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

Figure 4

Sorbitol modulated PDE4/PKA/CREB signaling: (a) upper panel: CaCO₂ 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: CaCO₂ 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, ; (d) IF was used to detect M cells mature marker GP2 expression and RANKL expression in the indicated group.

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