N-Acetylcysteine and Allopurinol Synergistically Enhance Cardiac Adiponectin Content and Reduce Myocardial Reperfusion Injury in Diabetic Rats

Background: Hyperglycemia-induced oxidative stress plays a central role in the development of diabetic myocardial complications. Adiponectin (APN), an adipokine with anti-diabetic and anti-ischemic effects, is decreased in diabetes. It is unknown whether or not antioxidant treatment with N-acetylcysteine (NAC) and/or allopurinol (ALP) can attenuate APN deficiency and myocardial ischemia reperfusion (MI/R) injury in the early stage of diabetes. Methodology/Principal Findings: Control or streptozotocin (STZ)-induced diabetic rats were either untreated (C, D) or treated with NAC (1.5 g/kg/day) or ALP (100 mg/kg/day) or their combination for four weeks starting one week after STZ injection. Plasma and cardiac biochemical parameters were measured after the completion of treatment, and the rats were subjected to MI/R by occluding the left anterior descending artery for 30 min followed by 2 h reperfusion. Plasma and cardiac APN levels were decreased in diabetic rats accompanied by decreased cardiac APN receptor 2 (AdipoR2), reduced phosphorylation of Akt, signal transducer and activator of transcription 3 (STAT3) and endothelial nitric oxide synthase (eNOS) but increased IL-6 and TNF-α (all P<0.05 vs. C). NAC but not ALP increased cardiac APN concentrations and AdipoR2 expression in diabetic rats. ALP enhanced the effects of NAC in restoring cardiac AdipoR2 and phosphorylation of Akt, STAT3 and eNOS in diabetic rats. Further, NAC and ALP, respectively, decreased postischemic myocardial infarct size and creatinine kinase-MB (CK-MB) release in diabetic rats, while their combination conferred synergistic protective effects. In addition, exposure of cultured rat cardiomyocytes to high glucose resulted in significant reduction of cardiomyocyte APN concentration and AdipoR2 protein expression. APN supplementation restored high glucose induced AdipoR2 reduction in cardiomyocytes. Conclusions/Significance: NAC and ALP synergistically restore myocardial APN and AdipoR2 mediated eNOS activation. This may represent the mechanism through which NAC and ALP combination greatly reduces MI/R injury in early diabetic rats. © 2011 Wang et al.published_or_final_versio

Fluid Flow and Inclusion Motion in Horizontal Continuous Casting Tundishes

The configuration of the tundish for a two-strand horizontal continuous caster were designed and optimized through water modeling, mathematical modeling and industrial trials. Three designs of the tundish were studied: the original tundish without any flow control devices, the tundish with a turbulence inhibitor at the bottom, and the tundish with an inlet launder and a tilted dam. In the water modeling, the residence time, the fraction of the dead zone, and fluid flow pattern were investigated. In the mathematical modeling, fluid flow, heat transfer and inclusion motion and removal were calculated. In the industrial trials, the total oxygen, nitrogen, and inclusions in the steel samples from the tundish and the billets were investigated. The results indicated that the tundish with an inlet launder and a tilted dam was the best of the three designs

Involvement of AP-1 and C/EBPβ in upregulation of endothelin B (ETB) receptor expression in a rodent model of glaucoma.

