Genetic Inhibition of Plppr5 Aggravates Hypoxic-Ischemie-Induced Cortical Damage and Excitotoxic Phenotype.

Hypoxia-ischemia (HI) is the most common acute brain threat in neonates and a leading cause of neurodevelopmental impairment. Exploring the new molecular mechanism of HI brain injury has important clinical translational significance for the next clinical intervention research. Lipid phosphatase-related proteins (PLPPRs) are regulators of mitochondrial membrane integrity and energy metabolism. We recently found that Plppr5 knockout exacerbated HI impairment in some aspects and partially attenuated the neuroprotective effects of melatonin, suggesting that Plppr5 may be a novel intervention target for HI. The present study aimed to determine the long-term effects of gene knockout of Plppr5 on HI brain injury, focusing on the neuronal excitability phenotype, and to determine the effect of Plppr5 gene silencing on neuronal zinc metabolism and mitochondrial function in vitro. 10-day-old wild type (WT) mice and Plppr5-deficient (Plppr5 -/-) mice were subjected to hypoxia-ischemia. Lesion volumes and HI-induced neuroexcitotoxic phenotypes were quantified together with ZnT1 protein expression in hippocampus. In addition, HT22 (mouse hippocampal neuronal cells) cell model was established by oxygen-glucose deprivation/reoxygenation (OGD/R) treatment and was treated with medium containing LV-sh_Plppr5 or control virus. Mitochondrial oxidative stress indicator ROS, mitochondrial ZnT1 protein expression and zinc ion content were detected. Results: Plppr5-deficient mice subjected to hypoxia-ischemia at postnatal day 10 present significantly higher cerebral infarction. Plppr5-deficient mice were endowed with a more pronounced superexcitability phenotype at 4 weeks after HI, manifested as a reduced seizure threshold. ZnT1 protein was also found reduced in Plppr5-deficient mice as well as in mice subjected to HI excitotoxicity. Plppr5 knockout in vivo exacerbates HI brain injury phenotypes, including infarct volume and seizure threshold. In addition, knockout of the Plppr5 gene reduced the MFS score to some extent. In vitro Plppr5 silencing directly interferes with neuronal zinc metabolism homeostasis and exacerbates hypoxia-induced mitochondrial oxidative stress damage. Taken together, our findings demonstrate for the first time that Plppr5-deficient mouse pups exposed to neuronal hypoxia and ischemia exhibit aggravated acute brain injury and long-term brain excitability compared with the same treated WT pups, which may be related to the disruption of zinc and mitochondria-dependent metabolic pathways in the hippocampus. These data support further investigation into novel approaches targeting Plppr5-mediated zinc and mitochondrial homeostasis in neonatal HIE.

PRG5 Knockout Precipitates Late-Onset Hypersusceptibility to Pilocarpine-Induced Juvenile Seizures by Exacerbating Hippocampal Zinc Signaling-Mediated Mitochondrial Damage.

INTRODUCTION: Epileptogenesis is understood as the plastic process that produces a persistent reorganization of the brain's neural network after a precipitating injury (recurrent neonatal seizures, for instance) with a latent period, finally leading to neuronal hyperexcitability. Plasticity-related genes (PRGs), also known as lipid phosphate phosphatase-related proteins (PLPPRs), are regulators of mitochondrial membrane integrity and energy metabolism. This study was undertaken to determine whether PRG5 gene knockout contributes to the delayed hypersensitivity induced by developmental seizures and the aberrant sprouting of hippocampal mossy fibers, and to determine whether it is achieved through the mitochondrial pathway. Here, we developed a "twist" seizure model by coupling pilocarpine-induced juvenile seizures with later exposure to penicillin to test the long-term effects of PRG5 knockout on seizure latency through comparison with wild-type (WT) mice. Hippocampal mossy fiber sprouting (MFS) was detected by Timm staining. In order to clarify the mechanism of the adverse reactions triggered by PRG5 knockout, hippocampal HT22 neuronal cultures were exposed to glutamate, with or without PRG5 interference. Mitochondrial function, oxidative stress indicators and zinc ion content were detected. RESULTS: PRG5 gene knockout significantly reduced the seizure latency, and aggravated the lowered seizure threshold induced by developmental seizures. Besides, knockout of the PRG5 gene reduced the MFS scores to a certain extent. Furthermore, PRG5 gene silencing significantly increases the zinc ion content in hippocampal neurons, impairs neuronal activity and mitochondrial function, and exacerbates glutamate-induced oxidative stress damage. CONCLUSION: In summary, PRG5 KO is associated with significantly greater hypersusceptibility to juvenile seizures in PRG5(-/-) mice compared with WT mice. These effects may be related to the hippocampal zinc signaling. The effects do not appear to be related to changes in MFS because KO mice with juvenile seizures had the shortest seizure latencies but exhibited less MFS than WT mice with juvenile seizures.

