Table 1.
CIA scoring system.
Table 2.
Histopathological analysis of joint tissues of CIA mice.
Table 3.
RT-qPCR primers used in this study.
Fig 1.
Co-administration effect of LMT-28 and kaempferol on collagen-induced arthritis mouse model.
(A) Arthritis scores of mice were measured every 3 days starting at 21 days after the first immunization. (B) DBA1/J mice showed deformation and swelling of the toe and ankle joints. (C) Paraffin-embedded sections of CIA mice joint were stained with hematoxylin and eosin. (D) Histological analysis was performed based on the analysis criteria: inflammation, synovial hyperplasia, pannus formation, and erosion of cartilage and bone. (E) The protein levels of IL-6, IL-1β and IL-17 in CIA mouse serum were measured using ELISA. Data are presented as mean ± SEM. #p<0.05 vs. normal group, *p<0.05, **p<0.01, and ***p<0.005 vs. CIA control group.
Fig 2.
Effect of LMT-28 and kaempferol on T cell differentiation.
Flow cytometry dot plots (left) and percentage comparisons (right) of (A) Treg cell and (B) Th17 cell population in splenocytes of CIA mice. (C) Dot plots and percentage comparisons of Th17 cell population differentiated from sorted splenocytes. Data are presented as mean ± SEM. #p<0.05 vs. normal group or non-treated group, *p<0.05, **p<0.01, and ***p<0.005 vs. control group.
Fig 3.
Combination effect of LMT-28 and kaempferol on RANKL-induced osteoclastogenesis.
BMMs isolated from normal mice were stimulated with M-CSF (20 ng/mL) and/or RANKL (40 ng/mL) and treated with LMT-28 (25 μM) and/or kaempferol (12.5 μM). (A) Microscopic image (200× magnification) of osteoclasts stained for TRAP activity and (B) quantification of the number of TRAP+ multinucleated osteoclasts (≥3 nuclei) in each treatment group are presented. (C) The mRNA levels of c-fos, nfatc1, ctsk and trap in osteoclasts were measured. Data are presented as mean ± SEM. #p<0.05 vs. M-CSF-treated group; *p<0.05, **p<0.01, and ***p<0.005 vs. M-CSF- and RANKL-treated control group.
Fig 4.
Effect of LMT-28 and kaempferol combination on IL-6-induced signaling in RA-FLS.
The human RA-FLS cell line MH7A was pre-treated with LMT-28 (25 μM) and/or kaempferol (12.5 μM) for 1 h, and stimulated with hyper IL-6 (20 ng/mL) for 5 min. Total proteins were extracted from MH7A cell homogenates and analyzed using western blotting. (A) Densitometric analysis of western blot bands and (B) expression level graphs of p-gp130, p-STAT3, p-ERK and p-AKT in MH7A cells are presented. Data are presented as mean ± SEM. #p<0.05 vs. non-treated group; *p<0.05 vs. hyper IL-6 treated group.
Fig 5.
Effect of LMT-28 and kaempferol co-treatment on RA-FLS activation.
MH7A cells were stimulated with hyper IL-6 (20 ng/mL) and treated with LMT-28 (25 μM) and/or kaempferol (12.5 μM). (A) MH7A cell proliferation was measured using the CCK-8 assay. The migration activity of MH7A cells was evaluated by wound-healing assay. MH7A cells were scratched using a sterile 200 μL pipette tip, followed by treatment with LMT-28 and/or Kaempferol for 48 h. (B, left) Images were captured at 200× magnification, and (B, right) the migration rate of MH7A cells was measured at 0, 24 and 48 h after treatment. Matrigel invasion assay was used to determine invasion after 48 h in LMT-28 and kaempferol-treated MH7A cells. (C, left) Microscopic images (200× magnification) of Matrigel and (C, right) the number of invading cells are presented. Relative (D) mRNA expression and (E) protein levels of MMPs in MH7A cells were measured. Data are presented as mean ± SEM. #p<0.05 vs. non-treated control group; *p<0.05, **p<0.01, and ***p<0.005 vs. hyper IL-6 group.
Fig 6.
Combined effect of LMT-28 and kaempferol on chondrocyte activation.
The human chondrocyte cell line C28/I2 was stimulated with hyper IL-6 (20 ng/mL) and treated with LMT-28 (25 μM) and/or kaempferol (12.5 μM). (A) C28/I2 cell proliferation was measured using CCK-8 assay. The migration activity of C28/I2 cells was evaluated by the wound-healing assay. C28/I2 cells were scratched using a sterile 200 μL pipette tip, followed by treatment with LMT-28 and/or kaempferol for 48 h. (B, left) Images were captured at 200× magnification, and (B, right) the migration rate of C28/I2 cells was measured at 0, 24 and 48 h after treatment. Matrigel invasion assay was used to determine invasion after 48 h in LMT-28 and kaempferol treated C28/I2 cells. (C, left) Microscopic images (200× magnification) of Matrigel and (C, right) the number of invading cells are presented. Data are presented as mean ± SEM. #p<0.05 vs. non-treated control group; *p<0.05, **p<0.01, and ***p<0.005 vs. hyper IL-6 group.