SerpinA3N limits cartilage destruction in osteoarthritis by inhibiting macrophage-derives leukocyte elastase

Inflammatory mediators such as interleukin 6 (IL-6) are known to activate catabolic responses in chondrocytes during osteoarthritis (OA). This study aimed to investigate the downstream targets of IL-6 in murine chondrocytes. RNA-sequencing was performed in murine articular chondrocytes treated with IL-6 in hypoxia (1%O2) or normoxia (21%O2). RNA-sequencing revealed that SerpinA3N is a major target of IL-6 in articular chondrocytes. The expression of SerpinA3N was increased in OA cartilage and further investigated. Mouse primary chondrocytes were cultured with 21% (normoxia) or 1% (hypoxia) oxygen in 10% FCS DMEM as described, then stimulated or not with IL-6 or IL-6R (100 ng/ml each) for 12 hr in serum-free DMEM. Total RNA was isolated by using TRIzol reagent (Life Technologies) and further purified (RNeasy Mini kit, Qiagen). At each step, RNA was checked on a bioanalyzer (RNA 6000 Pico Kit, Agilent) and quantified on Qubit. Purification was performed with the Ribo-Zero Magnetic Gold kit (Tebu Bio) to remove ribosomal RNA, followed by another purification with the RNeasy MinElute Cleanup Kit (Qiagen). cDNA libraries were constructed by using the TruSeq RNA Sample Prep kit (Illumina). Three barcoded cDNA libraries were made for each condition, at each time point, and RNA-seq (50-bp single-end reads) was performed on an Illumina HiSeq sequencer. Reads were aligned to the mouse genome (mm10) with Tophat2, then reads were counted with HTSeq. The DESeq2 package was used for analysis of differential gene expression, and Benjamini-Hochberg adjusted p values were calculated to adjust for multiple testing.