Caspase-1 Assay Kit (Colorimetric)

Osteoarthritis (OA) affects more than 300 million people worldwide and it is about to become the first disabling disease. OA is characterized by the progressive degradation of the articular cartilage but is a disease of the whole joint. Articular innate immune responses (IIR) associated with tissue degradation contribute to its progression. However, no treatment is available to block these IIRs. Through data text mining and computational pharmacology, we identified two clinical available drugs, naloxone, and thalidomide, with potential inhibitory properties on toll-like receptor 4 (TLR4), a major activator of these IIR. Proteome analysis confirmed that activation of this receptor or the IL1 receptor generated OA-like and gout-like proteomic changes in human primary chondrocytes. Both compounds were found to block TLR4 complex and inhibit TLR4 and IL1R-mediated IIR in OA chondrocytes, osteoblasts, and synoviocytes. Furthermore, naloxone and thalidomide inhibitory effects involved the downregulation of the NLRP3 inflammasome pathway, which is downstream of TLR4/IL1R signaling. We demonstrated that these compounds, within a therapeutic range of concentrations, exhibited anti-inflammatory and anti-catabolic properties in joint primary OA cells without any toxic effect. This data underpins naloxone & thalidomide repurpose to treat OA-associated inflammatory responses.

Trillium tschonoskii Maxim (TTM), a traditional Chinese medicine, has been demonstrated to have a potent anti-tumor effect. Recently, polyphyllin VI (PPVI), a main saponin isolated from TTM, was reported by us to significantly suppress the proliferation of non-small cell lung cancer (NSCLC) via the induction of apoptosis and autophagy in vitro and in vivo. In this study, we further found that the NLRP3 inflammasome was activated in PPVI administrated A549-bearing athymic nude mice. As is known to us, pyroptosis is an inflammatory form of caspase-1-dependent programmed cell death that plays an important role in cancer. By using A549 and H1299 cells, the in vitro effect and action mechanism by which PPVI induces activation of the NLRP3 inflammasome in NSCLC were investigated. The anti-proliferative effect of PPVI in A549 and H1299 cells was firstly measured and validated by MTT assay. The activation of the NLRP3 inflammasome was detected by using Hoechst33324/PI staining, flow cytometry analysis and real-time live cell imaging methods. We found that PPVI significantly increased the percentage of cells with PI signal in A549 and H1299, and the dynamic change in cell morphology and the process of cell death of A549 cells indicated that PPVI induced an apoptosis-to-pyroptosis switch, and, ultimately, lytic cell death. In addition, belnacasan (VX-765), an inhibitor of caspase-1, could remarkably decrease the pyroptotic cell death of PPVI-treated A549 and H1299 cells. Moreover, by detecting the expression of NLRP3, ASC, caspase-1, IL-1β, IL-18 and GSDMD in A549 and h1299 cells using Western blotting, immunofluorescence imaging and flow cytometric analysis, measuring the caspase-1 activity using colorimetric assay, and quantifying the cytokines level of IL-1β and IL-18 using ELISA, the NLRP3 inflammasome was found to be activated in a dose manner, while VX-765 and necrosulfonamide (NSA), an inhibitor of GSDMD, could inhibit PPVI-induced activation of the NLRP3 inflammasome. Furthermore, the mechanism study found that PPVI could activate the NF-κB signaling pathway via increasing reactive oxygen species (ROS) levels in A549 and H1299 cells, and N-acetyl-L-cysteine (NAC), a scavenger of ROS, remarkably inhibited the cell death, and the activation of NF-κB and the NLRP3 inflammasome in PPVI-treated A549 and H1299 cells. Taken together, these data suggested that PPVI-induced, caspase-1-mediated pyroptosis via the induction of the ROS/NF-κB/NLRP3/GSDMD signal axis in NSCLC, which further clarified the mechanism of PPVI in the inhibition of NSCLC, and thereby provided a possibility for PPVI to serve as a novel therapeutic agent for NSCLC in the future.