Fig 1.
GLM reduced the severity of CIA.
Three weeks after the first immunization, CII-induced DBA/1J mice were injected daily with GLM (500 mg/kg) or MTX (5 mg/kg), with oral administration of corn oil (200 μL) serving as a control. (A) Arthritis development was assessed based on the arthritis score and incidence. Representative images from one of two independent experiments are shown. (B) Histological staining of joint tissue sections with hematoxylin/eosin (original magnification, 40×) and safranin O (original magnification, 200×). Representative images are shown. The graphs depict the degree of inflammation, bone damage, and cartilage damage. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2.
GLM suppressed the expression of inflammatory cytokines in joints.
Three weeks after the first immunization, CII-induced DBA/1J mice were injected daily with GLM (500 mg/kg) or MTX (5 mg/kg), or orally administered corn oil (200 μL) as a control. (A) At 10 weeks after the first immunization, the expression levels of TNF-α, IL-1β, IL-6, IL-17, and TRAP in the ankle joints were determined by immunohistochemical staining. Representative images from one of two independent experiments are shown. Bars are percentage positive areas per field. Data are means ± SEM. *P < 0.05, **P < 0.01.
Fig 3.
GLM suppressed the Th17 proliferation.
Three weeks after the first immunization, CII-induced DBA/1J mice were injected daily with GLM (500 mg/kg) or MTX (5 mg/kg), or orally administered corn oil (200 μL) as a control. (A) At 10 weeks after the first immunization, the number of CD4+ IL-17+ cells in ex vivo splenocytes was analyzed by flow cytometry. Representative flow cytometry plots from one of two independent experiments are shown. (B) Confocal images of CD4+ IL-17+ cells in the spleen. Bars are percentages of co-localization of CD4 (green) and IL-17 (red). Data are means ± SEM. *P < 0.05, **P < 0.01.
Fig 4.
Effect of GLM on the osteoclastogenesis of mouse cells.
Bone marrow-derived monocytes/macrophages from normal C57BL/6 mice were cultured with M-CSF (10 ng/mL) and RANKL (50 ng/mL). (A) After 5 days, cells were stained for TRAP. Representative images from one of two independent experiments are shown. The number of multinucleated TRAP+ cells was determined. (B) mRNA levels of TRAP, cathepsin K, carbonic anhydrase, calcitonin receptor, NFATC1, and MMP9 determined by real-time PCR. Data are means ± SD. *P < 0.05, **P < 0.01.
Fig 5.
Effect of GLM on osteoclastogenesis in human cells.
Human PBMC-derived monocytes were cultured with M-CSF (10 ng/mL) and RANKL (30 ng/mL). (A) After 10 days, cells were stained for TRAP. Representative images from one of two independent experiments are shown. The number of multinucleated TRAP+ cells was determined. (B) mRNA levels of TRAP, RANK, cathepsin K, calcitonin receptor, NFATC1, and MMP9 determined by real-time PCR. Data are means ± SD. *P < 0.05.