Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid.

Background: Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells. Methods: Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed. Results: The C3 deposition level in GTKO pPBMCs or pECs in GVB++ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs. Conclusion: TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.

Authentication of tejocote (Crataegus mexicana) dietary supplements based on DNA barcoding and chemical profiling.

Tejocote (Crataegus mexicana, Mexican hawthorn), known as a weight-loss supplement, has been marketed online and is easily available for overseas direct purchase. Alipotec (brand name) is known as one of the most popular products containing tejocote in Mexico and other countries. However, adverse effects have been reported by users of these supplements. Therefore it is necessary to find the reason for the side effect. Dietary supplement samples labelled as containing tejocote were analysed using mass spectrometry and DNA barcoding analysis. Our results demonstrate that Alipotec samples contained ingredients from different species, yellow oleander instead of tejocote. The rpoB barcode region was able to differentiate between tejocote and yellow oleander species. Moreover, it was also observed that three compounds, including thevetin B, neriifolin, and digitoxigenin, clearly distinguish between tejocote and yellow oleander samples. This is the first and preliminary investigation to use an integrated approach of both chemical and genomic profiling for the authentication of dietary supplement containing tejocote.

ULK1 prevents cardiac dysfunction in obesity through autophagy-meditated regulation of lipid metabolism.

AIMS: Autophagy is essential to maintain tissue homeostasis, particularly in long-lived cells such as cardiomyocytes. Whereas many studies support the importance of autophagy in the mechanisms underlying obesity-related cardiac dysfunction, the role of autophagy in cardiac lipid metabolism remains unclear. In the heart, lipotoxicity is exacerbated by cardiac lipoprotein lipase (LPL), which mediates accumulation of fatty acids to the heart through intravascular triglyceride (TG) hydrolysis. METHODS AND RESULTS: In both genetic and dietary models of obesity, we observed a substantial increase in cardiac LPL protein levels without any change in messenger ribonucleic acid (mRNA). This was accompanied by a dramatic down-regulation of autophagy in the heart, as revealed by reduced levels of unc-51 like kinase-1 (ULK1) protein. To further explore the relationship between cardiac LPL and autophagy, we generated cardiomyocyte-specific knockout mice for ulk1 (Myh6-cre/ulk1fl/fl), Lpl (Myh6-cre/Lplfl/fl), and mice with a combined deficiency (Myh6-cre/ulk1fl/flLplfl/fl). Similar to genetic and dietary models of obesity, Myh6-cre/ulk1fl/fl mice had a substantial increase in cardiac LPL levels. When these mice were fed a high-fat diet (HFD), they showed elevated cardiac TG levels and deterioration in heart function. However, with combined deletion of LPL and ULK1 in Myh6-cre/ulk1fl/flLplfl/fl mice, HFD feeding did not lead to alterations in levels of TG or diacylglycerol, or in cardiac function. To further elucidate the role of autophagy in cardiac lipid metabolism, we infused a peptide that enhanced autophagy (D-Tat-beclin1). This effectively lowered LPL levels at the coronary lumen by restoring autophagy in the genetic model of obesity. This decrease in cardiac luminal LPL was associated with a reduction in TG levels and recovery of cardiac function. CONCLUSION: These results provide clear evidence of the critical role of modulating cardiac LPL activity through autophagy-mediated proteolytic clearance as a potential novel strategy to overcome obesity-related cardiomyopathy.

Identification of OCTN2 variants and their association with phenotypes of Crohn's disease in a Korean population.

Crohn's disease (CD) is a chronic inflammatory bowel disease and a genetic variant in the OCTN2, g.-207G > C is significantly associated with CD susceptibility. This study was aimed to identify novel OCTN2 functional promoter variants and their roles in transcriptional regulation using various in vitro assays. In addition, we investigated the association between OCTN2 genotypes and CD through genetic analysis using DNA samples from 193 patients with CD and 281 healthy controls. Among the three major promoter haplotypes of OCTN2 identified, one haplotype, H3, showed a significant decrease in promoter activity: two polymorphisms in H3 were associated with a significant reduction in promoter activity. In particular, we found that the reduced transcriptional activity of those two polymorphisms results from a reduction in the binding affinity of the activators, NF-E2 and YY1, to the OCTN2 promoter. The functional haplotype of the OCTN2 promoter was associated with clinical course of CD such as the disease behavior and need for surgery. However, genetic variants or haplotypes of OCTN2 did not affect the susceptibility to CD. Our results suggest that a common promoter haplotype of OCTN2 regulates the transcriptional rate of OCTN2 and influences the clinical course of CD.

