Abstract Silkworm excrement (SE), is used as a traditional antirheumatic medicine in China. The present study was designed to investigate the therapeutic efficacy of water fraction of SE (ST) and ethanol fraction of SE (CT) at two different doses on adjuvant induced arthritis (AA) rats. Arthritis severity was evaluated by body weight, paw thickness, histological changes and index of paws oedema and spleen. Serum samples were collected for estimation of biochemical indicators and cytokines. In addition, a metabonomic method based on the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) had been established to investigate the holistic efficacy of SE by serum and urine. Multivariate statistical approaches, such as partial least-squares discriminant analysis (PLS-DA) and orthogonal projection to latent structures squares-discriminant analysis (OPLS-DA) were built to evaluate the therapeutic effects of SE and find potential biomarkers and metabolic pathways. Administration with SE significantly ameliorated the AA severity, including body weight loss, paw swelling, histological changes and the levels of biochemical index. 33 endogenous metabolites had been identified (10 in serum and 23 in urine) in the AA rats. Urinary and serum metabolic profiling revealed that the metabolites underpin the metabolic pathway including nicotinate and nicotinamide metabolism; pentose and glucuronate interconversions; TCA cycle; beta-Alanine metabolism; purine metabolism and glycolysis or gluconeogenesis. The altered metabolites could be regulated closer to normal level after SE intervention. The results suggested SE possesses substantial anti-arthritic activity and demonstrated that metabonomics is a powerful tool to gain insight in the mechanism of SE formula in therapy. Keywords: silkworm excrement, rheumatoid arthritis, biomarker, metabolomics 1. Introduction Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disease that affects different parts of the body, inevitably causing pain, swelling and loss of function in joints [[34]1]. RA significantly impacts quality of life, leading to severe disability in the patient. Current conventional therapies for RA patients, including disease-modifying antirheumatic drugs (DMARDs) and biologics, are not satisfactory. To the best of our knowledge, there is no unified theory about the pathogenesis of RA. Therefore, it is necessary to find an early diagnostic marker with high sensitivity and specificity. Metabolomics is one of the newest methods focused on the association between disease and metabolic profile. Several analytical techniques have been widely used to determine the metabolites, including mass spectroscopy, ^1H-NMR spectroscopy and liquid chromatography-mass spectroscopy [[35]2]. Notably, emerging evidence indicates that RA susceptibility may be involved in the perturbation of metabolism [[36]3,[37]4]. The metabolomics approach can provide insight into the entire metabolism process and identify disparities in the metabolites and related metabolic pathways [[38]5]. Recent evidence has also demonstrated that metabolomics approach is an effective tool in characterizing the metabolic changes of RA [[39]6,[40]7]. Traditional Chinese Medicines (TCMs) have been used in China for centuries and have shown efficacy in RA treatment [[41]8]. Silkworm excrement, a classical traditional Chinese medicine, is commonly used in Chinese medicine as an anti-rheumatic drug. It was described to have the ability of “expelling wind and eliminating dampness” by Li Shizhen (during the Chinese Tang Dynasty) in his treatise “Compendium of Materia Medica” [[42]9]. The previous study also exhibited that silkworm excrement has significant anti-inflammatory effect and analgesic effect [[43]10]. In this study, we designed to investigate the therapeutic effect on adjuvant induced arthritis (AA) in rats and explain the metabolic mechanism of the anti-arthritic of SE by setting up an integrated platform of