ZnT-1, ZnT-3, CaMK II,PRG-1 expressions in hippocampus following neonatal seizure-induced cognitive deficit in rats

Epilepsy in children is associated with a broad spectrum of cognitive deficits, which is associated with hippocampal mossy fiber sprouting. The underlying molecular mechanisms involved in mossy fiber sprouting in hippocampus following developmental seizures are not completely known. We studied the timing of cognitive dysfunction following neonatal seizures and the relation of this cognitive impairment to zinc transporter 1 (ZnT-1), 3 (ZnT-3), calcium/calmodulin-dependent protein kinase II (CaMK II), plasticity-related gene 1 (PRG-1) expression in hippocampus. A seizure was induced by inhalant flurothyl daily in neonatal Sprague-Dawley rats from postnatal day 6 (P6). Rats were assigned into the single-seizure group (SS), the recurrent-seizure group (RS, seizures induced in six consecutive days), and the control group. During P41–P46 and P85–P90, the rats were tested for spatial learning and memory abilities with automatic Morris water maze task. At P90, mossy fiber sprouting and gene expression in hippocampus were determined subsequently by Timm staining and RT-PCR methods. The escape latencies from the water maze were significantly longer in rats of RS group than those of the control and SS groups at d4 of the first maze test and at d3, d4 of the second maze test. As far as Spatial Probe Test was concerned, the frequency of passing through the platform quadrant was significantly decreased in RS group than that in control and SS groups in the entire two probe tests. In rats with recurrent seizures (RS group), there was an increased distribution of Timm granules in both the supragranular region of the dentate gyrus and the stratum pyramidale of CA3 subfield in RS group, while remaining barely visible in control and SS groups; the Timm scores in CA3 and dentate gyrus in the RS animals were significantly higher than that in the control and SS groups. RT-PCR densitometry analysis showed that the ratios of hippocampal ZnT-1 to β-actin of SS and RS group were decreased significantly compared with that of control group. Meanwhile, CaMK II to β-actin of RS group was markedly lower compared with those of SS and control groups. Our results suggest that the long-term adverse effects of recurrent neonatal seizures on cognition and mossy fiber sprouting may be associated with the down-regulated expression of ZnT-1 and CaMK II in hippocampus.     

Expression profiles of hippocampal regenerative sprouting-related genes and their regulation by E-64d in a developmental rat model of penicillin-induced recurrent epilepticus

E-64d (a calpain and autophagy inhibitor) has previously been shown safe for the treatment of Alzheimer's disease in humans. In the present study, the potential protective mechanism of E-64d on hippocampal aberrant mossy fiber sprouting was examined in a developmental rat model of penicillin-induced recurrent epilepticus. A seizure was induced by penicillin every other day in Sprague–Dawley rats from postnatal day 21 (P21). The rats were randomly assigned into the control group (CONT1), the control plus E-64d (CONT2), the seizure group (EXP1) and the seizure plus E-64d (EXP2). On P51, mossy fiber sprouting and related gene expression in hippocampus were assessed by Timm staining and real-time RT-PCR methods, respectively. To validate the RT-PCR results, western blot analysis was performed on selected genes. E-64d obviously suppressed the aberrant mossy fiber sprouting in the supragranular region of dentate gyrus and CA3 subfield of hippocampus. Among the total twelve genes, six genes were strongly up- (MT-3, ACAT1, clusterin and ApoE) or down- (ZnT-1 and PRG-3) regulated by developmental seizures (EXP1) compared with that in the CONT1. Up-regulation of ApoE and Clusterin was blocked by pretreatment with E-64d both in mRNA and protein levels. Further, E-64d-pretreated seizure rats (EXP2) showed a significant downregulation of mRNA expression of PRG-1, PRG-3 and PRG-5, cathepsin B and ApoE, as well as up-regulated nSMase and ANX7 in hippocampus when compared with EXP1 rats. The results of the present study suggest that E-64d, an elective inhibitor of calpain and autophagy, is potentially useful in the treatment of developmental seizure-induced brain damage both by regulating abnormal zinc signal transduction and through the modulation of altered lipid metabolism via ApoE/clusterin pathway in hippocampus.     

