Tonicity-responsive enhancer binding protein haplodeficiency attenuates seizure severity and NF-κB-mediated neuroinflammation in kainic acid-induced seizures.

Kainic acid (KA)-induced seizures followed by neuronal death are associated with neuroinflammation and blood-brain barrier (BBB) leakage. Tonicity-responsive enhancer binding protein (TonEBP) is known as a transcriptional factor activating osmoprotective genes, and in brain, it is expressed in neuronal nuclei. Thus dysregulation of TonEBP may be involved in the pathology of KA-induced seizures. Here we used TonEBP heterozygote (+/-) mice to study the roles of TonEBP. Electroencephalographic study showed that TonEBP (+/-) mice reduced seizure frequency and severity compared with wild type during KA-induced status epilepticus. Immunohistochemistry and western blotting analysis showed that KA-induced neuroinflammation and BBB leakage were dramatically reduced in TonEBP (+/-) mice. Similarly, TonEBP-specific siRNA reduced glutamate-induced death in HT22 hippocampal neuronal cells. TonEBP haplodeficiency prevented KA-induced nuclear translocation of NF-κB p65 and attenuated inflammation. Our findings identify TonEBP as a critical regulator of neuroinflammation and BBB leakage in KA-induced seizures, which suggests TonEBP as a good therapeutic target.

Reduction of experimental diabetic vascular leakage and pericyte apoptosis in mice by delivery of αA-crystallin with a recombinant adenovirus.

AIMS/HYPOTHESIS: The study aimed to evaluate the efficacy of recombinant adenovirus expressing αA-crystallin (Ad-αAc-Gfp) in reducing pericyte loss within retinal vasculature in early diabetes. METHODS: Diabetes was induced by streptozotocin injection into C57BL/6 mice. Ad-αAc-Gfp was delivered by intravitreous injection to the right eyes of mice 2 weeks before induction of diabetes. Vascular leakage was determined by fluorescent angiography, Evans Blue leakage assay and leucocyte adhesion test. Production of αA-crystallin was analysed by immunoblotting and double immunostaining and pericyte loss was analysed by pericyte count. RESULTS: Vessel leakage and pericyte loss were observed in the streptozotocin-induced diabetic retina. Decreased abundance of αA-crystallin in retinas 2 and 6 months after the induction of diabetes was confirmed by two-dimensional electrophoretic analysis, immunoblotting and RT-PCR. Double immunofluorescence staining for αA-crystallin and NG2 chondroitin sulphate proteoglycan revealed that αA-crystallin was predominantly produced in the retinal pericyte and that the number of αA-crystallin-producing pericytes decreased in the diabetic retina. Retinal infection with Ad-αAc-Gfp led to decreased pericyte loss and vascular leakage compared with control. CONCLUSIONS/INTERPRETATION: Intravitreal delivery of Ad-αAc-Gfp protects against vascular leakage in the streptozotocin-induced model of diabetes. This effect is associated with the inhibition of diabetic retinal pericyte loss in early diabetes, suggesting that αA-crystallin has a role in preventing the pathogenesis of early diabetic retinopathy.

Aged red garlic extract reduces lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages and acute pulmonary inflammation through haeme oxygenase-1 induction.

AIM: It is known that garlic has antioxidative and anti-inflammatory properties. Aged red garlic (ARG), a novel aged garlic formulation, has higher antioxidant effects than fresh raw garlic. This study was performed to examine the anti-inflammatory effects of ARG extract (ARGE). METHODS: The anti-inflammatory effects of ARGE were evaluated in the lipopolysaccharide (LPS)-treated Raw 264.7 macrophages and acute lung inflammatory mice. NO production was determined by the Griess method, and iNOS, HO-1 and COX-2 expressions were measured using Western blot analysis. Histology and inflammation extent of lung were analysed using haematoxylin-eosin staining and immunohistochemistry. RESULTS: ARGE treatment markedly reduced LPS-induced nitrite production in RAW 264.7 macrophages and reduced inducible nitric oxide synthase (iNOS) expression. Treatment of cells with ARGE led to a significant increase in haeme oxygenase-1 (HO-1) protein expression, which was mediated by stimulating the expression of nuclear factor erythroid 2-related factor 2 (Nrf2). Treatment with zinc protoporphyrin, a selective inhibitor of HO-1, significantly reversed the ARGE-mediated inhibition of nitrite production (P < 0.05). In LPS-induced inflammatory mice, ARGE treatment down-regulated iNOS and COX-2 expressions, while it up-regulated HO-1 expression. CONCLUSION: These results show that ARGE reduces LPS-induced nitric oxide production in RAW 264.7 macrophages through HO-1 induction and suggest that ARGE may have potential effects on prevention and treatment of acute inflammatory lung injury.

The relaxation effect and mechanism of action of higenamine in the rat corpus cavernosum.