LC–Q-TOF-MS. 2. Results 2.1. Basic Physical Parameters Test During the experiment, body weights of control group significantly increased, while the model group increased slightly. Treatment with ST and CT could restore the body weights compared with the AA models, especially the CT groups ([44]Table 1). In the model group, the paw swelling degrees were remarkable more serious compared with the control group. The paw swelling degrees of rats in the groups treated with ST and CT were significantly decreased compared with the AA models ([45]Figure 1). On Day 12, the effect of CH on paw swelling degrees began to surpass the positive control group. The index of hind paw oedema and the index of spleen also exhibited an obvious recovery from model group under the treatment with CT ([46]Figure 2). The spleen is an important immune organ. The improved effect of silkworm excrement on the spleen index indicates that it has a certain degree of immunosuppressive effect and can improve the rheumatoid arthritis symptoms by inhibiting the body’s immune response. Table 1. The results of ST and CT on body weight gain in AA rats. Day 0 5 10 15 20 C 304.43 ± 13.83 343.33 ± 13.25 363.17 ± 15.08 378.50 ± 15.96 398.67 ± 24.14 M 290.57 ± 15.09 ^## 320.29 ± 13.28 ^## 341.29 ± 16.39 ^# 353.14 ± 16.20 ^# 368.00 ± 11.66 ^# Y 294.14 ± 15.26 330.14 ± 15.32 348.71 ± 15.63 364.29 ± 15.13 384.86 ± 16.60 SL 295.00 ± 18.85 332.00 ± 19.45 353.00 ± 20.22 367.11 ± 19.22 393.71 ± 20.47 * SH 295.20 ± 13.11 328.30 ± 10.12 348.00 ± 8.46 369.00 ± 9.10 388.13 ± 13.60 * CL 298.22 ± 15.20 334.14 ± 17.35 * 360.17 ± 13.98 * 369.67 ± 14.06 * 390.44 ± 14.99 * CH 296.70 ± 11.75 335.00 ± 12.38 * 358.22 ± 10.28 * 370.00 ± 13.37 * 389.20 ± 12.81 ** [47]Open in a new tab Values are presented as mean ± SD, n = 8. ** p < 0.01 compared with the AA model group. * p < 0.05 compared with the AA model group. ^## p < 0.01 compared with the control group. ^# p < 0.05 compared with the control group. Figure 1. [48]Figure 1 [49]Open in a new tab Effects of ST and CT on thickness of hind paw in AA rats. Values are presented as mean ± SD, n = 8. ** p < 0.01 compared with the AA model group; * p < 0.05 compared with the AA model group. ^### p < 0.001 compared with control group; ^## p < 0.01 compared with control group. Figure 2. [50]Figure 2 [51]Open in a new tab Effects of ST and CT on index of paws oedema and spleen in AA rats. Values are presented as mean ± SD, n = 8. ** p < 0.01 compared with the AA model group; * p < 0.05 compared with the AA model group. ^### p < 0.001 compared with the control group; ^# p < 0.05 compared with the control group. 2.2. Effect of ST and CT on Histopathological Changes in Ankle Joint The ankle joints of rats in the model group exhibited synovial hyperplasia, mononuclear cell infiltration in the surrounding tissue, cartilage erosion and joint cavity narrow compared with the normal group. The AA rats received ST, CT treatment showed slightly synovial hyperplasia, prevented the infiltration of inflammatory cells and erosion of bone, and remedied joint stenosis. The rats in high dose of CT group exhibited a remarkable reduction in all pathological damages above compared with AA rats ([52]Figure 3). Figure 3. [53]Figure 3 [54]Open in a new tab Effects of ST, CT on histopathological changes of ankle joints in AA rat (×100, HE staining). All images are from one of eight in each group. (A) vehicle control, (B) AA model, (C) positive control, (D) ST (low dose), (E) ST (high dose), (F) CT (low dose), (G) CT (high dose). 