism. |
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