Polyenylpyrrole Derivatives Inhibit NLRP3 Inflammasome Activation and Inflammatory Mediator Expression by Reducing Reactive Oxygen Species Production and Mitogen-Activated Protein Kinase Activation

10.1371/journal.pone.0076754PLoS ONE810-POLN

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Bamboo vinegar decreases inflammatory mediator expression and NLRP3 inflammasome activation by inhibiting reactive oxygen species generation and protein kinase C-α/δ activation.

Bamboo vinegar (BV), a natural liquid derived from the condensation produced during bamboo charcoal production, has been used in agriculture and as a food additive, but its application to immune modulation has not been reported. Here, we demonstrated that BV has anti-inflammatory activities both in vitro and in vivo. BV reduced inducible nitric oxide synthase expression and nitric oxide levels in, and interleukin-6 secretion by, lipopolysaccharide-activated macrophages without affecting tumor necrosis factor-α secretion and cyclooxygenase-2 expression. The mechanism for the anti-inflammatory effect of BV involved decreased reactive oxygen species production and protein kinase C-α/δ activation. Furthermore, creosol (2-methoxy-4-methylphenol) was indentified as the major anti-inflammatory compound in BV. Impaired cytokine expression and NLR family, pyrin domain-containing 3 (NLRP3) inflammasome activation was seen in mice treated with creosol. These findings provide insights into how BV regulates inflammation and suggest that it may be a new source for the development of anti-inflammatory agents or a healthy supplement for preventing and ameliorating inflammation- and NLRP3 inflammasome-related diseases, including metabolic syndrome

Effect of compound 1h on ROS production and MAPK phosphorylation in LPS-activated macrophages.

<p>In (<b>A</b>), RAW 264.7 macrophages (5 × 10⁵/ml; 1 ml) were incubated for 30 min with compound <b>1h</b> (20 µM), N-acetyl cysteine (NAC; 10 mM), or DMSO (vehicle), then 2’, 7’-dichlorofluorescein diacetate (2 µM) was added for 30 min, followed by LPS (1 µg/ml) stimulation for the indicated time, then ROS levels were measured by detection of the mean fluorescence intensity (MFI) of the fluorophore carboxyl-DCF and expressing this value relative to that at time zero. In (<b>B</b>), RAW 264.7 macrophages (5 × 10⁵/ml; 1 ml) were incubated for 30 min with compound <b>1h</b> (20 µM) or DMSO, then LPS (1 µg/ml) was added and incubation continued for 0-60 min, then phosphorylation of ERK1/2, JNK1/2, and p38 was analyzed by Western blotting and expressed relative to actin expression and as a fold increase compared to the control group at 0 time. In (<b>C</b>), J774A.1 macrophages (5 × 10⁵/ml; 1 ml) were incubated for 30 min with 10-40 µM compound <b>1h</b> or DMSO, then LPS (1 µg/ml) was added and incubation continued for 20 min, then phosphorylation of ERK1/2, JNK1/2, and p38 was analyzed as in B. In (<b>A</b>), the data are expressed as the mean ± SD for three separate experiments, while, in (<b>B</b>) and (<b>C</b>), the results are representative of those obtained in three different experiments. * indicates a significant difference at the level of <i>p</i> < 0.05 compared to the DMSO/LPS group.</p

Effect of compound 1h on NF-κB activation in LPS-activated macrophages.

<p>(<b>A</b>) RAW 264.7 macrophages or (<b>B</b>) J774A.1 macrophages (both 5 × 10⁵/ml; 1 ml) were incubated for 30 min with 10-40 µM compound <b>1h</b> or DMSO, then LPS (1 µg/ml) was added and incubation continued for 20 min, then levels of phosphorylated and total IKK-α and IκB-α were measured by Western blotting. (<b>C</b>) RAW 264.7 macrophages or (<b>D</b>) J774A.1 macrophages (both 5 × 10⁵/ml; 1 ml) were treated as in A and B, then nuclear translocation of NF-κB was analyzed by ELISA. (<b>E</b>) RAW-Blue^TM cells (5 × 10⁵/ml; 1 ml) were incubated for 30 min with 2.5-40 µM compound <b>1h</b> or DMSO, then LPS (1 µg/ml) was added and incubation continued for 24 h, then SEAP activity was measured by the QUANTI-Blue^TM assay and expressed as a percentage of that in the absence of compound <b>1h</b>. In (<b>A</b>) and (<b>B</b>), the results are representative of those obtained in three different experiments. In (<b>C</b>-<b>E</b>), the data are expressed as the mean ± SD for three separate experiments. *, **, and *** indicate a significant difference at the level of <i>p</i> < 0.05, <i>p</i> < 0.01, and <i>p</i> < 0.001, respectively, compared to the DMSO/LPS group.</p

Effect of polyenylpyrrole derivatives on the expression of inflammatory mediators in LPS-stimulated RAW 264.7 macrophages.

