Huang-Lian-Jie-Du-Tang (HLJDT) is a traditional Chinese medicine (TCM) with anti-inflammatory activity. The present study used a metabolomic approach based on LC-Q-TOF-MS to profile rheumatoid-arthritis- (RA-) related metabolic changes and to investigate the interventional mechanisms of HLJDT in collagen-induced arthritis rats. Forty male Wistar rats were randomly divided into five groups: (1) a model group, (2) a normal control group, (3) a dexamethasone group, (4) a HLJDT group, and (5) a group that received 13 components of HLJDT. Plasma samples were collected 8, 15, and 22 days after the rats were injected with bovine type II collagen. By combining variable importance in the projection values with partial least squares discriminant analysis, 18 potential biomarkers were identified in the plasma samples. The biomarkers were primarily involved in glycerophospholipid metabolism, fatty acid metabolism, tryptophan metabolism, linoleic acid metabolism, phenylalanine metabolism, purine metabolism, arachidonic acid metabolism, and bile acid biosynthesis. Using the potential biomarkers as a screening index, the results suggest that HLJDT can potentially reverse the process of RA by partially regulating fatty acid oxidation and arachidonic acid metabolism. This study demonstrates that a metabolomic strategy is useful for identifying potential RA biomarkers and investigating the underlying mechanisms of a TCM in RA treatment.
Rheumatoid arthritis (RA) is an autoimmune disease characterized by persistent synovitis, systemic inflammation, and autoantibodies [
Metabolomics focuses on the comprehensive measurement of all small molecular weight compounds, including endogenous and exogenous species, which are present in a biological system. Furthermore, it provides a functional readout of abnormal, disease-related physiological states in the human body and may provide new insights into the global effects of disease related to metabolic pathways [
Huang-Lian-Jie-Du-Tang is an aqueous extract that consists of four herbal materials:
HPLC-grade methanol and acetonitrile were purchased from J.T. Baker (NJ, USA). Ultrapure water (18.2 MΩ) was prepared with a Milli-Q water purification system (Millipore, MA, USA). The following HLJDT components were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China): geniposide, coptisine, phellodendrine, jatrorrhizine, magnoflorine, palmatine, berberine, baicalin, chlorogenic acid, crocin, wogonoside, baicalein, and wogonin. The following standard metabolites were obtained from Sigma-Aldrich (St. Louis, MO, USA): choline, carnitine, L-phenylalanine, arachidonic acid, hippuric acid, uric acid, allantoin, 5-hydroxy tryptophan, and L-tryptophan.
Adult male Wistar rats (140–160 g) were purchased from the SLAC Laboratory Animal Co. (Shanghai, China). Rats were kept in SPF-grade Experimental Animal Houses (the Second Military Medical University, Shanghai) with free access to food and water under standard temperature conditions (22°C) and a 12 h light/dark cycle. The animal experiments were conducted in strict accordance to the National Institutes of Health’s Guide to the Care and Use of Laboratory Animals. The animal experiments were approved by the local institutional review board at the authors’ affiliated institutions.
Type II collagen (Chondrex, Redmond, WA, USA) was emulsified with incomplete Freund’s adjuvant at a 1 : 1 ratio. Rats were intradermally injected with 2 mg/kg of collagen-IFA suspension at the base of the tail (day 0). A boost injection with 1 mg/kg of the collagen-IFA suspension was given on day 7 in the same manner.
Forty rats were randomly divided into 5 groups of 8 rats each: (1) rats without CIA immunization (normal control group, NG), (2) rats with CIA immunization (CIA model group, MG), (3) CIA rats treated with 270 mg/kg of HLJDT (HLJDT group, HG), (4) CIA rats treated with the 13 main components of HLJDT (components group, CG), and (5) rats treated with 0.05 mg/kg of dexamethasone (Sine Phama Lab Co., Ltd., Shanghai, China) (positive control group, DG). A dry powder of HLJDT was dissolved in 0.5% carboxymethyl cellulose sodium (CMC-Na), stirred at 37°C for 1 h and administered orally to the CIA rats. This 270 mg/kg dose was explored in the animal experiment and is considered within the MTD (2 g/kg) for oral administration. Based on the quantitative analysis of HLJDT [
After the second immunization, the rats were checked for the development of arthritis based on the extent of edema and/or erythema in their paws. The incidence and severity of arthritis were evaluated by observing changes in their arthritis scores every 2 days, measuring hind paw volumes every 4 days and measuring body weight every 3 days (only when arthritic signs were present). The observed severity of the arthritis was assessed by a semiqualitative score as follows: 0, normal, with no macroscopic signs of arthritis or swelling; 1, mild but distinct redness and swelling of the ankle or apparent redness and swelling of the individual digits, regardless of the number of affected digits; 2, moderate redness and swelling of the ankle; 3, redness and swelling of the entire paw, including the digits; and 4, maximally inflamed limb with the involvement of multiple joints. In these studies, the maximum score was 8, which represents the sum of the scores of both hind paws in each animal. The hind paw volumes were measured with a plethysmometer (7140UGO, Basile, Comerio, Italy) and were recorded as the mean volume displacement of both hind paws in each rat. A precision balance (Sartorius AG, Goettingen, Germany) was used to monitor changes in body weight.
