J Rheum Dis.  2016 Jun;23(3):161-173. 10.4078/jrd.2016.23.3.161.

The Therapeutic Effect of Sauchinone on Inflammatory Arthritis in Mice

• Jung SM
• Lee J
• Lee J
• Baek SY
• Park SH
• Kwok SK

• KMID: 2326731
• DOI: http://doi.org/10.4078/jrd.2016.23.3.161

Abstract

OBJECTIVE
Rheumatoid arthritis (RA) is a chronic inflammatory disease, mainly involving joints and bones. Sauchinone is an anti-inflammatory agent isolated from Saururus chinensis, which was used in oriental medicine. The aim of this study was to evaluate the therapeutic effect of sauchinone on inflammatory arthritis and underlying mechanism of anti-arthritic effect.
METHODS
Mice with collagen induced arthritis (CIA) was intraperitoneally injected with sauchinone (20 mg/kg) or vehicle. The clinical and histological evaluations were performed with arthritis scoring and hematoxylin-eosin staining, respectively. CD4+ interleukin (IL) 17+ T cells were determined under Th17 skewing condition treated with sauchinone. To evaluate the effect of sauchinone on osteoclastogenesis, mice bone marrow macrophages (BMMs) and human peripheral blood mononuclear cells (PBMCs) were cultured with macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand in the absence or presence of sauchinone.
RESULTS
Sauchinone significantly attenuated the inflammatory arthritis in CIA mice both clinically and histologically. The proportion of Th17 cells were decreased with treatment with sauchinone in vivo and in vitro. The expressions of Th17 cell markers (IL-17 and retinoic acid receptor-related orphan receptor gamma t) and B cell markers (activation-induced cytidine deaminase) were downregulated in the presence of sauchinone. Sauchinone also suppressed the formation of tartrate-resistant acid phosphatase positive cells from mice BMMs and human PBMCs, and the expression of osteoclastogenic markers.
CONCLUSION
Sauchinone alleviates inflammatory arthritis in mice through inhibition of Th17 differentiation and osteoclastogenesis. Sauchinone, one of traditional herbal medicine, could be a therapeutic candidate for the treatment of RA.

Keyword

Rheumatoid arthritis; Sauchinone; Collage induced arthritis; Experimental arthritis

MeSH Terms

Acid Phosphatase
Animals
Arthritis*
Arthritis, Rheumatoid
Bone Marrow
Child
Child, Orphaned
Collagen
Cytidine
Herbal Medicine
Humans
In Vitro Techniques
Interleukins
Joints
Macrophages
Medicine, East Asian Traditional
Mice*
Saururaceae
T-Lymphocytes
Th17 Cells
Tretinoin
Acid Phosphatase
Collagen
Cytidine
Interleukins
Tretinoin

Figure

• 

  Figure 1. Sauchinone alleviates the inflammatory arthritis in mice. Mice with collagen induced arthritis were intraperitoneally injected with sauchinone (20 mg/kg) or vehicle for six weeks immediately after second immunization. (A) Inflammatory arthritis was assessed with clinical scoring by three independent examiners. (B) The serum level of IgG and IgG2a was measured with ELISA. (C) Histological analysis was performed with hematoxylin-eosin staining of mice paws. Bar in the left corner of figure indicates 100 μ m. (D) Histologic score for synovial inflammation of mice paws was determined by three independent examiners. (E) The composition of mice splenocytes were determined by flowcytometry. The percentages of CD4＋ IL-17＋ T cells, CD4＋ IFN-γ＋ T cells, and CD4＋ CD25＋ FOXP3＋ T cells were compared between two groups. (F) The production of TNF-α, IFN-γ, and IL-17 from mice splenocytes was evaluated with ELISA of culture supernatant after stimulation with anti-CD3 antibodies. Values are presented as three independent experiments±standard error of mean. aCD3: stimulation with anti-CD3 antibodies, ELISA: enzyme-linked immunosorbent assay, IFN: interferon, Ig: immunoglobulin, IL: interleukin, Nil: no stimulation, NS: not significant, TNF: tumor necrosis factor. *p<0.05, ***p<0.001.