Effects of neuropeptide S on seizures and oxidative damage induced by pentylenetetrazole in mice

Neuropeptide S (NPS) and its receptor were recently discovered in the central nervous system. In rodents, NPS promotes hyperlocomotion, wakefulness, anxiolysis, anorexia, and analgesia and enhances memory when injected intracerebroventricularly (i.c.v.). Herein, NPS at different doses (0.01, 0.1 and 1 nmol) was i.c.v. administered in mice challenged with pentylenetetrazole (PTZ; 60 mg/kg) repeatedly injected. Aiming to assess behavioral alterations and oxidative damage to macromolecules in the brain, NPS was injected 5 min prior to the last dose of PTZ. The administration of NPS only at 1 nmol increased the duration of seizures evoked by PTZ, without modifying frequency and latency of seizures. Biochemical analysis revealed that NPS attenuated PTZ-induced oxidative damage to proteins and lipids in the hippocampus and cerebral cortex. In contrast, the administration of NPS to PTZ-treated mice increased DNA damage in the hippocampus, but not cerebral cortex. In conclusion, this is the first evidence of the potential proconvulsive effects of NPS in mice. The protective effects of NPS against lipid and protein oxidative damage in the mouse hippocampus and cerebral cortex evoked by PTZ-induced seizures are quite unexpected. The present findings were discussed analyzing the paradoxical effects of NPS: facilitation of convulsive behavior and protection against oxidative damage to lipids and proteins.     

Cannabidiol prevents a post-ischemic injury progressively induced by cerebral ischemia via a high-mobility group box1-inhibiting mechanism

We examined the cerebroprotective mechanism of cannabidiol, the non-psychoactive component of marijuana, against infarction in a 4-h mouse middle cerebral artery (MCA) occlusion model. Cannabidiol was intraperitoneally administrated immediately before and 3 h after cerebral ischemia. Infarct size and myeloperoxidase (MPO) activity, a marker of neutrophil, monocyte/macropharge, were measured at 24 h after cerebral ischemia. Activated microglia and astrocytes were evaluated by immunostaining. Moreover, high-mobility group box1 (HMGB1) was also evaluated at 1 and 3 days after MCA occlusion. In addition, neurological score and motor coordination on the rota-rod test were assessed at 1 and 3 days after cerebral ischemia. Cannabidiol significantly prevented infarction and MPO activity at 20 h after reperfusion. These effects of cannabidiol were not inhibited by either SR141716 or AM630. Cannabidiol inhibited the MPO-positive cells expressing HMGB1 and also decreased the expression level of HMGB1 in plasma. In addition, cannabidiol decreased the number of Iba1- and GFAP-positive cells at 3 days after cerebral ischemia. Moreover, cannabidiol improved neurological score and motor coordination on the rota-rod test. Our results suggest that cannabidiol inhibits monocyte/macropharge expressing HMGB1 followed by preventing glial activation and neurological impairment induced by cerebral ischemia. Cannabidiol will open new therapeutic possibilities for post-ischemic injury via HMGB1-inhibiting mechanism.     

Different induction of metallothioneins and Hsp70 and presence of the membrane transporter ZnT-1 in HepG2 cells exposed to copper and zinc

Eukaryotic cells respond to stressful environmental stimuli, such as toxic concentrations of heavy metals, by rapidly synthesising defence proteins: the metallothioneins (MT) and the heat shock protein 70 (Hsp70). In this study we have analysed how the human hepatoblastoma cell line HepG2 responds to exposure to excess copper (30 μg/ml) and zinc (50 μg/ml) for long exposure times (48 and 72 h). Accumulation of the two metals, as measured by ICP-AES, was time-dependent reaching a plateau after 72 h. HepG2 cells responded by dramatically increasing levels of MT during stress, mostly during zinc exposure. A time lag in Hsp70 induction was observed as the levels of this protein increased only after removal of the stress from culture medium (recovery) for 24 h, thus suggesting that the two defence mechanisms are not coordinated in a metal-induced stress response. Moreover in HepG2 cells, immunochemical and fluorescence techniques showed the presence and the localisation of the zinc membrane exporter ZnT-1 as a further mechanism of defence/homeostasis against zinc toxicity.     

