Follistatin alleviates articular cartilage degradation induced by carrageenan

INTRODUCTION: Activins, which belong to the Transforming Growth Factor-beta/ Bone Morphogenetic Protein (TGF/BMP) superfamily, are circulating cytokines involved in the process of inflammation of various tissues [1]. Follistatin is an endogeneous extracellular inhibitor for activins which binds and interfere the interaction between activins and their receptors. Jones et al showed that administration of follistatin reduced inflammatory response mediated by activins and mortality in endotoxemia in mice [2]. These data enhance the importance of activin signals in the pathogenesis of inflammation-mediated diseases and follistatin as a therapeutic target for them. Carrageenan is a sulphatedmucopolysaccharide derived from the Irish moss Chondruscrispus or from red Scottish seaweed. It is known for its remarkable capacity to stimulatelocal inflammation dominated by intense macrophage aggregationand by fibroblastic proliferation. It is reported that single intra-articular injection of carrageenan initiates a localized synovial inflammatory response, which results in a decrease in both the proteoglycancontent and in the rate of proteoglycan synthesis in the articular cartilage[3].Here we report that follistatin alleviatesarticular cartilage degradation induced by carrageenan. METHODS: This study was approved and conducted in accordance with the guideline of the animal committee of Tokyo Medical and Dental University. Male C57Bl/6J mice (12weeks old) were purchased from ORIENTAL YEAST co.,Ltd (Tokyo, Japan). They were housed under a 12-h light-dark cycle and allowed food and water ad libitum. Twelve mice were randomly divided into two groups (n=6/group). Mice were anesthetized by the inhalation of 5% isoflurane in oxygen. Under deep anesthesia, a solution of 30μg lamda-carrageenan (Sigma-Aldrich) in 5μL saline was injected into the left knee joint through the lateral margin of the patella tendon. Recombinant mouse follistatin (25ng in 5μl in physiological saline, Sigma-Aldrich) was injected into the left knee at 30 min before carrageenan challenge. The animals were sacrificed at 3 days after the challenge. Knee joints were dissected, fixed in 4% paraformaldehyde, decalcified in 20% EDTA (pH 7.4), and embedded in paraffin. Five μm-thick sagittal sections (medial condyle) were prepared and stained with safranin-O/fast green to evaluate the extent of cartilage proteoglycan loss. To evaluate the loss of proteoglycan from articular cartilage, we developed semi-quantitative scoring system indicated in table 1. Histological sores for femurs and tibiae were evaluated independently and total scores were compared between the two groups. The results were presented as mean +/SD. Statistical analyses were performed using Mann-Whitney’s U test and p values <0.05 were considered significant. RESULTS: As shown previously, dyeabilityof articular cartilage by safranin-o was significantly reduced by the singleintra-articular injection of carrageenan at 3 days (Fig1 upper panel). However, we did not observe obvious alteration in the articular surface structure at this stage. Interestingly, such a loss of dyeability was not observedby the preinjection of follistatin(Fig1 lower panel).We repeated this experiment six times and observed a similar result in each experiment. To evaluate the anti-catabolic effect of follistatinsemi-quantitatively, we employed a scoring system shown in table1. As shown in Fig2, preinjection of follistatinsignificantly rescued a loss of dyeability induced by carrageenan (p<0.05).