Compound K, a Metabolite of Ginsenosides, Attenuates Collagen-induced Arthritis in Mice

Yun Jong Lee; Kye Yong Song; Eun Young Lee; Heun Soo Kang; Yeong Wook Song

doi:10.4078/jrd.2015.22.3.154

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Lee, Song, Lee, Kang, and Song: Compound K, a Metabolite of Ginsenosides, Attenuates Collagen-induced Arthritis in Mice

Original Article

J Rheum Dis 2015;22(3):154-166.

Published online: 31 October 2015

DOI: https://doi.org/10.4078/jrd.2015.22.3.154

Compound K, a Metabolite of Ginsenosides, Attenuates Collagen-induced Arthritis in Mice

Yun Jong Lee^1,², Kye Yong Song³, Eun Young Lee^2,⁴, Heun Soo Kang⁵, Yeong Wook Song^4,⁶

¹Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Korea

²Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea

³Department of Dermatology and Histopathology, College of Medicine, Chung-Ang University, Seoul, Korea

⁴Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea

⁵Metabolab Inc., Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea

⁶Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Medical Research Center, Seoul National University, Seoul, Korea

Corresponding to: Yeong Wook Song, Department of Internal Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Korea. E-mail: ysong@snu.ac.kr

Received 5 December 2014 Revised 31 January 2015 Accepted 2 February 2015

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

Abstract

Objective

Although several ginsenosides have been reported to have anti-arthritic activity, few in vivo studies of the anti-ar-thritic effects of compound K (CK), a major metabolite of ginsenosides, have been conducted. Therefore, we investigated the preventative and therapeutic effects of CK on collagen-induced arthritis (CIA).

Methods

CK was administered to CIA mice pre-ventively and therapeutically and post-treatment bone microarchitectural characteristics, histopathological changes, and serum levels of anti-collagen antibodies, tumor necrosis factor-α, and interleukin (IL)-17 were investigated. We also examined cytokine production by type II collagen (CII)-stimulated splenocytes and mRNA expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinase (TIMP)-1, receptor activator of nuclear factor-κ B ligand (RANKL), and osteoprotegerin (OPG) in the joint tissues.

Results

CK reduced the severity of CIA preventively and therapeutically (all p＜0.05). Additionally, CK dose-dependently decreased histopathological signs of arthritis and improved microarchitectural characteristics (all p ＜0.05) at 10 to 20 mg/kg/d in CIA mice. CK treatment significantly decreased the serum levels of anti-CII immunoglobulin G (p＜0.01) and the secretion of interferon-γ and IL-2 from stimulated splenocytes (all p＜0.05). Furthermore, MMP-3/TIMP-1 and RANKL/OPG ratios were suppressed in CK treated mice (all p＜0.01).

Conclusion

CK attenuated CIA via suppression of the humoral immune response and modulation of joint-destructive mediators. These results suggest that CK has therapeutic potential in rheumatoid arthritis.

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Keywords: Panax, Ginsenoside M1, Experimental arthritis, Rheumatoid arthritis

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Figure 1.

The effect of compound K (CK) on collagen-induced arthritis (CIA) disease activity. CK (0, 5, 10, or 20 mg/kg/d) was administered 1 day (preventive trial) or 14 days (therapeutic trial) after the boosting immunization of type II collagen (A). Preventive (B, n=9) and therapeutic (C, n=9) administration of CK significantly decreased arthritis scores along the disease course. However, the suppressive effects of CK were not dose-dependent in the dose range tested (5 to 20 mg/kg/d). Error bars represent the standard error of the mean. *p＜0.05 by Kruskal-Wallace test with Dunn's multiple comparison test.

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Figure 2.

Representative sections of joint tissues (hindfoot and knee joints) stained with hematoxylin-eosin (H&E) and Masson's Trichrome (MT) in therapeutic models (scale bar, 50 μ m; A). Control group showed typical findings of collagen-induced arthritis (CIA); severe inflammatory cells infiltration, synovial hypertrophy, joint space narrowing, and bone and cartilage damage. These arthritic findings were decreased in mice therapeutically administered with compound K (CK). The histologic arthritis scores in knee joints significantly decreased with increasing dose of CK (B, n=6). Error bars represent the standard error of the mean. ^† p-val-ue was calculated by Jonckheere's trend test.

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Figure 3.

Microarchitectural analysis using micro-computed tomography (micro-CT) in therapeutic models (n=4 in each group, respectively). Representative micro-CT images of the hind paws (A) showed that the erosion in the metatarsophalangeal joints was decreased in compound K (CK)-treated groups. (B, C) Microarchitectural parameters included bone volume/total tissue volume (BV/TV), bone surface/bone volume (BS/BV), cross-sectional thickness (Cs.th), and trabecular thickness (Tb.th). Therapeutic CK administration significantly increased BV/TV, Cs.th, and Tb.th values and significantly decreased BS/BV values. Error bars represent the standard error of the mean. *p＜0.05 by Kruskal-Wallace test with Dunn's multiple comparison test; ^†p-values were calculated by Kruskal-Wallis test.