Proteomics for Studying the Effects of Ketogenic Diet Against Lithium Chloride/Pilocarpine Induced Epilepsy in Rats.

The ketogenic diet (KD) demonstrates antiepileptogenic and neuroprotective efficacy, but the precise mechanisms are unclear. Here we explored the mechanism through systematic proteomics analysis of the lithium chloride-pilocarpine rat model. Sprague-Dawley rats (postnatal day 21, P21) were randomly divided into control (Ctr), seizure (SE), and KD treatment after seizure (SE + KD) groups. Tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectroscopy (LC-MS/MS) were utilized to assess changes in protein abundance in the hippocampus. A total of 5,564 proteins were identified, of which 110 showed a significant change in abundance between the SE and Ctr groups (18 upregulated and 92 downregulated), 278 between SE + KD and SE groups (218 upregulated and 60 downregulated), and 180 between Ctr and SE + KD groups (121 upregulated and 59 downregulated) (all p < 0.05). Seventy-nine proteins showing a significant change in abundance between SE and Ctr groups were reciprocally regulated in the SD + KD group compared to the SE group (i.e., the seizure-induced change was reversed by KD). Of these, five (dystrobrevin, centromere protein V, oxysterol-binding protein, tetraspanin-2, and progesterone receptor membrane component 2) were verified by parallel reaction monitoring. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that proteins of the synaptic vesicle cycle pathway were enriched both among proteins differing in abundance between SE and Ctr groups as well as between SE + KD and SE groups. This comprehensive proteomics analyze of KD-treated epilepsy by quantitative proteomics revealed novel molecular mechanisms of KD antiepileptogenic efficacy and potential treatment targets.

Acute Phase Serum Leptin, Adiponectin, Interleukin-6, and Visfatin Are Altered in Chinese Children With Febrile Seizures: A Cross-Sectional Study.

Adipokines, including leptin, visfatin, adiponectin, and interleukin-6 (IL)-6, play multiple roles in the pathophysiology of epilepsy and febrile seizures (FS). We aimed to investigate the associations among plasma adipokines, mainly leptin, visfatin, adiponectin, or IL-6, and the prognosis of FS. This prospective cross-sectional study was conducted from January 2017 to December 2018 at the Wuxi Second People' Hospital China. The levels of serum leptin, visfatin, adiponectin, and IL-6 in 55 children with FS (FS group) were compared with 42 febrile children without seizure (FC group) and 48 healthy children (HC group) in an acute phase. The correlation with clinical indicators was determined by logistic regression analysis. Serum adiponectin and IL-6 levels were significantly higher in the FS group than in the FC and HC groups (p < 0.05), but there was no statistical difference between the FC and HC groups. In addition, logistic regression analysis showed that high concentrations of adiponectin and IL-6 were significantly associated with the occurrence of FS. For leptin and visfatin, they were significantly lower in the FS and FC groups than in the normal control group, but there was no statistical difference between the FS and FC groups. Our results suggest that higher plasma levels of IL-6 and adiponectin may serve as an additional biomarker in the early treatment or follow-up of the FS children.

Reduction of Mitophagy-Related Oxidative Stress and Preservation of Mitochondria Function Using Melatonin Therapy in an HT22 Hippocampal Neuronal Cell Model of Glutamate-Induced Excitotoxicity.

Recent evidence indicates that autophagy-mediated mitochondrial homeostasis is crucial for oxidative stress-related brain damage and repair. The highest concentration of melatonin is in the mitochondria of cells, and melatonin exhibits well-known antioxidant properties. We investigated the impact and mechanism involved in mitochondrial function and the mitochondrial oxidative stress/autophagy regulator parameters of glutamate cytotoxicity in mouse HT22 hippocampal neurons. We tested the hypothesis that melatonin confers neuroprotective effects via protecting against mitochondrial impairment and mitophagy. Cells were divided into four groups: the control group, melatonin alone group, glutamate injury group, and melatonin pretreatment group. We found that glutamate induced significant changes in mitochondrial function/oxidative stress-related parameters. Leptin administration preserved mitochondrial function, and this effect was associated with increased superoxide dismutase, glutathione (GSH), and mitochondrial membrane potential and decreased GSSG (oxidized glutathione) and mitochondrial reactive oxygen species. Melatonin significantly reduced the fluorescence intensity of mitophagy via the Beclin-1/Bcl-2 pathway, which involves Beclin-1 and Bcl-2 proteins. The mitophagy inhibitor CsA corrected these glutamate-induce changes, as measured by the fluorescence intensity of Mitophagy-Tracker Red CMXROS, mitochondrial ROS, and mitochondrial membrane potential changes. These findings indicate that melatonin exerts neuroprotective effects against glutamate-induced excitotoxicity by reducing mitophagy-related oxidative stress and maintaining mitochondrial function.

