Metabolomics Approach to Explore the Effects of Rebamipide on Inflammatory Arthritis Using Ultra Performance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry

Su-Jin Moon; Soo Hyun Lee; Byung-Hwa Jung; Jun-Ki Min

doi:10.4078/jrd.2017.24.4.192

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Moon, Lee, Jung, and Min: Metabolomics Approach to Explore the Effects of Rebamipide on Inflammatory Arthritis Using Ultra Performance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry

Original Article

J Rheum Dis 2017;24(4):192-202.

Published online: 31 October 2017

DOI: https://doi.org/10.4078/jrd.2017.24.4.192

Metabolomics Approach to Explore the Effects of Rebamipide on Inflammatory Arthritis Using Ultra Performance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry

Su-Jin Moon^1,^∗, Soo Hyun Lee^2,^∗, Byung-Hwa Jung³, Jun-Ki Min¹

¹Division of Rheumatology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea

²Department of Medical Records and Health Information Management, College of Nursing and Health, Kongju National University, Gongju, Korea

³Molecular Recognition Research Center, Korea Institute of Science and Technology, Seoul, Korea

Corresponding to: Jun-Ki Min, Division of Rheumatology, Department of Internal Medicine, Bucheon St. Mary's Hospital, College of Medicine, The Catholic University of Korea, 327 Sosa-ro, Wonmi-gu, Bucheon 14647, Korea. E-mail:min6403@catholic.ac.kr Byung-Hwa Jung, Molecular Recognition Research Center, Korea Institute of Science and Technology, 5, Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Korea. E-mail: jbhluck@kist.re.kr

^* These authors contributed equally to this work.

Received 25 January 2017 Revised 21 April 2017 Accepted 22 April 2017

This is a Open Access article, which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objective

Rebampide is a gastroprotective agent used to treat gastritis. It possesses anti-inflammatory and anti-arthritis effects, but the mechanisms of these effects are not well understood. The objective of this study was to explore mechanisms underlying the therapeutic effects of rebamipide in inflammatory arthritis.

Methods

Collagen-induced arthritis (CIA) was induced in DBA/1J mice. DBA/1J mice were immunized with chicken type II collagen, then treated intraperitoneally with rebamipide (10 mg/kg or 30 mg/kg) or vehicle (10% carboxymethylcellulose solution) alone. Seven weeks later, plasma samples were collected. Plasma metabolic profiles were analyzed using ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry-based metabolomics study and metabolite biomarkers were identified through multivariate data analysis.

Results

Low dose rebamipide treatment reduced the clinical arthritis score compared with vehicle treatment, whereas high dose rebamipide in CIA aggravated arthritis severity. Based on multivariate analysis, 17 metabolites were identified. The plasma levels of metabolites associated with fatty acids and phospholipid metabolism were significantly lower with rebamipide treatment than with vehicle. The levels of 15-deoxy-^Δ12,14 prostaglandin J2 and thromboxane B3 decreased only in high dose-treated groups. Certain peptide molecules, including enterostatin (VPDPR) enterostatin and bradykinin dramatically increased in re-bamipide-treated groups at both doses. Additionally, corticosterone increased in the low dose-treated group and decreased in the high dose-treated group.

Conclusion

Metabolomics analysis revealed the anti-inflammatory effects of rebamipide and suggested the potential of the drug repositioning in metabolism- and lipid-associated diseases.

Keywords: Rebamipide, Arthritis, Metabolomics, Antioxidant

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Figure 1.

Treatment with rebamipide (10 mg/kg) suppresses inflammatory arthritis in mice with collagen-induced arthritis (CIA). CIA was induced in DBA/1J mice by immunization with type II collagen (CII) in adjuvant. Changes in arthritis score in rebamipide-treated mice compared with vehicle-treated mice. Rebamipide dissolved in 10% carboxymethylcellulose solution (vehicle) was given intraperitoneally to 2 different groups (each receiving 10 or 30 mg/kg; n=10 mice per group) daily for 4 weeks, starting after booster immunization. A third group (n=10) received vehicle alone. WT: wild-type, D: day.

Figure 2.

(A) Principal component analysis (PCA) score plots and (B) partial least square-discriminant analysis (PLS-DA) score plots based on plasma metabolic profiling. (a) Positive ionization mode. (b) Negative ionization mode. In PCA, the score plot was obtained with the two PCs presenting 47.2% (PC1) and 14.5% (PC2) variance in positive ionization mode and that was with three PCs presenting 23.3% (PC1), 18.7% (PC2), and 13.2% (PC3) in negative ionization mode. In PLS-DA, the score plot was obtained with the two PCs presenting 43.2% (PC1) and 30.5% (PC2) variance in positive ionization mode and that was with three PCs presenting 53.7% (PC1), 38.6% (PC2), and 23.3% (PC3) in negative ionization mode.

Figure 3.

