NMR-based untargeted metabolomics approach to investigate the systemic lipid metabolism regulation of norisoboldine in collagen-induced arthritis rats.

Norisoboldine (NOR), an isoquinoline alkaloid, has previously been shown to ameliorate collagen-induced arthritis (CIA) by modulating the function of multiple cells such as T lymphocytes and fibroblast-like synoviocytes. To further study its anti-arthritis mechanism, the effect of NOR on the systemic metabolism regulation was investigated using an NMR-based untargeted metabolomics approach. CIA model rats were orally administered with NOR (30 mg/kg) for 14 consecutive days. The alterations of endogenous metabolites in the urine samples were quantified by 1H NMR. While NOR significantly mitigated CIA in rats as evidenced by the reduced clinical scores and histopathological changes, the results indicated that the treatment restored the levels of 22 metabolites that were significantly changed by arthritis, and most of which were related to lipid metabolism. Further studies demonstrated that NOR up-regulated the expression of carnitine palmitoyltransferase 1 (CPT-1) and down-regulated the expression of fatty acid synthase (FASN) in the spleens and the synovial tissues of CIA rats. Together these results revealed a strong association between RA and the system in metabolic disorders. The differential metabolites and their related pathways may also serve as novel therapeutic targets for RA.

Identification of the metabolites in normal and AA rat plasma, urine and feces after oral administration of Daphne genkwa flavonoids by LC-Q-TOF-MS spectrometry.

Daphne genkwa Sieb. et Zucc., as a traditional oriental herb, has been widely distributed and employed in China. The major bioactive components in D. genkwa are flavonoid compounds, which showed pharmacological activities such as anti-inflammatory, analgesic, anti-tumor and immunomodulatory activities. In this study, we analyzed total flavonoids in D. genkwa and their metabolites in normal and adjuvant arthritis (AA) rat plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). A total of 4 metabolites in plasma, 9 metabolites in urine and 15 metabolites in feces were characterized respectively by LC-Q-TOF-MS technology in normal rat. And 9 of the metabolites were observed in the AA rat urine, while there was no prototype drug or its metabolites detected in plasma and fecal samples. The metabolic pathway mainly involves hydroxylation, methylation, glucuronide, sulfate conjugation, oxidation and reduction, during the phase I and phase II biotransformation pathway. All the information gained here will be greatly helpful in elucidating the potential biological and pharmacological mechanism of flavonoid in D. genkwa, thus providing new ideas for drug development.

Application of GC/MS-Based Metabonomic Profiling in Studying the Therapeutic Effects of Aconitum carmichaeli with Ampelopsis japonica Extract on Collagen-Induced Arthritis in Rats.

Aconitum carmichaeli with Ampelopsis japonica (AA) is a classical traditional Chinese medicine (TCM) formula. There are a lot of examples showing that AA can be used to treat rheumatoid arthritis, but its mechanism of action is still not completely clear. In this research, collagen-induced arthritis (CIA) was chosen as a rheumatoid arthritis (RA) model. Rats of treated groups were continuously administered Aconitum carmichaeli (AC), Ampelopsis japonica (AJ) and Aconitum carmichaeli + Ampelopsis japonica (AA) orally once a day from the day after the onset of arthritis (day 7) until day 42. The results showed that AA not only significantly reduced paw swelling, but also improved the levels of TNF-α and IL-6 in serum. GC-MS-based urine metabonomics was established to analysis metabolic profiles and 21 biomarkers of RA rats were identified by the Partial Least Squares Discriminant Analysis (PLS-DA) and Support Vector Machine (SVM) methods. The prediction rate of the SVM method for the 21 biomarkers was 100%. Twenty of 21 biomarkers, including D-galactose, inositol and glycerol, gradually returned to normal levels after administration of AA. Metabolomic Pathway Analysis (MetPA) generated three related metabolic pathways-galactose metabolism, glycerolipid metabolism and inositol phosphate metabolism-which explain the mechanism of AA treatment of rheumatoid arthritis. This research provides a better understanding of the therapeutic effects and possible therapeutic mechanism of action of a complex TCM (AA) on rheumatoid arthritis.

