Possible relationship among resveratrol, PPARα and PDE.

<p>These diagrams present our hypothesis about short- and long-term effects of resveratrol, as shown in the text.</p

Docking models and analysis of PPARα residues required for binding to resveratrol.

<p>(A) The four docking modes of resveratrol predicted using the GOLD 3.0 docking program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120865#pone.0120865.ref024" target="_blank">24</a>] with protein co-ordination data from the PPARα-GW409544 complex structure (PDB ID: 1K7L) and a standard docking protocol. (B) Superimposition of docking mode II of resveratrol (orange) on the structure of PPARα bound to GW409544, a potent PPARα agonist (green). Only the amino acids located near to GW409544 are displayed. The hydrogen bonds of Tyr314 and Tyr464 are shown as dashed green lines. (C) Binding free energies (∆∆Gbind (kcal/mol)) of the indicated PPARα amino acid residues, calculated by alanine scanning using data for the four predicted docking modes. (D) Activation of wild-type (WT) PPARα and its mutants by 5, 50 μM resveratrol or Wy-14643. BAECs were transiently transfected with PPRE-luc, wild-type or mutant GS-hPPARα, and pSV-β-gal. The data are presented as relative luciferase activities normalized to those of the β-galactosidase standard and as 1 for cells treated with DMSO (control), and represent the mean ± SD of three independent wells of cells. Similar results were obtained by two additional experiments. The data were calculated the relative luciferase activity in cells transfected with wild-type PPARα.</p

Inhibition of PDE enhances the activation of PPARα by resveratrol, especially at higher doses.

<p>(A) The dose-dependent activation of PPARα by resveratrol, T4HS and 4-PAP in BAECs transiently transfected with PPRE-luc, GS-hPPARα, and pSV-β-gal. Following transfection, the cells were incubated for 24 h with resveratrol, T4HS or 4-PAP at the indicated concentrations. Data were normalized to the β-galactosidase standard and represent the mean ± SD of three independent wells of cells. The right graph corresponds to the lower area marked by a dashed rectangle in left graph. (B) cAMP-dependent enhancement of PPARα activation by resveratrol, T4HS or 4-PAP. BAECs transiently transfected with PPRE-luc, GS-hPPARα, and pSV-β-gal were incubated for 24 h with 5 μM compounds in the presence or absence of 25 μM rolipram, a PDE4 inhibitor, or 25 μM forskolin, an adenylate cyclase activator. Luciferase data were normalized to the β-galactosidase standard and represent the mean ± SD of three independent wells. *<i>p</i> < 0.05, ***<i>p</i> < 0.001 (unpaired <i>t</i>-test) compared with control cells treated with the same compound. (C) The inhibition of PDE by resveratrol, T4HS, and rolipram. Data represent the mean ± SD of three independent wells of cells. Similar results were obtained by two additional experiments. The IC₅₀ values are shown in the Table. ***<i>p</i> < 0.001 (unpaired <i>t</i>-test) compared with rolipram. ^###<i>p</i> < 0.001 (unpaired <i>t</i>-test) compared with resveratrol. Similar results were obtained by two additional experiments in (A-C).</p

Autonomic nervous system activity in healthy controls and patients with IBS.

Autonomic nervous system activity in healthy controls and patients with IBS.</p

Relationship among sympathetic nerve activity at 2 min before defecation, symptoms, and quality of life.

Sympathetic nerve activity was evaluated by the sum of Δlow frequency/high frequency (LF/HF). (a) Intensity of abdominal pain, (b) satisfaction levels of defecation, (c) gastrointestinal symptom rating scale (GSRS) diarrhea score, (d) GSRS constipation score, (e) the Japanese version of the 8-item Short-Form Health Survey (SF-8) role physical, and (f) the SF-8 role emotional score were significantly correlated with the sum of ΔLF/HF at 2 min before defecation. Data were analyzed by Spearman test.</p

The graph of obtained data and measuring method of ANS activity.

(a) The real graph of obtained data about autonomic nervous system (ANS) activity and life events. The results of low frequency (LF) (yellow line) and high frequency (HF) (green line) represented in a graph. Life events, such as defecation and eating (the enclosed area with orange line) and period with positive symptoms (the enclosed area with pink line), were recorded. (b) Measuring method of ANS activity. The baseline LF/HF was defined as the value included in the range of the mean ± 2 standard deviations (dashed line) of LF/HF measured in the period with no symptoms. The sum of LF/HF was the area under the curve of LF/HF measured in the period with positive symptoms (upward diagonal). The sum of ΔLF/HF was the sum of variation from the mean value of baseline measured in the period with positive symptoms (dots). The maximum variation of ΔLF/HF indicated the maximum variation from the mean value of baseline measured in the period with positive symptoms (arrow). HF was analyzed in the same way.</p

Autonomic nervous activities before and after defecation.

(a, b) Changes in sympathetic nerve activities before and after defecation were evaluated by the sum of Δlow frequency/high frequency (LF/HF) and the sum of LF/HF, respectively. (c, d) Changes in parasympathetic nerve activities before and after defecation were evaluated by the sum of ΔHF and the sum of HF, respectively. Data represents the median. Statistical significance was calculated by Mann–Whitney test (*p < 0.05).</p

Background characteristics.

Background characteristics.</p

T-shirt-type wearable device and smartphone application software.

Heart rate variability (HRV) was measured using a T-shirt wearable device attached to a transmitter in front of the T-shirt. The HRV data were transferred and recorded on a smartphone using Bluetooth. At the same time, life events, such as abdominal symptoms, defecation, eating, and awakening or sleep, were recorded in real time using a smartphone application software during HRV recording.</p

Hypothesized mechanism of exacerbated symptoms and increased sympathetic nerve activity before defecation.

The stress of abdominal pain activates the amygdala to release corticotrophin-releasing hormone (CRH), resulting in further exacerbation of abdominal pain by increasing colonic motility. These cascades “exacerbate the circle of pain.” Two minutes before defecation, the stress also activates sympathetic nerves as a stress response against abdominal pain. Increased sympathetic nerve activity can decrease colonic motility, which may have a protective role to hold back the defecation. This situation may be under competition between the accelerator of colonic motility caused by CRH and the brake caused by sympathetic nerve activation.</p