• 

  Figure 2. Sauchinone inhibits Th17 differentiation. (A) Cytotoxicity of sauchinone was evaluated with cell viability assay using cell counting kit-8 after treatment with 0 to 20 μ M of sauchinone. The dose of sauchinone with more than 80% of cell viability was considered as a tolerable dose. (B) CD4＋ T cells were differentiated into Th17 cells in the presence of sauchinone. The proportions of CD4＋IL-17＋ cells were determined with flowcytometry. The number of right bottom indicates the proportion of CD4＋ IL-17＋ cells. (C) The production of IL-17 from CD4＋ T cells under Th17 skewing condition was measured with ELISA of culture supernatant after pretreatment of sauchinone. (D) The expression of IL-17 and ROR-γ t was determined with qRT-PCR of CD4＋ T cells under Th17 differentiation condition after pretreatment with sauchinone. Values are presented as three independent experiments±standard error of mean. aCD3: stimulation with anti-CD3 antibodies, FITC: fluorescein isothiocyanate, IFN: interferon, IL: interleukin, ROR-γt: retinoic acid receptor-related orphan receptor gamma t, PE: phycoerythrin, Th17: Th17 skewing condition, Th17＋ S: Th17 skewing condition in the presence of sauchinone. *p<0.05.

• 

  Figure 3. Sauchinone affects B cell maturation. (A) The expression of Blimp-1 and AID was evaluated with qRT-PCR of CD19＋ B cells stimulated with LPS in the presence of sauchinone. (B) The production of IgG from CD19＋ B cells stimulated with LPS was measured with ELISA of culture supernatant after pretreatment with sauchinone. Values are presented as three independent experiments±standard error of mean. AID: activation-induced cytidine deaminase, Blimp-1: B lymphocyte-induced maturation protein-1, Ig: immunoglobulin, LPS: lipopolysaccharide, Nil: no stimulation, NS: not significant. *p<0.05, **p<0.01.

• 

  Figure 4. Sauchinone suppresses osteoclastogenesis in a dose-dependent manner. Mice bone marrow macrophages were cultured with M-CSF and RANKL to induced osteoclastogenesis. Sauchinone was added to the culture dishes to evaluate the effect of sauchinone on osteoclastogenesis. (A) The numbers of TRAP positive cells were counted in culture dishes at various doses of sauchinone.(B) The expression of osteoclastogenic markers were determined by qRT-PCR in the absence or presence of sauchinone. (C) The expression of c-fos, a transcription factor associated with osteoclastogenesis, was evaluated with Western blot of cells cultured with or without sauchinone. Values are presented as three independent experiments±standard error of mean. M: culture with M-CSF, M＋ R: culture with M-CSF and RANKL, M-CSF: macrophage-colony stimulating factor, MMP: matrix metalloproteinase, NS: not significant, OSCAR: osteoclast-associated receptor, qRT-PCR: quantitative reverse transcription-polymerase chain reaction, RANKL: receptor activator of nuclear factor-κ B ligand, TRAP: tartrate-resistant acid phosphatase. *p<0.05, **p<0.01.

• 

  Figure 5. Saucinone regulates Th17 differentiation and osteoclastogenesis in human mononuclear cells. To confirm the inhibitory effect of sauchinone on human cells, pripheral blood mononuclear cells isolated from healthy controls were cultured in the absence or presence of sauchinone. (A) The proportion of CD4＋ IL17＋ T cells and the production of IL-17 under Th17 skewing condition with or without sauchinone was evaluated by flowcytometry and ELISA, respectively. (B) The number of TRAP positive cells was measured after culture with M-CSF and RANKL in the absence or presence of sauchinone. (C) The expression of osteoclastogenic markers was compared between cells differentiated to osteoclast with or without sauchinone. Values are presented as three independent experiments±standard error of mean. M: culture with M-CSF, M＋ R: culture with M-CSF and RANKL, M-CSF: macro-phage-colony stimulating factor, MMP: matrix metalloproteinase, NFATc1: nuclear factor of activated T-cells, cytoplasmic 1, NS: not significant, RANKL: receptor activator of nuclear factor-κ B ligand, Th17: Th17 skewing condition, Th17＋ S: Th17 skewing condition in the presence of sauchinone, TRAP: tartrate-resistant acid phosphatase. *p<0.05.

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