Journal: International Journal of Neuropsychopharmacology

Article Title: Upregulation of Phosphodiesterase 7A Contributes to Concurrent Pain and Depression via Inhibition of cAMP-PKA-CREB-BDNF Signaling and Neuroinflammation in the Hippocampus of Mice

doi: 10.1093/ijnp/pyae040

Figure Lengend Snippet: Partial sciatic nerve ligation (PSNL) or complete Freund’s adjuvant (CFA) leads to increased phosphodiesterase 4B (PDE4B) and PDE7A and decreased cyclic AMP (cAMP) expression in mouse hippocampus. Mice were treated as described in the schedule of . The expression of PDE2, PDE4, PDE5, and PDE7 in the hippocampus of mice subjected to PSNL or CFA were quantified by western blotting analysis (A–G), and the levels of cAMP and cyclic GMP (cGMP) were analyzed by ELISA assays (H–I) 33 days after surgery or injection. The optic densities of PDE2A, PDE4A, PDE4B, PDE4D, PDE5A, PDE7A, and PDE7B were normalized to those of sham (or saline) controls. Data shown are mean ± SEM (n = 5). * P < .05, ** P < .01, *** P < .001 vs corresponding sham or saline.

Article Snippet: The membrane was incubated overnight at 4°C with primary antibodies (diluted 1:1000) against the following proteins: PDE2A (Proteintech, Chicago, IL, USA), PDE4A (Proteintech), PDE4B (Cell Signaling Technology, Danvers, MA, USA), PDE4D (Abcam, Cambridge, UK), PDE5A (Abcam), PDE7A (Santa Cruz Biotechnology, Shanghai, China), PDE7B (Abcam), total PKA or phosphorylated PKA (p-PKA; Cell Signaling Technology), total CREB or phosphorylated CREB (p-CREB; Abcam), BDNF (Abcam), the p65 subunit of NF-kB (Cell Signaling Technology), or β-actin (Zsbio, Beijing, China).

Techniques: Ligation, Adjuvant, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Saline

Journal: Mediators of Inflammation

Article Title: Sorbitol Destroyed Intestinal Microfold Cells (M Cells) Development through Inhibition of PDE4-Mediated RANKL Expression

doi: 10.1155/2024/7524314

Figure Lengend Snippet: Sorbitol regulated M-cell differentiation through PDE4/PKA/CREB signaling-mediated RANKL: (a) after establishing M-cell model, the total protein was extracted from M-cell model treated with 100 mM mannitol or 100 mM sorbitol for 24 hr, western blotting was used to detect RANKL; (b) the M cells was established and treated described in (a), and the concentration of RANKL level in supernatant was measured in indicated group through ELISA assay. Data presented as the mean ± s.e.m. of three independent experiments and were analyzed by t test, ∗∗∗∗ p < 0.001; (c) intestinal organoids culture was performed to visualize the effect of PDE4 inhibition by DIP on sorbitol-mediated M-cell development. Bar: 50 μ m. Data presented as the mean ± s.e.m. and were analyzed by one ANOVA, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: RANKL monoclonal antibody (Proteintech, 66610-1-Ig); phospho-PDE4 (Immunoway, YP0668) was from Immunoway (Jiangsu, China), PKACA (Proteintech, 67491-1-Ig), CREB1 polyclonal antibody (Proteintech, 12208-1-AP); Phospho-CREB1 (Ser133) polyclonal antibody (Proteintech, 28792-1-AP), Lamin A/C polyclonal antibody (Proteintech, 10298-1-AP) and α -tubulin monoclonal antibody (Proteintech, 66031-1-Ig) were from Proteintech; phospho-PKA (Thr197) (CST, 5661) was from cell signaling technology (Danvers, MA, USA); peroxidase-affiniPure goat anti-rabbit IgG (H + L) (111-035-003) and peroxidase-affiniPure goat anti-mouse IgG (H + L) (115-035-003)were purchased from Jackson.

