Loss of Ninjurin1 alleviates acetaminophen-induced liver injury via enhancing AMPKα-NRF2 pathway.

Acetaminophen (APAP), a widely used pain and fever reliever, is a major contributor to drug-induced liver injury, as its toxic metabolites such as NAPQI induce oxidative stress and hepatic necrosis. While N-acetylcysteine serves as the primary treatment for APAP-induced liver injury (AILI), its efficacy is confined to a narrow window of 8-24 h post-APAP overdose. Beyond this window, liver transplantation emerges as the final recourse, prompting ongoing research to pinpoint novel therapeutic targets aimed at enhancing AILI treatment outcomes. Nerve injury-induced protein 1 (Ninjurin1; Ninj1), initially recognized as an adhesion molecule, has been implicated in liver damage stemming from factors like TNFα and ischemia-reperfusion. Nonetheless, its role in oxidative stress-related liver diseases, including AILI, remains unexplored. In this study, we observed up-regulation of Ninj1 expression in the livers of both human DILI patients and the AILI mouse model. Through the utilization of Ninj1 null mice, hepatocyte-specific Ninj1 KO mice, and myeloid-specific Ninj1 KO mice, we unveiled that the loss of Ninj1 in hepatocytes, rather than myeloid cells, exerts alleviative effects on AILI irrespective of sex dependency. Further in vitro experiments demonstrated that Ninj1 deficiency shields hepatocytes from APAP-induced oxidative stress, mitochondrial dysfunctions, and cell death by bolstering NRF2 stability via activation of AMPKα. In summary, our findings imply that Ninj1 likely plays a role in AILI, and its deficiency confers protection against APAP-induced hepatotoxicity through the AMPKα-NRF2 pathway.

Tat-heat shock protein 10 ameliorates age-related phenotypes by facilitating neuronal plasticity and reducing age-related genes in the hippocampus.

We investigated the effects of heat shock protein 10 (HSP10) protein on memory function, hippocampal neurogenesis, and other related genes/proteins in adult and aged mice. To translocate the HSP10 protein into the hippocampus, the Tat-HSP10 fusion protein was synthesized, and Tat-HSP10, not HSP10, was successfully delivered into the hippocampus based on immunohistochemistry and western blotting. Tat-HSP10 (0.5 or 2.0 mg/kg) or HSP10 (control protein, 2.0 mg/kg) was administered daily to 3- and 21-month-old mice for 3 months, and observed the senescence maker P16 was significantly increased in aged mice and the treatment with Tat-HSP10 significantly decreased P16 expression in the hippocampus of aged mice. In novel object recognition and Morris water maze tests, aged mice demonstrated decreases in exploratory preferences, exploration time, distance moved, number of object contacts, and escape latency compared to adult mice. Treatment with Tat-HSP10 significantly improved exploratory preferences, the number of object contacts, and the time spent swimming in the target quadrant in aged mice but not adults. Administration of Tat-HSP10 increased the number of proliferating cells and differentiated neuroblasts in the dentate gyrus of adult and aged mice compared to controls, as determined by immunohistochemical staining for Ki67 and doublecortin, respectively. Additionally, Tat-HSP10 treatment significantly mitigated the reduction in sirtuin 1 mRNA level, N-methyl-D-aspartate receptor 1, and postsynaptic density 95 protein levels in the hippocampus of aged mice. In contrast, Tat-HSP10 treatment significantly increased sirtuin 3 protein levels in both adult and aged mouse hippocampus. These suggest that Tat-HSP10 can potentially reduce hippocampus-related aging phenotypes.

Okadaic Acid Is at Least as Toxic as Dinophysistoxin-1 after Repeated Administration to Mice by Gavage.