Effects of ursolic acid on glucose metabolism, the polyol pathway and dyslipidemia in non-obese type 2 diabetic mice.

Ursolic acid (UA) is a pentacyclic triterpenoid compound that naturally occurs in fruits, leaves and flowers of medicinal herbs. This study investigated the dose-response efficacy of UA (0.01 and 0.05%) on glucose metabolism, the polyol pathway and dyslipidemia in streptozotocin/nicotinamide-induced diabetic mice. Supplement with both UA doses reduced fasting blood glucose and plasma triglyceride levels in non-obese type 2 diabetic mice. High-dose UA significantly lowered plasma free fatty acid, total cholesterol and VLDL-cholesterol levels compared with the diabetic control mice, while LDL-cholesterol levels were reduced with both doses. UA supplement effectively decreased hepatic glucose-6-phosphatase activity and increased glucokinase activity, the glucokinase/glucose-6-phosphatase ratio, GLUT2 mRNA levels and glycogen content compared with the diabetic control mice. UA supplement attenuated hyperglycemia-induced renal hypertrophy and histological changes. Renal aldose reductase activity was higher, whereas sorbitol dehydrogenase activity was lower in the diabetic control group than in the non-diabetic group. However, UA supplement reversed the biochemical changes in polyol pathway to normal values. These results demonstrated that low-dose UA had preventive potency for diabetic renal complications, which could be mediated by changes in hepatic glucose metabolism and the renal polyol pathway. High-dose UA was more effective anti-dyslipidemia therapy in non-obese type 2 diabetic mice.

Fabrication of a Ni nano-imprint stamp for an anti-reflective layer using an anodic aluminum oxide template.

Aluminum anodizing can alter pore diameter, density distribution, periodicity and layer thickness in a controlled way. Because of this property, porous type anodic aluminum oxide (AAO) was used as a template for nano-structure fabrication. The alumina layer generated at a constant voltage increased the pore size from 120 nm to 205 nm according to an increasing process time from 60 min to 150 min. The resulting fabricated AAO templates had pore diameters at or less than 200 nm. Ni was sputtered as a conductive layer onto this AAO template and electroplated using DC and pulse power. Comparing these Ni stamps, those generated from electroplating using on/reverse/off pulsing had an ordered pillar array and maintained the AAO template morphology. This stamp was used for nano-imprinting on UV curable resin coated glass wafer. Surface observations via electron microscopy showed that the nano-imprinted patterned had the same shape as the AAO template. A soft mold was subsequently fabricated and nano-imprinted to form a moth-eye structure on the glass wafer. An analysis of the substrate transmittance using UV-VIS/NIR spectroscopy showed that the transmittance of the substrate with the moth-eye structure was 5% greater that the non-patterned substrate.

Retinal attenuates inflammatory arthritis by reciprocal regulation of IL-17-producing T cells and Foxp3(+) regulatory T cells and the inhibition of osteoclastogenesis.

Retinoids (e.g., vitamin A and its derivatives) can regulate immune responses. The aim of this study was to determine whether all-trans retinaldehyde (retinal), a vitamin A derivative, can inhibit inflammatory responses and joint destruction in DBA/1J mice with collagen-induced arthritis (CIA). The arthritis score and incidence of arthritis were lower in mice treated with retinal compared to those treated with cottonseed oil. Histopathologic evidence of joint damage was lower in mice treated with retinal, corresponding with a reduction in the infiltration of immune cells in mice treated with retinal type II collagen (CII)-stimulated spleen cells. In addition, the expression of proinflammatory cytokines, oxidative stress proteins, and osteoclast markers were significantly reduced in mice treated with retinal. In vitro, retinal induced increased Foxp3 expression and inhibited Th17 development. The proportion of Foxp3(+) Treg cells was increased in the spleens of mice treated with retinal, whereas the proportion of Th17 cells was reduced. In both mice and a human culture system, tartrate-resistant acid phosphatase (TRAP) positive mononuclear cells and multinucleated cells were significantly reduced after treatment with retinal. The expression of osteoclast differentiation markers was dramatically decreased upon addition of retinal. This is the first study to demonstrate the therapeutic effect of retinal on an autoimmune arthritis model in mice through reciprocal regulation of Th17 and regulatory T cells and protection of differentiation and activation of osteoclasts. Taken together, our findings indicate that retinal has profound immunoregulatory functions and potential value for the treatment of autoimmune inflammatory disorders.

Anti-inflammatory mechanism of compound K in activated microglia and its neuroprotective effect on experimental stroke in mice.