Metabolomics Analysis Reveals Therapeutic Effects of α-Mangostin on Collagen-Induced Arthritis in Rats by Down-regulating Nicotinamide Phosphoribosyltransferase.

α-Mangostin (MAN) is a bioactive compound isolated from pericarp of mangosteen (Garcinia mangostana Linn.) with significant anti-rheumatic potentials. The purpose of this study was to explore the mechanisms underlying its therapeutic effects on collagen-induced arthritis (CIA) in rats with metabolomics approaches. Therapeutic effects of MAN on CIA were assessed by radiographic, histological, and immunohistochemical methods. Metabolic profiles of rats were characterized based on UPLC-MS/MS analysis of urine samples, followed by verification in HFLS-RA cells using a variety of toxicological and biochemical assays. We found that MAN treatment protected joint structures in CIA rats and caused a decrease of nicotinamide mononucleotide (NMN) in urine. The levels of nicotinamide phosphoribosyltransferase (NAMPT) were reduced in fibroblast-like synoviocytes by MAN both in vivo and in vitro, which was accompanied with a decline in nicotinamide adenine dinucleotide (NAD) production. Secretion of extracellular NAMPT (eNAMPT) in HFLS-RA cells was also decreased upon MAN treatment, which lagged behind the changes of its intracellular counterpart (iNAMPT). Co-treatment with NMN raised the secretion of eNAMPT and restored the decline of p-p65 and TNF-α induced by MAN in vitro. Sirt1 expression was down-regulated under MAN treatments too. These results suggest that MAN treatment suppressed NAD production by inhibiting iNAMPT expression, which in turn decreased eNAMPT secretion and alleviated NF-κB-mediated inflammations in CIA rats.

Metabolomic Analysis of Biochemical Changes in the Serum and Urine of Freund's Adjuvant-Induced Arthritis in Rats after Treatment with Silkworm Excrement.

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.

1H NMR-based metabolomics approach to investigate the urine samples of collagen-induced arthritis rats and the intervention of tetrandrine.

Tetrandrine is an effective ingredient isolated from the roots of a frequently used medicinal plant Stephania tetrandra S. Moore. It has been used for the management of arthritis in China, but the precise mechanism remains unclear. In the present study, a metabolomics method based on the 1H NMR was constituted to quantify the alterations of the endogenous metabolites in the urines of collagen-induced arthritis (CIA) rats treated with tetrandrine. Data showed that tetrandrine treatment could alleviate the ankle joint swelling and ameliorate histopathological changes in rats. The metabonomic analysis indicated that 23 potential biomarkers in urine were affiliated with CIA. They mainly participated in energy metabolism, amino acid metabolism, lipid metabolism and gut microbe metabolism. Moreover, our results implied that tetrandrine could reverse the pathological process of CIA through adjusting the unbalanced metabolic pathways. Thus, these metabolic pathways and potential biomarkers might be the potential therapeutic targets of tetrandrine, and these findings supplied new visions into the protective effect of tetrandrine against arthritis in rats.

Metabolomics analysis of Danggui Sini decoction on treatment of collagen-induced arthritis in rats.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by persistent joint inflammation leading to bone and cartilage damage and even disability. However, the pathogenesis of RA is multi-factorial and to a large degree, remains unknown. Danggui Sini decoction (DSD), a traditional Chinese medicine (TCM) formula, has been widely used as a remedy for rheumatoid arthritis (RA) in recent years. In our study, 1H-nuclear magnetic resonance (1H NMR) based metabolomics analysis of 7 potential biomarkers, including taurine (1), urea (2), betaine (3), pyruvate (4), hippurate (5), succinate (6) and acetone (7) was performed to investigate the progression of RA and assess the efficacy of DSD in collagen-induced arthritis (CIA) rats. According to pathway analysis using identified metabolites and correlation construction, taurine and hypotaurine metabolism, gut microbiota metabolism, pyruvate metabolism, glycolysis/gluconeogenesis, the citrate cycle (TCA cycle) and lipid metabolism were recognized as being the most influenced metabolic pathways associated with RA. As a result, deviations of metabolites 1, 3, 4, 5, 6 and 7 in CIA rats were improved by DSD, which suggested that DSD mediated the abnormal metabolic pathways synergistically. In summary, the efficacy and its underlying therapeutic mechanisms of DSD on RA were systematically investigated and expect to provide a new insight in relevant studies of other TCM formulas.

