Celastrol reduces the number of T cells and B cells present in the synovial membrane, and suppresses synovial cell proliferation.

<p>(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.</p

Histological images of joints after celastrol treatment.

<p>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.</p

(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.

<p>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.</p

Celastrol suppresses arthritic inflammation and tissue damage locally in the joints of AIA rats.

<p>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.</p

Celastrol reduces the serum levels of IL-6 in arthritic rats.

<p>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.</p

Celastrol reduces the number of synovial CD68+ macrophages.

<p>(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.</p

Serum quantification of IL6.

<p>Serum samples collected at day 11 and 22 post disease induction were analyzed by ELISA technique. IL6 was increased in arthritic rats at day 11 and 22 (p = 0.0003 and p<0.0001vs healthy controls, respectively). Differences were considered statistically significant for p-values<0.05, according to the Mann–Whitney tests Healthy D11 N = 11, Healthy D22 N = 21, Arthritic D11 N = 16 and Arthritic D22 N = 23.</p

Bone turnover markers quantification.

<p>Serum samples collected at day11 and 22 post disease induction were analyzed by ELISA technique. Bone resorption marker, CTX-I (A) and bone formation marker, P1NP (B) were increased in arthritic rats at day 22 (p<0.0001 and p = 0.0007, respectively). Results also demonstrate increased values of CTX-I in arthritic rats at day 11 when compared with healthy controls (p = 0.0218). Differences were considered statistically significant for p-values<0.05, according to the Mann–Whitney tests. Healthy D11 N = 11, Healthy D22 N = 18, Arthritic D11 N = 16 and Arthritic D22 N = 18.</p

Histological images of joints after 11 and 22 days of disease induction.

<p>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 50X. Bar: 100 μm.</p

Micro-computed tomography (micro-CT)—Trabecular analysis of tibiae rat sample.

<p>MicroCT images from healthy and arthritic tibiae rats (A). Images acquired with SkyScan 1272, Bruker microCT, Belgium. Results showed decreased values of the ratio bone volume/tissue volume (B), trabecular thickness (C) and number (D) in arthritic group at day 11 and 22 post disease induction. Trabecular bone also showed increased values of trabecular separation (E), porosity (F) and structural model index in both arthritic groups (G). Differences were considered statistically significant for p-values<0.05, according to the Mann–Whitney tests. Healthy D11 N = 11, Healthy D22 N = 30, Arthritic D11 N = 16 and Arthritic D22 N = 31.</p