Microglial activation plays a pivotal role in the pathogenesis of various neurologic disorders, such as cerebral ischemia, Alzheimer's disease, and Parkinson's disease. Thus, controlling microglial activation is a promising therapeutic strategy for such brain diseases. In the present study, we found that a ginseng saponin metabolite, compound K [20-O-D-glucopyranosyl-20(S)-protopanaxadiol], inhibited the expressions of inducible nitric-oxide synthase, proinflammatory cytokines, monocyte chemotactic protein-1, matrix metalloproteinase-3, and matrix metalloproteinase-9 in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and primary cultured microglia. Subsequent mechanistic studies revealed that compound K suppressed microglial activation via inhibiting reactive oxygen species, mitogen-activated protein kinases, and nuclear factor-κB/activator protein-1 activities with enhancement of heme oxygenase-1/antioxidant response element signaling. To address the anti-inflammatory effects of compound K in vivo, we used two brain disease models of mice: sepsis (systemic inflammation) and cerebral ischemia. Compound K reduced the number of Iba1-positive activated microglia and inhibited the expressions of tumor necrosis factor-α and interleukin-1ß in the LPS-induced sepsis brain. Furthermore, compound K reduced the infarct volume of ischemic brain induced by middle cerebral artery occlusion and suppressed microglial activation in the ischemic cortex. The results collectively suggest that compound K is a promising agent for prevention and/or treatment of cerebral ischemia and other neuroinflammatory disorders.

Anti-inflammatory mechanism of ginsenoside Rh1 in lipopolysaccharide-stimulated microglia: critical role of the protein kinase A pathway and hemeoxygenase-1 expression.

Microglia activation plays a pivotal role in neurodegenerative diseases, and thus controlling microglial activation has been suggested as a promising therapeutic strategy for neurodegenerative diseases. In the present study, we showed that ginsenoside Rh1 inhibited inducible nitric oxide synthase, cyclooxygenase-2, and pro-inflammatory cytokine expression in lipopolysaccharide (LPS)-stimulated microglia, while Rh1 increased anti-inflammatory IL-10 and hemeoxygenase-1 (HO-1) expression. Suppression of microglial activation by Rh1 was also observed in the mouse brain following treatment with LPS. Subsequent mechanistic studies revealed that Rh1 inhibited LPS-induced MAPK phosphorylation and nuclear factor-κB (NF-κB)-mediated transcription without affecting NF-κB DNA binding. As the increase of pCREB (cAMP responsive element-binding protein) is known to result in suppression of NF-κB-mediated transcription, we examined whether Rh1 increased pCREB levels. As expected, Rh1 increased pCREB, which was shown to be related to the anti-inflammatory effect of Rh1 because pre-treatment with protein kinase A inhibitors attenuated the Rh1-mediated inhibition of nitric oxide production and the up-regulation of IL-10 and HO-1. Furthermore, treatment of HO-1 shRNA attenuated Rh1-mediated inhibition of nitric oxide and reactive oxygen species production. Through this study, we have demonstrated that protein kinase A and its downstream effector, HO-1, play a critical role in the anti-inflammatory mechanism of Rh1 by modulating pro- and anti-inflammatory molecules in activated microglia.

Anti-inflammatory mechanism of ginseng saponins in activated microglia.

In the present study, we investigated the effect of ginseng extract (KRG) and total saponins (GTS) on microglial activation. KRG and GTS inhibited LPS-induced expression of iNOS, MMP-9 and proinflammatory cytokines in microglial cells. Suppression of microglial activation by ginseng was also observed in the mouse brain inflamed by LPS. Furthermore, KRG and GTS significantly suppressed NF-kappaB and MAP kinase activities, which are upstream signaling molecules in inflammation. Among the individual ginsenosides tested, Rh2, Rh3 and compound K significantly inhibited LPS-induced iNOS and cytokine expressions. Therefore, the inhibition of microglial activation by ginseng saponins may a good potential therapeutic modality for neurodegenerative diseases.

The beneficial effect of high loading dose of rosuvastatin before percutaneous coronary intervention in patients with acute coronary syndrome.