Alterations in the Neurobehavioral Phenotype and ZnT3/CB-D28k Expression in the Cerebral Cortex Following Lithium-Pilocarpine-Induced Status Epilepticus: the Ameliorative Effect of Leptin.

Zinc transporter 3 (ZnT3)-dependent "zincergic" vesicular zinc accounts for approximately 20% of the total zinc content of the mammalian telencephalon. Elevated hippocampal ZnT3 expression is acknowledged to be associated with mossy fiber sprouting and cognitive deficits. However, no studies have compared the long-term neurobehavioral phenotype with the expression of ZnT3 in the cerebral cortex following status epilepticus (SE). The aim of this study was to investigate changes in the long-term neurobehavioral phenotype as well as the expression of ZnT3 and calcium homeostasis-related CB-D28k in the cerebral cortex of rats subjected to neonatal SE and to determine the effects of leptin treatment immediately after neonatal SE. Fifty Sprague-Dawley rats (postnatal day 6, P6) were randomly assigned to two groups: the pilocarpine hydrochloride-induced status epilepticus group (RS, n = 30) and control group (n = 20). Rats were further divided into the control group without leptin (Control), control-plus-leptin treatment group (Leptin), RS group without leptin treatment (RS), and RS-plus-leptin treatment group (RS + Leptin). On P6, all rats in the RS group and RS + Leptin group were injected intraperitoneally (i.p.) with lithium chloride (5 mEq/kg). Pilocarpine (320 mg/kg, i.p.) was administered 30 min after the scopolamine methyl chloride (1 mg/kg) injection on P7. From P8 to P14, animals of the Leptin group and RS + Leptin group were given leptin (4 mg/kg/day, i.p.). The neurological behavioral parameters (negative geotaxis reaction reflex, righting reflex, cliff avoidance reflex, forelimb suspension reflex, and open field test) were observed from P23 to P30. The protein levels of ZnT3 and CB-D28k in the cerebral cortex were detected subsequently by the western blot method. Pilocarpine-treated neonatal rats showed long-term abnormal neurobehavioral parameters. In parallel, there was a significantly downregulated protein level of CB-D28k and upregulated protein level of ZnT3 in the cerebral cortex of the RS group. Leptin treatment soon after epilepticus for 7 consecutive days counteracted these abnormal changes. Taken together with the results from our previous reports on another neonatal seizure model, which showed a significant positive inter-relationship between ZnT3 and calcium/calmodulin-dependent protein kinase IIα (CaMKIIα), the data here suggest that ZnT3/CB-D28k-associated Zn (2+)/Ca(2+) signaling might be involved in neonatal SE-induced long-term brain damage in the aspects of neurobehavioral impairment. Moreover, consecutive leptin treatment is effect at counteracting these hyperexcitability-related changes, suggesting a potential clinical significance.

Zinc/CaMK II Associated-Mitophagy Signaling Contributed to Hippocampal Mossy Fiber Sprouting and Cognitive Deficits Following Neonatal Seizures and Its Regulation by Chronic Leptin Treatment.