Nicotine decreases the activity of glutamate transporter type 3

Nicotine, the main ingredient of tobacco, elicits seizures in animal models and cigarette smoking is regarded as a behavioral risk factor associated with epilepsy or seizures. In the hippocampus, the origin of nicotine-induced seizures, most glutamate uptake could be performed primarily by excitatory amino acid transporter type 3 (EAAT3). An association between temporal lobe epilepsy and EAAT3 downregulation has been reported. Therefore, we hypothesized that nicotine may elicit seizures through the attenuation of EAAT3 activity. We investigated chronic nicotine exposure (72 h) cause reduction of the activity of EAAT3 in a Xenopus oocyte expression system using a two-electrode voltage clamp. The roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) were also determined. Nicotine (0.001–1 μM) resulted in a time- and dose-dependent decrease in EAAT3 activity with maximal inhibition at nicotine concentrations of 0.03 μM or higher and at an exposure time of 72 h. V_max on the glutamate response was significantly reduced in the nicotine group (0.03 μM for 72 h), but the K_m value of EAAT3 for glutamate was not altered. When nicotine-exposed oocytes (0.03 μM for 72 h) were pretreated with phorbol-12-myristate-13-acetate (PMA, a PKC activator), the nicotine-induced reduction in EAAT3 activity was abolished. PKC inhibitors (staurosporine, chelerythrine, and calphostin C) significantly reduced basal EAAT3 activity, but there were no significant differences among the PKC inhibitors, nicotine, and PKC inhibitors + nicotine groups. Similar response patterns were observed among PI3K inhibitors (wortmannin and LY294002), nicotine, and PI3K inhibitors + nicotine. In conclusion, this study suggests that nicotine decreases EAAT3 activity, and that this inhibition seems to be dependent on PKC and PI3K. Our results may provide an additional mechanism for nicotine-induced seizure.     

雷公藤内酯醇抑制大鼠脑局灶性缺血再灌注后脑组织IL-1βmRNA表达的研究

目的　观察雷公藤内酯醇 (Triptolide ,TL)对局灶性脑缺血再灌注大鼠脑组织内IL 1βmRNA水平变化的影响 ,探讨雷公藤内酯醇对大鼠局灶性缺血再灌注脑损伤保护作用的机制。方法　建立大鼠局灶性脑缺血再灌注模型 ,观察应用雷公藤内酯醇 (0 2和 0 4mg·kg-1)对大鼠脑组织微血管内附壁中性粒细胞计数 ,以及神经功能受损程度的影响。应用RT PCR技术 ,检测大中动脉闭塞侧脑组织IL 1βmRNA表达水平的变化情况。结果　与对照组比较 ,雷公藤内酯醇两治疗组大脑中动脉闭塞侧微血管内附壁中性粒细胞计数、脑组织IL 1βmRNA水平均明显下降。神经功能受损程度也明显改善。结论　雷公藤内酯醇具有明显抑制缺血再灌注大鼠脑组织内IL 1βmRNA表达、降低IL 1β含量和抑制白细胞浸润的作用。这可能是雷公藤内酯醇对大鼠缺血再灌注脑损伤保护作用的机制之一     

Diesel exhaust particles modulate vascular endothelial cell permeability: Implication of ZO-1 expression