Okadaic acid (OA) and its analogues cause diarrhetic shellfish poisoning (DSP) in humans, and risk assessments of these toxins require toxicity equivalency factors (TEFs), which represent the relative toxicities of analogues. However, no human death by DSP toxin has been reported, and its current TEF value is based on acute lethality. To properly reflect the symptoms of DSP, such as diarrhea without death, the chronic toxicity of DSP toxins at sublethal doses should be considered. In this study, we obtained acute oral LD50 values for OA and dinophysistoxin-1 (DTX-1) (1069 and 897 µg/kg, respectively) to set sublethal doses. Mice were treated with sublethal doses of OA and DTX-1 for 7 days. The mice lost body weight, and the disease activity index and intestinal crypt depths increased. Furthermore, these changes were more severe in OA-treated mice than in the DTX-1-treated mice. Strikingly, ascites was observed, and its severity was greater in mice treated with OA. Our findings suggest that OA is at least as toxic as DTX-1 after repeated oral administration at a low dose. This is the first study to compare repeated oral dosing of DSP toxins. Further sub-chronic and chronic studies are warranted to determine appropriate TEF values for DSP toxins.

Purpurin ameliorates D-galactose-induced aging phenotypes in mouse hippocampus by reducing inflammatory responses.

Purpurin, an anthraquinone, has potent anti-oxidant and anti-inflammatory effects in various types of brain damage. In a previous study, we showed that purpurin exerts neuroprotective effects against oxidative and ischemic damage by reducing pro-inflammatory cytokines. In the present study, we investigated the effects of purpurin against D-galactose-induced aging phenotypes in mice. Exposure to 100 mM D-galactose significantly decreased cell viability in HT22 cells, and purpurin treatment significantly ameliorated the reduction of cell viability, formation of reactive oxygen species, and lipid peroxidation in a concentration-dependent manner. Treatment with 6 mg/kg purpurin significantly improved D-galactose-induced memory impairment in the Morris water maze test in C57BL/6 mice and alleviated the reduction of proliferating cells and neuroblasts in the subgranular zone of the dentate gyrus. In addition, purpurin treatment significantly mitigated D-galactose-induced changes of microglial morphology in the mouse hippocampus and the release of pro-inflammatory cytokines such as interleukin-1ß, interleukin-6, and tumor necrosis factor-α. In addition, purpurin treatment significantly ameliorated D-galactose-induced phosphorylation of c-Jun N-terminal kinase and cleavage of caspase-3 in HT22 cells. These results suggest that purpurin can delay aging by reducing the inflammatory cascade and phosphorylation of the c-Jun N-terminal in the hippocampus.

Tat-malate dehydrogenase fusion protein protects neurons from oxidative and ischemic damage by reduction of reactive oxygen species and modulation of glutathione redox system.

Malate dehydrogenase (MDH) plays an important role in the conversion of malate to oxaloacetate during the tricarboxylic acid cycle. In this study, we examined the role of cytoplasmic MDH (MDH1) in hydrogen peroxide (H2O2)-induced oxidative stress in HT22 cells and ischemia-induced neuronal damage in the gerbil hippocampus. The Tat-MDH1 fusion protein was constructed to enable the delivery of MDH1 into the intracellular space and penetration of the blood-brain barrier. Tat-MDH1, but not MDH1 control protein, showed significant cellular delivery in HT22 cells in a concentration- and time-dependent manner and gradual intracellular degradation in HT22 cells. Treatment with 4 µM Tat-MDH1 significantly ameliorated 200 µM H2O2-induced cell death, DNA fragmentation, and reactive oxygen species formation in HT22 cells. Transient increases in MDH1 immunoreactivity were detected in the hippocampal CA1 region 6-12 h after ischemia, but MDH1 activity significantly decreased 2 days after ischemia. Supplementation of Tat-MDH1 immediately after ischemia alleviated ischemia-induced hyperlocomotion and neuronal damage 1 and 4 days after ischemia. In addition, treatment with Tat-MDH1 significantly ameliorated the increases in hydroperoxides, lipid peroxidation, and reactive oxygen species 2 days after ischemia. Tat-MDH1 treatment maintained the redox status of the glutathione system in the hippocampus 2 days after ischemia. These results suggest that Tat-MDH1 exerts neuroprotective effects by reducing oxidative stress and maintaining glutathione redox system in the hippocampus.

Novel NF-κB reporter mouse for the non-invasive monitoring of inflammatory diseases.

