Heme-oxygenase-1 induction and carbon monoxide-releasing molecule inhibit lipopolysaccharide (LPS)-induced high-mobility group box 1 release in vitro and improve survival of mice in LPS- and cecal ligation and puncture-induced sepsis model in vivo. Mol Ph

ABSTRACT We examined our hypothesis that heme-oxygenase-1 (HO-1)-derived carbon monoxide (CO) inhibits the release of highmobility group box 1 (HMGB1) in RAW264.7 cells activated with lipopolysaccharide (LPS) in vitro and in LPS-or cecal ligation and puncture (CLP)-induced septic mice in vivo, so that HO-1 induction or CO improves survival of sepsis in rodents. We found that pretreatment with HO-1 inducers (hemin, cobalt protoporphyrin IX) or transfection of HO-1 significantly inhibited HMGB1 release, which was blocked by HO-1 small interfering RNA, in cells activated by LPS. Carbon monoxide-releasing molecule 2 (CORM-2) but not bilirubin or deferoxamine inhibited HMGB1 release in LPS-activated macrophages. Oxyhemoglobin reversed the effect of HO-1 inducers on HMGB1 release. Translocation of HMGB1 from nucleus to cytosol was significantly inhibited by HO-1 inducers, CORM-2, or HO-1 transfection. Neutralizing antibodies to tumor necrosis factor (TNF)-␣, interleukin (IL)-1␤, interferon-␤, and N -nitro-L-arginine methyl ester hydrochloride but not N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398) significantly inhibited HMGB1 release in LPS-activated cells. Production of TNF-␣, IL-1␤ , and IFN-␤ was significantly reduced by pretreatment of HO-1 inducers, CORM-2, or HO-1 transfection in LPS-activated cells. Plasma levels of HMGB1 in mice challenged with LPS or CLP were significantly reduced by the administration of HO-1 inducers or CORM-2, which was accompanied by either reduction (pretreatment) or no change (delayed administration) of serum TNF-␣ and IL-1␤ levels. Regardless of pretreatment or delayed administration, CORM-2 and hemin rescued mice from lethal endotoxemia and sepsis induced by LPS or CLP. Taken together, we concluded that HO-1-derived CO reduces HMGB1 release in LPS-activated cells and LPS-or CLP-induced animal model of sepsis. Sepsis is defined as a systemic inflammatory response syndrome from a microbial infection that results from excessive stimulation of the host immune system by pathogen components to produce various proinflammatory cytokines, and their overproduction causes systemic inflammation that can lead to the lethal multiple organ damage ABBREVIATIONS: HMGB1, high-mobility group box 1; HO-1, heme-oxygenase-1; CORM-2, carbon monoxide-releasing molecule II; LPS, lipopolysaccharide; CLP, cecal ligation and puncture; CoPPIX, cobalt protoporphyrin IX; COX, cyclooxygenase; DFO, deferoxamine mesylate; L-NAME, N -nitro-L-arginine methyl ester hydrochloride; iNOS, inducible nitric-oxide synthase; methane sulfonamide; siRNA, small interfering RNA; TNF-␣, tumor necrosis factor-␣; IL-1␤, interleukin-1␤; INF-␤, interferon-␤; ELISA, enzymelinked immunosorbent assay; HbO 2 , oxyhemoglobin; DMSO, dimethyl sulfoxide

Comparative proteomic analysis of malformed umbilical cords from somatic cell nuclear transfer-derived piglets: implications for early postnatal death

Background: Somatic cell nuclear transfer (scNT)-derived piglets have high rates of mortality, including stillbirth and postnatal death. Here, we examined severe malformed umbilical cords (MUC), as well as other organs, from nine scNT-derived term piglets. Results: Microscopic analysis revealed complete occlusive thrombi and the absence of columnar epithelial layers in MUC (scNT-MUC) derived from scNT piglets. scNT-MUC had significantly lower expression levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) and angiogenesis-related genes than umbilical cords of normal scNT piglets (scNT-N) that survived into adulthood. Endothelial cells derived from scNT-MUC migrated and formed tubules more slowly than endothelial cells from control umbilical cords or scNT-N. Proteomic analysis of scNT-MUC revealed significant down-regulation of proteins involved in the prevention of oxidative stress and the regulation of glycolysis and cell motility, while molecules involved in apoptosis were significantly up-regulated. Histomorphometric analysis revealed severe calcification in the kidneys and placenta, peliosis in the liver sinusoidal space, abnormal stromal cell proliferation in the lungs, and tubular degeneration in the kidneys in scNT piglets with MUC. Increased levels of apoptosis were also detected in organs derived from all scNT piglets with MUC. Conclusion: These results suggest that MUC contribute to fetal malformations, preterm birth and low birth weight due to underlying molecular defects that result in hypoplastic umbilical arteries and/or placental insufficiency. The results of the current study demonstrate the effects of MUC on fetal growth and organ development in scNT-derived pigs, and provide important insight into the molecular mechanisms underlying angiogenesis during umbilical cord development

Moringa Extract Attenuates Inflammatory Responses and Increases Gene Expression of Casein in Bovine Mammary Epithelial Cells

