Analyses of Transcriptomics upon IL-1-Stimulated Mouse Chondrocytes and the Protective Effect of Catalpol through the NOD2/NF-B/MAPK Signaling Pathway

The overall objective of this study was to investigate the mechanism of inflammation on chondrocyte injury and the protective effect of catalpol on chondrocytes in an inflammatory environment. Chondrocytes were isolated and cultured from the knee joints of three-day-old newborn mice. Alcian Blue staining and the immunocytochemistry staining of type II collagen were used to identify the purity of chondrocytes. Primary chondrocytes were stimulated by IL-1 beta (10 ng/mL) and subjected to transcriptome analysis. Differentially expressed genes (DEGs) were further analyzed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. In this experimental study, we performed the viability assay to determine the effects of different concentrations of catalpol on the cell viability of chondrocytes. Chondrocytes were seeded in six-well plates and exposed to 10 mu M catalpol 2 h prior to treatment with IL-1 beta (10 ng/mL). Quantitative real-time (qPCR) and Western blotting were performed to evaluate the RNA and protein expression, respectively. Based on the results of transcriptomics analysis, we found the NOD2 signaling pathway, the NF-kappa B signaling pathway, and the MAPK signaling pathway showed significant changes in chondrocyte damage caused by inflammation. Catalpol (10 mu M and 100 mu M) could significantly reduce NO, IL-6, IL-1 beta, and TNF-alpha in supernatant of chondrocytes. Catalpol significantly inhibited the mRNA expression of IL-1, IL-6, and IL-12 in chondrocytes induced by IL-1 beta. Catalpol markedly inhibited MMP3, MMP13 mRNA, and protein levels. Catalpol could significantly reduce TNF-alpha mRNA levels in inflammatory chondrocytes. Inflammation causes significant increases in mRNA levels and protein levels of NOD2, mRNA levels, and protein levels were markedly suppressed by catalpol. In addition, catalpol could significantly increase IKB alpha protein levels and significantly lower intranuclear P65 levels. Catalpol significantly lowered the phosphorylation protein levels of ERK, p38, and JNK. Our transcriptomic analysis demonstrated that the activation of NOD2 and its downstream pathways, NF-kappa B and MAPK, is an important cause of the inflammatory injury to chondrocytes induced by IL-1 beta. Catalpol inhibited the activation of the NOD2 signaling pathway, which reduced the phosphorylation of ERK, p38, and JNK, inhibited the degradation of I kappa B alpha, inhibited p65 translocation into the nucleus, reduced the release of inflammatory cytokines, and attenuated the inflammatory damage to chondrocytes.