The role of leptin in the pathogenesis of epilepsy is getting more and more attention in clinical and basic research. Although there are data indicating neuroprotective effects of elevated serum/brain leptin levels following acute seizures, no study to date has dealt with the impact of chronic leptin treatment on long-term brain injury following developmental seizures. The aim of this study was to evaluate whether chronic leptin treatment may have neuroprotective effects on cognitive and hippocampal mossy fiber sprouting following flurothyl-induced recurrent neonatal seizures and whether these effects are mediated by the zinc/CaMKII-associated mitophagy signaling pathway. Forty Sprague-Dawley rats (postnatal day 6, P6) were randomly assigned into two groups: neonatal seizure group and control group. At P13, they were further divided into control group, seizure group (RS), control + leptin (leptin, i.p., 2 mg/kg/day for 10 days), seizure+leptin group (RS+Leptin, 2mg/kg/day, i.p., for 10 consecutive days). Morris water maze test was performed during P27-P32. Subsequently, Timm staining and Western blotting were used to detect the mossy fiber sprouting and protein levels in hippocampus. Flurothyl-induced seizures (RS group) significantly down-regulated mitophagy markers PINK, Drp1, PHB, and memory marker CaMK II alpha while up-regulating zinc transporters ZnT3, ZnT4, ZIP7, and autophagy execution molecular cathepsin-E, which were paralleled with hippocampal aberrant mossy fiber sprouting and cognitive dysfunction. However, these changes were restored by chronic leptin treatment (RS+Leptin group). The results showed that leptin had neuroprotective effect on hippocampal pathological damage and cognitive deficits induced by neonatal seizures and suggested that Zinc/CaMK II associated-mitophagy signaling pathway in hippocampus may be a new target of leptin's neuroprotection, with potential value of translational medicine.

Leptin-regulated autophagy plays a role in long-term neurobehavioral injury after neonatal seizures and the regulation of zinc/cPLA2 and CaMK II signaling in cerebral cortex.

Metabolic disorders play an important role in the pathogenesis of many neurological diseases. Recent evidence suggests that leptin levels in peripheral blood and brain are lower in patients with epilepsy. Leptin is an energy-regulating hormone that plays a neuroprotective role in neurodegenerative diseases and brain trauma. However, little is known about the effects and molecular mechanisms of leptin treatment on long-term neurobehavioral impairment caused by developmental seizures. The present study evaluated whether chronic leptin treatment protected against neurobehavioral impairments induced by recurrent seizures in newborns treated with flurothyl. We also examined the effect of leptin on the expression of zinc/cPLA2-related autophagy signaling molecules and CaMKII in the cerebral cortex. Twenty Sprague-Dawley rats (6 days after birth, P6) were randomly divided into two groups, a neonatal seizure group and control group. Rats were subdivided on P13 into control, control + leptin (leptin, 2 mg/kg/day, continuous 10 days), seizure (RS), and seizure + leptin group (RS + leptin, 2 mg/kg/day for 10 consecutive days). Neurological behavioral parameters (negative geotaxis reaction reflex, righting reflex, cliff avoidance reflex, forelimb suspension reflex and open field test) were observed from P23 to P30. mRNA and protein levels in the cerebral cortex were detected using real-time RT-PCR and Western blotting, respectively. Flurothyl-induced seizures (RS group) produced long-term abnormal neurobehavior, which was improved with leptin treatment. Chronic leptin treatment restored several expression parameters affected by neonatal seizures, including seizure-induced up-regulated zinc transporter ZnT1/ZIP7, lipid membrane injury-related cPLA2, autophagy marker beclin-1/bcl2, LC3II/LC3I, and its execution molecule cathepsin-E, and down-regulated memory marker CaMK II alpha. Our results suggest that the early use of leptin after neonatal recurrent seizures may exert neuroprotective effects and antagonize the long-term neurobehavioral impairment caused by seizures. Autophagy-mediated Zn/cPLA2 and CaMK II signaling in the cerebral cortex may be involved in the neuroprotective effect of leptin. Our results provide new clues for anti-epileptogenetic treatment.

Leptin Maintained Zinc Homeostasis Against Glutamate-Induced Excitotoxicity by Preventing Mitophagy-Mediated Mitochondrial Activation in HT22 Hippocampal Neuronal Cells.