Bovine mastitis is a common inflammatory disease in the udder of dairy cows that causes economic loss to dairy industries. The development of alternative strategies, especially the utilization of natural products, e.g., Moringa oleifera, has gained a lot of interests. The objective of the current study was to investigate the protective effects of moringa extract (ME) in bovine mammary epithelial cells (MAC-T) in in vitro settings. Radical scavenging capacities and anti-inflammatory properties of ME were examined using lipopolysaccharide (LPS)-challenged MAC-T cells. ME showed significant radical scavenging activities. In addition, ME decreased reactive oxygen species produced by LPS in cells. ME also attenuated inflammatory cyclooxygenase-2 expression induced by LPS by down-regulating NF-κB signaling cascade. Moreover, ME ameliorated LPS-induced pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1β, and interleukin-6. Furthermore, ME up-regulated mRNA expression levels of heme oxygenase-1, NAD(P)H: quinone oxidoreductase-1, and thioredoxin reductase 1. Importantly, ME promoted differentiated MAC-T cells by increasing mRNA expression levels of α-casein S1, α-casein S2, and β-casein. In conclusion, ME has beneficial effects in bovine mammary epithelial cells through its anti-inflammatory, antioxidant, and casein production properties. Our study provides evidence that ME could be a good candidate for a feed supplement to decrease inflammatory responses due to bovine mastitis

Effect of Roasting Time and Cryogenic Milling on the Physicochemical Characteristics of Dried Ginseng Powder

This study aimed to evaluate the effect of reduced particle size of ginseng by roasting and cryogenic milling on increasing its water solubility and physiological activity. The samples were roasted for different times (9–21 min) and generated in different sizes (10–50, and >50 μm). All roasted samples revealed significantly smaller particle sizes than did non-roasted samples, based on Sauter mean diameter (D [3,2], p < 0.05). Furthermore, the particle sizes of roasted samples decreased until roasting up to 15 min. In terms of the water solubility index (WSI), antioxidant activity, total polyphenol content (TPC), and total polysaccharides according to particle size, 10–20 μm-sized samples showed the highest values when compared with >50 μm-sized samples. Based on roasting time, WSI values of all samples roasted for up to 15 min were higher than those of the control (not roasted) (p < 0.05). Antioxidant activity and TPC also increased with increasing roasting time. Total polysaccharide content was the highest upon roasting for 15 min, except for the 10–20 μm sample. Ginsenoside content of roasted samples >20 μm size was higher than that of the control (not roasted) except after 15 min of roasting. Therefore, roasting and cryogenic milling are effective in producing ginseng root powder

In Vitro Studies of Fermented Korean Chung-Yang Hot Pepper Phenolics as Inhibitors of Key Enzymes Relevant to Hypertension and Diabetes

This study was investigated to evaluate the antioxidant activity, the angiotensin I-converting enzyme (ACE) inhibition effect, and the α-amylase and α-glucosidase inhibition activities of hot pepper water extracts both before and after their fermentation. The fermented pepper water extract (FP) showed significantly higher total phenol content, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical inhibition effect, metal chelating activity and ACE inhibition activity compared to the non-fermented raw pepper water extract (RP) (p < 0.05). Meanwhile, the FP showed lower α-amylase and higher α-glucosidase inhibitory activities, but the RP showed similar levels of α-amylase and α-glucosidase inhibitory activities. Taken together, these results suggested that fermented pepper extract using water should be expected to have potentially inhibitory effects against both hyperglycemia and hypertension

Ginsenoside Re Mitigates 6-Hydroxydopamine-Induced Oxidative Stress through Upregulation of GPX4

Ginsenosides are active components found abundantly in ginseng which has been used as a medicinal herb to modify disease status for thousands of years. However, the pharmacological activity of ginsenoside Re in the neuronal system remains to be elucidated. Neuroprotective activity of ginsenoside Re was investigated in SH-SY5Y cells exposed to 6-hydroxydopamine (6-OHDA) to induce cellular injury. Ginsenoside Re significantly inhibited 6-OHDA-triggered cellular damage as judged by analysis of tetrazolium dye reduction and lactose dehydrogenase release. In addition, ginsenoside Re induced the expression of the antioxidant protein glutathione peroxidase 4 (GPX4) but not catalase, glutathione peroxidase 1, glutathione reductase, or superoxide dismutase-1. Furthermore, upregulation of GPX4 by ginsenoside Re was mediated by phosphoinositide 3-kinase and extracellular signal-regulated kinase but not by p38 mitogen-activated protein kinase or c-Jun N-terminal kinase. Ginsenoside Re also suppressed 6-OHDA-triggered cellular accumulation of reactive oxygen species and peroxidation of membrane lipids. The GPX4 inhibitor (1S,3R)-RSL3 reversed ginsenoside Re-mediated inhibition of cellular damage in SH-SY5Y cells exposed to 6-OHDA, indicating that the neuronal activity of ginsenoside Re is due to upregulation of GPX4. These findings suggest that ginsenoside Re-dependent upregulation of GPX4 reduces oxidative stress and thereby alleviates 6-OHDA-induced neuronal damage