Exposure to air pollutants increases the incidence of cardiovascular disease. Recent toxicity studies revealed that ultra-fine particles (UFP, d_p < 100–200 nm), the major portion of particulate matter (PM) by numbers in the atmosphere, induced atherosclerosis. In this study, we posited that variations in chemical composition in diesel exhausted particles (DEP) regulated endothelial cell permeability to a different extent. Human aortic endothelial cells (HAEC) were exposed to well-characterized DEP (d_p < 100 nm) emitted from a diesel engine in either idling mode (DEP1) or in urban dynamometer driving schedule (UDDS) (DEP2). Horse Radish Peroxidase-Streptavidin activity assay showed that DEP2 increased endothelial permeability to a greater extent than DEP1 (control = 0.077 ± 0.005, DEP1 = 0.175 ± 0.003, DEP2 = 0.265 ± 0.006, n = 3, p < 0.01). DEP2 also down-regulated tight junction protein, Zonular Occludin-1 (ZO-1), to a greater extent compared to DEP1. LDH and caspase-3 activities revealed that DEP-mediated increase in permeability was not due to direct cytotoxicity, and DEP-mediated ZO-1 down-regulation was not due to a decrease in ZO-1 mRNA. Hence, our findings suggest that DEP1 vs. DEP2 differentially influenced the extent of endothelial permeability at the post-translational level. This increase in endothelium permeability is implicated in inflammatory cell transmigration into subendothelial layers with relevance to the initiation of atherosclerosis.     

Differential role of nitric oxide (NO) in acute and chronic stress induced neurobehavioral modulation and oxidative injury in rats

The present study evaluated the effects of acute and chronic restraint stress (RS 1 h or 6 h), and their modulation by nitrergic agents on neurobehavioral and oxidative stress markers in rats. Acute RS (1 h or 6 h) reduced open arm entries (OAE) and open arm time (OAT) in the elevated plus maze test — which were attenuated by the NO precursor, L-arginine but not influenced appreciably by the NO synthase inhibitor, L-NAME. These behavioral changes were associated with differential changes in brain NO metabolites (NOx) but consistently reduced GSH and raised MDA levels in comparison to the control group. Following RS 1 h × 10 the neurobehavioral suppression and changes in brain oxidative stress markers were less pronounced as compared to the acute RS (1 h) group indicating adaptation. L-arginine pretreatment facilitated this adaptation to chronic RS (1 h). Interestingly RS 6 h × 10, induced severe behavioral suppression and aggravation of MDA and NOx levels and L-NAME pretreatment tended to protect against these chronic RS induced aggravations. These results suggest that acute and chronic RS induces duration/intensity dependent neurobehavioral and oxidative injury which are under the differential regulatory control of NO.     

Tissue-specific alterations in expression and function of P-glycoprotein in streptozotocin-induced diabetic rats

Aim:

To investigate the changes of expression and function of P-glycoprotein (P-GP) in cerebral cortex, hippocampus, liver, intestinal mucosa and kidney of streptozocin-induced diabetic rats.

Methods:

Diabetic rats were prepared via a single dose of streptozocin (65 mg/kg, ip). Abcb1/P-GP mRNA and protein expression levels in tissues were evaluated using quantitative real time polymerase chain reaction (QRT-PCR) analysis and Western blot, respectively. P-GP function was investigated via measuring tissue-to-plasma concentration ratios and body fluid excretion percentages of rhodamine 123.

Results:

In 5- and 8-week diabetic rats, Abcb1a mRNA levels were significantly decreased in cerebral cortices and intestinal mucosa, but dramatically increased in hippocampus and kidney. In liver, the level was increased in 5-week diabetic rats, and decreased in 8-week diabetic rats. Abcb1b mRNA levels were increased in cerebral cortex, hippocampus and kidney, but reduced in liver and intestinal mucosa in the diabetic rats. Western blot results were in accordance with the alterations of Abcb1a mRNA levels in most tissues examined. P-GP activity was markedly decreased in most tissues of diabetic rats, except kidney tissues.

Conclusion:

Alterations in the expression and function of Abcb1/P-GP under diabetic conditions are tissue specific, Abcb1 specific and diabetic duration-dependent.     