Developmental seizure-induced long-term neuronal hyperexcitation is partially mediated by regenerative mossy fiber sprouting in hippocampus. Yet, there are no effective drugs available to block this pathological process. Recently, leptin has been shown to prevent the sprouting of hippocampal mossy fibers and abnormalities in the neurobehavioral parameters. However, their underlying molecular mechanisms are largely unknown. The purpose of this study was to determine the effect of glutamate on the parameters of zinc homeostasis, mitochondrial functions, and mitophagy regulating factors, as well as to investigate the protective effects of leptin against cytotoxicity of glutamate in murine HT22 hippocampal neuronal cells. Cells were assigned to one of the four groups as follows: control group, leptin alone group, glutamate injury group, and leptin pretreatment group. Our results demonstrated that glutamate induced a decrease in superoxide dismutase, GSH (glutathione), and mitochondrial membrane potential and an increase in GSSG (oxidized glutathione), mitochondrial reactive oxygen species, and supplementation of leptin blocked the toxic effect of glutamate on cell survival. The glutamate-induced cytotoxicity was associated with an increase in mitophagy and intracellular zinc ion levels. Furthermore, glutamate activated the mitophagy markers PINK1, Parkin, and the ratio of LC3-II/LC3-I, as well as increased the expression of zinc transporter 3 (ZnT3). Leptin corrected these glutamate-caused alterations. Finally, the mitophagy inhibitor, CsA, significantly reduced intracellular zinc ion content and ZnT3 expression. These results suggest that mitophagy-mediated zinc dyshomeostasis and mitochondrial activation contributed to glutamate-induced HT22 neuronal cell injury and that leptin treatment could counteract these detrimental effects, thus highlighting mitophagy-mediated zinc homeostasis via mitochondrial activation as a potential strategy to counteract neuroexcitotoxicity.

Terrabacter ginsengisoli sp. nov., isolated from ginseng cultivating soil.

A Gram-positive, strictly aerobic, nonmotile, yellowish, coccus-rod-shaped bacterium (designated Gsoil 653T) isolated from ginseng cultivating soil was characterized using a polyphasic approach to clarify its taxonomic position. The strain Gsoil 653T exhibited optimal growth at pH 7.0 on R2A agar medium at 30°C. Phylogenetic analysis based on 16S rRNA gene sequence similarities, indicated that Gsoil 653T belongs to the genus Terrabacter of the family Humibacillus, and was closely related to Terrabacter tumescens DSM 20308T (98.9%), Terrabacter carboxydivorans PY2T (98.9%), Terrabacter terrigena ON10T (98.8%), Terrabacter terrae PPLBT (98.6%), and Terrabacter lapilli LR-26T (98.6%). The DNA G + C content was 70.5 mol%. The major quinone was MK-8(H4). The primary polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidyl-ethanolamine. The predominant fatty acids were iso-C15:0, iso-C16:0, iso-C14:0, and anteiso-C15:0, as in the case of genus Terrabacter, thereby supporting the categorization of strain Gsoil 653T. However, the DNA-DNA relatedness between Gsoil 653T and closely related strains of Terrabacter species was low at less than 31%. Moreover, strain Gsoil 653T could be both genotypically and phenotypically distinguished from the recognized species of the genus Terrabacter. This isolate, therefore, represents a novel species, for which the name Terrabacter ginsengisoli sp. nov. is proposed with the type strain Gsoil 653T (= KACC 19444T = LMG 30325T).

Leptin treatment prevents long-term abnormalities in cognition, seizure threshold, hippocampal mossy fiber sprouting and ZnT3/CB-D28k expression in a rat developmental "twist" seizure model.

The mechanism of linking neonatal seizures with long-term brain damage is unclear, and there is no effective drug to block this long-term pathological process. Recently, the fat-derived hormone leptin has been appreciated for its neuroprotective function in neurodegenerative processes, although less is known about the effects of leptin on neonatal seizure-induced brain damage. Here, we developed a "twist" seizure model by coupling pilocarpine-induced neonatal status epilepticus (SE) with later exposure to penicillin to test whether leptin treatment immediately after neonatal SE would exert neuroprotective effects on cognition, seizure threshold and hippocampal mossy fiber sprouting, as well if leptin had any influence on the expression of zinc transporter 3 (ZnT3) and calcium homeostasis-related CB-D28k in the hippocampus. Fifty Sprague-Dawley rats (postnatal day 6, P6) were randomly assigned to four groups: control (n = 10), control with intraperitoneal (i.p.) injection of leptin (Leptin, n = 10), pilocarpine-induced neonatal SE (RS), and RS i.p. leptin injection (RS+Leptin). At P6, all the rats in the RS group and RS+Leptin group were injected with lithium chloride i.p. (5 mEq/kg). Pilocarpine (320 mg/kg, i.p.) was administered 30 min after scopolamine methyl chloride (1 mg/kg) injection at P7 to block the peripheral effect of pilocarpine. From P8 to P14, the animals in the Leptin group and RS+Leptin group were given leptin (4 mg/kg, i.p.). The Morris water maze test was performed during P28-P33. Following routine seizure threshold detection and Timm staining procedures, Western blot analysis was performed for each group. Pilocarpine-induced neonatal SE severely impaired learning and memory abilities, reduced seizure threshold, and induced aberrant hippocampal CA3 mossy fiber sprouting. In parallel, there was a significantly down-regulated protein level of CB-D28k and an up-regulated protein level of ZnT3 in the hippocampus of the RS group. Furthermore, leptin treatment soon after neonatal SE for seven consecutive days counteracted these hyperexcitability-related alterations. These novel findings established that leptin has a neuroprotective role in the model of cholinergic neonatal SE and highlights ZnT3/CB-D28k associated-Zn (2+)/Ca (2+) signaling as a promising therapeutic target.