Dose-response characteristics of Clematis triterpenoid saponins and clematichinenoside AR in rheumatoid arthritis rats by liquid chromatography/mass spectrometry-based serum and urine metabolomics.

Clematidis Radix et Rhizoma is a traditional Chinese medicine widely used for treating arthritic disease. Clematis triterpenoid saponins (TS) and clematichinenoside AR (C-AR) have been considered to be responsible for its antiarthritic effects. However, the underling mechanism is still unclear because of their low bioavailability. To address of this issue, metabolomics tools were performed to determine metabolic variations associated with rheumatoid arthritis (RA) and responses to Clematis TS, C-AR and positive drug (Triptolide, TP) treatments. This metabolomics investigation of RA was conducted in collagen-induced arthritis (CIA) rats. Liquid chromatography/mass spectrometry and multivariate statistical tools were used to identify the alteration of serum and urine metabolites associated with RA and responses to drug treatment. As a result, 45 potential metabolites associated with RA were identified. After treatment, a total of 24 biomarkers were regulated to normal like levels. Among these, PC(18:0/20:4), 9,11-octadecadienoic acid, arachidonic acid, 1-methyladenosine, valine, hippuric acid and pantothenic acid etc, were reversed in Clematis TS and C-AR groups. Tetrahydrocortisol was regulated to normal levels in Clematis TS and TP groups, while 3,7,12-trihydroxycholan-24-oic acid was regulated in C-AR and TP groups. Biomarkers like citric acid, p-cresol glucuronide, creatinine, cortolone were reversed in TP group.

Urinary metabolite profiling provides potential differentiation to explore the mechanisms of adjuvant-induced arthritis in rats.

To explore the pathogenesis of rheumatoid arthritis (RA) from the perspective of metabolomics, gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) technology was used to observe changes in the metabolic profiles of urine output from rats with adjuvant-induced arthritis (AA). Sprague-Dawley rats were randomly divided into a control group and an experimental group, with eight in each. Rats in the experimental group were induced by intracutaneous innoculation of 0.1 mL Freund's complete adjuvant to right paws. On day 20 after immunization, the metabolic profiles between rat control and experimental groups were compared by combining GC-TOF/MS technology with multivariate statistical approaches, including principal component analysis, partial least squares discriminant analysis and orthogonal projections to latent structures-discriminant analysis. Nine potential biomarkers were identified, including 2,2-dimethylsuccinic acid, tartronic acid, dehydroshikimic acid, hippuric acid, adenine, phenaceturic acid, l-dopa, 1,4-dihydroxy-2-naphthoic acid and melibiose. The findings indicate that the rats with AA are disturbed in metabolism of purine, amino acid, fat and energy. This study also demonstrates that the dysfunction in a range of biosynthetic and catabolic pathways, which leads to increased oxygen free radicals and inflammation, could cause underlying pathogenesis of RA. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolomic analysis of biochemical changes in the plasma and urine of collagen-induced arthritis in rats after treatment with Huang-Lian-Jie-Du-Tang.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Lian-Jie-Du-Tang (HLJDT oren-gedoku-to in Japanese), a classical traditional Chinese medicine (TCM) formula, is well known for the treatment of inflammatory-related diseases such as gastritis, dermatitis, and ulcerative colitis. Our previous studies have indicated that HLJDT has therapeutic potential in rheumatoid arthritis treatment. To investigate the therapeutic mechanism of a traditional Chinese medicine formula Huang-Lian-Jie-Du-Tang (HLJDT oren-gedoku-to in Japanese) and its constituents combination for collagen-induced arthritis in rats using a metabolomics approach. MATERIALS AND METHODS: Rats were divided into 9 groups, and drugs were administered from on the day after the onset of arthritis (day 12) until day 31 of the experiment once daily continuously. Urine and plasma were analyzed by reversed-phase liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). Partial least-squares discriminate analysis (PLS-DA) models were built to evaluate the therapeutic effects of HLJDT and its constituents combination. 15 identified CIA biomarkers were investigated to explain its therapeutic mechanism. RESULTS: Administration of HLJDT and its constituents combination in CIA rats not only significantly reduced arthritic scores and serum levels of IL-1ß but also improved histopathologic changes in joint architecture. Urinary and plasma metabolic profiling revealed that perturbation of energy metabolism, lipid metabolism, oxidative injury and some amino acids metabolism occurred in collagen-induced arthritis (CIA). Our results also indicated that the disturbed urinary levels of succinic acid, citric acid, creatine, uridine, pantothenic acid, carnitine, phenylacetylglycine, allantoin and plasma levels of phenylpyruvic acid in model rats were gradually restored to normal after administration of HLJDT. The treatment of constituents combination of HLJDT group was able to restore to normal the disturbed urinary levels of citric acid, creatine, pantothenic acid, carnitine, pantothenic acid, phenylacetylglycine and plasma levels of uric acid, L-histidine, and l-phenylalanine in model rats. CONCLUSIONS: Our study indicates that HLJDT and its constituents combination treatment can ameliorate CIA through partially regulating the perturbed energy metabolism. Our work demonstrated that metabonomics-based approach is a promising new tool to evaluate the therapeutic effects and mechanism of complex TCM prescriptions.