The plasma samples were obtained by centrifuging blood samples for 10 min at 3500 rpm and 4°C. The supernatant was used in the subsequent bioassays. Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) assays were performed using commercially available kits according to their manufacturer’s instructions (Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the lipid peroxide content was determined by measuring the concentration of thiobarbituric-acid- (TBA-) reactive substances. The TBA-reactive content was expressed in terms of MDA content using l,l,3,3-tetraethoxypropane as a standard. The absorbance was measured at 532 nm and the values were expressed as nmol of MDA per mg of protein. The SOD assay was based on SOD’s inhibitory effects on the spontaneous autoxidation of 6-hydroxydopamine. One IU of SOD is required to inhibit the initial rate of 6-hydroxydopamine autoxidation by 50%. The GSH-Px activity assay is based on measurements of decreasing absorbance at 340 nm due to the consumption of NADPH.
The plasma samples were collected from the NG, MG, DG, HG, and CG on days 8, 15, and 22. The samples were stored at −80°C prior to analysis. One-hundred-microliter aliquots of plasma were diluted with 300
LC-Q-TOF-MS analysis was performed on an Agilent-1290 LC system (Agilent Technologies, Palo Alto, CA, USA) coupled with an electrospray ionization (ESI) source and an Agilent-6530 Q-TOF mass spectrometer. Chromatographic separation was performed on a Zorbax SB-C18 column (1.8
Positive and negative ion modes were used in mass detection. The source parameters were set as follows: drying gas flow rate, 11 L/min; gas temperature, 350°C; pressure of nebulizer gas, 45 psig; Vcap, 4000 V in positive mode and 3000 V in negative mode; fragmentor, 120 V; skimmer, 45 V; and scan range,
The MS spectra were processed using Agilent’s Mass Hunter Qualitative Analysis Software (Version B.03.01, Agilent Technologies, USA) for peak detection. A list of detected peak intensities was generated using the retention time
All quantitative data were expressed as the mean ± SD as indicated. The comparisonsbetween the two groups were analyzed by an unpaired Student
Immunization with bovine type II collagen (coadministered with incomplete Freund’s adjuvant) started producing severe arthritis 10 days after primary immunization and reached a peak on day 22 in the model group (Figure
(a) Time schedule for CIA immunization, pathogenesis, peak incidence, drug administration, and sample collection for examinations. Wistar rats were immunized with bovine type II collagen with incomplete Freund’s adjuvant and randomly divided into normal control, model, dexamethasone (0.05 mg/kg), HLJDT (270 mg/kg), and its 13-component groups on the day of arthritis onset (day 0,
Immunization with bovine type II collagen caused a significant decrease in the activities of SOD (
Effects of HLJDT and its components on MDA levels and antioxidant enzymes’ activities on day 22.
Groups | MDA (nmol/mL) | SOD (U/mL)a | GSH-Px (U/mL)b |
---|---|---|---|
Normal control | 2.71 ± 0.34 | 4.56 ± 0.24 | 7.72 ± 0.71 |
Model control | 4.98 ± 0.53## | 2.95 ± 0.22## | 4.97 ± 1.56# |
Dexamethasone | 2.62 ± 0.28** | 4.23 ± 0.33** | 7.69 ± 1.38* |
HLJDT | 2.99 ± 0.38** | 4.74 ± 0.29** | 7.30 ± 1.13* |
Components of HLJDT | 2.91 ± 0.42** | 5.27 ± 0.22** | 6.82 ± 0.91* |
SOD: superoxide dismutase; GSH-Px: glutathione peroxidase. *
aOne unit of SOD activity is defined as amount of SOD when SOD inhibition ratio reaches 50% in 1 mL reaction solution.
bOne unit of GSH-Px activity is defined as amount of enzyme required to degrade 1
The repeatability and stability of the LC-Q-TOF-MS method were validated by analyzing 6 injections of identical QC samples that were prepared according to the same protocol. The relative standard deviations of the peak retention times and areas were less than 1.0% and 5.0%, respectively. Thus, the precision and repeatability of the proposed method were satisfactory for metabolomic analysis.