Effects of postnatal exposure to methylmercury on spatial learning and memory and brain NMDA receptor mRNA expression in rats

The extreme vulnerability of developing nervous system to methylmercury (MeHg) is well documented. Still unclear is the consequence of different postnatal period exposure to MeHg. We investigated the critical postnatal phase when MeHg induced neurotoxicity in rats and the underlying mechanism. Rats were given 5 mg/(kg day) methylmercury chloride (MMC) orally on postnatal day (PND) 7, PND14, PND28, and PND60 for consecutive 7 days. A control group was treated with 0.9% sodium chloride solution 5 ml/(kg day) instead. On PND69, spatial learning and memory was evaluated by Morris water maze test. Behavior deficits were found in MMC-treated rats of PND7 and PND14 groups (p < 0.01). N-methyl-d-aspartate (NMDA) receptor 2 subunits mRNA expressions were evaluated 3 days after the last administration. In hippocampus, the mRNA expression of NR2A and NR2B decreased, but the NR2C expression increased in PND14 group following MMC-treatment (p < 0.01). In cerebral cortex, mRNA expression of NR2A decreased, with NR2C expression elevating in PND14 group following MMC-treatment (p < 0.05). These observations suggest that the postnatal exposure to MeHg during PND7–20 could cause neurobehavioral deficits which extend to adulthood. Furthermore, the abnormal expression of NMDAR 2 subunits might associate with the impairment.     

Regulation of metallothioneins and ZnT-1 transporter expression in human hepatoma cells HepG2 exposed to zinc and cadmium

Essential and non-essential metals can affect vital cellular processes, when over-accumulated within the cells. For this reason, cells have evolved multiple protein sensors, transporters, and other type of proteins to regulate and control free metal homeostasis. Among these, metallothioneins (MT) and ZnT-1 transporter play a key role in the regulation of free Zn concentrations.Herewith, MT expression in Zn (170 μM) and Cd (0.1 and 10 μM) exposed HepG2 cells is analyzed and compared. In addition, the modulation and localization of the membrane transporter ZnT-1 has been investigated. MT-I and MT-II were up-regulated in response to both Zn and Cd exposure and, as expected, Cd represented the most potent inducer. Namely, 0.1 μM Cd was able to up-regulate MT-I, and -II in a way comparable to 170 μM Zn. This is in agreement with MT general function of metal-chelating protein, acting with higher tolerance to essential metals than to non-essential ones. ZnT-1 protein, a plasma membrane specific Zn transporter, was up-regulated as well by both Zn and Cd, although in the same way. Immunofluorescence technique provided evidence that high levels of ZnT-1 measured by biochemical techniques, are related to an increased localization of the transporter at the plasma membrane.     

ABCG2/BCRP decreases the transfer of a food-born chemical carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in perfused term human placenta

We have studied the role of ATP binding cassette (ABC) transporters in fetal exposure to carcinogens using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) a known substrate for ABC transporters as a model compound. In perfusion of human term placenta, transfer of ¹⁴C-PhIP (2 μM) through the placenta resulted in fetal-to-maternal concentration ratio (FM ratio) of 0.72 ± 0.09 at 6 h. The specific ABCG2 inhibitor KO143 increased the transfer of ¹⁴C-PhIP from maternal to fetal circulation (FM ratio 0.90 ± 0.08 at 6 h, p < 0.05) while the ABCC1/ABCC2 inhibitor probenecid had no effect (FM ratio at 6 h 0.75 ± 0.10, p = 0.84). There was a negative correlation between the expression of ABCG2 protein in perfused tissue and the FM ratio of ¹⁴C-PhIP (R = − 0.81, p < 0.01) at the end of the perfusion. The expression of ABCC2 protein did not correlate with FM ratio of PhIP (R: − 0.11, p = 0.76). In addition, PhIP induced the expression of ABC transporters in BeWo cells at mRNA level. In conclusion, our data indicates that ABCG2 decreases placental transfer of ¹⁴C-PhIP in perfused human placenta. Also, PhIP may modify ABC transporter expression in choriocarinoma cells.     

