Fig 1: Curcumenol inhibits the inflammatory pathways, especially the TNF signaling pathway in NP cells in vitro and lumbar spine instability mouse model in vivo. In general, it inhibits the up-regulation of TRAF3 induced by TNFa, and the subsequent phosphorylation and activation of I?Ba with P65. Finally, it prevents the following activation of inflammatory factors like MMP families. Created with BioRender.com
Fig 2: Curcumenol showed little cytotoxicity in NP cells and down-regulated inflammatory pathways based on RNA-seq in vitro. (A) Chemical structure of Curcumenol. (B–D) Cell Counting Kit-8 assay results of NP cells stimulated with Curcumenol at different concentrations (0, 12.5, 25, and 50 µM) and different time periods (ranging from 24 to 72 h). (E) Ratio of up-regulated mRNA in NP cells treated with Curcumenol (50 µM) versus DMSO (1:1000) using KEGG pathway analyses (3 paired biological replicates). (F) Ratio of changed mRNA in NP cells treated with Curcumenol (50 µM) versus DMSO (1:1000) using Volcano Plot analyses (3 paired biological replicates). (G) Heat Map of changed mRNA in NP cells treated with Curcumenol (50 µM) versus DMSO (1:1000) using Volcano Plot analyses (3 paired biological replicates). (H) Western blot analysis of CXCL10 and TRAF3 expression in NP cells stimulated with TNFa (10 ng/ml) or/and 50 µM Curcumenol for 24 h (I) RT-qPCR analysis of the relative mRNA expression levels of CXCL10 and TRAF3 expression in NP cells stimulated with TNFa (10 ng/ml) or/and 50 µM Curcumenol for 24 h. All data are presented as mean ± SD from three replicates. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.
Fig 3: IRAK4 is not involved in TLR4 endocytosis or TRAF3 and IRF3 activation(A and B) Study of TLR4 endocytosis in WT, Irak4 Ki, and Irak4-/- BMDMs and control Cd14-/- immortalized BMDMs, stimulated with LPS for up to 60 min. (A) Histogram of surface TLR4 in the indicated strains unstimulated, stimulated with LPS for 60 min, or unstimulated cells stained with isotype control antibody. (B) Quantification of surface TLR4 after LPS stimulation for up to 60 min. (C) Immunoblot of ubiquitinated TRAF3 pulled down from indicated BMDMs treated with LPS for 30 min.(D and E) Kinetic study of IRF3 phosphorylation by immunoblot analysis of whole cell lysates from WT, Irak4 Ki, Irak4-/-, and Trif-/- BMDMs (D) and densitometric analysis (E).(F) Quantification of CCL5 produced by WT, Irak4 Ki, and Irak4-/-, MyD88-/-, Trif-/-, and MyD88-/-Trif-/- BMDMs after LPS stimulation for 24 h. All stimulations were done with LPS 100 ng mL-1. *p < 0.05 in comparison with WT (one-way analysis of variance with Tukey’s multiple comparisons test). (A) Data are representative of three independent experiments. Data from two (E) or three (B, F) independent experiments (mean and SEM). (C and D) Images are representative of two independent experiments.
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