Dysregulation of zinc/lipid metabolism­associated genes in the rat hippocampus and cerebral cortex in early adulthood following recurrent neonatal seizures.

Although it has been established that recurrent or prolonged clinical seizures during infancy may cause lifelong brain damage, the underlying molecular mechanism is still not well elucidated. The present study, to the best of our knowledge, is the first to investigate the expression of twenty zinc (Zn)/lipid metabolism­associated genes in the hippocampus and cerebral cortex of rats following recurrent neonatal seizures. In the current study, 6­day­old Sprague­Dawley rats were randomly divided into control (CONT) and recurrent neonatal seizure (RS) groups. On postnatal day 35 (P35), mossy fiber sprouting and gene expression were assessed by Timm staining and reverse transcription­quantitative polymerase chain reaction, respectively. Of the twenty genes investigated, seven were significantly downregulated, while four were significantly upregulated in the RS group compared with CONT rats, which was observed in the hippocampus but not in the cerebral cortex. Meanwhile, aberrant mossy fiber sprouting was observed in the supragranular region of the dentate gyrus and Cornu Ammonis 3 subfield of the hippocampus in the RS group. In addition, linear correlation analysis identified significant associations between the expression of certain genes in the hippocampus, which accounted for 40% of the total fifty­five gene pairs among the eleven regulated genes. However, only eight gene pairs in the cerebral cortex exhibited significant positive associations, which accounted for 14.5% of the total. The results of the present study indicated the importance of hippocampal Zn/lipid metabolism­associated genes in recurrent neonatal seizure­induced aberrant mossy fiber sprouting, which may aid the identification of novel potential targets during epileptogenesis.

Neuroprotective effects of autophagy inhibition on hippocampal glutamate receptor subunits after hypoxia-ischemia-induced brain damage in newborn rats.

Autophagy has been suggested to participate in the pathology of hypoxic-ischemic brain damage (HIBD). However, its regulatory role in HIBD remains unclear and was thus examined here using a rat model. To induce HIBD, the left common carotid artery was ligated in neonatal rats, and the rats were subjected to hypoxia for 2 hours. Some of these rats were intraperitoneally pretreated with the autophagy inhibitor 3-methyladenine (10 mM in 10 µL) or the autophagy stimulator rapamycin (1 g/kg) 1 hour before artery ligation. Our findings demonstrated that hypoxia-ischemia-induced hippocampal injury in neonatal rats was accompanied by increased expression levels of the autophagy-related proteins light chain 3 and Beclin-1 as well as of the AMPA receptor subunit GluR1, but by reduced expression of GluR2. Pretreatment with the autophagy inhibitor 3-methyladenine blocked hypoxia-ischemia-induced hippocampal injury, whereas pretreatment with the autophagy stimulator rapamycin significantly augmented hippocampal injury. Additionally, 3-methyladenine pretreatment blocked the hypoxia-ischemia-induced upregulation of GluR1 and downregulation of GluR2 in the hippocampus. By contrast, rapamycin further elevated hippocampal GluR1 levels and exacerbated decreased GluR2 expression levels in neonates with HIBD. Our results indicate that autophagy inhibition favors the prevention of HIBD in neonatal rats, at least in part, through normalizing GluR1 and GluR2 expression.

Autophagy-regulated AMPAR subunit upregulation in in vitro oxygen glucose deprivation/reoxygenation-induced hippocampal injury.

Autophagy has been implicated to mediate experimental cerebral ischemia/reperfusion-induced neuronal death; the underlying molecular mechanisms, though, are poorly understood. In this study, we investigated the role of autophagy in regulating the expression of AMPAR subunits (GluR1, GluR2, and GluR3) in oxygen glucose deprivation/reperfusion (OGD/R)-mediated injury of hippocampal neurons. Our results showed that, OGD/R-induced hippocampal neuron injury was accompanied by accumulation of autophagosomes and autolysosomes in cytoplasm alongside a dramatic increase in expression of autophagy-related genes, LC3 and Beclin 1 and increased intracellular Ca2+ levels. Pre-treatment with autophagy inhibitor 3-methyladenine (3-MA) significantly reduced this effect. Moreover, the OGD/R-induced upregulation of mRNA and protein expressions of GluR1, GluR2, and GluR3 were also effectively reversed in cells pretreated with 3-MA. Our findings indicate that OGD/R induced the expression of GluRs by activating autophagy in in vitro cultured hippocampal neurons, which could be effectively reversed by the administration of 3-MA.

