Fig 2: Redd1 expression activates NF-?B signaling in senescent cardiomyocytes. (A, B) Western blot analysis of total and phosphorylated p65 and p38 MAPK in Dox-treated H9c2 cardiomyocytes following Redd1 overexpression and knockdown. (C) Immunofluorescent detection of nuclear translocation of p65 in Dox-treated H9c2 cardiomyocytes following Redd1 overexpression and knockdown. (D) Quantification of data from p65 nuclear translocation assays (n = 4 per group). *p < 0.05 vs. Lv-shNC group. Data are mean ± SEM. *p < 0.05 vs. control group. &p < 0.05 vs. Lv-Redd1 group.

Fig 3: TWEAK activates canonical NF-?B signalling and Notch activation and mediates the response of CD163-/- mice to HLI.(a) Analysis of plasma from WT and CD163-/- mice before and after femoral ligation for TWEAK and sCD163 levels by ELISA (n=5 per group). (b) Laser doppler analysis of CD163-/- mice 3, 7, 14, 21 and 28 days after femoral ligation administered an isotype control or TWEAK blocking antibody (n=5 per group). (c) Immunoblotting of CD163-/- IL at 14 days after femoral ligation with administration of isotype control or TWEAK blocking antibody (n=5 per group) with graphs showing quantitation of densitometry for phosphorylated/total p65 and NICD below. (d) Immunostaining for VE-cadherin (green) and ß-dystroglycan (red) in CD163-/- limbs with 14 day administration of TWEAK (5 or 25 µg kg-1 day-1) or saline (control) (n=5 per group). Scale bars, 100 µm. The graphs show the number of VE-cadherin positive cells/myofibre and the average muscle fibre area. (e) Immunoblotting of CD163-/- limbs with 14 day administration of TWEAK (5 or 25 µg kg-1 day-1) or saline (control) (n=5 per group) with graphs showing quantitation of densitometry for phosphorylated p65/total p65, p52 and NICD. All bars show mean±s.e.m. *P<0.05 versus WT in a; versus CD163-/- control in b,c and e. P<0.05 versus other groups in d,e. Comparisons between groups were achieved using a two-sided student's t-test. For multiple group comparisons, we utilized a one-way ANOVA. If the variance ratio test (F-test) was significant, a more detailed post hoc analysis of differences between groups was made using a Tukey–Kramer honest significance difference test.

Fig 4: PLPs, Caffeic acid, and P-coumaric acid inhibited the secretion of pro-inflammatory factors and the activation of NF-?B cell signaling in LPS-stimulated RAW264.7 cells. RAW264.7 cells were stimulated with LPS (1 µg/mL) and treated with PLPs (20 µg/mL), Caffeic acid (10 µg/mL), and P-coumaric acid (10 µg/mL) for 12 h (A–F). p-p65, p65, and IL-1ß were detected by Western blot (A–C). IL-6, IL-1ß, and TNF-a mRNA in colon tissue were determined by real-time PCR (D–F). Nor: normal control cells; LPS: lipopolysaccharide-induced control cells; PLP20: pineapple leaf phenols-treated (20 µg/mL) LPS-induced cells; Caffeic acid: Caffeic acid-treated (10 µg/mL) LPS-induced cells; P-coumaric acid: P-coumaric acid-treated (10 µg/mL) LPS-induced cells. Data are expressed as the mean ± SD, and the results are representative of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the cells treated with DSS alone.

Fig 5: AAV-shRNA treatment improves liver tissue histology and alleviates inflammation by attenuating the NF-?B signaling pathway and reducing serum IL-6 levels. Mice received AAV-shRNA treatment, and liver samples were collected at 6 months after injection. (A) Liver sections stained with H&E. (B) LX-2 cells were transfected with pSSV9-HBV with or without pAAV-shRNA. Phospho-NF-?B (Ser468)/NF-?B p65 levels were evaluated in mice. (C) IL-6 concentrations in serum were determined by ELISA. ***P<0.05. Scale bar, 50 µm. Data of proteins are expressed as the means ± SD (n=3). AAV, adeno-associated virus; shRNA, short hairpin RNA; NF-?B, nuclear factor-?B; IL-6, interleukin-6; H&E, hematoxylin and eosin; ELISA, enzyme linked immunosorbent assay.