Fingerprints of the plasma samples were acquired in positive and negative modes. After comparing our results between both nodes, we observed higher noise, fewer peaks, and a matrix effect in the negative mode, whereas the total ionic chromatogram (TIC) of the positive mode was more suitable for analysis (Figure
Representative base peak intensity chromatogram of the rat plasma obtained in ESI negative mode (a) and ESI positive mode (b) based on LC-Q-TOF-MS.
Ions were generated in the LC-Q-TOF-MS analysis. PLS-DA, a supervised method, is frequently used to classify groups that show metabolic differences and to extract potential biomarkers. After PLS-DA processing, the CIA model group was clearly separated from the normal control group on day 22 (Figure
Results of multiple pattern recognition of plasma biomarkers between normal control group and model group on day 22. PLS-DA score plot (
The three steps to identify these biomarkers were as follows. First, the MS2 spectrum of significantly different metabolic ions was obtained using a targeted MS/MS mode. Next, several online databases, such as METLIN (
Identification of a selected biomarker (
Following the identification process, 18 unique metabolites were identified (Table
Potential biomarkers in response to RA and their metabolic pathways.
Mode | Number |
|
|
Formula | Identification | Folda |
|
Related pathway |
---|---|---|---|---|---|---|---|---|
1 | 1.16 | 104.1076 | C5H14NO | Cholineb | 26.11 | 0.000 | Glycerophospholipid metabolism | |
2 | 1.19 | 162.1129 | C7H15NO3 | Carnitineb | −0.84 | 0.000 | Fatty acid metabolism, oxidative injury | |
3 | 1.20 | 258.1108 | C8H21NO6P | Glycerophosphocholinec | 25.39 | 0.000 | Glycerophospholipid metabolism | |
4 | 1.73 | 204.1235 | C9H17NO4 | Acetylcarnitinec | 0.819 | 0.006 | Fatty acid metabolism, oxidative injury | |
5 | 3.06 | 166.0869 | C9H11NO2 | L-Phenylalanineb | 0.81 | 0.005 | Phenylalanine metabolism | |
ESI(+) | 6 | 5.07 | 180.0661 | C9H9NO3 | Hippuric acidb | 25.27 | 0.007 | Phenylalanine metabolism |
7 | 5.30 | 194.0820 | C10H11NO3 | Phenylacetylglycinec | −0.93 | 0.004 | Phenylalanine metabolism | |
8 | 10.13 | 357.2796 | C24H36O2 | DHA ethyl esterc | 3.19 | 0.002 | Alpha linolenic acid and linoleic acid metabolism | |
9 | 14.89 | 400.3427 | C23H45NO4 | Palmitoyl-L-carnitinec | 0.57 | 0.003 | Fatty acid metabolism | |
10 | 16.58 | 305.2481 | C20H32O2 | Arachidonic acidb | 0.94 | 0.002 | Arachidonic acid metabolism | |
11 | 16.79 | 271.2637 | C17H34O2 | Palmitic acid methyl esterc | 4.09 | 0.000 | Fatty acid metabolism | |
| ||||||||
12 | 1.24 | 157.0361 | C4H6N4O3 | Allantoinb | 2.13 | 0.000 | Purine metabolism, oxidative injury | |
13 | 1.74 | 167.0207 | C5H4N4O3 | Uric acidb | −1.16 | 0.004 | Purine metabolism, oxidative injury | |
14 | 2.19 | 219.0775 | C11H12N2O3 | 5-Hydroxy tryptophanb | 0.99 | 0.008 | Tryptophan metabolism | |
ESI(−) | 15 | 4.51 | 203.0831 | C11H12N2O2 | L-Tryptophanb | 1.61 | 0.003 | Tryptophan metabolism |
16 | 8.34 | 212.0025 | C8H7NO4S | Indoxyl sulfatec | 1.27 | 0.003 | Tryptophan metabolism | |
17 | 6.21 | 464.3024 | C26H43NO6 | Glycocholic acidc | −0.38 | 0.022 | Bile acid biosynthesis | |
18 | 10.14 | 391.2855 | C24H40O4 | Deoxycholic acidc | −0.44 | 0.046 | Bile acid biosynthesis |
aFold changes (calculated as log2 (average peak intensity of model group/average peak intensity of normal control group) and
bMetabolites validated with standards.
cMetabolites putatively annotated.