[Establishment and validation of a neonatal pig model of hemolytic jaundice].

OBJECTIVE: To establish a neonatal pig model of hemolytic jaundice. METHODS: Twelve seven-day-old purebred Yorkshire pigs were randomly divided into an experimental group and a control group (n=6 each). Immunization of New Zealand white rabbits was used to prepare rabbit anti-porcine red blood cell antibodies, and rabbit anti-porcine red blood cell serum was separated. The neonatal pigs in the experimental group were given an intravenous injection of rabbit anti-porcine red blood cell serum (5 mL), and those in the control group were given an intravenous injection of normal saline (5 mL). Venous blood samples were collected every 6 hours for routine blood test and liver function evaluation. RESULTS: The experimental group had a significantly higher serum bilirubin level than the control group at 18 hours after the injection of rabbit anti-porcine red blood cell serum (64±30 µmol/L vs 20±4 µmol/L; P<0.05). In the experimental group, the serum bilirubin level reached the peak at 48 hours (275±31 µmol/L), and decreased significantly at 96 hours after the injection (95±17 µmol/L), but all significantly higher than that in the control group (P<0.05). At 18 hours after the injection, the experimental group had a significantly lower red blood cell (RBC) count than the control group [(4.58±0.32)×10(12)/L vs (5.09±0.44)×10(12)/L; P<0.05]; at 24 hours, the experimental group showed further reductions in RBC count and hemoglobin level and had significantly lower RBC count and hemoglobin level than the control group [RBC: (4.21±0.24)×10(12)/L vs (5.11±0.39)×10(12)/L, P<0.05; hemoglobin: 87±3 g vs 97±6 g, P<0.05]. The differences in RBC count and hemoglobin level between the two groups were largest at 36-48 hours. CONCLUSIONS: The neonatal pig model of hemolytic jaundice simulates the pathological process of human hemolytic jaundice well and provides good biological and material bases for further investigation of neonatal hemolysis.

Molecular targeting of the oncoprotein PLK1 in pediatric acute myeloid leukemia: RO3280, a novel PLK1 inhibitor, induces apoptosis in leukemia cells.

Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor; however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.

Early B-cell factor 3 (EBF3) is a novel tumor suppressor gene with promoter hypermethylation in pediatric acute myeloid leukemia.

BACKGROUND: Pediatric acute myeloid leukemia (AML) comprises up to 20% of all childhood leukemia. Recent research shows that aberrant DNA methylation patterning may play a role in leukemogenesis. The epigenetic silencing of the EBF3 locus is very frequent in glioblastoma. However, the expression profiles and molecular function of EBF3 in pediatric AML is still unclear. METHODS: Twelve human acute leukemia cell lines, 105 pediatric AML samples and 30 normal bone marrow/idiopathic thrombocytopenic purpura (NBM/ITP) control samples were analyzed. Transcriptional level of EBF3 was evaluated by semi-quantitative and real-time PCR. EBF3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BGS). The molecular mechanism of EBF3 was investigated by apoptosis assays and PCR array analysis. RESULTS: EBF3 promoter was hypermethylated in 10/12 leukemia cell lines. Aberrant EBF3 methylation was observed in 42.9% (45/105) of the pediatric AML samples using MSP analysis, and the BGS results confirmed promoter methylation. EBF3 expression was decreased in the AML samples compared with control. Methylated samples revealed similar survival outcomes by Kaplan-Meier survival analysis. EBF3 overexpression significantly inhibited cell proliferation and increased apoptosis. Real-time PCR array analysis revealed 93 dysregulated genes possibly implicated in the apoptosis of EBF3-induced AML cells. CONCLUSION: In this study, we firstly identified epigenetic inactivation of EBF3 in both AML cell lines and pediatric AML samples for the first time. Our findings also showed for the first time that transcriptional overexpression of EBF3 could inhibit proliferation and induce apoptosis in AML cells. We identified 93 dysregulated apoptosis-related genes in EBF3-overexpressing, including DCC, AIFM2 and DAPK1. Most of these genes have never been related with EBF3 over expression. These results may provide new insights into the molecular mechanism of EBF3-induced apoptosis; however, further research will be required to determine the underlying details. Our findings suggest that EBF3 may act as a putative tumor suppressor gene in pediatric AML.