BACKGROUND: Statin therapy prior to percutaneous coronary intervention (PCI) is associated with reduced mortality and periprocedural myocardial injury after PCI. We studied whether single high dose statin loading is beneficial on the outcome of patients with acute coronary syndrome (ACS) underwent PCI. METHODS: Consecutive 445 patients with ACS who underwent PCI were randomly assigned to either the group of no statin treatment before PCI (Control group: n=220, 63+/-11 years, male 62%) or the group of 40 mg rosuvastatin loading before PCI (Rosuvastatin group: n=225, 64+/-10 years, male 60%). Incidence of periprocedural myocardial injury was assessed by analysis of creatinine kinase-MB (CK-MB) and cardiac troponin T before PCI, at 6 h and the next morning after PCI. RESULT: There were no significant differences in clinical characteristics between the two groups. After PCI, incidence of periprocedural myocardial injury was higher in control than in rosuvastatin group (11.4% versus 5.8%, p=0.035). Mean preprocedural CK-MB and high sensitivity C-reactive protein were similar between the two groups, whereas after PCI, peak values of both markers were elevated significantly higher in control than in rosuvastatin group. Multivariate analysis revealed that no prior use of statin (OR=2.2; 95% CI=1.1-4.6; p=0.029), procedural complication (OR=3.1; 95% CI=1.4-6.9; p=0.007) and multi-vessel disease (OR=2.6; 95% CI=1.0-6.6; p=0.039) were the independent predictors for periprocedural myocardial infarction. CONCLUSION: Single high dose of rosuvastatin prior to PCI reduces periprocedural myocardial injury in patients with ACS.

Identification and molecular characterization of two hepcidin genes from black rockfish (Sebastes schlegelii).

Antimicrobial peptides (AMPs) are important mediators of the immune response against bacteria and cysteine-rich peptide hepcidin is a 20-26 residues member with functions in iron regulation and antimicrobial activity. Here, we have identified two different types of hepcidin cDNA from the Black rockfish, Sebastes schlegelii, by EST analysis. Both hepcidin genes (hepcidin I and II) consist of two introns and three exons that encode a prepropeptide (88 amino acids). A TATA box and several consensus-binding motifs for transcription factors were found in the upstream of the transcriptional start site. Semi-quantitative RT-PCR analysis suggested that hepcidin I transcripts were detected in various tissues, while hepcidin II was only expressed in the liver. During the bacterial challenge with the fish pathogen, Streptococcus iniae, two hepcidin genes were differentially expressed. Hepcidin I and II dramatically increased at 24 h post-injection, then gradually declined at 3 days in hepcidin II, while hepcidin I expression continued at 3 days after challenge.

Neural network-based analysis of thiol proteomics data in identifying potential selenium targets.

Generation of a monomethylated selenium metabolite is critical for the anticancer activity of selenium. Because of its strong nucleophilicity, the metabolite can react directly with protein thiols to cause redox modification. Here, we report a neural network-based analysis to identify potential selenium targets. A reactive thiol specific reagent, BIAM, was used to monitor thiol proteome changes on 2D gel. We constructed a dynamic model and evaluated the relative importance of proteins mediating the cellular responses to selenium. Information from this study will provide new clues to unravel mechanisms of anticancer action of selenium. High impact selenium targets could also serve as biomarkers to gauge the efficacy of selenium chemoprevention.

Du-zhong (Eucommia ulmoides Oliv.) cortex water extract alters heme biosynthesis and erythrocyte antioxidant defense system in lead-administered rats.

This study examined the ameliorative effect of a Du-zhong (Eucommia ulmoides Oliv.) cortex water extract (DzCw) on heme biosynthesis and erythrocyte antioxidant enzyme activities in lead (Pb)-administered rats. Male rats were divided into three groups: normal control group, Pb control group (Pb), and DzCw-administered Pb group (Pb + DzCw). The Pb (25 mg/kg of body weight) was administered orally once a week for 4 weeks, while the DzCw was administered orally at a dosage of 0.139 g of DzCw/kg of body weight/day. DzCw administration significantly lowered plasma Pb concentration compared with the Pb group. Furthermore, the blood hematocrit and hemoglobin levels were significantly higher in the Pb + DzCw group than in the Pb group. Although the blood and hepatic delta-aminolevulinic acid dehydratase (ALAD) activities were significantly lower in the Pb group compared with the normal control group, both ALAD activities was normalized with the administration of DzCw. The erythrocyte superoxide dismutase and catalase activities were significantly higher in the Pb group than in the normal control group, whereas the glutathione peroxidase activity and glutathione level were lowered by Pb administration compared with the normal group. However, the administration of DzCw was found to enhance the antioxidant defense system and significantly lower lipid peroxidation levels in erythrocytes compared with the Pb group. These results indicate that the DzCw administration alleviated the Pb-induced oxidative stress in the erythrocytes through elevating the blood and hepatic ALAD activity and enhancing the antioxidant enzyme activities.

Two cinnamate derivatives produce similar alteration in mRNA expression and activity of antioxidant enzymes in rats.