2.3. Effect of ST and CT on Biochemical Parameters and Cytokines of Serum in AA Rats A significant increase in the MDA, NO and OH· levels was observed in AA rats. Meanwhile, the concentration of SOD was significantly lower in AA rats than normal animals. All these showed a higher level of oxidative stress and a lower level of antioxidant capacity. ST and CT treatments produced a significant reduction in the serum MDA, NO and OH· levels as compared to the model group, while the SOD level was also restored ([55]Figure 4). Figure 4. [56]Figure 4 [57]Figure 4 [58]Open in a new tab Determination of MDA, NO, ·OH, SOD, AKP, ALT, IL-1β, IL-6, TNF-α and SA in serum among all groups. Values are presented as mean ± SD, n = 8. *** p < 0.001 compared with the AA model group; ** p < 0.01 compared with the AA model group; * p < 0.05 compared with the AA model group. ^### p < 0.001 compared with the control group; ^## p < 0.01 compared with the control group; ^# p < 0.05 compared with the control group. All the model animals showed an increase in serum AKP, ALT and SA levels. The treatments of ST, CT and indomethacin reverted all these indexes. The decreases of AKP, ALT and SA were significant compared with AA rats. The treatment of ST and CT produced a dose dependent reduction and were better than indomethacin ([59]Figure 4). Serum IL-6, IL-1βand TNF-α levels showed a similar effect. IL-6, IL-1βand TNF-αlevels of model group were significantly increased compared with control group. The treatment with ST, CT reverted the up-regulated levels of IL-6, IL-1βand TNF-α. While, the ST showed better effects on IL-6, IL-1βlevels ([60]Figure 4). 2.4. Metabolomics Results 2.4.1. QC Samples Analysis The relatively tight clustering of QC samples ([61]Figure 5) and relative standard deviations (RSD%) of ion intensity ([62]Table 2) demonstrated the quality of QC data. The trend plot showed the variation over all observations with respect to run order ([63]Figure 5B). Ten ions chromatographic peaks were selected to method validation. The repeatability of method was evaluated through six replicates of QC sample. From the PCA results ([64]Figure 5), it can be seen that the QC samples are tightly clustered together, indicating that the experimental results have little difference and the instrument stability is good. These results provided the repeatability and stability of the method were well. Figure 5. [65]Figure 5 [66]Open in a new tab Assessment of QC samples (A) PCA score plot (PC1 versus PC2) of test samples and QC samples; (B) Trend plot showing the variation of t[1] over all observations. QC samples were colored as red boxes and test samples were colored as black triangle. X axis numbers represented sample number (53 injections). Y axis was arbitrary. Table 2. Coefficient of variation of ion intensity of selected ions present in the QC samples covering the range of retention times. T[R_]m/z Pairs QC1 QC2 QC3 QC4 QC5 QC6 RSD% 1.58_218.1055 21.69666 22.13039 19.78364 19.24688 19.91056 20.79174 5.56 2.02_160.0429 21.94678 21.72565 19.82855 19.83975 19.87191 21.20539 4.84 3.01_336.0743 86.66117 88.21009 87.51105 88.2303 83.3309 86.05141 2.13 4.3_113.0266 94.79924 94.10457 94.61891 96.31335 94.83017 94.60155 0.79 5.99_417.1196 98.49249 98.61852 103.366 97.91074 104.4012 96.68017 3.17 7.48_357.1018 199.4248 198.0985 198.6274 190.8936 201.4087 194.3293 1.94 8.26_355.1405 126.9033 118.2539 121.2757 123.4014 127.2227 120.4814 2.93 9.59_291.1269 21.91479 21.79148 21.69425 22.98142 21.70019 22.18444 2.24 11.28_450.2643 11.66441 13.14806 12.26132 12.96975 13.07599 12.1941 4.79 13.24_681.2932 18.93239 20.19889 18.84712 18.31389 20.96083 18.7799 5.24 [67]Open in a new tab 2.4.2. Multivariate Statistical Analysis The data of serum and urine samples were analyzed by OPLS-DA and PLS-DA in both positive and negative modes. The score plots of OPLS-DA presented notable separation between control and model groups both in serum and urine metabolic profiles ([68]Figure 6(A1–A4)). R2Y and Q2of the OPLS-DA model in positive and negative modes were both above 0.75; suggesting that the OPLS-DA models presented excellent classification and prediction ability. The potential metabolic markers between control and model groups could be identified from the Splot of OPLS-DA ([69]Figure 6(B1–B4)), combining with the retention time, the standard references, precise molecular mass and MS/MS data.