All the potential biomarkers in response to RA detected by cluster analysis. The columns show the expression levels and each row represents a biomarker. The red color indicates upregulated biomarkers compared with normal control group, while the green color represents downregulated biomarkers compared with normal control group. NG, normal control group; MG, model group;
Overproduction of oxidants leads to oxidative tissue damage at the molecular level. A growing number of reports have provided evidence that implicates oxidative injury as a major pathogenic mechanism in RA [
In summary, 18 potential biomarkers were identified. These markers are mainly associated with glycerophospholipid metabolism, fatty acid metabolism, tryptophan metabolism, linoleic acid metabolism, phenylalanine metabolism, purine metabolism, arachidonic acid metabolism, and bile acid biosynthesis and reveal RA regulating network
PCA, an unsupervised pattern recognition method, was used to observe trends in mean metabolite pattern changes across various time points (Figure
Dynamic PCA scores’ plots of plasma metabolites impacted by different groups from day 0 to 22. (a) HLJDT group, (b) dexamethasone group, (c) components group, and (d) model group. HG, HLJDT group; DG, dexamethasone group; CG, components group; MG, model group.
Comparison of PLS-DA scores plots of rat plasma data of different groups on days 8, 15, and 22. (a) Day 8 (
Nine metabolites were reversed by HLJDT, and 7 were reversed by its components (Table
Summary of potential biomarkers in HLJDT and its components’ groups on day 22.
Biomarkers | HLJDT | Components of HLJDT | ||
---|---|---|---|---|
Folda |
|
Foldb |
|
|
Choline | −20.24 | 0.041 | — | — |
Carnitine | 0.40 | 0.000 | 0.18 | 0.003 |
Glycerophosphocholine | −19.87 | 0.001 | −16.79 | 0.009 |
Acetylcarnitine | −0.76 | 0.003 | −0.15 | 0.002 |
L-Phenylalanine | −0.28 | 0.012 | −0.19 | 0.043 |
Hippuric acid | −20.17 | 0.004 | −18.87 | 0.006 |
Palmitoyl-L-carnitine | −0.56 | 0.002 | −0.01 | 0.022 |
Allantoin | −0.12 | 0.018 | — | — |
Arachidonic acid | −0.24 | 0.002 | −0.14 | 0.003 |
aFold changes calculated as log2 (average peak intensity of HLJDT group/average peak intensity of model group) and
bFold changes calculated as log2 (average peak intensity of components of HLJDT group/average peak intensity of model group) and
The results from a lipid peroxide assay, antioxidant enzyme activity assays, and metabolomic analysis demonstrate that HLJDT and its components have extensive effects in RA treatment by regulating the pathway disruptions associated with oxidative injury and arachidonic acid metabolism.
In this study, metabolomic analysis with LC-Q-TOF-MS was used to profile RA-related metabolic changes in the plasma and to investigate the interventional mechanisms of HLJDT and its components. After multiple levels of statistical analysis, 18 significant biomarkers (11 metabolites detected in the positive mode and 7 metabolites detected in the negative mode) were identified. These biomarkers are primarily involved in glycerophospholipid metabolism, fatty acid metabolism, tryptophan metabolism, linoleic acid metabolism, phenylalanine metabolism, purine metabolism, arachidonic acid metabolism, and bile acid biosynthesis. Potential biomarkers-related glycerophospholipid metabolism and fatty acid metabolism, namely, carnitine, acetylcarnitine, allantoin, uric acid, choline, and glycerophosphocholine, appear to have diagnostic and/or prognostic values for RA and require further investigation in clinical studies. Using the potential biomarkers identified in this study as a screening index, we hypothesize that HLJDT and its components can limit the pathological process of RA by partially reversing metabolite levels and regulating pathway disruptions. The metabolomic results presented here provide a systemic view of the development and progression of RA as well as a theoretical basis for the prevention or treatment of RA.
Rheumatoid arthritis
Collagen-induced arthritis
Liquid chromatography quadrupole time-of-flight mass spectrometry
Huang-Lian-Jie-Du-Tang formula
Normal group
Model group
Dexamethasone group
HLJDT group
Components group
Traditional Chinese medicine
Principal component analysis
Partial least squares discriminant analysis
Glutathione peroxidase
Superoxide dismutase
Malondialdehyde
Thiobarbituric acid.
The authors declare that they have no conflict of interests.
R. Yue and L. Zhao contributed equally to this paper.
Thid work was supported by NSFC (81102865, 81230090, and 30725045), partially supported by Global Research Network for Medicinal Plants (GRNMP), Shanghai Leading Academic Discipline Project (B906), FP7-PEOPLE-IRSES-2008 (TCMCANCER Project 230232), Key Laboratory of Drug Research for Special Environments, PLA, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300), the Scientific Foundation of Shanghai China (09DZ1975700, 09DZ1971500, and 10DZ1971700), National Major Project of China (2011ZX09307-002-03), and the Twelfth Five-Year National Science and Technology Support Program (2012BAI29B06).