Bioluminescence imaging is useful for non-invasively monitoring inflammatory reactions associated with disease progression, and since NF-κB is a pivotal transcription factor that alters expressions of inflammatory genes, we generated novel NF-κB luciferase reporter (NF-κB-Luc) mice to understand the dynamics of inflammatory responses in whole body, and also in various type of cells by crossing NF-κB-Luc mice with cell-type specific Cre expressing mice (NF-κB-Luc:[Cre]). Bioluminescence intensity was significantly increased in NF-κB-Luc (NKL) mice exposed to inflammatory stimuli (PMA or LPS). Crossing NF-κB-Luc mice with Alb-cre mice or Lyz-cre mice generated NF-κB-Luc:Alb (NKLA) and NF-κB-Luc:Lyz2 (NKLL) mice, respectively. NKLA and NKLL mice showed enhanced bioluminescence in liver and macrophages, respectively. To confirm that our reporter mice could be utilized for the non-invasive monitoring of inflammation in preclinical models, we conducted a DSS-induced colitis model and a CDAHFD-induced NASH model in our reporter mice. In both models, our reporter mice reflected the development of these diseases over time. In conclusion, we believe that our novel reporter mouse can be utilized as a non-invasive monitoring platform for inflammatory diseases.

Cu,Zn-Superoxide Dismutase has Minimal Effects Against Cuprizone-Induced Demyelination, Microglial Activation, and Neurogenesis Defects in the C57BL/6 Mouse Hippocampus.

Cuprizone causes consistent demyelination and oligodendrocyte damage in the mouse brain. Cu,Zn-superoxide dismutase 1 (SOD1) has neuroprotective potential against various neurological disorders, such as transient cerebral ischemia and traumatic brain injury. In this study, we investigated whether SOD1 has neuroprotective effects against cuprizone-induced demyelination and adult hippocampal neurogenesis in C57BL/6 mice, using the PEP-1-SOD1 fusion protein to facilitate the delivery of SOD1 protein into hippocampal neurons. Eight weeks feeding of cuprizone-supplemented (0.2%) diets caused a significant decrease in myelin basic protein (MBP) expression in the stratum lacunosum-moleculare of the CA1 region, the polymorphic layer of the dentate gyrus, and the corpus callosum, while ionized calcium-binding adapter molecule 1 (Iba-1)-immunoreactive microglia showed activated and phagocytic phenotypes. In addition, cuprizone treatment reduced proliferating cells and neuroblasts as shown using Ki67 and doublecortin immunostaining. Treatment with PEP-1-SOD1 to normal mice did not show any significant changes in MBP expression and Iba-1-immunoreactive microglia. However, Ki67-positive proliferating cells and doublecortin-immunoreactive neuroblasts were significantly decreased. Simultaneous treatment with PEP-1-SOD1 and cuprizone-supplemented diets did not ameliorate the MBP reduction in these regions, but mitigated the increase of Iba-1 immunoreactivity in the corpus callosum and alleviated the reduction of MBP in corpus callosum and proliferating cells, not neuroblasts, in the dentate gyrus. In conclusion, PEP-1-SOD1 treatment only has partial effects to reduce cuprizone-induced demyelination and microglial activation in the hippocampus and corpus callosum and has minimal effects on proliferating cells in the dentate gyrus.

CHIP ameliorates neuronal damage in H2O2-induced oxidative stress in HT22 cells and gerbil ischemia.

Carboxyl terminus of Hsc70-interacting protein (CHIP) is highly conserved and is linked to the connection between molecular chaperones and proteasomes to degrade chaperone-bound proteins. In this study, we synthesized the transactivator of transcription (Tat)-CHIP fusion protein for effective delivery into the brain and examined the effects of CHIP against oxidative stress in HT22 cells induced by hydrogen peroxide (H2O2) treatment and ischemic damage in gerbils by 5 min of occlusion of both common carotid arteries, to elucidate the possibility of using Tat-CHIP as a therapeutic agent against ischemic damage. Tat-CHIP was effectively delivered to HT22 hippocampal cells in a concentration- and time-dependent manner, and protein degradation was confirmed in HT22 cells. In addition, Tat-CHIP significantly ameliorated the oxidative damage induced by 200 µM H2O2 and decreased DNA fragmentation and reactive oxygen species formation. In addition, Tat-CHIP showed neuroprotective effects against ischemic damage in a dose-dependent manner and significant ameliorative effects against ischemia-induced glial activation, oxidative stress (hydroperoxide and malondialdehyde), pro-inflammatory cytokines (interleukin-1ß, interleukin-6, and tumor necrosis factor-α) release, and glutathione and its redox enzymes (glutathione peroxidase and glutathione reductase) in the hippocampus. These results suggest that Tat-CHIP could be a therapeutic agent that can protect neurons from ischemic damage.

