Fig 2: Ripk3-mediated signals are indispensable for persisting synovitis in hTNFtg Ikk2Ms-KO mice. a Representative histological images of H&E-, TB- and TRAP-stained ankle joint sections of 8-week-old hTNFtg Ikk2f/f Ripk3−/− (n = 11) and hTNFtg Ikk2Ms-KO Ripk3−/− mice (n = 13) in comparison to hTNFtg Ikk2Ms-KO and Ikk2f/f mice. Note the differences in synovium and adjacent tissues in respective areas indicated by the arrows. b Survival rates of Ikk2Δ/Δ Ripk3−/− and hTNFtg Ikk2Δ/Δ Ripk3−/− SF cultures treated with TNF (T), zVAD (Z), and nec-1 (N) as indicated. c Representative immunodetection of pMlkl, Mlkl and Ripk3 levels in the cell extracts of Ikk2Δ/Δ Ripk3−/− and hTNFtg Ikk2Δ/Δ Ripk3−/− SFs treated as indicated compared to control Ikk2Δ/Δ Ripk3+/+ and hTNFtg Ikk2Δ/Δ Ripk3+/+ samples, respectively (n = 4 per genotype, 2 experiments). d Representative immunodetection of Casp8 (C8), Cleaved Casp3 (CC3), Xiap, cFLIP(s) and IKK2 levels in cell extracts of Ikk2Δ/Δ Ripk3−/− and hTNFtg Ikk2Δ/Δ Ripk3−/− SFs treated as indicated compared to control Ikk2f/f Ripk3−/− and hTNFtg Ikk2f/f Ripk3−/− samples, respectively (the non-hTNFtg control cultures are indicated as WT) (n = 4 per genotype, 2 experiments). Scale bar: 500 μm. Data are presented as the mean ± SEM. ***P < 0.001 one-way ANOVA test (b). Unprocessed original scans of blots are shown in Supplementary Fig. 8

Fig 3: Molecular docking results. (A,B) Schematic diagram of the binding mode of chrysin to ALOX12 protein (left panels) and details of the binding pose (right panels). (C,D) Schematic diagram of the binding mode of 7,8-DHF to IKKB protein (left panels) and details of the binding pose (right panels). (E,F) Schematic diagram of the binding mode of DHC to TRPV1 protein (left panels) and details of the binding pose (right panels).

Fig 4: Validation of the effects of DHC, chrysin, and 7,8-DHF on inflammation-related proteins. RAW264.7 cells were exposed to LPS and treated with DHC, chrysin, or 7,8-DHF for 12 h. (A,B) The effects of DHC on the expressions of TRPV1, NLRP3, Caspase-1, p-NF-κB, Bip, and ATF4 were examined by Western blot (n = 3). (C,D) The effects of chrysin on the expressions of ALOX12, p-IκBα, IκBα, p-NF-κB, NLRP3, Caspase-1, Bip, and ATF4 were detected by Western blot. (n = 3). (E,F) Western blot was employed to detect the effects of 7,8-DHF on the expressions of IKKB, p-IκBα, IκBα, p-NF-κB, NLRP3 and Caspase-1 (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. Control groups; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs. LPS-treated groups.

Fig 5: Enhanced osteogenic differentiation and matrix mineralization in IKKβ-deficient MC3T3–E1 cells. (A) Phosphorylation of p65 was examined by Western blot analysis in steady state. The phosphorylation of p65 at Ser536, an important site of IKKβ action was significantly inhibited. (B) The wildtype (WT) control and Ikbkb−/− clone 19, 24, 26 were cultured in the control medium or osteogenic medium for 8 days. ALP activity was analyzed using ALP staining. (C) Microscopy images of Alizarin Red S staining of WT control and Ikbkb−/− cells induced by osteogenic medium. (B,C) Results were representatives of three independent experiments. (D) Mineralization described in (C) was quantified by de-staining Alizarin Red S with 10% cetylpyridinium chloride and the absorbance was measured at 560 nm (n = 3), the level of mineralization in Ikbkb−/− cells was compared with WT cells. Statistics by one-way ANOVA. (E) FACS plots showing non-transfected control and cells transfected with Ikbkb targeting plasmids. We sorted 10,000 DsRed2/ECFP double-positive cells as Ikbkb−/− cells bulk, and subjected to phenotype experiment. Alizarin Red S staining (F) and quantification (G) of Ikbkb−/− cells bulk induced by osteogenic medium for 16 days compared with WT cells (n = 3). Statistics by two-tailed t-test. (H) The deletion of IKKβ increased mRNA levels of osteoblast differentiation marker genes as determined by real-time polymerase chain reaction (n = 3), compared between WT and IKKβ-deficient cells. Statistics by one-way ANOVA. Scale bars: 500μm. Error bars represent ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant.