NecroX-5

NecroX-5 ameliorates bleomycin-induced pulmonary fibrosis via inhibiting NLRP3-mediated epithelial-mesenchymal transition

Background: Pulmonary fibrosis is a progressive and usually lethal pulmonary disease. Despite considerable research efforts, no effective therapeutic strategy for pulmonary fibrosis has been developed. NecroX-5 has been reported to possess anti-inflammatory, anti-oxidative and anti-tumor activities. In the present study, we aimed to determine whether NecroX-5 exhibits antifibrotic property in bleomycin (BLM)-induced pulmonary fibrosis. Results: We found that pre-treatment with NecroX-5 alleviated inflammatory response, reduced oxidative stress, inhibited epithelial-mesenchymal transition (EMT), and ameliorated pulmonary fibrosis in vivo and in vitro. Our data further indicated that NecroX-5 substantially reduced activation of NLRP3 inflammasome and TGF-β1/Smad2/3 signaling in vivo and in vitro. Additionally, NLRP3 overexpression significantly reversed the protective effects of NecroX-5 in lung epithelial cells exposed to BLM. Conclusions: Overall, our results demonstrate the potent antifibrotic properties of NecroX-5 and its therapeutic potential for pulmonary fibrosis.

NecroX-5 alleviate lipopolysaccharide-induced acute respiratory distress syndrome by inhibiting TXNIP/NLRP3 and NF-κB

The activation of NLRP3 inflammasome and NF-κB pathway, associating with oxidativestress, have been implicated in the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). NecroX-5 has been reported to exhibit theeffectsofanti-oxidation and anti-stress in various diseases. However, the role of NecroX-5 in ALI has not been explicitly demonstrated. The aim of this study was to explore the therapeutic effects and potential mechanism action of NecroX-5 on ALI. Here, we found that NecroX-5 pretreatment dramatically diminished the levels of IL-1β, IL-18 and ROS in in RAW264.7 cells challenged with LPS and ATP. Furthermore, NecroX-5 suppressed the activation of NLRP3 inflammasome and NF-κB signalpathway. In addition, NecroX-5 also inhibited the thioredoxin-interacting protein (TXNIP) expression. In vivo, NecroX-5 reduced the LPS-induced lung histopathological injury, the number of TUNEL-positive cells, lung wet/dry (W/D) ratio, levels of total protein and inflammatory cytokines in the bronchoalveolar lavage fluid (BALF) in mice. Additionally, LPS-induced upregulation of myeloperoxidase (MPO), ROS production and malondialdehyde (MDA) were inhibited by NecroX-5 administration. Thus, our results demonstrate that NecroX-5 protects against LPS-induced ALI by inhibiting TXNIP/NLRP3 and NF-κB.

NecroX-5 Can Suppress Melanoma Metastasis by Reducing the Expression of Rho-Family GTPases

NecroX-5 (NX-5) is a cell-permeable necrosis inhibitor with cytoprotective effects. Although it has been reported to inhibit lung and breast cancer metastasis by modulating migration, its therapeutic effect on melanoma metastasis is still unknown. In this study, we examined the anti-metastatic effect of NX-5 on melanoma cell lines and its related therapeutic mechanism. The anti-metastatic effect of NX-5 on melanoma cell lines was determined using a transwell migration assay. We performed a quantitative real-time polymerase chain reaction and western blot analysis to measure changes in the expression of mRNA and protein, respectively, for major mediators of Rho-family GTPases after NX-5 treatment in melanoma cells. In addition, after constructing the 3D melanoma model, the expression of Rho-family GTPases was measured by immunohistochemistry. NX-5 (10 μM and 20 μM) treatment significantly reduced melanoma cell migration (p < 0.01). Additionally, NX-5 (20 μM) treatment significantly decreased the mRNA and protein expression levels of Cdc42, Rac1, and RhoA in melanoma cells compared with the untreated group (p < 0.001 and p < 0.05, respectively). Immunohistochemistry for our 3D melanoma model showed that Cdc42, Rac1, and RhoA were constitutively expressed in the nuclei of melanoma cells of the untreated group, and NX-5 treatment decreased their expression. These results demonstrate that NX-5 can suppress melanoma metastasis by reducing the expression of Rho-family GTPases.

NecroX-5 ameliorates inflammation by skewing macrophages to the M2 phenotype

This study aimed to evaluate the role of NecroX-5, a powerful anti-inflammatory agent, on the functional plasticity of macrophages and the possible underlying mechanism using RAW264.7 cells, thioglycollate-elicited peritoneal macrophages from C57BL/6 mice, and a murine model of dextran sodium sulfate (DSS)-induced colitis. The change in cell morphology was examined by scanning electron microscopy. The expression of CD206, arginase (Arg)-1, and inducible nitric oxide synthase (iNOS) were examined by western blotting. The production of inflammatory cytokines was detected by enzyme-linked immunosorbent assays and statistical comparisons were made. The results showed that treatment of RAW264.7 cells with NecroX-5 caused an elongated shape in comparison to non-treated cells. The expression levels of macrophage mannose receptor CD206 and Arg-1, specific markers of M2 cells, were significantly upregulated by NecroX-5 treatment, while those of iNOS (M1 macrophages) was decreased. In addition, NecroX-5 significantly reduced the secretion of inflammatory cytokines, while interleukin (IL)-4 and IL-13 secretion in the supernatant was significantly enhanced. Treatment with NecroX-5 considerably ameliorated the progression of DSS-induced colitis and significantly inhibited the mRNA expression of pro-inflammatory cytokines, including tumor necrosis factor-α and IL-1β. Taken together, our findings demonstrated that NecroX-5 might dampen inflammation by switching the M1 phenotype to the M2 phenotype due to IL-4 and IL-13 induction.

NecroX-5 protects mitochondrial oxidative phosphorylation capacity and preserves PGC1α expression levels during hypoxia/reoxygenation injury

Although the antioxidant and cardioprotective effects of NecroX-5 on various in vitro and in vivo models have been demonstrated, the action of this compound on the mitochondrial oxidative phosphorylation system remains unclear. Here we verify the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity during hypoxia-reoxygenation (HR). Necrox-5 treatment (10 ?M) and non-treatment were employed on isolated rat hearts during hypoxia/reoxygenation treatment using an ex vivo Langendorff system. Proteomic analysis was performed using liquid chromatography-mass spectrometry (LC-MS) and non-labeling peptide count protein quantification. Real-time PCR, western blot, citrate synthases and mitochondrial complex activity assays were then performed to assess heart function. Treatment with NecroX-5 during hypoxia significantly preserved electron transport chain proteins involved in oxidative phosphorylation and metabolic functions. NecroX-5 also improved mitochondrial complex I, II, and V function. Additionally, markedly higher peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC1α) expression levels were observed in NecroX-5-treated rat hearts. These novel results provide convincing evidence for the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity and in preserving PGC1α during cardiac HR injuries.