Rapid-resolution liquid chromatography TOF-MS for urine metabolomic analysis of collagen-induced arthritis in rats and its applications.

AIM OF THE STUDY: Rheumatoid arthritis (RA) is a kind of autoimmune diseases characterized by persistent synovitis, systemic inflammation and autoantibodies. Huang-Lian-Jie-Du-Tang (HLJDT) is a traditional Chinese medicine (TCM) with anti-inflammatory activity. In the present study, a rapid-resolution liquid chromatography tandem time-of-flight mass spectrometry (RRLC-TOF-MS) based metabolomic study was developed to obtain a systematic view of the progression of RA and assess the efficacy of HLJDT and its components in collagen-induced arthritis (CIA) rats. MATERIALS AND METHODS: Forty male Wistar rats were randomly divided into five groups, including model group, normal control group, dexamethasone group, HLJDT group and the mixture of 13 components of HLJDT group after immunized with bovine type II collagen. Urine samples for metabolomic study were collected on 8, 15, 22 day during the animal experiment. RESULTS: The pharmacological changes (swelling paws and arthritis scores) showed that prophylactic treatment with HLJDT and its components significantly suppressed the swelling of rats' paws. By combining with partial least squares discriminant analysis, 24 potential biomarkers were identified and primarily involved in 12 metabolism pathways, such as tricarboxylic acids cycle metabolism, lipid metabolism, tryptophan metabolism and phenylalanine metabolism, which revealed a new insight into the RA network in vivo. These potential metabolites identified in CIA model need to be further investigated to prove their diagnostic and/or prognostic values for RA. Taking potential biomarkers found in the study as screening indexes, it revealed that HLJDT and its components could reverse the pathological process of RA through partly regulating the disturbed metabolic pathways. CONCLUSIONS: This study indicated that the metabolomic strategy based on RRLC-TOF-MS is a useful tool to search potential biomarkers related to RA and to dissect the underlying mechanisms of TCM in treating RA.

Prevention of the progression of adjuvant induced arthritis by oral supplementation of Indian fresh water mussel (Lamellidens marginalis) aqueous extract in experimental rats.

