And (C,D) myometrium were incubated with or without 10 mg/mL
And (C,D) myometrium were incubated with or without 10 mg/mL of LPS in the absence or presence 200 mM of nobiletin for 20 h (n = 6 patients per group). (A,C) MMP-9 mRNA expression was analysed by qRT-PCR and normalised to GAPDH mRNA expression. The relative fold change was calculated relative to LPS and data presented as mean 6 SEM. *P,0.05 vs. LPS (one-way ANOVA). (B,C) The incubation medium was assayed for pro MMP-9 levels by gelatin zymography. The relative fold change was calculated relative to LPS and data presented as mean 6 SEM. *P,0.05 vs. LPS (one-way ANOVA). Zymography from one patient per tissue type is also shown. doi:10.1371/journal.pone.0108390.gdietary supplementation may be more plausible. Indeed, supplements containing citrus polymethoxyflavones have shown promising cardioprotective effects in humans [74]. Human studies focussed on dose, bioavailability, efficacy and safety are, however, required to propel the use of these promising therapeutic agents into the clinical arena. Of promise are the studies using Sytrinol for heart disease. Sytrinol is a patented dietary supplement containing the citrus flavones nobiletin and tangeretin at 1:1 ratio. These studies highlight the translation potential of our findings. Preterm birth is a global issue that affects the lives of millions of SIRT3 Purity & Documentation families every year and causes over a million deaths every year [75]. Currently there are no long-term treatments, with most only capable in delaying birth by hours [19]. Inflammation has a central role in the genesis of preterm birth and the adverse neonatal sequelae that follow [3,768]. Thus, a safe and effective agent that could block the inflammatory response would be an ideal therapeutic. In this study, we report the beneficial actions of the citrus flavone nobiletin in reducing P2Y1 Receptor manufacturer pro-inflammatory and pro-labour mediators in the presence of bacterial endotoxin LPS. Significantly, we were also able to show that nobiletin can reduce the inflammation already present in the preterm fetal membranes. These studies support the numerous epidemiological studies that high fruit and vegetable intake in pregnancy is associated with a decreased risk of adverse pregnancy outcomes [206].AcknowledgmentsThe authors gratefully acknowledge the assistance of the Clinical Research Midwives Genevieve Christophers, Gabrielle Fleming, Debra Jinks, Rachel Murdoch and Renee Grant; and the Obstetrics and Midwifery staff of the Mercy Hospital for Women for their co-operation.Author ContributionsConceived and designed the experiments: ML. Performed the experiments: CJM ML. Analyzed the data: CJM ML. Contributed reagents/materials/ analysis tools: ML. Contributed to the writing of the manuscript: CJM ML.PLOS ONE | plosone.orgAnti-Inflammatory Actions of NobiletinFigure 6. Effect of nobiletin on pro-inflammatory cytokines in preterm fetal membranes. Preterm fetal membranes with histological chorioamnionitis and following spontaneous preterm labour were incubated for 20 h with or without 200 mM nobiletin (n = 9 patients). (A ) TNF-a, IL-1b, IL-6 and IL-8 mRNA expression was analysed by qRT-PCR and normalised to GAPDH mRNA expression. The fold change was calculated relative to basal expression, which was set at 1. Data is displayed as mean 6 SEM (one-way ANOVA). *P,0.05 vs. basal expression. (E, F) The incubation medium was assayed for concentration of IL-6 and IL-8 by ELISA. Data was normalised to untreated (basal) levels, which was set at 1. Each bar represents mean 6 SE.
