Indirubin protects chondrocytes and alleviates OA by inhibiting the MAPK and NF-ΚB pathways

Pharmacological relevance: Indirubin (IR) is a key active ingredient in the traditional Chinese medication QingDai, also called indigo naturalis, which are extensively used in China to treat chronic diseases, such as inflammation and cancer. However, the function of IR in reducing chondrocyte inflammation in osteoarthritis (OA) is still unclear.Aim of the study: The aim of this research was to examine how IR inhibits arthritis and to highlight some of its cellular-level processes. Materials and methods: Chondrocytes from the knee joint of C57 mice were gathered and grown for in vitro tests and used to determine the toxicity of IR toward chondrocytes using a CCK8 kit. Chondrocytes were treated with IL-1 beta and IR or with IL-1 beta alone, and western blotting was used to determine the expression levels of inflam-matory mediators. Meanwhile, through the identification and examination of pertinent markers via quantitative PCR. By using PCR assays, western blotting, toluidine blue staining and safranin O staining, the expression of proteoglycan (AGG) and type II collagen (collagen II) was investigated. Furthermore, western blotting was used to detect activation of the NF-Kappa B and MAPK signaling pathways, and immunofluorescence was used to detect p65 nuclear translocation. In an in vivo experiment, C57BL/6 mice were subjected to destabilization of the medial meniscus (DMM) surgery to produce an OA model, and IR was injected into the articular cavity for 8 weeks. Eventually, the mice were killed, and samples of the knee joints were obtained for histological examination and analysis.Results: IR significantly reduced the expression of inflammatory regulators in chondrocytes treated with IL-1 beta, including iNOS and COX-2. Inhibition of IL-1 beta induced production of the key catabolic enzymes MMP3, MMP13 and A5. Additionally, an improvement in the downregulation of collagen II and AGG expression was observed. Moreover, IR prevented the aberrant IL-1 beta-induced activation of the NF-Kappa B and MAPK signaling pathways, which resulted in downregulation of p65 and p38 expression. Compared to the DMM group, the severity of cartilage injury in animals was dramatically lessened and OARSI scores were lower in the treated groups. Conclusion: According to the above findings, IR is quite effective in preventing arthritis both in vivo and in vitro, suggesting that IR may be employed as a possible anti-arthritis drug.