Previous studies showed that the endothelin B receptor (ETB) expression was upregulated and played a key role in neurodegeneration in rodent models of glaucoma. However, the mechanisms underlying upregulation of ETB receptor expression remain largely unknown. Using promoter-reporter assays, the 1258 bp upstream the human ETB promoter region was found to be essential for constitutive expression of ETB receptor gene in human non-pigmented ciliary epithelial cells (HNPE). The -300 to -1 bp and -1258 to -600 bp upstream promoter regions of the ETB receptor appeared to be the key binding regions for transcription factors. In addition, the crucial AP-1 binding site located at -615 to -624 bp upstream promoter was confirmed by luciferase assays and CHIP assays which were performed following overexpression of c-Jun in HNPE cells. Overexpression of either c-Jun or C/EBPβ enhanced the ETB receptor promoter activity, which was reflected in increased mRNA and protein levels of ETB receptor. Furthermore, knock-down of either c-Jun or C/EBPβ in HNPE cells was significantly correlated to decreased mRNA levels of both ETB and ETA receptor. These observations suggest that c-Jun and C/EBPβ are important for regulated expression of the ETB receptor in HNPE cells. In separate experiments, intraocular pressure (IOP) was elevated in one eye of Brown Norway rats while the corresponding contralateral eye served as control. Two weeks of IOP elevation produced increased expression of c-Jun and C/EBPβ in the retinal ganglion cell (RGC) layer from IOP-elevated eyes. The mRNA levels of c-Jun, ETA and ETB receptor were upregulated by 2.2-, 3.1- and 4.4-fold in RGC layers obtained by laser capture microdissection from retinas of eyes with elevated IOP, compared to those from contralateral eyes. Taken together, these data suggest that transcription factor AP-1 plays a key role in elevation of ETB receptor in a rodent model of ocular hypertension

The −615 to −624 bp region of the ET_B receptor promoter is the AP-1 binding site in HNPE cells.

<p><b>A.</b> 0.5 µg of ET_B receptor promoter constructs and 0.5 µg of either the empty vector or c-Jun expressing plasmid DNA were co-transfected into HNPE cells in each well of a 12-well plate. Luciferase assays were performed 24 hours after transfection. In the absence of c-Jun overexpression, luciferase activity obtained from the mutant construct was 54% of the value from wild-type promoter construct. Following c-Jun overexpression, a similar trend of decrease (57% of the wild-type promoter) in promoter activity was observed in the mutant construct. Bars represented mean ± SEM, n = 3. <b>B.</b> CHIP assays were performed in HNPE cells, which were transfected with vector control or c-Jun overexpression plasmid DNA. DNA fragments were immunoprecipitated by incubation with or without c-Jun antibody. The data obtained by real-time PCR showed that there was a 4.1 fold increase of AP-1 binding to this specific region of ET_B receptor promoter in c-Jun overexpression group compared to vector-transfected control (*p<0.05, student’s <i>t</i>-test). The positive control, promoter region of cyclooxygenase-2 (COX-2) was increased 9.6 fold in c-Jun overexpression group (**p<0.005, student’s <i>t</i>-test), whereas promoter region of glyceraldehyde 3-phosphate dehydrogenase (<i>GAPDH)</i> which served as a negative control showed no appreciable change. Bars represented relative fold increased in data obtained from real-time PCR, n = 4–6. A representative agarose gel shows the amplified DNA from regular PCR at the end of 31 cycles.</p

Promoter activities of ET_B receptor promoter constructs with mutations in different AP-1 binding sites in HNPE cells.

<p>Six AP-1 binding sites were identified in the full-length human ET_B receptor promoter region. Corresponding mutations in the different AP-1 binding sites were introduced using site-directed mutagenesis. Luciferase assays were used to determine promoter activities of the full length ET_B receptor promoter and six mutant constructs. The deletion at −615 to −624 bp region of promoter abolished the luciferase activity by 45.3% compared to the wild-type ET_B receptor promoter (*p<0.05, One Way ANOVA, Student-Newman-Keuls). An apparent decrease in activity was also shown in mutations at −88 to −90 bp, −835 to −838 bp. The results indicated that the −615 to −624 bp AP-1 binding site was very important for ET_B receptor promoter activity in HNPE cells. Bars represented mean ± SEM, n = 3.</p

Promoter-reporter activities of promoter truncations and effect of overexpression of c-Jun and C/EBPβ on ET_B receptor promoter activity in human non-pigmented ciliary epithelial cells (HNPE).