Phosphoglycerate kinase 1 protects against ischemic damage in the gerbil hippocampus.

Phosphoglycerate kinase 1 (PGK1) is a metabolic enzyme that converts 1,3-diphosphoglycerate to 3-phosphoglycerate. In the current study, we synthesized a PEP-1-PGK1 fusion protein that can cross the blood-brain barrier and cell membrane, and the effects of PEP-1-PGK1 against oxidative stress were investigated HT22 cells and ischemic gerbil brain. The PEP-1-PGK1 protein and its control protein (Con-PGK1) were treated and permeability was evaluated HT22 cells. The PEP-1-PGK1 was introduced into HT22 cells depending on its concentration and incubation time and was gradually degraded over 36 h after treatment. PEP-1-PGK1, but not Con-PGK1, significantly ameliorated H2O2-induced cell damage and reactive oxygen species formation in HT22 cells. Additionally, PEP-1-PGK1, but not Con-PGK1, mitigated ischemia-induced hyperlocomotion 1 d after ischemia and 4 d after ischemia of neuronic cell death. PEP-1-PGK1 treatment significantly alleviated the raised lactate and succinate dehydrogenase activities in the early (15 min to 6 h) and late (4 and 7 d) stages of ischemia, respectively. In addition, PEP-1-PGK1 treatment ameliorated the decrease in ATP and pH levels in the late stage (2-7 d) of ischemia. Nuclear factor erythroid-2-related factor 2 (Nrf2) levels accelerated the ischemia-induced increase in the hippocampus 1 d after ischemia after PEP-1-PGK1 treatment. Neuroprotective and ameliorative effects were prominent at a low concentration (0.1 mg/kg), but not at a high concentration (1 mg/kg), of PEP-1-PGK1. Collectively, low concentrations of PEP-1-PGK1 prevented neuronal stress by increasing energy production.

Deficiency of Ninjurin1 attenuates LPS/D-galactosamine-induced acute liver failure by reducing TNF-α-induced apoptosis in hepatocytes.

Nerve injury-induced protein 1 (Ninjurin1, Ninj1) is a membrane protein that mediates cell adhesion. The role of Ninj1 during inflammatory response has been widely investigated in macrophages and endothelial cells. Ninj1 is expressed in various tissues, and the liver also expresses high levels of Ninj1. Although the hepatic upregulation of Ninj1 has been reported in human hepatocellular carcinoma and septic mice, little is known of its function during the pathogenesis of liver diseases. In the present study, the role of Ninj1 in liver inflammation was explored using lipopolysaccharide (LPS)/D-galactosamine (D-gal)-induced acute liver failure (ALF) model. When treated with LPS/D-gal, conventional Ninj1 knock-out (KO) mice exhibited a mild inflammatory phenotype as compared with wild-type (WT) mice. Unexpectedly, myeloid-specific Ninj1 KO mice showed no attenuation of LPS/D-gal-induced liver injury. Whereas, Ninj1 KO primary hepatocytes were relatively insensitive to TNF-α-induced caspase activation as compared with WT primary hepatocytes. Also, Ninj1 knock-down in L929 and AML12 cells and Ninj1 KO in HepG2 cells ameliorated TNF-α-mediated apoptosis. Consistent with in vitro results, hepatocyte-specific ablation of Ninj1 in mice alleviated LPS/D-gal-induced ALF. Summarizing, our in vivo and in vitro studies show that lack of Ninj1 in hepatocytes diminishes LPS/D-gal-induced ALF by alleviating TNF-α/TNFR1-induced cell death.

The neuroprotective effects of phosphoglycerate mutase 5 are mediated by decreasing oxidative stress in HT22 hippocampal cells and gerbil hippocampus.

Phosphoglycerate mutase 5 (PGAM5), a glycolytic enzyme, plays an important role in cell death and regulation of mitochondrial dynamics. In this study, we investigated the effects of PGAM5 on oxidative stress in HT22 hippocampal cells and ischemic damage in the gerbil hippocampus to elucidate the role of PGAM5 in oxidative and ischemic stress. Constructs were designed with a PEP-1 expression vector to facilitate the intracellular delivery of PGAM5 proteins. We observed time- and concentration-dependent increases in the intracellular delivery of the PEP-1-PGAM5 protein, but not its control protein (PGAM5), in HT22 cells, and morphologically demonstrated the localization of the transduced protein, which was stably expressed in the cytoplasm after 12 h of PEP-1-PGAM5 treatment. PEP-1-PGAM5 treatment significantly ameliorated cell death, reactive oxygen species formation, DNA fragmentation, and the reduction of cell proliferation induced by H2O2 treatment in HT22 cells. In addition, PEP-1-PGAM5 was effectively delivered to the gerbil hippocampus 8 h after treatment, and ischemia-induced hyperlocomotion and neuronal death in the hippocampal CA1 region were significantly alleviated 1 and 4 days after ischemia, respectively. Ischemia-induced microglial activation was also mitigated by treatment with 1.0 mg/kg PEP-1-PGAM5. At 3 h after ischemia, PEP-1-PGAM5 treatment significantly ameliorated the increase in lipid peroxidation, as assessed by malondialdehyde and hydroperoxide levels, and decreased glutathione levels (increases in glutathione disulfide, the oxidized form of glutathione) in the hippocampus. Two days after ischemia, treatment with PEP-1-PGAM5 significantly alleviated the ischemia-induced reduction in glutathione peroxidase activity and further increased superoxide dismutase activity in the hippocampus. The neuroprotective effects of PEP-1-PGAM5 are partially mediated by a reduction in oxidative stress, such as the formation of reactive oxygen species, and increases in the activity of antioxidants such as glutathione peroxidase and superoxide dismutase.

Neuroprotective Effects of Purpurin Against Ischemic Damage via MAPKs, Bax, and Oxidative Stress Cascades in the Gerbil Hippocampus.

Purpurin has various effects, including anti-inflammatory effects, and can efficiently cross the blood-brain barrier. In the present study, we investigated the effects of purpurin on oxidative stress in HT22 cells and mild brain damage in the gerbil hippocampal CA1 region induced by transient forebrain ischemia. Oxidative stress induced by H2O2 was significantly ameliorated by treatment with purpurin, based on changes in cell death, DNA fragmentation, formation of reactive oxygen species, and pro-apoptotic (Bax)/anti-apoptotic (Bcl-2) protein levels. In addition, treatment with purpurin significantly reduced the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK), and p38 signaling in HT22 cells. Transient forebrain ischemia in gerbils led to a significant increase in locomotor activity 1 day after ischemia and significant decrease in number of surviving cells in the CA1 region 4 days after ischemia. Administration of purpurin reduced the travel distance 1 day after ischemia and abrogates the neuronal death in the hippocampal CA1 region 4 days after ischemia based on immunohistochemical and histochemical staining for NeuN and Fluoro-Jade C, respectively. Purpurin treatment significantly decreased the activation of microglia and astrocytes as well as the increases of nuclear factor kappa-light-chain-enhancer of activated B cells p65 in the hippocampal CA1 region 4 days after ischemia and ameliorated the ischemia-induced transient increases of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the hippocampus 6 h after ischemia. In addition, purpurin significantly alleviated the ischemia-induced phosphorylation of JNK, ERK, and p38 in the hippocampus 1 day after ischemia. Furthermore, purpurin treatment significantly mitigated the increases of Bax in the hippocampus 1 day after ischemia and the lipid peroxidation based on malondialdehyde and hydroperoxides levels 2 days after ischemia. These results suggest that purpurin can be one of the potential candidates to reduce neuronal damage and inflammatory responses after oxidative stress in HT22 cells or ischemic damage in gerbils.

Comparison of the Effects of Cuprizone on Demyelination in the Corpus Callosum and Hippocampal Progenitors in Young Adult and Aged Mice.

Cuprizone is commonly used to induce neuronal demyelination in mice. In the present study, we compared the cuprizone-induced demyelination in the corpus callosum and investigated the effects of cuprizone on proliferating cells and neuroblasts in the dentate gyrus of young adult and aged mice. 5-week- and 23-month-old mice were fed a normal diet or a 0.2% cuprizone-enriched diet for 5 weeks. Mice fed a cuprizone-supplemented diet showed a significant reduction in myelin basic protein-positive structures in the corpus callosum, with the reduction in myelinated fibers being confirmed by electron microscopic analysis. In addition, we observed a marked increase in Ki67-positive proliferating cells and doublecortin-immunoreactive neuroblasts in young adult mice in response to cuprizone treatment, although not in aged mice, as the basal levels of these cells were significantly lower in these older mice. Furthermore, Ser133-phosphorylated cAMP response element-binding protein (pCREB)-positive nuclei and brain-derived neurotrophic factor (BDNF) protein levels were significantly reduced in young adult mice following cuprizone treatment in young adult, although again not in the aged mice. However, in both young adult and aged mice, there were no significant reductions in hippocampal mature neurons in response to cuprizone treatment. These observations indicate that in the mice of both age groups a cuprizone-supplemented diet contributes to an increase in demyelination in the corpus callosum and neural progenitor cells in the dentate gyrus, although the damage is more pronounced in young adult mice. This demyelination and reduction in neural progenitor cells may be associated with changes in the levels of BDNF and pCREB in the dentate gyrus.

Spatial and temporal changes in the PGE2 EP2 receptor in mice hippocampi during postnatal development and its relationship with cyclooxygenase-2.

OBJECTIVES: Prostaglandin E2 E-prostanoid 2 receptor (PGE2 EP2), downstream of cyclooxygenase-2 (COX-2), plays an important role in inflammatory responses, but there are some reports about synaptic functions of COX-2 and PGE2 EP2 in the hippocampus. MATERIALS AND METHODS: C57BL/6J mice were sacrificed at postnatal days (P) 1, 7, 14, 28, and 56 for immunohistochemical staining for EP2 and doublecortin as well as western blot for EP2. In addition, COX-2 knockout and its wild-type mice were euthanized for immunohistochemical staining for EP2. RESULTS: EP2 immunoreactivity was observed in the majority of the cells in the dentate gyrus at P1 and P7, while at P14, it was detected in the outer granule cell layer and was confined to its subgranular zone at P28 and P56. EP2 protein levels in the hippocampal homogenates were also highest at P7 and lowest at P56. EP2 immunoreactivity was partially colocalized, with doublecortin (DCX)-immunoreactive neuroblasts appearing in the mid-zone of the granule cell layer at P14 and in the subgranular zone of the dentate gyrus at P28. Co-localization of EP2 and DCX was significantly decreased in the dentate gyrus in the P28 group compared with that in the P14 group. In COX-2 knockout mice, EP2 immunoreactivity was significantly decreased in the hippocampal CA1 region (P=0.000165) and dentate gyrus (P=0.00898). CONCLUSION: EP2 decreases with age, which is expressed in DCX-immunoreactive neuroblasts in the dentate gyrus. This suggests that EP2 is closely linked to structural lamination and adult neurogenesis in the dentate gyrus.

Tat-p27 Ameliorates Neuronal Damage Reducing α-Synuclein and Inflammatory Responses in Motor Neurons After Spinal Cord Ischemia.

p27Kip1 (p27) regulates the cell cycle by inhibiting G1 progression in cells. Several studies have shown conflicting results on the effects of p27 against cell death in various insults. In the present study, we examined the neuroprotective effects of p27 against H2O2-induced oxidative stress in NSC34 cells and against spinal cord ischemia-induced neuronal damage in rabbits. To promote delivery into NSC34 cells and motor neurons in the spinal cord, Tat-p27 fusion protein and its control protein (Control-p27) were synthesized with or without Tat peptide, respectively. Tat-p27, but not Control-27, was efficiently introduced into NSC34 cells in a concentration- and time-dependent manner, and the protein was detected in the cytoplasm. Tat-p27 showed neuroprotective effects against oxidative stress induced by H2O2 treatment and reduced the formation of reactive oxygen species, DNA fragmentation, and lipid peroxidation in NSC34 cells. Tat-p27, but not Control-p27, ameliorated ischemia-induced neurological deficits and cell damage in the rabbit spinal cord. In addition, Tat-p27 treatment reduced the expression of α-synuclein, activation of microglia, and release of pro-inflammatory cytokines such as interleukin-1ß and tumor necrosis factor-α in the spinal cord. Taken together, these results suggest that Tat-p27 inhibits neuronal damage by decreasing oxidative stress, α-synuclein expression, and inflammatory responses after ischemia.

Establishment of particulate matter-induced lung injury model in mouse.

BACKGROUND: Particulate matter (PM) is one of the principal causes of human respiratory disabilities resulting from air pollution. Animal models have been applied to discover preventive and therapeutic drugs for lung diseases caused by PM. However, the induced severity of lung injury in animal models using PM varies from study to study due to disparities in the preparation of PM, and the route and number of PM administrations. In this study, we established an in vivo model to evaluate PM-induced lung injury in mice. RESULTS: PM dispersion was prepared using SRM2975. Reactive oxygen species were increased in MLE 12 cells exposed to this PM dispersion. In vivo studies were conducted in the PM single challenge model, PM multiple challenge model, and PM challenge with ovalbumin-induced asthma using the PM dispersion. No histopathological changes were observed in lung tissues after a single injection of PM, whereas mild to moderate lung inflammation was obtained in the lungs of mice exposed to PM three times. However, fibrotic changes were barely seen, even though transmission electron microscopy (TEM) studies revealed the presence of PM particles in the alveolar macrophages and alveolar capillaries. In the OVA-PM model, peribronchial inflammation and mucous hypersecretion were more severe in the OVA+PM group than the OVA group. Serum IgE levels tended to increase in OVA+PM group than in OVA group. CONCLUSIONS: In this study, we established a PM-induced lung injury model to examine the lung damage induced by PM. Based on our results, repeated exposures of PM are necessary to induce lung inflammation by PM alone. PM challenge, in the presence of underlying diseases such as asthma, can also be an appropriate model for studying the health effect of PM.

Gynura procumbens Root Extract Ameliorates Ischemia-Induced Neuronal Damage in the Hippocampal CA1 Region by Reducing Neuroinflammation.

Gynura procumbens has been used in Southeast Asia for the treatment of hypertension, hyperglycemia, and skin problems induced by ultraviolet irradiation. Although considerable studies have reported the biological properties of Gynura procumbens root extract (GPE-R), there are no studies on the effects of GPE-R in brain damages, for example following brain ischemia. In the present study, we screened the neuroprotective effects of GPE-R against ischemic damage and neuroinflammation in the hippocampus based on behavioral, morphological, and biological approaches. Gerbils received oral administration of GPE-R (30 and 300 mg/kg) every day for three weeks and 2 h after the last administration, ischemic surgery was done by occlusion of both common carotid arteries for 5 min. Administration of 300 mg/kg GPE-R significantly reduced ischemia-induced locomotor hyperactivity 1 day after ischemia. Significantly more NeuN-positive neurons were observed in the hippocampal CA1 regions of 300 mg/kg GPE-R-treated animals compared to those in the vehicle-treated group 4 days after ischemia. Administration of GPE-R significantly reduced levels of pro-inflammatory cytokines such as interleukin-1ß, -6, and tumor necrosis factor-α 6 h after ischemia/reperfusion. In addition, activated microglia were significantly decreased in the 300 mg/kg GPE-R-treated group four days after ischemia/reperfusion compared to the vehicle-treated group. These results suggest that GPE-R may be one of the possible agents to protect neurons from ischemic damage by reducing inflammatory responses.

Changes in the expression of the B subunit of vacuolar H+-ATPase, in the hippocampus, following transient forebrain ischemia in gerbils.

Objectives: Vacuolar H+-ATPase is a highly conserved enzyme that plays an important role in maintaining an acidic environment for lysosomal function and accumulating neurotransmitters in synaptic vesicles. In the present study, we investigated the time-dependent changes in the expression of vacuolar H+-ATPase V1B2 (ATP6V1B2), a major neuronal subtype of vacuolar H+-ATPase located in the hippocampus, after 5 min of transient forebrain ischemia in gerbils. We also examined the pH and lactate levels in the hippocampus after ischemia to elucidate the correlation between ATP6V1B2 expression and acidosis. Materials and Methods: Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 5 min and animals were sacrificed at various time points after ischemia for immunohistochemical staining of ATP6V1B2 and measurements of pH and lactate levels in the hippocampus. Results: ATP6V1B2 immunoreactivity was found to be transiently increased in the hippocampal CA1 region and dentate gyrus 12-24 hr after ischemia when the pH and lactate levels were decreased. In addition, ATP6V1B2 immunoreactivity significantly increased in the hippocampal CA3 and dentate gyrus, regions relatively resistant to ischemic damage, 4 days after ischemia, when the NeuN-positive, mature neuron numbers were significantly decreased in the hippocampal CA1 region. Conclusion: These results suggest that ATP6V1B2 expression is transiently increased in the hippocampus following ischemia, which may be intended to compensate for ischemia-related dysfunction of ATP6V1B2 in the hippocampus.

Entacapone promotes hippocampal neurogenesis in mice.

Entacapone, a catechol-O-methyltransferase inhibitor, can strengthen the therapeutic effects of levodopa on the treatment of Parkinson's disease. However, few studies are reported on whether entacapone can affect hippocampal neurogenesis in mice. To investigate the effects of entacapone, a modulator of dopamine, on proliferating cells and immature neurons in the mouse hippocampal dentate gyrus, 60 mice (7 weeks old) were randomly divided into a vehicle-treated group and the groups treated with 10, 50, or 200 mg/kg entacapone. The results showed that 50 and 200 mg/kg entacapone increased the exploration time for novel object recognition. Immunohistochemical staining results revealed that after entacapone treatment, the numbers of Ki67-positive proliferating cells, doublecortin-positive immature neurons, and phosphorylated cAMP response element-binding protein (pCREB)-positive cells were significantly increased. Western blot analysis results revealed that treatment with tyrosine kinase receptor B (TrkB) receptor antagonist significantly decreased the exploration time for novel object recognition and inhibited the expression of phosphorylated TrkB and brain-derived neurotrophic factor (BDNF). Entacapone treatment antagonized the effects of TrkB receptor antagonist. These results suggest that entacapone treatment promoted hippocampal neurogenesis and improved memory function through activating the BDNF-TrkB-pCREB pathway. This study was approved by the Institutional Animal Care and Use Committee of Seoul National University (approval No. SNU-130730-1) on February 24, 2014.

Physical Stress Induced Reduction of Proliferating Cells and Differentiated Neuroblasts Is Ameliorated by Fermented Laminaria japonica Extract Treatment.

Laminaria japonica is widely cultivated in East Asia, including South Korea. Fucoidan, a main component of L. japonica, protects neurons from neurological disorders such as ischemia and traumatic brain injury. In the present study, we examined the effects of extract from fermented L. japonica on the reduction of proliferating cells and neuroblasts in mice that were physically (with electric food shock) or psychologically (with visual, auditory and olfactory sensation) stressed with the help of a communication box. Vehicle (distilled water) or fermented L. japonica extract (50 mg/kg) were orally administered to the mice once a day for 21 days. On the 19th day of the treatment, physical and psychological stress was induced by foot shock using a communication box and thereafter for three days. Plasma corticosterone levels were significantly increased after exposure to physical stress and decreased Ki67 positive proliferating cells and doublecortin immunoreactive neuroblasts. In addition, western blot analysis demonstrated that physical stress as well as psychological stress decreased the expression levels of brain-derived neurotrophic factor (BDNF) and the number of phosphorylated cAMP response element binding protein (pCREB) positive nuclei in the dentate gyrus. Fermentation of L. japonica extract significantly increased the contents of reduced sugar and phenolic compounds. Supplementation with fermented L. japonica extract significantly ameliorated the increases of plasma corticosterone revels and decline in the proliferating cells, neuroblasts, and expression of BDNF and pCREB in the physically stressed mice. These results indicate that fermented L. japonica extract has positive effects in ameliorating the physical stress induced reduction in neurogenesis by modulating BDNF and pCREB expression in the dentate gyrus.