AIM OF THIS STUDY: Mussel is well accepted as food all over India. Beside for its nutritive value, people residing in Kosi river basin, Bihar, India, consume a preparation of soup, made from the footpad of molluscan species, with the belief that it gives relief from signs and symptoms of joint pain and related problems. This study was designed to explore the preventive activity of Indian fresh water mussel (Lamellidens marginalis) aqueous extract oral supplementation in experimental arthritis model. MATERIALS AND METHODS: Arthritis was induced in male albino rats by intradermal injection of Freund's complete adjuvant in right hind footpad. Lamellidens marginalis extract (LME1, 500 mg/kg/day and LME2, 1 g/kg/day) peroral supplementation started from the 1st day after adjuvant injection and was continued for the subsequent 13 days. Severity of arthritis was evaluated from paw diameter, ankle diameter, paw weight, urinary hydroxyproline, glucosamine level, serum interleukin-1ß, IL6, IL10, CINC1, TNFα level, lysosomal enzyme levels and from histopathological assessment. RESULTS: Lamellidens marginalis extract supplementation significantly (p<0.05) decreased paw diameter, ankle diameter, and paw weight in treated groups (LME1, 500 mg/kg/day and LME2, 1 g/kg/day) as compared with arthritic group. Urinary hydroxyproline, glucosamine level, serum IL1ß, IL6, CINC1, TNFα, IL10 and lysosomal enzyme levels were restored significantly (p<0.05) in treated groups (LME1, 500 mg/kg/day and LME2, 1 g/kg/day) as compared to arthritic group. Synovial membrane damage and neutrophil infiltration in histopathological examination was restored significantly by LME supplementation as compared to arthritic group. CONCLUSIONS: Thus, it might be concluded that experimental animals supplemented with Lamellidens marginalis extract were protected against the severity of disease progression in adjuvant induced arthritis.

Methotrexate and erythro-9-(2-hydroxynon-3-yl) adenine therapy for rat adjuvant arthritis and the effect of methotrexate on in vivo purine metabolism.

The objectives were: (1) to test the association of methotrexate (MTX) efficacy in rat adjuvant arthritis (rat AA) with interference of purine biosynthesis and adenosine metabolism and (2) to test the efficacy of erythro-9-(2-hydroxynon-3-yl) adenine (EHNA), an inhibitor of adenosine deaminase, and the efficacy of aminoimidazolecarboxamide (AICA) riboside plus MTX in rat AA. Radiographic and histologic examinations of the hind limbs were measures of efficacy. Urinary excretions of AICA and adenosine were markers of AICA ribotide transformylase inhibition (i.e., blockage of purine biosynthesis) and interference with adenosine metabolism, respectively. AICA and adenosine excretions increased during the day of MTX dosing (treatment day) compared to the previous baseline day in animals responding well to MTX (i.e., low radiographic and histologic scores). Based on radiographic and histologic scores, adjuvant injected rats were separated into two disease categories (i.e., no/mild and moderate/severe). Only AICA excretion was significantly elevated on the treatment day in rat AA with no/mild disease (i.e., those responding well to MTX therapy). AICA (not adenosine) excretion was significantly correlated with the above scores. EHNA was not efficacious, even at toxic levels, while AICA riboside potentiated the efficacy of MTX. The data suggests that efficacious MTX therapy in rat AA (1) blocks purine biosynthesis; (2) increases in in vivo AICA levels. Also adenosine accumulation and blockage of adenosine deaminase (i.e., by EHNA) appear to be less critical to MTX efficacy. Increased levels of AICA metabolites may suppress the immune response in rat AA.

Urinary collagen type II C-telopeptide fragments are sensitive markers of matrix metalloproteinase-dependent cartilage degradation in rat adjuvant-induced arthritis.

OBJECTIVE: To assess the relevance of collagen type II C-telopeptide fragments (CTX-II) as markers of cartilage degradation during adjuvant-induced arthritis in rats. METHODS: Rats were injected with Freund's adjuvant on day 0 and treated orally for 21 days twice a day with vehicle or 10 or 20 mg/kg of a newly designed matrix metalloproteinase inhibitor (MMP-Inh). Urine samples were collected for 24 h between days 19 and 20 and the concentration of the cartilage-derived CTX-II was measured with a 2-site, sandwich-type ELISA. To assess arthritis, inflammatory scores were determined, and changes in paw volumes were measured by plethysmography. RESULTS: On day 21, the inflammation was generalized in rats injected with Freund's adjuvant. The urinary concentration of CTX-II was significantly higher in arthritic rats than in control non-injected rats. Oral treatment of arthritic rats with MMP-Inh dramatically decreased the concentration of CTX-II in urine, with values returning to those of controls. Treatment simultaneously reduced the clinical variables of the disease. CONCLUSION: These results demonstrate that fragments of type II collagen in urine can be used as a measure of cartilage degradation in arthritic rats as well as potent non-invasive markers of the efficacy of chondroprotective treatments.

