Suppression of Collagen-Induced Arthritis by Oral Administration of the Citrus Flavonoid Hesperidin

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

The preventive and therapeutic effects of the Citrus flavonoid hesperidin (HES) on the development of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA), were investigated. Mice were administered orally HES three times a week starting from either the onset (day 21) of secondary immunization or on day 31, when the CIA development had reached a plateau. In both cases, treatment with HES resulted in a significant suppression of clinical scores and improvement of histological features. These results suggest that oral administration of HES could be effective for treating human RA patients.

The production of TNF-α and IL-1β is considered to be the hallmark of the pathogenesis of RA, an autoimmune disease with chronic inflammation in the synovial membranes of the joint, followed by erosion of cartilage and bone [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. In previous studies, we demonstrated that HES, shown in Fig. [1], suppressed LPS-induced TNF-α production in vivo [11], and that pretreatment with HES also blocked endotoxin shock caused by infection with Salmonella typhimurium [12]. However, the suppressive effect was not observed in mice which were treated with HES after LPS administration or Salmonella infection. This limitation suggests that treatment with HES might be more effective in chronic inflammatory responses than in acute-phase ones. The regulatory potential of HES on autoimmune diseases with chronic inflammation is not understood as yet. We therefore attempted to clarify whether HES is effective for improvement of the chronic inflammation model CIA. To determine preventive effects on CIA development, HES was orally administered to mice, which were immunized with type II collagen emulsified with Freund’s complete adjuvant (FCA), three times a week from the onset (day 21 after priming) and thereafter. Treatment with 150 mg/kg HES caused a significant improvement of clinical scores on days 42, 45 and 47 (Table [1]).

To assess the therapeutic effect, HES was orally given to CIA mice three times a week starting on day 31 after priming. HES significantly improved the clinical scores at late stages, but the activity of HES was weaker than that of prednisolone, daily administered at 1 mg/kg (Fig. [2]). Furthermore, the efficacy of HES was proved by histological evaluation of knee joints of CIA mice on day 49. The knee joints of CIA mice show representative features such as damage of interchondral structures, increases of synovial cells, infiltration of inflammatory cells and pannus formation (Fig. [3]). Treatment with HES reduced the damage of interchondral joints and significantly suppressed the increases of synovial cells, infiltration of inflammatory cells and pannus formation (Fig. [4]).

Since TNF-α plays a critical role in the pathogenesis of joints of CIA mice, TNF-α mRNA expression in lesions was analyzed by RT-PCR. In CIA controls, strong mRNA expression was observed in lesions (Fig. [5]). Treatment with HES caused a down-regulation of TNF-α mRNA expression in lesions.

In conclusion, we have clearly demonstrated that HES has preventive and therapeutic effects on CIA development, suggesting that administration of HES could be effective for treating human RA patients.

Materials and Methods

Mice were housed under specific pathogen-free conditions. The experiments were approved by the Animal Use Committee. Type II collagen (chicken sternal cartilage, Sigma-Aldrich Inc., St. Louis, MO, USA) was dissolved in 10 mM acetic acid (2 mg/mL) and mixed with an equal volume of FCA, consisting of 5 mg heat-killed Mycobacterium tuberculosis H37Ra in 1 mL of Freund’s incomplete adjuvant (Difco Laboratories, Detroit, MI, USA). The emulsified antigen was injected intradermally to the tail base of 9-week-old DBA/1J mice for priming (day 0) and similarly on day 21 for boosting. HES (Tokyo Kasei Kogyo Co., Tokyo, Japan) was suspended in water (Otsuka, Tokushima, Japan) and orally administered at a dose of 150 mg/kg three times a week either from day 21 to assess preventive effects, or from day 31 to assess therapeutic effects until the termination of the experiments. Prednisolone (Wako Pure Chemical Industries, Osaka, Japan) and water were used as positive and negative controls, respectively. Clinical scores were estimated by the following criteria; 0 = unchanged, 1 = rubor, 2 = slight swelling, 3 = medium swelling, 4 = severe swelling and 5 = loss of joint functions. The clinical score for each mouse is a cumulative value of four footpads.