Effect of 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one on oxidative stress in cerebral cortex of rats

The objective of this study was to verify the effect of the organochalcogen 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one on some parameters of oxidative stress in the brain of 10-day-old rats. Cerebral cortex was incubated for 1 h in the presence or absence of 1, 10 or 30 μM of 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one and thiobarbituric acid reactive substances (TBARS), carbonyl, sulfhydryl, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), nitric oxide (NO) production and the release of the cytosolic enzyme lactate dehydrogenase (LDH) were measured. The organotellurium was not capable to alter TBARS and carbonyl assays. In contrast, the compound at 10 and 30 μM provoked a reduced of protein thiol groups measured by the sulfhydryl assay. Furthermore, the activity of the antioxidant enzyme CAT (10 and 30 μM) and GPx (1, 10 and 30 μM) was reduced by the organochalcogen. On the other hand, the activity of SOD and GST were enhanced respectively by 1, 10 and 30 μM of the compound. Furthermore, NO production was also increased by 30 μM of this organochalcogen. Finally, we verified that the organotellurium was capable of enhance the LDH release at 30 μM concentration. Our findings indicate that this organotellurium compound induces in vitro oxidative stress in the cerebral cortex of rats being potentially toxic for the brain of rats.     

In vitro assay of the interaction between Rnc1 protein and Pmp1 mRNA by affinity capillary electrophoresis with a carboxylated capillary

The interaction between Rnc1, an RNA interactive protein, and a Pmp1 mRNA was investigated by affinity capillary electrophoresis (ACE). Prior to the ACE experiments, the column performances of three capillaries (an untreated fused silica capillary, a polybrene–polyacrylic acid (PB–PAA) double layer coating capillary, and a carboxylated capillary with a covalent modification) were studied with model proteins including ribonuclease B (RNase B) and bovine serum albumin (BSA). Using an untreated fused silica and a PB–PAA double layer coating capillaries, both of the protein peaks were broad and tailing. However, using a carboxylated capillary, the protein peaks were sharp and symmetric, and migration times were repeatable (RSD < 0.4%). Further, the proteins in human serum also gave sharp peaks and its repeatability was kept at a high level by pre-treatment of a capillary inner wall with 1 M sodium chloride solution before each run. An Rnc1 protein was analyzed by ACE with background electrolytes containing various concentrations of Pmp1 sense mRNA using a carboxylated capillary. Increase in the concentration of the mRNA was found to delay the migration time of the protein. But the migration time of the protein was kept constant with increasing Pmp1 anti-sense mRNA instead of Pmp1 sense mRNA. A straight line (r = 0.987) was obtained by plotting 1/(migration time shift) versus 1/(Pmp1 sense mRNA concentration) and the association constant of Rnc1 protein with Pmp1 sense mRNA could be estimated to be 4.15 × 10⁶ M⁻¹. These results suggest that the association constants of proteins with mRNAs as ligands were easily determined by the proposed method.     

Caffeine-induced inhibition of the activity of glutamate transporter type 3 expressed in Xenopus oocytes

Caffeine has been known to trigger seizures, however, the precise mechanism about the proconvulsive effect of caffeine remains unclear. Glutamate transporters play an important role to maintain the homeostasis of glutamate concentration in the brain tissue. Especially, dysfunction of excitatory amino acid transporter type 3 (EAAT3) can lead to seizures. We investigated the effects of caffeine on the activity of EAAT3 and the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K). Rat EAAT3 was expressed in Xenopus oocytes by injecting EAAT3 mRNA. l-Glutamate (30 μM)-induced inward currents were recorded via the two-electrode voltage clamp method. Caffeine decreased EAAT3 activity in a dose-dependent manner. Caffeine (30 μM for 3 min) significantly reduced V_max, but did not alter K_m value of EAAT3 for glutamate. When preincubated oocytes with phorbol-12-myristate-13-acetate (PMA, a PKC activator) were exposed to caffeine, PMA-induced increase in EAAT3 activity was abolished. Two PKC inhibitors (chelerythrine and staurosporine) significantly reduced basal EAAT3 activity. Whereas, there were no significant differences among the PKC inhibitors, caffeine, and PKC inhibitors + caffeine groups. In similarly fashion, wortmannin (a PI3K inhibitor) significantly decreased EAAT3 activity, however no statistical differences were observed among the wortmannin, caffeine, and wortmannin + caffeine groups. Our results demonstrate that caffeine attenuates EAAT3 activity and this reducing effect of caffeine seems to be mediated by PKC and PI3K.