A comparative study on eating habits and mental health of Korean middle school students according to their bedtime across regions: using data from the 2020-2022 Korea Youth Risk Behavior Survey.

BACKGROUND/OBJECTIVES: The objective of this study was to compare dietary habits and mental health among middle school students in urban and rural areas based on bedtime, and to provide evidence supporting appropriate bedtime for Korean middle school students in relation to their healthy dietary habits and mental well-being. SUBJECTS/METHODS: The study population consisted of 25,681 second-year middle school students who participated in the Korea Youth Risk Behavior Survey in 2020-2022. Participants were asked about their bedtime and wake-up time during the past 7 days and were classified into five categories. The study compared the general characteristics, academic factors, dietary habits, and mental health of urban and rural students based on their bedtime. RESULTS: Bedtime was found to be later in the following order: urban female students, rural female students, urban male students, and rural male students. As bedtime got later, the rates of smoking and alcohol consumption increased. