Anti-inflammatory and pro-anabolic effects of 5-aminosalicylic acid on human inflammatory osteoarthritis models

Abstract

Background: Osteoarthritis (OA) is the most common degenerative joint disease, mainly affecting the elderly worldwide, for which the drug treatment remains a major challenge. Low-grade inflammation plays a pivotal role in OA onset and progression. Exploration of notable anti-inflammatory and disease-modifying drugs on human samples could facilitate the evaluation of therapeutic strategies for OA.

Methods: The anti-inflammatory drug 5-aminosalicylic acid (5-ASA) is a first-line drug for ulcerative colitis (UC), however no study has explored the effects of 5-ASA on articular chondrocytes. In this work, both in vitro (chondrocyte pellets) and ex vivo (osteochondral explants) human inflammatory OA models were applied to evaluate the effects of 5-ASA.

Results: In the inflammatory pellet model, 5-ASA remarkably downregulated the gene expression of interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2) while upregulating proteoglycan 4 (PRG4) and cartilage oligomeric matrix protein (COMP) gene expression. Total glycosaminoglycan (GAG) synthesis by pellets was markedly increased in 5-ASA-treated groups compared with the inflammatory group. In conditioned medium, inflammatory mediators (IL-8, nitric oxide) were markedly inhibited upon 5-ASA treatment. Moreover, histological staining showed 5-ASA retained proteoglycan content and inhibited degradation of extracellular matrix (ECM) core components, aggrecan (ACAN) and collagen type II (COL2). In the inflammatory explant model, 5-ASA mitigated signs of OA development by reducing inflammatory mediators and GAG loss.

Conclusions: These findings suggest that 5-ASA has anti-inflammatory and pro-anabolic effects on human chondrocyte pellet and osteochondral explant inflammatory OA models.

The translational potential of this article: Disease-modifying OA drugs are an unmet clinical need for the treatment of OA. Our study explored and demonstrated the anti-inflammatory and protective effects of 5-ASA on in vitro and ex vivo human inflammatory OA models, showing its translational potential for OA treatment.

Keywords: 5-aminosalicylic acid; Chondrocyte pellet; Inflammatory model; Osteoarthritis; Osteochondral explant; Treatment.

Figure 1

Experimental design and cell viability of chondrocytes treated with 5-ASA. A. Molecular strucutre of 5-ASA. B. Expeimental design of in vitro (chondrocyte pellet) inflammatory OA model. C. Experimental design of ex vivo (osteochondral explant) inflammatory OA model. D. Cell viability of monolayer-cultured chondrocytes treated with various concentrations of 5-ASA (one way ANOVA, n ​= ​12). Data were presented with mean ​+ SD. ∗∗∗p ​< ​0.001, ∗∗∗∗p ​< ​0.0001. B and C were made with Biorender.

Figure 2

5-ASA regulated anabolism- and inflammation-related gene expression levels of chondrocytes in pellet OA model after 3-day treatment (COX-2, COMP, one-way ANOVA; IL-6, IL-8, ACAN, PRG4, COL2A1, Kruskal–Wallis test; n ​= ​7). Data were presented as boxplot; center line, median; box limits, 25th to 75th percentiles; whiskers, min to max. ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001, ∗∗∗∗p ​< ​0.0001.

Figure 3

5-ASA inhibited the release of inflammatory markers in pellet OA model. Cumulative content of IL-6 (A), IL-8 (B) and Nitric oxide (NO) (C) in the conditioned medium (IL-6, Kruskal–Wallis test; IL-8, NO, one-way ANOVA; n ​= ​4). Data were presented as mean ​+ SD. a∗, p ​< ​0.05 compared with Control group; b∗, p ​< ​0.05 compared with OA group; c∗, p ​< ​0.05 compared with 5-ASA 10 ​mM group; d∗, p ​< ​0.05 compared with 5-ASA 20 ​mM group.

Figure 4

5-ASA promoted GAG synthesis after treatment for 14 days in pellet OA model. GAG content (A), DNA content (B), GAG/DNA (C) per pellet. D, E. Cumulative GAG content released in medium. F. Total GAG synthesis by pellets after 14-day treatment period. G. Percentage of GAG in medium to total GAG synthesis. Data were shown as mean ​+ SD. n ​= ​3. Statistical analysis was performed using one-way ANOVA. ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001, ∗∗∗∗p ​< ​0.0001.

Figure 5

5-ASA preserved PG in pellet OA model. A. Safranin O/Fast Green staining of pellets after 5-ASA treatment for 3, 8, 14 days respectively. Scale bars, 100 ​μm. B, C. Semi-quantitative analysis of Safranin O staining on Day 8 and Day 14 (Day 8, one-way ANOVA; Day 14, Kruskal–Wallis test; n ​= ​3–4). Data were shown as mean ​+ SD. ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001, ∗∗∗∗p ​< ​0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Figure 6

5-ASA reduced aggrecan degradation in pellet OA model. A. Immunohistochemistry staining of ACAN after 5-ASA treatment for 8 and 14 days. Scale bars, 100 ​μm. Neg, negative control. B, C. Semi-quantitative analysis of ACAN immunostaining on Day 8 and Day 14 (Day 8, one-way ANOVA; Day 14, Kruskal–Wallis test; n ​= ​3–4). Data were shown as mean ​+ SD. ∗p ​< ​0.05, ∗∗p ​< ​0.01.

Figure 7

5-ASA reduced collagen type II (COL2) degradation in pellet OA model. A. Immunohistochemistry staining of COL2 after 5-ASA treatment of 8 and 14 days. Scale bars, 100 ​μm. Neg, negative control. B, C. Semi-quantitative analysis of COL2 immunostaining on Day 8 and Day 14 was performed using one-way ANOVA (n ​= ​3–4). Data were shown as mean ​+ SD. ∗p ​< ​0.05, ∗∗p ​< ​0.01.

Figure 8

5-ASA upregulated anabolic gene expression and downregualated inflammatory gene expression of chondrocytes in explant OA model after 7 days treatment (n ​= ​3–4). Statistical analysis was performed using Kruskal–Wallis test. Data were presented as boxplot; center line, median; box limits 25th to 75th percentiles; whiskers, min to max. ∗p ​< ​0.05.

Figure 9

5-ASA attentuated OA progerssion in inflammatory OA model established with human osteochondral explants. A. Cumulative content of IL-6, IL-8, and NO released in conditioned medium (n ​= ​3–4). B. Representative images of Safranin O/Fast Green staining of the cartilage after 7 days. Scale bars, 200 ​μm. Yellow dotted line separates the non- or weakly stained superficial area from the intensely stained deep area in cartilage. C. Semi-quantitative analysis of Safranin O staining (n ​= ​3). Statistical analysis was performed using one-way ANOVA. Data were shown as mean ​+ SD. a∗, p ​< ​0.05 compared with Control group; b∗, p ​< ​0.05 compared with OA group; c∗, p ​< ​0.05 compared with 5-ASA 10 ​mM group; d∗, p ​< ​0.05 compared with 5-ASA 20 ​mM group. ∗p ​< ​0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)