OH-CATH30 targets CD40 to suppress NF-κB signaling and promote arachidonic acid metabolic reprogramming in acute pancreatitis

Figure 6 CD40 is a functional target of OH-CATH30: Validation of CD40-tumor necrosis factor receptor-associated factor 6-transforming growth factor beta-activated kinase 1-nuclear factor-kappa B axis. A: Co-immunoprecipitation assay confirming binding between FITC-labeled OH-CATH30 and CD40 protein. Cell lysates were immunoprecipitated with anti-FITC antibody or control IgG, followed by immunoblotting with anti-CD40 antibody; B: Western blotting analysis of CD40 pathway proteins (CD40, tumor necrosis factor receptor-associated factor 6, transforming growth factor beta-activated kinase 1, nuclear factor-kappa B p65) in 266-6 cells under indicated conditions: Control (untreated); cholecystokinin (CCK) (100 nM CCK for 24 hours); OH-CATH30 (100 nM OH-CATH30 after CCK injury); OH-CATH30 + CD40 (LV-CD40 transfection prior to CCK injury and OH-CATH30 treatment); sh-CD40 (LV-sh-CD40 knockdown prior to CCK injury). Glyceraldehyde-3-phosphate dehydrogenase functioned as loading control; C: Western blotting analysis of CD40 pathway proteins in mouse pancreatic tissues from indicated groups: Control (phosphate-buffered saline only); acute pancreatitis (AP) (cerulein-induced); OH-CATH30 (30 μg/kg after AP induction); OH-CATH30 + CD40 (AAV-CD40 tail vein injection prior to AP induction and OH-CATH30 treatment); sh-CD40 (AAV-sh-CD40 injection prior to AP induction without OH-CATH30 treatment); D and E: Representative images (D) and quantification (E) of EdU incorporation assay to assess cell proliferation in 266-6 cells. Scale bar = 50 μm; F and G: Representative images (F) and quantification (G) of TUNEL staining to detect apoptosis in 266-6 cells. Scale bar = 50 μm; H and I: Representative hematoxylin and eosin-stained pancreatic sections (H) and corresponding histological scores (I) from mice. Scale bar = 100 μm. All data are presented as mean ± SD [n = 3 independent experiments (A-G) or n = 6 mice per group (H and I)]. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. ^aP < 0.05 vs control, ^bP < 0.05 vs cholecystokinin or acute pancreatitis group, ^cP < 0.05 vs OH-CATH30 group, ^dP < 0.05 vs OH-CATH30 + CD40 group. TRAF6: Tumor necrosis factor receptor-associated factor 6; TAK1: Transforming growth factor beta-activated kinase 1; NF-κB: Nuclear factor-kappa B; CCK: Cholecystokinin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; AP: Acute pancreatitis.