| Background and Aims:Rheumatoid arthritis (RA) is an autoimmune disease which exhibits complicate pathogenic conditions, is difficult to cure and has extremely high disability rate. As the cause of this disease is not clear yet, it is often treated mainly with symptomatic therapy in Western medicine; the treatment concept of "single target" used in Western medicine seems straitened in the face of complex pathological mechanisms. Traditional Chinese medicine (TCM) has certain curative effect on RA, and its advantage lies in the method of multi-target intervention of the disease. Bizhongxiao decoction (BZXD), a compound prescription summarized by Professor Liang Qinghua from our department based on many years’of clinical experience, has achieved a good effect in treatment of RA. The treatment effect of a Chinese herbal compound prescription is the result of combined action of multiple ingredients; different active ingredients act on a single target or multiple targets. However, since the effective ingredients of TCM are complicated, modern researches on TCM often start from its chemical ingredients. Oldenlandia diffusa (OD) is the monarch drug in BZXD prescription; we attempted to unveil the action mechanism of TCM starting from this drug. In this experiment, the ultra performance liquid chromatography (UPLC) was used to determine the contents of multiple active ingredients in OD, and absorbable ingredients in the plasma of rats after intragastric administration were detected; the absorbable ingredients detected in the plasma were used to intervene against the rat collagen-induced arthritis (CIA) model, and the intervention results were observed. After demonstrating that the active ingredients in OD could inhibit the inflammatory responses of rats with CIA, the method of quantitative proteomics was used to study the change of protein expression in the synovial tissues of rats with CIA, and the signal transduction network graph was drawn to find out the major nodes. Through investigation of the effects of these nodes on change of protein expression in the synovial tissues of rats with CIA, we attempted to find out part of the pathogenesis of RA and also to lay the foundation for explanation of the mechanism in which the active ingredients in OD inhibit CIA in rats.Methods:1. Quantitative analysis of active ingredients in OD decoction and qualitative analysis of absorbable ingredients in the plasma of rats intragastrically administrated with OD decoctionThe UPLC-PDA method was adopted to determine the contents of active ingredients in OD decoction. Meanwhile, the standard samples of active ingredients were detected, and standard curves were drawn; the precision, recovery rate, sample stability and repeatability were verified. The UPLC-PDA method was adopted to determine the contents of active ingredients in OD decoction The normal SD rats were intragastrically administered with OD decoction;30min later, plasma was taken from them; the UPLC-PDA method was used for qualitative detection of the absorbable active ingredients in rat plasma.2. Study on the anti-inflammatory effect of BZX and its active ingredient ferulic acid in the process of intervention against CIA in ratsDifferent doses of standard solution of ferulic acid were used to intervene against CIA in rats; the ELISA method was adopted to detect the serum levels of inflammatory cytokines IL-1(3and TNF-a, and dose-response curves were obtained so that the optimum dose of ferulic acid for intervention against CIA in rats was determined. The SD rats were injected with bovine collagen type II emulsified in complete Freund’s adjuvant at multiple points to establish the rat CIA model. The rats were intragastrically administered with the standard solution of ferulic acid at the above optimum dose to intervene against CIA in rats; dynamic observation was carried out on the general conditions, weight gain, arthritis index score, degree of joint swelling, X-ray films of joints and pathological changes in synovial tissues of joints in rats; also, the levels of IL-1β and TNF-a in each group of rats were detected by means of ELISA. The anti-inflammatory effect of ferulic acid on rats with CIA was observed by the above method.3. Quantitative proteomics study of BZX and its active ingredient FA in intervention against CIA in ratsSynovial tissues of rats in each group were extracted on the28th and42nd days, respectively; total protein was extracted, which further underwent enzymatic digestion; different iTRAQ reagents were respectively used for labeling, and the labeling results were detected. The proteins were went through SCX/RPLC separation; the LC-MS/MS technique was used to identify differentially expressed proteins; Ingenuity Pathway Analysis (IPA) software was utilized to analyze the bioinformation of the differential proteins, including biological function, canonical pathway, drawing protein interaction network graph, etc., thus to select the target proteins related to the pathological mechanism of RA. In the meantime, the western blot (WB) and the immunohistochemistry techniques were used to verify the quantitative and qualitative accuracy of the target proteins. Last, through retrieval of relevant literature and materials, further analyses were conducted on the function and significance of these target proteins as the targets in the process of intervention against CIA in rats based on the anti-inflammatory effect of ferulic acid. Results:1. Quantitative analysis of active ingredients in OD decoction and qualitative analysis of absorbable ingredients in the plasma of rats intragastrically administrated with OD decoction With the UPLC-PDA method, it was observed that the active ingredients p-coumaric acid and ferulic acid in OD decoction achieved good separation in the chromatogram at the time of10.2min, with no interfering peaks. These two ingredients exhibited good linear regression (R2>0.9998,0.9994) within the detection range; their standard samples showed good within-day and day-to-day precision; the relative standard