Fig 2: Intestinal indicators reflect the effect of roflumilast on LPS-induced cells. (A) Reverse transcription-quantitative PCR analysis and (B) western blotting were conducted to detect the expression levels of PDE4A and PDE4B in cells treated with LPS or LPS + roflumilast. (C) LDH, DAO and iFABP levels in cells were detected using respective assay kits. **P<0.01, ***P<0.001; n≥3. PDE4, phosphodiesterase 4; LPS, lipopolysaccharide; LDH, lactate dehydrogenase; DAO, D-amino acid oxidase; iFABP, intestinal fatty acid binding protein.

Fig 3: PDE4 expression in small intestinal cells. (A) IEC-6 cells were treated with different concentrations of LPS for 24 h to detect its effect on cell viability. (B) Reverse transcription-quantitative PCR analysis and (C) western blotting were performed to detect the expression levels of PDE4A, PDE4B and PDE4D in cells treated with increasing doses of LPS. *P<0.05, ***P<0.001; n≥3. PDE4, phosphodiesterase 4; LPS, lipopolysaccharide.

Fig 4: Opposing effects of ethanol and synaptamide on the cAMP system in LPS-induced neuro-inflammation. WT and GPR110 KO mice were given 3 g/kg ethanol through oral gavage and LPS (1 mg/kg, i.p.) was injected at 4 h after ethanol administration. Synaptamide (5 mg/kg, i.p.) was injected immediately after LPS administration. The expression of mRNA and protein in brain tissues was measured for isoforms of AC (ADCY) and pde4 at 2 and 24 h after LPS injection, respectively. The mRNA expression of ADCY8 (A) and PDE4B (B) were perturbed by LPS which was potentiated by ethanol. Synaptamide GPR110-dependently restored the reduced expression of ADCY8 caused by LPS and EtOH + LPS and reduced the PDE4B expression elevated by LPS and EtOH + LPS. The western blot analysis (C, D) showed an LPS-induced increase in PDE4B protein which was further elevated by ethanol pretreatment but was suppressed by synaptamide in a GPR110-dependent manner. Values are presented as mean ± SEM (n = 4 for A, B; n = 3 for C, D), representing two independent experiments. ns, the difference of means is not statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. Maltose group

Fig 5: Timeless deletion alters PDE4B expression, cAMP levels, and synaptic transmission in hippocampus(A) TIMELESS upregulates Pde4b transcription and thereby PDE4B protein levels in hippocampus. At ZT12, Pde4b mRNA levels and PDE4B protein levels are reduced in Timeless CKO relative to CTRL (qRT-PCR for mRNA: CTRL n = 9, CKO n = 5; ***p < 0.001, Mann-Whitney test. Western blot for protein: CTRL n = 7, CKO n = 7; **p = 0.014, Mann-Whitney test).(B) TIMELESS negatively regulates cAMP concentration in hippocampus. ELISA showed that cAMP concentration was elevated in hippocampus of CKO compared with CTRL (CTRL n = 5, CKO n = 6; *p = 0.027, Mann-Whitney test).(C) Schematic of PDE4B/cAMP signaling cascade in neurons.(D and E) Pharmacological treatments that target cAMP activation to promote synaptic potentiation demonstrated that TIMELESS can affect synaptic transmission. (D) CKO mice showed a moderate potentiation induced by bath application of 50 μM FSK (an adenylate cyclase activator) compared with the CTRL group, which displayed a dramatic transient decrease in neurotransmission. PDE4B-mediated cAMP degradation to AMP and generation of adenosine via 5′-nucleotidase can lead to suppression of neurotransmitter release,55,56 as seen in CTRL mice. Reduced PDE4B activity in CKO may explain the absence of transient depression in neurotransmission. (E) Quantification of mean fEPSP slope at 30–35 min of recording (CTRL n = 5 slices/4 animals; CKO n = 7 slices/4 animals; **p = 0.005, Mann-Whitney test).(F) Timeless deletion led to a reduced chemical potentiation induced by co-application of 50 μM FSK and 100 nM DPCPX (an A1 receptor antagonist) in nominal magnesium (FSK, DPCPX, 0 Mg2+) in slices from CKO mice relative to CTRL slices.(G) Quantification of mean fEPSP slope at 60–65 min of recording (CTRL n = 6 slices/4 animals; CKO n = 7 slices/4 animals; *p = 0.035, Mann-Whitney test). Individual points represent replicates from the number of animals indicated (n). All samples were collected and experiments performed at ZT12. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. In (C), AC denotes adenylate cyclase, ATP denotes adenosine triphosphate, AMP denotes adenosine monophosphate, cAMP denotes cyclic AMP, PDE4B denotes phosphodiesterase 4B, FSK denotes forskolin, 5′NTE denotes 5′-nucleotidase, and DPCPX stands for the selective A1 adenosine receptor antagonist. In (D) and (F), representative traces of baseline fEPSPs are in light color and after treatment in dark color. The number 1 denotes baseline and 2 denotes treatment.