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Figure 4.

Serum levels of anti-type II collagen (CII) antibodies in therapeutic models. Therapeutical administration of compound K (CK) produced a significant reduction of anti-CII antibody IgG₂ a (A) and IgG₂ b (B, n=6 in each group). Error bars represent the standard error of the mean. *p＜0.05 by Kruskal-Wallace test with Dunn's multiple comparison test; ^† p-values were calculated by Kruskal-Wallis test.

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Figure 5.

Cytokine production from the type II collagen stimulated splenocytes in therapeutic models. Splenocytes were isolated at the time of sacrifice after 4 weeks of compound K (CK) treatment and were stimulated with bovine CII for 48 h (n=6 in each group). When tumor necrosis factor (TNF)-α (A), interleukin (IL)-2 (B), interferon (IFN)-γ (C), and IL-4 (D) levels were measured in the culture media, IL-2 and IFN-γ were dose-dependently decreased in CK-treated groups. However, no significant effects on TNF-α or IL-4 production were observed. Error bars represent the standard error of the mean. *p＜0.05 by Kruskal-Wallace test with Dunn's multiple comparison test; ^†p-values were calculated by Kruskal-Wallis test.

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Figure 6.

Serum levels of tumor necrosis factor (TNF)-α and interleukin (IL)-17 in therapeutic models (n=6 in each group). Therapeutic compound K (CK) administration did not significantly affect TNF-α (A) and IL-17 (B) levels. Error bars represent the standard error of the mean.

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Figure 7.

Effects of compound K (CK) on matrix metalloproteinase (MMP)-3 and MMP-13 mRNA expression in the hind feet of therapeutic models. When MMP-3 and MMP-13 mRNA levels in the hind foot tissue were analyzed using quantitative real-time polymerase chain reaction (n=6 in each group), therapeutic administration of CK significantly decreased expression of MMP-3 (A) and MMP-13 (B). The suppressive effect of CK on MMP-3 expression remained significant after adjustment for tissue inhibitors of metalloproteinase (TIMP)-1 mRNA (C, n=4 in each group). MMP-13/TIMP-1 ratios (D, n=4 in each group) tended to decrease in a dose dependent manner. Error bars represent the standard error of the mean. *p＜0.05 by Kruskal-Wallace test with Dunn's multiple comparison test; ^† p-values were calculated by Kruskal-Wallis test.

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Figure 8.

Effects of compound K (CK) on receptor activator of nuclear factor-κ B ligand (RANKL) and osteoprotegerin (OPG) mRNA expression in the hind feet of therapeutic models. When RANKL and OPG mRNA levels in the hind foot tissue were analyzed using quantitative real-time polymerase chain reaction (n=6 in each group), therapeutic CK administration significantly decreased RANKL mRNA expression (A) and tended to increase OPG mRNA (B). Consequently, the ratios of RANKL/OPG were significantly reduced in CK administered mice (C). Error bars represent the standard error of the mean. *p＜0.05 by Kruskal-Wallace test with Dunn's multiple comparison test; ^† p-values were calculated by Kruskal-Wallis test.

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Table 1.

Primers and probes used for quantitative real-time polymerase chain reaction

Gene (Accession No.)	Sequences of primers and probes (5′ → 3′)	Reference
GAPDH	Forward: TTCACCACCATGGAGAAGGC	[23]
(NM_001289726)	Reverse: GGCATGGACTGTGGTCATGA
	Probe: VIC ^TM-TGCATCCTGCACCACCAACTGCTTAG
MMP-3	Forward: GGAAATCAGTTCTGGGCTATACGA	[24]
(NM_010809)	Reverse: TAGAAATGGCAGCATCGATCTTC
	Probe: FAM-AGGTTATCCTAAAAGCATTCACACCCTGGGTCT
MMP-13	Forward: GGGCTCTGAATGGTTATGACATTC	[24]
(NM_008607)	Reverse: AGCGCTCAGTCTCTTCACCTCTT
	Probe: FAM-AAGGTTATCCCAGAAAAATATCTGACCTGGGATTC
TIMP-1	Forward: CATGGAAAGCCTCTGTGGATATG	[24]
(NM_001044384)	Reverse: AAGCTGCAGGCACTGATGTG
	Probe: FAM-CTCATCACGGGCCGCCTAAGGAAC
OPG	Forward: AGCTGCTGAAGCTGTGGAA	[25]
(NM_008764)	Reverse: TGTTCGAGTGGCCGAGAT
	Probe: FAM-CCAAGACATTGACCTCTGTGAAAGCA
RANKL	Forward: TGGAAGGCTCATGGTTGGAT	[25]
(NM_011613)	Reverse: CATTGATGGTGAGGTGTGCAA
	Probe: FAM-AGGCTTGCCTCGCTGGGCCAC

FAM: 6-carboxyfluorescein, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, MMP: matrix metalloproteinase, OPG: osteoprotegerin, RANKL: receptor activator of nuclear factor-κ B ligand, TIMP: tissue inhibitors of metalloproteinase, VIC ^TM:2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein.

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