Comparison of metabolites with significant changes involved in (A) peptide metabolism, (B) fatty acid metabolism, (C) phospholipid metabolism, (D) acylcarnitine (β-oxidation) metabolism, (E) prostaglandin metabolism, and (F) corticosteroid hormone metabolism. *p＜0.05, **p＜0.01, and ***p＜0.001 compared with vehicle control. HpODE: hydroxyoctadecadienoic acid, LysoPC: lysophosphatidylcholine, PGJ2: prostaglandin J2.

Table 1.

Identification of potential biomarkers

rRT_m/z	Molecular formula	Fragmentation	Identified metabolite	Related metabolism	Change trend
rRT_m/z	Molecular formula	Fragmentation	Identified metabolite	Related metabolism	W vs. V	L vs. V	H vs. V	H vs. L
0.34_317.18	C₂₃ H₄₀ N₈ O₆	C₁₇ H₁₇ N₃ O₂	Enterostatin (VPDPR)	Peptide	–	↑^∗	↑^∗	–
[M+H]		(− C₁₆ H₂₄ N₅ O₄)
1.04_354.19	C₅₀ H₇₃ N₁₅ O₁₁	C₃₂ H₅₀ N₁₀ O₇	Bradykinin		–	↑^∗	↑^∗	–
[M+H]		(− C₁₈ H₂₄ N₅ O₄)
1.94_305.25	C₂₀ H₃₂ O₂	C₁₇ H₂₅	Arachidonic acid	Fatty acid	–	↓	↓	–
[M+H]		(− C₃ H₆ O₂)
2.03_257.25	C₁₆ H₃₂ O₂	C₁₆ H₃₃ O	Palmitic acid		–	↓^∗	↓^∗	–
[M+H]		(− O)
2.07_283.27	C₁₈ H₃₄ O₂	C₁₇ H₃₅	Oleic acid		–	↓	↓	–
[M+H]		(− CO₂)
1.51_333.20	C₁₈ H₃₄ O₅	C₁₇ H₃₅ O₃	HpODE		–	↓	↓	–
[M+Na-2H]		(− CO₂)
1.56_468.31	C₂₂ H₄₆ NO₇ P	C₅ H₁₃ O₅ P	LysoPC (14:0)	Phospholipid	–	↓^∗	↓^∗	–
[M+H]		(− C₁₇ H₃₄ NO₂)
1.80_496.34	C₂₄ H₅₀ NO₇ P	C₅ H₁₃ O₅ P	LysoPC (16:0)		–	↓^∗	↓^∗	–
[M+H]		(− C₁₉ H₃₈ NO₂)
1.99_524.37	C₂₆ H₅₄ NO₇ P	C₅ H₁₃ O₅ P	LysoPC (18:0)		–	↓^∗	↓^∗	–
[M+H]		(− C₂₁ H₄₂ NO₂)
2.77_787.67	C₄₄ H₈₆ NO₈ P	C₅ H₁₃ O₅ P	PC (18:0/18:1)		–	↓	–	–
[M+H]		(− C₃₉ H₇₄ NO₃)
1.25-370.30	C₂₁ H₃₉ NO₄	C₈ H₁₄ O₃	Tetradecenoylcarnitine	Acylcarnitine	–	↓^∗	↓^∗	–
[M+H]		(− C₁₃ H₂₆ NO)		(β-oxidation)
1.34_372.31	C₂₁ H₄₁ NO₄	C₈ H₁₄ O₃	Tetradecanoylcarnitine		–	↓^∗	↓^∗	–
[M+H]		(− C₁₃ H₂₈ NO)
1.40_398.33	C₂₃ H₄₃ NO₄	C₈ H₁₄ O₃	Hexadecenoylcarnitine		–	↓^∗	↓^∗	–
[M+H]		(− C₁₅ H₃₀ NO)
1.53_400.43	C₂₃ H₄₅ NO₄	C₈ H₁₄ O₃	Palmitoylcarnitine		–	↓^∗	↓^∗	↓^∗
[M+H]		(− C₁₅ H₃₂ NO)
1.84_317.18	C₂₀ H₃₀ O₄	C₁₂ H₁₆ O₃	15-deoxy-^Δ12,14 PGJ2	Prostaglandin	–	–	↓	↓^∗
[M+H]		(− C₈ H₁₅ O)
2.21_369.23	C₂₀ H₃₂ O₆	C₁₂ H₁₇ O₅	Thromboxane B3		–	–	↓^∗	↓
[M+H]		(− C₈ H₁₅ O)
2.07_381.17	C₂₁ H₃₀ O₄	C₂₁ H₂₈ O₃	Corticosterone	Corticosteroid	–	↑^∗	↓	↓^∗
[M+Cl]		(− H₂ O)		hormone

rRT=retention time of metabolite/retention time of internal standard (IS) (reserpine). ↓: indicates decrease, ↑: indicates increase,–: indicates no significant changes, W: wild type, V: vehicle-treated CIA mice, L: low-dose rebamipide (10 mg/kg)-treated CIA mice, H: high-dose rebamipide (30 mg/kg)-treated CIA mice, HpODE: hydroxyoctadecadienoic acid, LysoPC: lysophosphatidylcholine, PGJ2: prostaglandin J2.

^* Indicates significant change (p＜0.01), the other indicates significant change (p＜0.05).

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