Anti-inflammation of isoliquiritigenin via the inhibition of NF-κB and MAPK in LPS-stimulated MAC-T cells.

BACKGROUND: The application of plant extracts has received great interest for the treatment of bovine mastitis. Isoliquiritigenin (ISL) is a rich dietary flavonoid that has significant antioxidative, anti-inflammatory and anticancer activities. This study was conducted to explore the protective efficacy and related mechanism of ISL against lipopolysaccharide (LPS)-stimulated oxidation and inflammation in bovine mammary epithelial cells (MAC-T) by in vitro experiments. RESULTS: Real-time PCR and ELISA assays indicated that ISL treatment at 2.5, 5 and 10 µg/mL significantly reduced the mRNA and protein expression of the oxidative indicators cyclooxygenase-2 and inducible nitric oxide synthase (P < 0.01), and of the inflammatory cytokines interleukin-6 (P < 0.05), interleukin-1ß (P < 0.01) and tumor necrosis factor-α (P < 0.01) in LPS-stimulated MAC-T cells. Moreover, Western blotting and immunofluorescence tests indicated that the phosphorylation levels of nuclear factor kappa (NF-κB) p65 and the inhibitor of NF-κB were significantly decreased by ISL treatment, thus blocking the nuclear transfer of NF-κB p65. In addition, ISL attenuated the phosphorylation levels of p38, extracellular signal-regulated kinase and c-jun NH2 terminal kinase. CONCLUSIONS: Our data demonstrated that ISL downregulated the LPS-induced inflammatory response in MAC-T cells. The anti-inflammatory and antioxidative activity of ISL involves the NF-κB and MAPK cascades.

Inhibition of PHLDA2 transcription by DNA methylation and YY1 in goat placenta.

Pleckstrin homology-like domain family A member 2 (PHLDA2) is essential for placental development in mammals. This study was conducted to investigate transcriptional regulation of goat PHLDA2 in the placenta. Real-time PCR and Western blot analyses showed different expression of the PHLDA2 in goat placentas during gestation with highest expression at 30 and 45 days post coitus (P < 0.05). Luciferase reporter assays demonstrated the highest promoter activity in the region of -1023/+20 (P < 0.05). A CpG island was defined within -631/+379 region, where lower level of CpG-methylation was detected with bisulfite sequencing PCR in the placenta than that in the spleen and liver (P < 0.05). Meanwhile, in vitro experiments showed that 5-AzaC enhanced the gene expression in a dose-dependent manner. Site-directed mutation in vitro demonstrated that transcription factor Ying-yang 1 (YY1) had an inhibitory effect on the PHLDA2 expression, and the inhibition was further confirmed with overexpression and siRNA constructs of YY1. ChIP and RE-ChIP analyses further identified the binding of YY1 to the PHLDA2 promoter by interaction with histone deacetylase 1 (HDAC1) and HDAC3. This study uncovers the negative regulation of the CpG-methylation and YY1 on goat PHLDA2 expression. YY1 prefers binding to CpG-methylation sequences, and inhibits goat PHLDA2 expression via recruiting HDAC1 and 3.

Promoter characterization and functional association with placenta of porcine MAGEL2.

MAGEL2 (melanoma antigen-like gene 2) is essential for circadian function, metabolism and reproduction in mammals. This study was conducted to investigate transcriptional regulation and functional importance in the placenta of porcine MAGEL2. Quantitative real-time PCR showed that MAGEL2 was highly expressed in porcine hypothalamus, pituitary and placenta (P<0.05). The gene was down-regulated in Meishan but up-regulated in Duroc placentas from 25 days post-coitum (dpc) to 105 dpc (P<0.01). Dual luciferase assay demonstrated that the region -151/+110 had the highest promoter activity. Of the g. -712C>G and g. -708T>C polymorphisms in MAGEL2 promoter, -712C and -708T were observed to be predominant in Large White, Landrace and Duroc populations, while -712G and -708C were predominant in Meishan and Rongchang populations. Moreover, -712C>G and -708T>C had significant effects on MAGEL2 transcription (P<0.05) and placental efficiency (P<0.01). In conclusion, -151/+110 harbors the basal promoter of porcine MAGEL2. The region upstream the basal promoter contains repressive cis-elements. And, MAGEL2 is essential in porcine placenta.