<p>In (<b>A</b>) and (<b>C</b>), the cells (5 × 10⁵/ml; 1 ml) were incubated for 30 min with 2.5-40 µM compound <b>1h</b>, <b>1i</b>, or <b>1n</b> or DMSO (vehicle), then LPS (1 µg/ml) was added and incubation continued for 24 h, then NO (<b>A</b>) or IL-6 or TNF-α (<b>C</b>) in the culture medium was assayed by the Griess reaction or ELISA, respectively. In (<b>B</b>), cells (5 × 10⁵/ml; 1 ml) were pretreated for 30 min with 2.5-40 µM compound <b>1h</b> or DMSO, then LPS (1 µg/ml) was added and incubation continued for 24 h, then expression of iNOS and COX-2 was measured by Western blotting. The fold increase is the intensity of the band of interest divided by that of the actin band normalized to the corresponding value for the 0 LPS/0 inhibitor control. In (<b>A</b>) and (<b>C</b>), the data are expressed as the mean ± SD for three separate experiments, while, in (<b>B</b>), the results are representative of those obtained in three different experiments and the histogram shows the quantification expressed as the mean ± SD for these 3 experiments. *, **, and *** indicate a significant difference at the level of <i>p</i> < 0.05, <i>p</i> < 0.01, or <i>p</i> < 0.001, respectively, compared to the DMSO/LPS group.</p

Effect of compound 1h on NLRP3 inflammasome activation in LPS+ATP-activated J774A.1 macrophages.

<p>(<b>A</b>) J774A.1 macrophages (1 × 10⁶/ml; 1 ml) or (<b>B</b>) peritoneal macrophages (1 × 10⁵/ml; 1 ml) were incubated with 10-40 µM compound <b>1h</b> or DMSO for 30 min, then LPS (1 µg/ml) was added and incubation continued for 5.5 h, then the cells were stimulated with ATP (5 mM) for an additional 30 min, then IL-1β in the culture medium was measured by ELISA (<b>A, upper panel; B</b>) and levels of active caspase-1 (p10) (<b>A, lower panel</b>) measured by Western blotting. In (<b>C</b>) and (<b>D</b>), J774A.1 macrophages (1 × 10⁶/ml; 1ml) were incubated with LPS (1 µg/ml) for 5.5 h, then with 10-40 µM compound <b>1h</b> or DMSO for 30 min in the continued presence of LPS, followed by stimulation with ATP (5 mM) for an additional 30 min, then IL-1β levels (<b>C, upper panel</b>) and IL-6 levels (<b>D</b>) in the culture medium were measured by ELISA and levels of active caspase-1 (p10) were measured by Western blotting (<b>C, lower panel</b>). In A and C, the fold increase is the intensity of the p10 band divided by that of the p45 band normalized to the corresponding value for the 0 LPS/0 inhibitor control. In (<b>E</b>), J774A.1 macrophages (1 × 10⁶/ml; 1 ml) were incubated for 30 min with DMSO or 1-40 µM compound <b>1h</b>, then LPS (1 µg/ml) was added and incubation continued for 6 h, then expression of NLRP3 and proIL-1β was measured by Western blotting. The fold increase is the intensity of the band of interest divided by that of the actin band normalized to the corresponding value for the 0 LPS/0 inhibitor control. In the ELISA studies, the data are expressed as the mean ± SD for three separate experiments, while, in the Western blot studies, the results shown are representative of those obtained in three different experiments and the histogram shows the quantification expressed as the mean ± SD. *, **, and *** indicate a significant difference at the level of <i>p</i> < 0.05, <i>p</i> < 0.01, and <i>p</i> < 0.001, respectively, compared to the DMSO/LPS/ATP group (<b>A</b>, <b>B</b>), LPS/DMSO/ATP group (<b>C</b>, <b>D</b>), or the DMSO/LPS group (<b>E</b>).</p

Effect of compound 1h on ROS production and PKC-α phosphorylation in ATP-activated macrophages.

