| Objective: To quantitatively observe the change of endogenous small molecule metabolites(i.e., a variety of amino acids),which may be the potential biomarkers, in serum and urine from the rats before and after the formation of rheumatoid arthritis(RA),the rat rheumatoid arthritis model and pre-column derivation HPLC-UV method should be set up. According to the variety of the potential biomarkers, the efficacy and action mechanism of the fruit of Capparis spinosa L. on the rheumatoid arthritis rats should be studied. Methods: 1) The extracts of strongest inflammatory activity from the fruit of Capparis spinosa L. was selected according to the results of anti-inflammation test of mouse. 2) The rheumatoid arthritis rat model induced by collagen should be established,and anti-inflammatory activity of selected extracts was verified. 3) The pre-column derivation HPLC-UV method should be set up, which was used to detect the contents of amino acids in serum and urine from the rats. According to the contents analysis of amino acids in serum and urine of the rats which were from normal group, RA model group, the positive group and extracts from the fruit of Capparis spinosa L., multivariate statistical analysis, principal component analysis(PCA) and partial least squares-discriminant analysis(PLS-DA) were used to find the potential biomarkers, the efficacy and action mechanism of the fruit of Capparis spinosa L. on the rheumatoid arthritis rats were discussed. Results: 1) According to the results of metatarsal swelling in mice, there is significant difference between the water extracts from the fruit of Capparis spinosa L.(WEFC) and blank control group(p<0.05), and there is no difference between the WEFC and the positive control group(p>0.05), which shows the WEFC has the obvious effect of inhibiting mouse foot swelling. According to the results of mouse ear swelling induced by xylene, there is significant difference between the WEFC and blank control group(p<0.05), and there is no difference between the WEFC and the positive control group(p>0.05), which shows the WEFC has the obvious effect of inhibiting mouse ear swelling.So the WEFC is the extracts of strongest anti-inflammatory activity. 2) The RA rat model induced by collagen was established successfully. Compared with the model group, there is significant difference of paw thickness swelling and paw volume swelling between positive group, high dose and middle dose group of WEFC, suggesting that high dose and middle dose group of WEFC could significantly inhibit the paw swelling rat induced by bovine collagen type II. Compared with the model group, there is significant difference in contents of TNF-α and IL-1β between positive group, high dose, middle dose and low dose group of WEFC(P<0.01), suggesting that WEFC could low the level of TNF-αand IL-1β and inhibit the paw swelling rat induced by bovine collagen type II. The results of determination index of immune organs showed that enhancing the cell immunity,promoting the body’s immune organs development may be a kind of action mechanism of the WEFC and positive drug. There is a similar X ray inspection results of foot among the positive group, high dose group of WEFC and blank control group. Pathological examination results showed that the high dose group of WEFC has effect of anti-inflammation on RA rats induced by bovine collagen II, and the effect is better than that of positive group. According to the results of verification test, WEFC is the extracts of strongest anti-inflammatory activity. 3) The pre-column derivation HPLC-UV method was set up, which can be used to detect the contents of 16 kinds of amino acids in serum and was in line with the requirements of methodology. According to the contents of six kinds of amino acids( glutamine, serine, alanine, arginine, lysine and tryptophan), there were significant differences between the model group and blank group. When model rats were given WEFC, seven kinds of amino acids were call back to normal levels, which may be the biomarkers for RA model induced by bovine collagen II. The results suggest that WEFC has effect of anti-inflammation on RA rats induced by bovine collagen II. 4)The pre-column derivation HPLC-UV method was set up, which can be used to detect the contents of 13 kinds of amino acids in urine and was in line with the requirements of methodology. Compared with model group rats, the rats in the blank group before modeling, peak and LAST data, there were significant differences according to the contents of eight kinds of amino acids(glycine, glutamine, arginine, alanine, methionine,leucine and kynurenine), which may be the biomarkers for the development of RA. When model rats were given WEFC, amino acids were call back to normal levels, suggesting that WEFC has effect of anti-inflammation on RA rats induced by bovine collagen II. Theresults of calling back of glutamine, glycine and leucine are consistent with the results of serum detection. Conclusions: 1) WEFC can help model rat’s metabolomics return to the control. The result of evaluation based on metabolomics is consistent with the results of evaluation of pharmacodynamics research. 2) Metabolomics methods can be used to detect the state of amino acid metabolism in the body circulation pathway, which is an effective means to evaluate the metabolic changes of RA in rats. The paper lays a foundation for the study of mechanisms of rheumatoid arthritis and comprehensive efficacy assessment of the fruit of Capparis spinosa L. at the molecular level. |
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