Techniques: Cell Differentiation, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Mediators of Inflammation

Article Title: Sorbitol Destroyed Intestinal Microfold Cells (M Cells) Development through Inhibition of PDE4-Mediated RANKL Expression

doi: 10.1155/2024/7524314

Figure Lengend Snippet: CREB is critical for sorbitol-mediated RANKL expression: (a) reporter gene containing RANKL promoter was transfected into CaCO 2 cells combined with pGL4.74 plasmid for 24 hr, CaCO 2 cell was treated with 100 mM mannitol and 100 mM sorbitol for further 24 hr, the relative luciferase unit was detected using dual-luciferase reporter assay system. Data presented as the mean ± s.e.m. of three independent experiments and were analyzed by two ANOVA, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; (b) after serum-free for 24 hr, CaCO 2 cells were stimulated for 1 hr, and the whole cell was fixed and lysed to incubate with an anti-CREB antibody, the chromosome fragments binding to CREB were amplified and quantified by real-time PCR with RANKL promoter primer. Data presented as the mean ± s.e.m. of three independent experiments and were analyzed by two ANOVA, ∗∗∗∗ p < 0.0001; (c) ELISA was employed to detect RANKL level in indicated group in CaCO 2 cells, data presented as the mean ± s.e.m. of five independent experiments and were analyzed by one ANOVA, ∗∗∗∗ p < 0.0001.

Article Snippet: RANKL monoclonal antibody (Proteintech, 66610-1-Ig); phospho-PDE4 (Immunoway, YP0668) was from Immunoway (Jiangsu, China), PKACA (Proteintech, 67491-1-Ig), CREB1 polyclonal antibody (Proteintech, 12208-1-AP); Phospho-CREB1 (Ser133) polyclonal antibody (Proteintech, 28792-1-AP), Lamin A/C polyclonal antibody (Proteintech, 10298-1-AP) and α -tubulin monoclonal antibody (Proteintech, 66031-1-Ig) were from Proteintech; phospho-PKA (Thr197) (CST, 5661) was from cell signaling technology (Danvers, MA, USA); peroxidase-affiniPure goat anti-rabbit IgG (H + L) (111-035-003) and peroxidase-affiniPure goat anti-mouse IgG (H + L) (115-035-003)were purchased from Jackson.

Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Binding Assay, Amplification, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: Sorbitol Destroyed Intestinal Microfold Cells (M Cells) Development through Inhibition of PDE4-Mediated RANKL Expression

doi: 10.1155/2024/7524314

Figure Lengend Snippet: Sorbitol modulated PDE4/PKA/CREB signaling: (a) upper panel: CaCO 2 cells were serum starved for 24 hr and stimulated with mannitol, sorbitol, and sorbitol+DIP for further 1 hr. Cell fraction was isolated and levels of cytosolic CREB were detected by western blotting. α -Tubulin and lamin A/C were used as internal controls for the cytosolic and nuclear fractions, respectively. Bottom panel: CaCO 2 cells were confluence, serum starved for 24 hr, then stimulated with 100 mM mannitol or sorbitol combined with or without DIP for 1 hr. Immunoprecipitation was performed with antibodies targeting endogenous PKA and immunoblotting was used to detect CREB; (b) after establishing M-cell model, the total protein was extracted from M-cell model treated with 100 mM mannitol or 100 mM sorbitol and 100 mM sorbitol + DIP for 24 hr, western blotting was used to detect RANKL, baseline and phosphorylation of PDE4/PKA/CREB. α -tubulin was served as an internal control; (c) intestinal organoids culture was performed to analyze the mannitol, sorbitol, sorbitol + DIP on differentiation. Data presented as the mean ± s.e.m. of five independent experiments and were analyzed by one ANOVA, ∗ p < 0.05; (d) IF was used to detect M cells mature marker GP2 expression and RANKL expression in the indicated group.

Article Snippet: RANKL monoclonal antibody (Proteintech, 66610-1-Ig); phospho-PDE4 (Immunoway, YP0668) was from Immunoway (Jiangsu, China), PKACA (Proteintech, 67491-1-Ig), CREB1 polyclonal antibody (Proteintech, 12208-1-AP); Phospho-CREB1 (Ser133) polyclonal antibody (Proteintech, 28792-1-AP), Lamin A/C polyclonal antibody (Proteintech, 10298-1-AP) and α -tubulin monoclonal antibody (Proteintech, 66031-1-Ig) were from Proteintech; phospho-PKA (Thr197) (CST, 5661) was from cell signaling technology (Danvers, MA, USA); peroxidase-affiniPure goat anti-rabbit IgG (H + L) (111-035-003) and peroxidase-affiniPure goat anti-mouse IgG (H + L) (115-035-003)were purchased from Jackson.

Techniques: Isolation, Western Blot, Immunoprecipitation, Phospho-proteomics, Control, Marker, Expressing