Knee joint specimens were taken on day 49, fixed with 4 % paraformaldehyde in 0.01 M phosphate buffer (pH 7.4), and decalcified with EDTA-Na solution. After embedding in paraffin, successive 3 μm sections were stained with hematoxylin and eosin (H&E). Total RNA was prepared from sliced specimens of knee joints using Trizol reagent. The first-strand cDNA was synthesized using each RNA sample (5 μg). The PCR reaction mix (50 μL) contained 2 μL of cDNA product, 5 μL of 10 × PCR buffer, 4 μL of dNTP mix, 20 pmol of forward/reverse primers and 2.5 units of Taq polymerase (Takara, Kyoto, Japan). The specific oligonucleotide primers used were as follows: 5′-GGCAGGTCTACTTTGGAGTCATTGC-3′ and 5′-ACATTCGAGGCTCCAGTGAATTCGG-3′ for TNF-α; 5′-GTTTGTTGTTGGATATGCCCTTGAC-3′ and 5′-GGGGACGCAGCAACTGACATTTCTA-3′ for HPRT. The PCR comprised 30 thermal cycles of 94 °C for 30 sec, 56.5 °C for 1.5 min, and 72 °C for 1 min. The product was separated on a 2 % agarose gel and stained with ethidium bromide.

Acknowledgements

We would like to thank Dr. Roland Ryll for a critical review of the manuscript and Mr. Masayuki Ito, Hirotoshi Aono and Ryo Miyake for their technical assistance. This work was supported in part by a grant-in-aid for collaboration study (3340) from Kitasato University to Y.K.

References

• 1 Feldmann M, Brennan F M, Chantry D, Haworth C, Turner M, Katsikis P. et al . Cytokine assays: role in evaluation of the pathogenesis of autoimmunity.  Immunol Rev. 1991;  119 105-23
  PubMedSearch in Google Scholar
• 2 Elson C J, Thompson S J, Westacott C I, Bhoola K D. Mediators of joint swelling and damage in rheumatoid arthritis and pristane induced arthritis.  Autoimmunity. 1992;  13 327-31
  PubMedSearch in Google Scholar
• 3 Klareskog L, McDevitt H. Rheumatoid arthritis and its animal models: the role of TNF-α and the possible absence of specific immune reactions.  Curr Opin Immunol. 1999;  11 657-62
  PubMedSearch in Google Scholar
• 4 Chu C Q, Field M, Feldmann M, Maini R N. Localization of tumor necrosis factor α in synovial tissues and at the cartilage-pannus junction in patients with rheumatoid arthritis.  Arthritis Rheum. 1991;  34 1125-32
  PubMedSearch in Google Scholar
• 5 Brahn E, Peacock D J, Banquerigo M L, Liu D Y. Effects of tumor necrosis factor α (TNF-α) on collagen arthritis.  Lymphokine Cytokine Res. 1992;  11 253-6
  PubMedSearch in Google Scholar
• 6 Mikuls T R, Weaver A L. Lessons learned in the use of tumor necrosis factor-α inhibitors in the treatment of rheumatoid arthritis.  Curr Rheumatol Rep. 2003;  5 270-7
  PubMedSearch in Google Scholar
• 7 Taylor P C. Anti-TNFα therapy for rheumatoid arthritis: an update.  Intern Med. 2003;  42 15-20
  PubMedSearch in Google Scholar
• 8 Shanahan J C, St Clair W. Tumor necrosis factor-α blockade: a novel therapy for rheumatic disease.  Clin Immunol. 2002;  103 231-42
  CrossrefPubMedSearch in Google Scholar
• 9 Graninger W B, Smolen J S. One-year inhibition of tumor necrosis factor-α: a major success or a larger puzzle.  Curr Opin Rheumatol. 2001;  13 209-13
  CrossrefPubMedSearch in Google Scholar
• 10 Feldmann M, Maini R N. Anti-TNF alpha therapy of rheumatoid arthritis: what have we learned?.  Annu Rev Immunol. 2001;  19 163-96
  CrossrefPubMedSearch in Google Scholar
• 11 Kawaguchi K, Kikuchi S, Hasegawa H, Maruyama H , Morita H, Kumazawa Y. Suppression of lipopolysaccharide-induced tumor necrosis factor-release and liver injury in mice by naringin.  Eur J Pharmacol. 1999;  368 245-50
  CrossrefPubMedSearch in Google Scholar
• 12 Kawaguchi K, Kikuchi S , Hasunuma R, Maruyama H , Yoshikawa T, Kumazawa Y. A citrus flavonoid hesperidin suppresses infection-induced endotoxin shock in mice.  Biol Pharm Bull. 2004;  27 679-83
  CrossrefPubMedSearch in Google Scholar