<p><b>A.</b> Diagram of the predicted AP-1 binding sites on human ET_B receptor promoter region. Six AP-1 binding sites and forty C/EBPβ sites (not shown in the diagram) were predicted by the Promo-3 software. <b>B.</b> Luciferase assay showed promoter activities using different size constructs of the ET_B receptor promoter. 1 µg of promoter construct plasmid DNA was transfected into HNPE cells and luciferase activity was measured using a Luciferase assay kit (Promega). The 300 bp and 600 bp upstream promoter constructs yielded 21.7- and 23.4-fold, respectively, increase in luciferase activity, whereas 1258 bp promoter produced an 89.2-fold increase, compared to the empty vector control without a promoter sequence. <b>C.</b> Promoter activities of full-length ET_B receptor promoter in presence of c-Jun or C/EBPβ co-expression in HNPE cells. 0.5 µg of the human ET_B receptor promoter construct was co-transfected with 0.5 µg of either c-Jun or C/EBPβ construct in HNPE cells. The results showed that overexpression of either c-Jun or C/EBPβ boosted the luciferase activity 2.7 and 3.1 fold respectively, compared to that of the ET_B receptor promoter construct alone (*p<0.05, One Way ANOVA, Student-Newman-Keuls). Bars represented mean ± SEM, n = 3.</p

Immunostaining of c-Jun and C/EBPβ was significantly increased in retinal ganglion cells in eyes with IOP elevation for two weeks.

<p>The Morrison’s glaucoma model was developed in Brown Norway rats and rats with IOP elevation were maintained for 2 weeks. Paraformaldehyde-fixed retina sections from these rats were stained using specific antibodies to c-Jun and C/EBPβ (Santa Cruz Biotech). Eight to ten images were captured using Z-scan in a Zeiss 510meta confocal microscope from each view and stacked. <b>A.</b> Stained RGCs are indicated by white arrows. Red: c-Jun; Green: C/EBPβ; Blue: DAPI. Intense staining of c-Jun and C/EBPβ (indicated by arrows) was detected mainly in RGC layer from retinas of rats and increased staining was observed in retinal sections from IOP elevated rat eyes. <b>B.</b> A representative plot from a Brown Norway rat which was subjected to IOP elevation and maintained for 2 weeks. IOP was measured twice a week and values were plotted as mean ± SD (n = 10) for each measurement. The IOP elevation generated 66.2 mm Hg-days of IOP exposure in this rat. <b>C.</b> Fluorescent intensity was measured at 10 different regions in the ganglion cell layers using NIH ImageJ. The fluorescent intensity values are shown as mean ± SEM, n = 10.</p

Regulated protein and mRNA levels of ET_A receptor, ET_B receptor in response to alteration of c-Jun, C/EBPβ expression.

<p><b>A.</b> Western blot analysis was used to detect the protein level of ET_B receptor in HNPE cells following overexpression of c-Jun or C/EBPβ. ET_B receptor protein was upregulated in membrane fractions of HNPE cell when either c-Jun or C/EBPβ was overexpressed. Calnexin, a housekeeping gene, served as a loading control. <b>B.</b> Relative mRNA expression of ET_A and ET_B receptors was analyzed using real-time PCR in HNPE transfected with c-Jun or C/EBPβ plasmid. Overexpression of c-Jun or C/EBPβ triggered an increase of ET_A and ET_B receptor mRNA level. Results showed as mean ± SEM, n = 3. <b>C.</b> siRNAs were employed to knock down c-Jun or C/EBPβ. Total RNA was extracted from HNPE cells transfected with c-Jun or C/EBPβ siRNA and 1 µg of total RNA was transcribed to cDNA. Cyclophilin A served as an internal control for normalization of equal loading of mRNA in the RT-PCR analysis. Knock-down of c-Jun or C/EBPβ significantly reduced the mRNA level of ET_A and ET_B receptor by more than 5 fold. Knock-down effect in protein level of c-Jun siRNA was also confirmed by Western blot, whereas knock-down effect of C/EBPβ was not detectable.</p