[Impact of neonatal exposure to different doses of bisphenol A on puberty in female rats].

OBJECTIVE: To evaluate the effects of neonatal exposure to different doses of bisphenol A (BPA) on the vaginal opening day (VOD), hypothalamic Kiss-1 mRNA expression, and ovarian estrogen receptor (ER) mRNA expression in female rats. METHODS: Neonatal female Sprague-Dawley (SD) rats were randomly divided into six groups: blank control, vehicle, 17ß-estradiol (17ß-estradiol, E2, 10 µg/d), low-dose BPA [25 µg(kg·d)], medium-dose BPA [50 µg(kg·d)], and high-dose BPA groups [250 µg(kg·d)]. The rats were subcutaneously injected with respective agents on postnatal days 0-6. The VOD was recorded, and each rat was sacrificed on the same day. The hypothalamus and ovary were taken and weighed, and the organ coefficients of hypothalamus and ovary were calculated. The hypothalamic Kiss-1 mRNA expression and ovarian ERα and ERß mRNA expression were measured by real-time PCR. RESULTS: Compared with the control group, the E2 and medium- and high-dose BPA groups had advanced VOD, and the E2 group had significantly reduced hypothalamic Kiss-1 mRNA expression and ovarian ERß mRNA expression (P<0.05). CONCLUSIONS: Neonatal exposure to medium- and high-dose BPA[50 and 250 µg/(kg·d)] can induce precocious puberty in rats, but it may not result from the change in hypothalamic Kiss-1 mRNA expression. Neonatal exposure to low-dose BPA [25 µg/(kg·d)] does not induce precocious puberty in rats.

[Diagnostic values of neopterin and S100b for central nervous system infections in children].

OBJECTIVE: To study the diagnostic values of cerebrospinal concentrations of neopterin (NPT) and S100b for central nervous system infections in children. METHODS: Enzyme-linked immunosorbent assay was used to determinate the cerebrospinal concentrations of NPT and S100b in children with central nervous system infections and control children. The two groups of children were compared in terms of the two indicators, and the diagnostic values of the two indicators were evaluated by ROC curve analysis. RESULTS: Children with viral encephalitis had significantly increased cerebrospinal concentrations of NPT and S100b compared with the control group and children with purulent meningitis (P<0.01); there was no difference in the cerebrospinal concentration of NPT between children with purulent meningitis and the control group, while the concentration of S100b in the purulent meningitis group was significantly higher than in the control group (P<0.01). According to the ROC curves, S100b was more valuable than NPT in the diagnosis of viral encephalitis; when cerebrospinal concentration was more than 0.384 ng/mL, S100b had a sensitivity of 93.3% and a specificity of 97.9%; a combination of the two indicators had a higher diagnostic value for viral encephalitis, with a sensitivity of 96.7% and a specificity of 97.9%. CONCLUSIONS: Both NPT and S100b have certain values in the diagnosis of central nervous system infections in children, and S100b is better than NPT.

[Changes of biological clock protein in neonatal rats with hypoxic-ischemic brain damage].

OBJECTIVE: To study the effects of biological clock protein on circadian disorders in hypoxic-ischemic brain damage (HIBD) by examining levels of CLOCK and BMAL1 proteins in the pineal gland of neonatal rats. METHODS: Seventy-two 7-day-old Sprague-Dawley (SD) rats were randomly divided into sham-operated and HIBD groups. HIBD model was prepared according to the modified Levine method. Western blot analysis was used to measure the levels of CLOCK and BMAL1 in the pineal gland at 0, 2, 12, 24, 36 and 48 hours after operation. RESULTS: Both CLOCK and BMAL levels in the pineal gland increased significantly 48 hours after HIBD compared with the sham-operated group (P<0.05). There were no significant differences in levels of CLOCK and BMAL proteins between the two groups at 0, 2, 12, 24 and 36 hours after operation (P>0.05). CONCLUSIONS: Levels of CLOCK and BMAL1 proteins in the pineal gland of rats increase significantly 48 hours after HIBD, suggesting that both CLOCK and BMAL1 may be involved the regulatory mechanism of circadian disorders in rats with HIBD.