Dr. Professor Yoshio Kumazawa

Department of Biosciences

School of Science and Graduate School of Fundamental Life Science

Kitasato University

1-15-1 Kitasato

Sagamihara 228-8555

Japan

Phone: +81-42-778-9534

Fax: +81-42-778-9534

Email: kumazawa@sci.kitasato-u.ac.jp

References

• 1 Feldmann M, Brennan F M, Chantry D, Haworth C, Turner M, Katsikis P. et al . Cytokine assays: role in evaluation of the pathogenesis of autoimmunity.  Immunol Rev. 1991;  119 105-23
  PubMedSearch in Google Scholar
• 2 Elson C J, Thompson S J, Westacott C I, Bhoola K D. Mediators of joint swelling and damage in rheumatoid arthritis and pristane induced arthritis.  Autoimmunity. 1992;  13 327-31
  PubMedSearch in Google Scholar
• 3 Klareskog L, McDevitt H. Rheumatoid arthritis and its animal models: the role of TNF-α and the possible absence of specific immune reactions.  Curr Opin Immunol. 1999;  11 657-62
  PubMedSearch in Google Scholar
• 4 Chu C Q, Field M, Feldmann M, Maini R N. Localization of tumor necrosis factor α in synovial tissues and at the cartilage-pannus junction in patients with rheumatoid arthritis.  Arthritis Rheum. 1991;  34 1125-32
  PubMedSearch in Google Scholar
• 5 Brahn E, Peacock D J, Banquerigo M L, Liu D Y. Effects of tumor necrosis factor α (TNF-α) on collagen arthritis.  Lymphokine Cytokine Res. 1992;  11 253-6
  PubMedSearch in Google Scholar
• 6 Mikuls T R, Weaver A L. Lessons learned in the use of tumor necrosis factor-α inhibitors in the treatment of rheumatoid arthritis.  Curr Rheumatol Rep. 2003;  5 270-7
  PubMedSearch in Google Scholar
• 7 Taylor P C. Anti-TNFα therapy for rheumatoid arthritis: an update.  Intern Med. 2003;  42 15-20
  PubMedSearch in Google Scholar
• 8 Shanahan J C, St Clair W. Tumor necrosis factor-α blockade: a novel therapy for rheumatic disease.  Clin Immunol. 2002;  103 231-42
  CrossrefPubMedSearch in Google Scholar
• 9 Graninger W B, Smolen J S. One-year inhibition of tumor necrosis factor-α: a major success or a larger puzzle.  Curr Opin Rheumatol. 2001;  13 209-13
  CrossrefPubMedSearch in Google Scholar
• 10 Feldmann M, Maini R N. Anti-TNF alpha therapy of rheumatoid arthritis: what have we learned?.  Annu Rev Immunol. 2001;  19 163-96
  CrossrefPubMedSearch in Google Scholar
• 11 Kawaguchi K, Kikuchi S, Hasegawa H, Maruyama H , Morita H, Kumazawa Y. Suppression of lipopolysaccharide-induced tumor necrosis factor-release and liver injury in mice by naringin.  Eur J Pharmacol. 1999;  368 245-50
  CrossrefPubMedSearch in Google Scholar
• 12 Kawaguchi K, Kikuchi S , Hasunuma R, Maruyama H , Yoshikawa T, Kumazawa Y. A citrus flavonoid hesperidin suppresses infection-induced endotoxin shock in mice.  Biol Pharm Bull. 2004;  27 679-83
  CrossrefPubMedSearch in Google Scholar

Dr. Professor Yoshio Kumazawa

Department of Biosciences

School of Science and Graduate School of Fundamental Life Science

Kitasato University

1-15-1 Kitasato

Sagamihara 228-8555

Japan

Phone: +81-42-778-9534

Fax: +81-42-778-9534

Email: kumazawa@sci.kitasato-u.ac.jp