<p>In (<b>A</b>), J774A.1 macrophages (1 × 10⁶/ml; 1 ml) were incubated with LPS (1 µg/ml) for 6 h, then with compound <b>1h</b> (20 µM), the NADPH oxidase inhibitor DPI (25 µM), or DMSO (vehicle) for 30 min in the continued presence of LPS, then 2’,7’-dichlorofluorescein diacetate (2 µM) was added for 30 min, followed by ATP (5 mM) for the indicated time, then ROS levels were determined by measuring the mean fluorescence intensity (MFI) of the fluorophore carboxyl-DCF and expressing this value relative to that at time zero. In (<b>B</b>), J774A.1 macrophages (1 × 10⁶/ml; 1 ml) were incubated with compound <b>1h</b> (20 µM), DPI (25 µM), or DMSO (vehicle) for 30 min, then LPS (1 µg/ml) was added for 6 h; the cells were then incubated with 2’,7’-dichlorofluorescein diacetate (2 µM) for 30 min, then with ATP (5 mM) for the indicated time and ROS levels were measured by detection of the fluorescence intensity of the fluorophore carboxyl-DCF and expressed relative to that at time zero. In (<b>C</b>), LPS-primed J774A.1 macrophages (1 × 10⁶/ml; 1 ml) were incubated for 30 min with 20 µM compound <b>1h</b> or DMSO (vehicle) followed by ATP (5 mM) stimulation for 0-60 min, then phosphorylation of PKC-α was analyzed by Western blotting and expressed as the fold increase measured as the intensity of the PKC-α band divided by that of the actin band normalized to the corresponding value for DMSO at 0 minutes. In (<b>A</b>) and (<b>B</b>), the data are expressed as the mean ± SD for three separate experiments, while, in (<b>C</b>), the results are representative of those obtained in three different experiments and the histogram shows the quantification expressed as the mean ± SD. * indicates a significant difference at the level of <i>p</i> < 0.05 compared to the DMSO/ATP group.</p

Effect of compound 1h on LPS-induced secretion of IL-6 and TNF-α by J774A.1 macrophages, peritoneal macrophages, and JAWSII dendritic cells.

<p>(<b>A</b>) J774A.1 macrophages, (<b>B</b>) peritoneal macrophages, or (<b>C</b>) JAWSII dendritic cells (all 4 × 10⁵/ml; 1 ml) were incubated for 30 min with 10-40 µM compound <b>1h</b> or DMSO, then LPS (1 µg/ml) was added and incubation continued for 24 h, then IL-6 levels (<b>left panels</b>) and TNF-α levels (<b>right panels</b>) in the culture medium were measured by ELISA. The data are expressed as the mean ± SD for three separate experiments. *, **, and *** indicate a significant difference at the level of <i>p</i> < 0.05, <i>p</i> < 0.01, or <i>p</i> < 0.001, respectively, compared to the DMSO/LPS group.</p

Effect of BV-4 fractions on NO generation and cell viability.

<p> In (A), RAW 264.7 macrophages (1×10⁶ in 2 ml of medium) were incubated for 30 min with or without the indicated concentrations of the neutral, acidic, or phenolic fraction of BV-4, then for 24 h with or without addition of 1 µg/ml of LPS, then NO generation in the culture medium was measured by the Griess reaction. In (B), RAW 264.7 macrophages (1×10⁶ in 2 ml of medium) were incubated for 30 min with or without the indicated concentration of the phenolic fraction of BV-4, then for 24 h with or without addition of 1 µg/ml of LPS, then NO generation in the culture medium was measured by the Griess reaction. In (C), RAW 264.7 macrophages (5×10⁴ in 1 ml of medium) were incubated for 30 min with or without the phenolic fraction of BV-4, then for 24 h with or without addition of 1 µg/ml of LPS, then cell viability was measured by the AlamarBlue® assay. The data are expressed as the mean ± SD for three separate experiments. *and # indicate a significant difference at the respective levels of <i>p</i><0.05 and <i>p</i><0.001 compared to the LPS-treated group.</p