deviation (RSD) fluctuated within the range of1.03-3.35%. P-coumaric acid and ferulic acid exhibited good stability and repeatability in OD decoction, and the RSD fluctuated within the range of1.53-2.41%. Meanwhile, both ingredients achieved good sample recovery rate in the decoction; the average sample recovery rate was97.9%-102.8%, and the RSD fluctuated within the range of1.26-2.41%. After detection by the UPLC-PDA method, it was obtained that the yields of p-coumaric acid and ferulic acid in OD were respectively0.527and0.231mg (effective ingredient)/g (crude drug). After detection by the UPLC-PDA method, it was observed that the active ingredients p-coumaric acid and ferulic acid were found in OD decoction. Through study of the characteristics of main chromatographic peaks of p-coumaric acid and ferulic acid, these two ingredients were successfully detected in the plasma30min after the normal SD rats were intragastrically administered with OD decoction.2. Study on the anti-inflammatory effect of BZX and its active ingredient ferulic acid in the process of intervention against CIA in ratsPrecious research findings pose no strong evidence to support the effect of p-coumaric acid in treatment of RA, while there is plenty of literature showing the anti-inflammatory effect of ferulic acid in inhibition of RA. Therefore, ferulic acid was chosen as the intervention means to investigate its anti-inflammatory effect in this research. Through the experiment, it was found that the standard solution of ferulic acid at dose3(1.28mg/kg-d) achieved a good effect in down-regulating the serum levels of IL-1β and TNF-α in rats with CIA, and this dose was the optimum dose in this experiment which made use of the anti-inflammatory effect of ferulic acid to interfere against CIA in rats. The SD rats were injected with bovine collagen type Ⅱ emulsified in complete Freund’s adjuvant at multiple points to successful copy the rat CIA model. On the14th day, the model was evaluated; it was found that the success rate reached90%. After the above optimum dose was used to interfere against CIA in rats, it was observed that the rats in the normal group were active and ate normally with lustrous and soft hair. After the14th day, the rats in the model group showed such symptoms as listlessness, inactivity, somnolence, sluggishness, less diet and redness and swelling of joints compared with the normal rats; after the28th day, the weight gain of rats with CIA was significantly lower than the normal rats, and some of the rats had swelling and deformation of joints, obvious tenderness, movement disorder or even stiffness or paralysis of both lower limbs. Compared with the rats in the model group, on the42nd day, those in the intervention group were relatively active with no obvious diet change; their arthritis index and paw swelling were significantly improved (P<0.05) and their average weight slightly increased compared with the rats in the model group. Besides, pathological observation of the joint tissues showed that inflammatory cell infiltration was obvious in the joint tissues of rats with CIA and a large number of pannuses were formed compared with the normal rats. As to the rats in the intervention group, their inflammation was gradually relieved over time, and the degree of inflammatory cell infiltration was lower than before. From the X-ray films of joints, it was observed that the rats with CIA showed obvious swelling of soft tissues around the joints, and the ankle joint spaces were vaguer and narrower than those in the normal group. Compared with the rats in the model group, those in the intervention group exhibited lower degree of swelling of soft tissues around the joints, and the pathological change of joint space was improved. The ELISA method was used to detect the serum levels of IL-1β and TNF-α in each group of rats on the28th and42nd days, respectively; it was found that the levels of inflammatory cytokines in the model group increased significantly compared with the normal group (P<0.05), and the average levels of IL-1β and TNF-α in the intervention group dropped compared with the model group (P<0.05).3. Quantitative proteomics study of BZX and its active ingredient FA in intervention against CIA in ratsThrough analysis with IPA software, it was found that there were145differentially expressed proteins in the synovial tissues of rats in the model group, of which,54proteins were down-regulated and89proteins up-regulated both on the28th and42nd days of the model compared with the normal group. On the28th and42nd days after intervention with ferulic acid, the expression of10proteins could be reversed, including6proteins with down-regulated expression and4proteins with up-regulated expression. On the28th and42nd days after intervention with OD, the expression of10proteins could be reversed, including7proteins with down-regulated expression and3proteins with up-regulated expression. Through analysis of the pathways of these proteins,32RA-related cell signaling pathways were found in the ferulic acid group, and57RA-related cell signaling pathways in the OD group; meanwhile, the protein interaction network graph was drawn. Conclusions:1. With the UPLC-PDA method, it can be successfully detected that p-coumaric acid and ferulic acid are active ingredients in OD decoction and are also ingredients that can be absorbed by rat plasma.2. BZX and its active ingredient ferulic acid have certain effect on up-regulation of the serum levels of IL-1β and TNF-α in rats with CIA, and ferulic acid may be a material basis of BZX in treatment of RA.3. The abnormal expression levels of multiple proteins and the abnormality of multiple signal transduction pathways are part of the pathogenesis of RA; after BZX and its active ingredient ferulic acid are used to intervene against CIA in rats, the expression of multiple proteins can be reversed, which will act on the key nodes in multiple signal transduction pathways and regulate the signal transduction pathways. |
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