The effect of methotrexate and 7-hydroxymethotrexate on rat adjuvant arthritis and on urinary aminoimidazole carboxamide excretion.

OBJECTIVE: To study the efficacy, toxicity, and antifolate activities of 7-hydroxymethotrexate (7-OH-MTX) versus methotrexate (MTX) in the treatment of rat adjuvant-induced arthritis. METHODS: Dose-dependent effects in rat adjuvant arthritis were determined by histologic and clinical examinations. Antifolate activity was determined by urinary levels of aminoimidazole carboxamide (AIC) as a marker for blockade of the folate-dependent enzyme, aminoimidazolecarboxamide ribotide transformylase (AICARTase). RESULTS: MTX was 8 times more efficacious than 7-OH-MTX and resulted in higher urinary AIC levels. Increased urinary AIC levels were correlated with suppression of rat adjuvant arthritis regardless of the drug or dose level used. CONCLUSION: The ability to metabolize MTX to 7-OH-MTX and the sensitivity of AICARTase to inhibition by 7-OH-MTX may at least partially account for the variability in response to MTX. Blocking of AICARTase may be important in the efficacy of these antifolates.

Inhibitory effects of JTE-522, a novel prostaglandin H synthase-2 inhibitor, on adjuvant-induced arthritis and bone changes in rats.

OBJECTIVE AND DESIGN: To investigate the effect of JTE-522, a novel selective prostaglandin H synthase (PGHS)-2 inhibitor, on adjuvant-induced arthritis and bone changes. SUBJECTS: Male Lewis rats at 8 weeks old were immunized with heat-killed mycobacteria. TREATMENT: JTE-522 (0.1-30 mg/kg) and indomethacin (0.1-3 mg/kg) were administered orally once-daily after immunization. METHODS: Paw swelling, bone changes in arthritic paws and vertebrae, urinary levels of deoxypyridinoline and pyridinium crosslinks, and the incidence of gastric lesions were determined in arthritic rats. RESULTS: JTE-522 (from 0.3 mg/kg) suppressed the development of paw swelling, and also reduced bone damage (score and bone mineral density) in arthritic paws and the urinary excretion of deoxypyridinoline and pyridinium crosslinks. However, JTE-522 did not cause gastric lesions even at 30 mg/kg in arthritic rats. CONCLUSIONS: These results suggest that JTE-522 possesses potent anti-arthritic activities and suppressive activity on inflammatory bone resorption without gastric side effects.

Collagen-induced arthritis in rhesus monkeys: evaluation of markers for inflammation and joint degradation.

The objective of this study was to analyse parameters in rhesus monkey collagen-induced arthritis (CIA) with which the inflammation and destruction of the joints can be described in quantitative terms. CIA was induced in genetically susceptible and resistant monkeys, which can be distinguished on the basis of the dominant resistance marker Mamu-A26. The disease course was monitored daily using a semiquantitative scoring system. Plasma samples were collected once or twice weekly and analysed for C-reactive protein (CRP). Urines were collected overnight once a week and analysed for excretion rates of the collagen cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP). The results show that periods of active CIA are characterized by substantial weight loss and increased plasma CRP levels, followed shortly thereafter by increased excretion rates of the collagen cross-links HP and LP. Remission of the disease can be recognized by a decline in plasma CRP levels and especially an increase in body weight. The highest CRP levels were found in the most severely arthritic monkeys, indicating a possible relationship of the absolute plasma CRP levels to the severity of inflammation. During periods of active arthritis, increased excretion rates of collagen cross-links HP and LP in the urine were found. In particular, the major collagen cross-link in articular cartilage, HP, showed a strong increase (9- to 15-fold). The excretion rates of LP, which is considered as a bone-specific degradation marker, only increased 4- to 6-fold, thus indicating predominant destruction of cartilage and less of bone. In conclusion, the severity of CIA can be monitored in a quantitative manner using plasma CRP levels, urinary excretion rates of HP and LP, and body weights, superimposed on semiquantitative clinical scores. The parameters also facilitate a more objective assessment of the effect of anti-arthritic drugs in the model than with the clinical scores alone.