Higenamine mediates cardiotonic, vascular relaxation and bronchodilator effects. The relaxation effects and the mechanism of action of higenamine on the rat corpus cavernosum (CC) were assessed to investigate the effect of higenamine on penile erection. Strips of CC and aorta were used in organ baths for isometric tension studies. Tension was measured with isometric force transducers, and muscle relaxation was expressed as the percent decrease in precontraction induced by phenylephrine (PE). The relaxation reactions were investigated in an endothelial-denuded group and groups pretreated with N(G)-nitro-L-arginine methyl ester (NO synthesis inhibitor), propranolol (ß-receptor blocker), indomethacin (COX inhibitor), glibenclamide (K(+)(ATP) channel inhibitor), 4-aminopyridine (membrane potential-dependent potassium channel inhibitor) and methylene blue (guanylyl cyclase inhibitor) for 30 min. Intracavernous pressure (ICP) was assessed in rats after the intravenous administration of higenamine, and changes in guanosine 3',5'-cyclic monophosphate and adenosine 3',5'-cyclic monophosphate (cAMP) concentrations were measured on the basis of the higenamine concentration. Also, the combined reaction of higenamine and the phosphodiesterase type-5 (PDE-5) inhibitors was assessed. Higenamine induced relaxation of the CC and the aortic strips precontracted with PE in a dose-dependent manner. The CC was significantly more relaxed than the aortic rings in response to the same higenamine concentration (P<0.05). The CC relaxation reaction was suppressed by the ß-receptor blocker propranolol. The cAMP concentration increased gradually with increased higenamine concentration (P<0.05). The ICP also increased with increased higenamine concentration in vivo (P<0.05). In the group pretreated with 10(-7) M higenamine, the relaxation reaction of CC induced by the PDE-5 inhibitor increased significantly, compared with CC exposed to the PDE-5 inhibitor but not pretreated with higenamine (P<0.05). In conclusion, higenamine induced relaxation of the rat CC in a dose-dependent manner. The effect may be mediated through ß-adrenoceptors. The results suggest that higenamine may be valuable as a new lead compound for treating erectile dysfunction.

Triamcinolone suppresses retinal vascular pathology via a potent interruption of proinflammatory signal-regulated activation of VEGF during a relative hypoxia.

We examined the effect of triamcinolone acetonide (TA), a corticosteroid, on the relationship between vascular pathophysiology and vascular endothelial growth factor (VEGF) activation in the retina of a rat model of oxygen-induced retinopathy (OIR). OIR was induced by exposure of hyperoxia (80% oxygen) to Sprague-Dawley (SD) rats from P2 to P14 and then returned to normoxic conditions. TA was intravitreal-injected once into the right eye of OIR rats at P15. Effects of TA on vascular pathophysiology or changes of various genes in response to hypoxia and/or proinflammation under hypoxic retina were assessed by the Evans-blue method, fluorescein isothiocyanate-dextran (FITC-D) infusion, immunoblotting, and ELIZA. TA not only reduced retinal neovascularization and vascular leakage in the OIR-rat retina, but also blocked the induction of hypoxia-response proinflammatory genes before it negatively controlled VEGF activation. These findings suggest a potential that TA suppresses retinal neovascular pathophysiology via proinflammation-mediated activation of VEGF during hypoxia.

Ethanol decreases the expression of mitochondrial cytochrome c oxidase mRNA in the rat.

To evaluate the molecular and cellular bases of effects of ethanol on the brain, we utilized a differential display-polymerase chain reaction. Several cDNA fragments were differentially expressed in the hippocampus of control vs. ethanol-treated rats. One of these genes was homologous to the rat mitochondrial cytochrome c oxidase mRNA. Northern blot analysis revealed that the expression of this message in the whole hippocampus was clearly lower after ethanol treatment. Using in situ hybridization, we also found that cytochrome c oxidase mRNA expression, especially in the CA1 and CA3 of the hippocampal regions, was significantly decreased by ethanol treatment. As cytochrome c oxidase is related to oxidative stress, the present study suggests that ethanol might affect the brain through modulation of an oxidative stress reaction.

Functional characteristics of two BKCa channel variants differentially expressed in rat brain tissues.

cDNAs encoding large-conductance Ca2+-activated K+ channel alpha-subunit (rSlo) were obtained from rat brain. From the DNA sequence of multiple rslo clones, we identified a specific sequence variation of 81 nucleotides, which is either absent from or present at the N-terminal region of a putative Ca2+-sensing domain of the channel. Transcripts containing such variations were detected in different ratios from several brain regions, and their functional significance was further examined. When heterologously expressed in Xenopus oocytes, both rSlo variants, named rSlo0 and rSlo27, generated Ca2+-activated and voltage-activated K+ currents characteristic of neuronal large-conductance Ca2+-activated K+ (BKCa) channels. Single-channel recordings of the two channels showed almost identical permeation characteristics and steady-state gating behavior. Noticeable differences between rSlo0 and rSlo27 were revealed when the macroscopic currents were measured at various voltages and intracellular Ca2+ concentrations. rSlo27 activated was more rapidly than rSlo0 in the presence of the same voltage stimulus, and the differences in these activation kinetics were dependent on the concentration of intracellular Ca2+. Despite their similar apparent affinities for Ca2+, rSlo0 and rSlo27 showed significant differences in their co-operative gating behavior. The Hill coefficient for intracellular Ca2+ was estimated to be about 3.7 for rSlo27 regardless of the membrane voltage, and that for rSlo0 was reduced from about 5 to 2 as the membrane voltage changed from 40 to 140 mV. As activation of BKCa channels is involved in rapid hyperpolarization of action potentials, the differential processing of rslo transcripts, and the generation of channels with different activation kinetics and Ca2+ cooperativity may be a mechanism for tuning the excitability of neurons in different brain regions.