kjpp The Korean Journal of Physiology & Pharmacology Korean J Physiol Pharmacol 1226-4512 2093-3827 Korean J Physiol Pharmacol 10.4196/kjpp.2010.14.4.199 kjpp-14-199 Original Article The Efficacy of Shikonin on Cartilage Protection in a Mouse Model of Rheumatoid Arthritis KimYoung Ock 1 HongSeung Jae 2 YimSung-Vin 3 Department of Herbal Crop Research, National Institute of Horticultural & Herbal Science, Rural Development Administration, Eumseong 369-873, Korea Department of Rheumatology, School of Medicine, Kyung Hee University, Seoul 130-701, Korea Department of Clinical Pharmacology, School of Medicine, Kyung Hee University, Seoul 130-701, Korea Corresponding to: Sung-Vin Yim, Department of Clinical Pharmacology, School of Medicine, Kyung Hee University, 1, Hoegi-dong, Dongdaemungu, Seoul 130-701, Korea. (Tel) 82-2-961-0303, (Fax) 82-2-3293-0305, (E-mail) ysvin@khu.ac.kr 022010 18022010 14 4 199 204 09042010 07082010 11082010 Copyright © 2010 Korean J Physiol Pharmacol 2010 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract

The potential therapeutic action of shikonin in an experimental model of rheumatoid arthritis (RA) was investigated. As a RA animal model, DBA/1J mice were immunized two times with type II collagen. After the second collagen immunization, mice were orally administered shikonin (2 mg/kg) once a day for 35 days, and the incidence, clinical score, bone mineral density (BMD), bone mineral content (BMC) and joint histopathology were evaluated. BMD in the proximal regions of the tibia largely increased in the shikonin treatment group compared with the control group. We also examined the effect of shikonin on inflammatory cytokines and cartilage protection. Shikonin treatment significantly reduced the incidence and severity of collagen-induced arthritis (CIA), markedly abrogating joint swelling and cartilage destruction. Shikonin also significantly inhibited the production of matrix metalloproteinase (MMP)-1 and up-regulated tissue inhibitors of metalloproteinase (TIMP)-1 in mice with CIA. In conclusion, shikonin exerted therapeutic effects through regulation of MMP/TIMP; these results suggest that shikonin is an outstanding candidate as a cartilage protective medicine for RA.

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Figures and Tables

Structure of shikonin.

Experimental schedule.

Histopathologies of hind paw sections from mice treated with shikonin. (A) Hematoxylin eosin staining of 2 mg/kg shikonintreated mice, (B) vehicle-treated mice, (C) 50 mg/kg Meloxicamtreated mice, (D) Normal group (non RA). ∗Pannus formation over articular cartilage.

Induction of paw edema and effect of shikonin treatment (2 mg/kg) after 2nd immunization. Paw edema measured in type II collagen-induced arthritic mice (n=10). Treated with vehicle (♦), Meloxicam (50 mg/kg ×), and shikonin (2 mg/kg ▪) after 2nd immunization. ∗∗p< 0.005 and ∗∗∗p< 0.001 compared with vehicle-treated mice with CIA.

Inhibitory effect of shikonin on disease progression in mice with collagen-induced arthritis (CIA). CIA was induced by primary (day 0) and secondary (day 20) immunizations with bovine type II collagen in Freund's complete adjuvant. (A) Arthritis score (clinical severity of arthritis). The maximum possible score is 16, as described in materials and methods. (B) Incidence of arthritis. Values are reported as the mean ± S.E.M. ∗p<0.05, ∗∗p< 0.005 and ∗∗∗p< 0.001 compared with vehicle-treated mice with CIA. Treated with vehicle (▪), Meloxicam (50 mg/kg ), and shikonin (2 mg/kg ▴).

Effect of shikonin on IL-1β and TNF-α levels (pg/mg protein, mean±S.E.M) Serum was obtained from four groups of mice: sham group (no arthritis + vehicle treatment, n=10), control group (arthritis + vehicle, n=10), meloxicam group (arthritis + meloxicam treatment at 50 mg/kg/day n=10), and shikonin group (arthritis + shikonin treatment at 2 mg/kg/day n=10). After shikonin treatment, serum IL-1β and TNF-α decreased significantly, ∗∗∗p< 0.001, compared to the vehicle control group.

The bone mineral density and bone mineral contents of tibia in vehicle, shikonin, and Meloxicam-treated arthritic mice. Values are reported as the mean±S.E.M. ∗p<0.05, ∗∗p<0.005 and ∗∗∗p<0.001, compared with vehicle-treated mice with CIA.

Effect of shikonin on serum concentrations of MMP-1 and TIMP-1 in CIA mice

Treatment n MMP-1 (pg/ml) TIMP-1 (pg/ml)
Control 6 112.7±10.27∗∗∗ 159±13.9∗∗
Vehicle with CIA 6 772.6±92.12 105.4±2.57
Meloxicam with CIA 6 381.7±125.9∗ 103.7±2.7
Shikonin with CIA 6 245.9±83.7∗∗∗ 124.1±8.1∗

Levels of MMP-1 and TIMP-1 were analyzed by ELISA. Data are reported as the mean±SEM.

p<0.05,

p< 0.005, and

p < 0.001 compared with vehicle-treated mice with CIA.