Cinnamate is a widespread secondary metabolite of phenolic compound synthesized by plants for defensive purposes. The current study was designed to investigate the effect of two structurally related cinnamate derivatives, 4-hydroxycinnamate and 3-(4-hydroxyphenyl)propionic acid (HPP), on the mRNA expression and activity of antioxidant enzymes in high-cholesterol-fed rats. Male rats were fed a 1 g/100 g high-cholesterol diet with supplements of either 4-hydroxycinnamate or HPP (0.135 mmol/100 g diet) for 6 weeks. The plasma paraoxonase activity was found to be higher in the cinnamate-derivative-supplemented groups than in the control group. The erythrocyte superoxide dismutase (SOD) and catalase (CAT) activities, plus glutathione (GSH) level, were all significantly higher in the 4-hydroxycinnamate- and HPP-supplemented groups than in the control group. However, both 4-hydroxycinnamate and HPP supplementation significantly lowered the hepatic activities and mRNA expression of CAT and glutathione peroxidase (GSH-Px) compared to the control group. The hepatic mRNA expression and activity of SOD did not differ between the groups. The hepatic thiobarbituric acid reactive substances (TBARS) level was significantly lowered by the 4-hydroxycinnamate and HPP supplementation. Accordingly, these results indicate that supplementation by 4-hydroxycinnamate and HPP would seem to enhance the antioxidative defense of erythrocyte. Both HPP and 4-hydroxycinnamate would appear to be beneficial in improving the function of antioxidative enzymes on a molecular level in high-cholesterol-fed rats.

Cinnamate supplementation enhances hepatic lipid metabolism and antioxidant defense systems in high cholesterol-fed rats.

This study investigated the effect of cinnamate, a phenolic compound found in cinnamon bark and other plant materials, on lipid metabolism and antioxidant enzyme activities in rats fed a high cholesterol diet. Three groups of rats were given a diet containing 1 g of cholesterol/kg for 6 weeks. The control group only received the high cholesterol diet, whereas the other two groups received a diet supplemented with lovastatin or cinnamate (0.1 g/100 g of diet). The plasma high-density lipoprotein-cholesterol levels were significantly higher in the cinnamate group than in either the control or lovastatin groups, and the atherogenic index was significantly lower in rats with cinnamate supplementation. Supplementation with cinnamate resulted in significantly lower hepatic cholesterol and triglyceride levels. Accumulation of hepatic lipid droplets was higher in the control group than in the rats supplemented with either cinnamate or lovastatin. Hepatic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity was significantly lower in the cinnamate group compared with the other groups, whereas only acyl-CoA:cholesterol acyltransferase activity was significantly lower in the lovastatin group compared with the control group. Cinnamate supplementation resulted in higher catalase and glutathione peroxidase activities, while hepatic thiobarbituric acid-reactive substances were significantly lower in both the cinnamate and lovastatin groups. The fecal acidic sterol was higher in the lovastatin group than in the control or cinnamate groups. These results suggest that dietary cinnamate inhibits hepatic HMG-CoA reductase activity, resulting in lower hepatic cholesterol content, and suppresses lipid peroxidation via enhancement of hepatic antioxidant enzyme activities.

Alternation of hepatic antioxidant enzyme activities and lipid profile in streptozotocin-induced diabetic rats by supplementation of dandelion water extract.

BACKGROUND: Dandelion water extract (DWE), an herbal medication, may have an effect on the activity and mRNA expression of hepatic antioxidant enzymes and lipid profile in streptozotocin (STZ)-induced diabetic rats. METHODS: Male Sprague-Dawley rats were divided into nondiabetic (control), diabetic, and diabetic-DWE-supplemented groups. Diabetes was induced by injecting streptozotocin (55 mg/kg BW, i.p.) in a citrate buffer. The extract was supplemented in 2.4 g of a DWE/kg diet. RESULTS: The DWE supplement significantly decreased the serum glucose concentration in the diabetic rats. The hepatic superoxide dismutase and catalase activities significantly increased and the GSH-Px activity decreased in the diabetic rats, compared with the control group. When the DWE supplement was given to the diabetic rats, the antioxidant enzyme activity reverted to near-control values. However, there was no difference in the mRNA expression concentrations of these enzymes between the groups. With regard to the hepatic lipid peroxidation product, the malondialdehyde (MDA) content was significantly higher in the diabetic group than in the nondiabetic group. However, the DWE supplement lowered the hepatic MDA concentration in the diabetic-induced rats. The DWE supplement also lowered the total cholesterol and triglyceride concentrations in the serum and hepatic tissue, while increasing the serum HDL-cholesterol in the diabetic rats. CONCLUSIONS: A DWE supplement can improve the lipid metabolism and is beneficial in preventing diabetic complications from lipid peroxidation and free radicals in diabetic rats.