Effects of tiaprofenic acid on urinary pyridinium crosslinks in adjuvant arthritic rats: comparison with doxycycline.

OBJECTIVE AND DESIGN: To study the effects of tiaprofenic acid and doxycycline on urinary pyridinium crosslinks and paw swelling in adjuvant arthritic rats, and to gain additional information on the drugs' inhibitory potential vs. in vitro targets, such as enzyme activity of matrix metalloproteinases and cytokine generation. MATERIAL: 124 male Wistar Lewis rats; for the in vitro studies human matrix metalloproteinases and human mononuclear cells were used. TREATMENT: Arthritis was induced by injection of complete Freund adjuvant. Drugs (2, 15, 50 mg tiaprofenic acid/kg; 5, 15, 30 mg doxycycline/kg) were administered daily p.o. until day 21. In the in vitro studies 10-1000 mumoles/l of these drugs were used. METHODS: Urinary levels of pyridinoline and deoxypyridinoline, determined by HPLC/fluorescence, and paw volumes were the measurements in the rat study. In the in vitro studies enzyme activities were assessed using fluorogenic peptide substrates; cytokines were determined by ELISA. RESULTS: On day 21 of disease crosslink excretion was about twofold higher compared to the healthy controls. After administering daily 15 or 30 mg/kg tiaprofenic acid p.o. this increase was almost completely prevented whereas the paw volumes were suppressed by about 50%. Up to 50 mg/kg doxycycline did not display significant suppressive effects on crosslinks and paw volumes. In vitro 50-100 mumol/l of both drugs inhibited the activities of selected metallo-proteinases, but only doxycycline suppressed the generation of IL-1 beta/TNF alpha in human mononuclear cells, whereas tiaprofenic acid was virtually inactive in that model. CONCLUSIONS: In arthritic rats tiaprofenic acid has not only the capability to suppress paw inflammation, but also to prevent with high potency the excretion of pyridinium crosslinks. Doxycycline without inherent antiinflammatory activity does not exhibit such preserving effects on collagen degradation in this model. Thus the mode of action of cartilage protecting drugs within the complex pathogenesis of arthritis will need further elucidation.

Changes in urinary deoxypyridinoline level and vertebral bone mass in the development of adjuvant-induced arthritis in rats.

This study was designed to determine the effect of bone resorption on the development of generalized osteopenia in adjuvant-induced arthritic rats. Thirty of a total of sixty male SD rats, 6 weeks of age, were injected with killed mycobacterium butyricum suspended in mineral oil into the right hind paw and assigned to six groups of 5 animals each. The other thirty animals served as the age-matched noninjected controls. Animals were sacrificed at 4, 7, 10, 14, 21, and 28 days post-injection after measuring the bilateral hind-paw volumes. Twenty-four-hour urinary samples were obtained before sacrifice and the levels of deoxypyridinoline (D-Pyr) and creatinine (CR) were measured. Plasma intact osteocalcin levels were measured by a sandwich enzyme immunoassay at the start, 14 and 28 days after injection. Bone mineral measurement and histomorphometrical analyses were performed on specimens of the third lumbar vertebral body. On the seventh day after injection, arthritic rats showed significant decreases in the values of bone mineral content (BMC) and density (BMD) when compared to controls. Urinary D-Pyr/Cr ratios, however, did not increase on the seventh day, showing a significant increase on the tenth day after injection. The serum osteocalcin level was significantly reduced on the fourteenth day. The trabecular bone volume (BV/TV) in the arthritic rats showed a significant decrease from the seventh day. The trabecular thickness (Tb.Th) value significantly decreased on the seventh day after injection.(ABSTRACT TRUNCATED AT 250 WORDS)