J Korean Med Sci.  2015 Mar;30(3):233-239. 10.3346/jkms.2015.30.3.233.

Eupatilin Ameliorates Collagen Induced Arthritis

• Kim J
• Kim Y
• Yi H
• Jung H
• Rim YA
• Park N
• Jung SM
• Park SH
• Ju JH

• KMID: 2133298
• DOI: http://doi.org/10.3346/jkms.2015.30.3.233

Abstract

Eupatilin is the main active component of DA-9601, an extract from Artemisia. Recently, eupatilin was reported to have anti-inflammatory properties. We investigated the anti-arthritic effect of eupatilin in a murine arthritis model and human rheumatoid synoviocytes. DA-9601 was injected into collagen-induced arthritis (CIA) mice. Arthritis score was regularly evaluated. Mouse monocytes were differentiated into osteoclasts when eupatilin was added simultaneously. Osteoclasts were stained with tartrate-resistant acid phosphatase and then manually counted. Rheumatoid synoviocytes were stimulated with TNF-alpha and then treated with eupatilin, and the levels of IL-6 and IL-1beta mRNA expression in synoviocytes were measured by RT-PCR. Intraperitoneal injection of DA-9601 reduced arthritis scores in CIA mice. TNF-alpha treatment of synoviocytes increased the expression of IL-6 and IL-1beta mRNAs, which was inhibited by eupatilin. Eupatilin decreased the number of osteoclasts in a concentration dependent manner. These findings, showing that eupatilin and DA-9601 inhibited the expression of inflammatory cytokines and the differentiation of osteoclasts, suggest that eupatilin and DA-9601 is a candidate anti-inflammatory agent.

Keyword

Eupatilin; Arthritis, Experomental; Arthritis, Rheumatoid; DA-9601

MeSH Terms

Animals
Anti-Inflammatory Agents/pharmacology/*therapeutic use
Arthritis, Experimental/chemically induced/*drug therapy
Arthritis, Rheumatoid/drug therapy/pathology
Cell Differentiation/*drug effects
Cells, Cultured
Collagen Type II
Cytokines/biosynthesis
Disease Models, Animal
Drugs, Chinese Herbal/therapeutic use
Female
Flavonoids/pharmacology/*therapeutic use
Humans
Inflammation/drug therapy/immunology
Interleukin-1beta/genetics/metabolism
Interleukin-6/genetics/metabolism
Lymph Nodes/cytology
Mice
Mice, Inbred DBA
Monocytes/cytology
Osteoclasts/*cytology
Plant Extracts/pharmacology
RNA, Messenger/biosynthesis
Synovial Membrane/cytology
T-Lymphocytes, Regulatory/cytology/immunology
Tumor Necrosis Factor-alpha/pharmacology
Anti-Inflammatory Agents
Collagen Type II
Cytokines
Drugs, Chinese Herbal
Flavonoids
Interleukin-1beta
Interleukin-6
Plant Extracts
RNA, Messenger
Tumor Necrosis Factor-alpha

Figure

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  Fig. 1 Chemical structure of eupatilin, drawn using ACD/ChemSketch free software (Advanced Chemistry Development, Inc, Tronto, Canada).

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  Fig. 2 Eupatilin suppresses mRNAs encoding inflammatory cytokines. (A) CCK assay of eupatilin cytotoxicity. FLS were seeded in 96 well plates, incubated with eupatilin for 24 hr, and incubated with CCK-8 solution for 2 hr. All values are expressed as mean±SEM. CTL (control), treatment with dimethyl sulfoxide. *P < 0.01 compared with CTL. (B) FLS were incubated with eupatilin for 24 hr and TNF-α (10 ng/mL) was added for 15 min. The amounts of IL-6 and IL-1β mRNAs were assayed by RT-PCR.

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  Fig. 3 Eupatilin (Eu) inhibition of osteoclast formation. (A) Mouse monocytes were treated with M-CSF (10 ng/mL) and RANKL (30 ng/mL), in the presence of eupatilin or DMSO (CTL), to induce their differentiation into osteoclasts, defined as cells with >10 nuclei. Values are expressed as mean ± SEM and *P < 0.001 compared with CTL. (B) Microscopic view of the final morphology of differentiated osteoclasts. Multinuclear cells were stained with tartrate-resistant acid phosphatase (magnification, ×100).

• 

  Fig. 4 Effect of DA-9601 on experimental arthritis. (A) Arthritis scores of mouse groups. Arthritis was induced by intradermal injection of a 1:1 emulsion of CII in CFA, followed 21 days later by an intraperitoneal injection of CII solution. The mice were injected with DA-9601 (100 mg/kg) every other day. The arthritis score represents the average degree of swelling of the four limbs. (B) Cytology of sectioned joints of mice. Hematoxylin and eosin staining shows decreased bone destruction and inflammation in the DA-9601 group compared with the CIA group. Safranin-O and toluidine blue staining show that cartilage damage by CIA was ameliorated by DA-9601. (C) Inflammation and joint destruction scores were evaluated by three investigators, as specified in the Methods section. All results are shown as mean ± SEM. *P < 0.001 for the DA-9601 compared with the CIA group. (D) The TRAP stained image of mice toe. The slide of murine toe was stained with TRAP solution. Compared with wild type, TRAP stained part of CIA group was increased. In DA-9601 group, TRAP stained part was fewer shown than CIA group (magnification, ×200).

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  Fig. 5 DA-9601 increases Treg populations in lymph nodes. (A) Single cells obtained from lymph nodes were stained with anti CD4 antibody conjugated with APC and permeabilized, followed by intracellular staining with anti-Foxp3-conjugated with FITC. (B) The Treg (CD4+FOXP3+) population in the DA-9601 group was higher than in the CIA group. (C) The Treg (CD4+FOXP3+) population of CD4+ cells was higher in the DA-9601 than in the CIA group.

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