Fig 1.
(A) Celastrol ameliorates inflammation throughout time. Notice that after 7 days of treatment celastrol early-treated rats presented minimal inflammatory activity, whereas arthritic rats started to increase the inflammatory manifestations sharply. Arrows indicate the beginning of treatment after 4 and 11 days of disease induction. (B) Celastrol improves the clinical outcome in adjuvant-induced arthritic rats. Inflammatory score in celastrol-treated AIA rats is maintained significantly diminished in comparison with arthritic rats. (C) Celastrol suppresses the progression of swelling in the left hind paw.
Left paw edema/swelling is markedly present in arthritic rats in contrast to celastrol-treated animals. Data are expressed as median with interquartile range. Differences were considered statistically significant for p-values<0.05, according to the Kruskal-Wallis (Dunn´s Multiple Comparison tests) and Mann–Whitney tests. Healthy N = 19, Arthritic N = 23, Celastrol early group N = 15 and Celastrol late group N = 15.
Fig 2.
Celastrol reduces the serum levels of IL-6 in arthritic rats.
Notice that celastrol treatment significantly reduces the systemic concentration of the proinflammatory cytokine IL-6 to levels similar to healthy controls. Data are expressed as median with interquartile range. Differences were considered statistically significant for p-values<0.05, according to the Kruskal-Wallis (Dunn´s Multiple Comparison tests) and Mann–Whitney tests. Healthy N = 21, Arthritic N = 23, Celastrol early group N = 15 and Celastrol late group N = 15.
Fig 3.
Histological images of joints after celastrol treatment.
These patterns are merely illustrative of the type of histological features observed. Black arrow indicates the absence/presence of ankle swelling in rat hind paws. C–calcaneus, E–edema or erosion, S–synovia, Tb–tibia, Ts–tarso. Magnification of 50×. Bar: 100 μm.
Fig 4.
Celastrol suppresses arthritic inflammation and tissue damage locally in the joints of AIA rats.
A semi-quantitative evaluation of histological sections was performed. Notice that celastrol has inhibited cellular infiltration (A), completely reversed the number of lining layer cells to the normal values (B) and prevented bone erosion occurrence (C), allowing for a normal joint structure comparable to healthy rats in both early and late treatment groups (D). Data are expressed as median with interquartile range. Differences were considered statistically significant for p-values<0.05, according to the Kruskal-Wallis (Dunn´s Multiple Comparison tests) and Mann–Whitney tests. Correlation analysis was performed using the Spearman test. Healthy N = 19, Arthritic N = 23, Celastrol early group N = 15 and Celastrol late group N = 15.
Fig 5.
Celastrol reduces the number of T cells and B cells present in the synovial membrane, and suppresses synovial cell proliferation.
(A) Representation of the immunohistochemical evaluation performed in paw sections at day 22 after celastrol treatment. Magnifications of 200×. Bar: 100 μm. (B) Immunohistochemical analysis was performed using a semi-quantitative score. Notice that both celastrol early and late-treated rats showed a significant reduction in the number of CD3 and CD19 positive cells as well as a reduction in the levels of synovial cell proliferation assessed by Ki67 marker in comparison with arthritic rats at day 22. Healthy N = 16, Arthritic N = 10, Celastrol early group N = 15 and Celastrol late group N = 15. (C) Immunohistochemical quantification was performed using an image analysis software written in MATLAB to identify and count the number of positive cells for each antibody in representative sections. Notice that both celastrol early and late-treated rats showed a significant reduction in the number of CD3, CD19 and Ki67 positive cells in comparison with arthritic rats at day 22. Healthy N = 5, Arthritic N = 5, Celastrol early group N = 5 and Celastrol late group N = 5. Data are expressed as median with interquartile range. Differences were considered statistically significant for p-values<0.05, according to the Kruskal-Wallis (Dunn´s Multiple Comparison tests) and Mann–Whitney tests.
Fig 6.
Celastrol reduces the number of synovial CD68+ macrophages.
(A) Representation of the immunohistochemical evaluation performed in paw sections at day 22 after celastrol treatment. Magnifications of 200×. Bar: 100 μm. (B) Immunohistochemical analysis was performed using a semi-quantitative score. Notice that both celastrol early and late-treated rats showed a significant reduction in the number of CD68 and CD163 positive cells in comparison with arthritic rats at day 22. Healthy N = 16, Arthritic N = 10, Celastrol early group N = 15 and Celastrol late group N = 15. (C) Immunohistochemical quantification was performed using an image analysis software written in MATLAB to identify and count the number of positive cells for each antibody in representative sections. Notice that both celastrol early and late-treated rats showed a significant reduction in the number of CD68 and CD163 positive cells in comparison with arthritic rats at day 22. Healthy N = 5, Arthritic N = 5, Celastrol early group N = 5 and Celastrol late group N = 5. Data are expressed as median with interquartile range. Differences were considered statistically significant for p-values<0.05, according to the Kruskal-Wallis (Dunn´s Multiple Comparison tests) and Mann–Whitney tests.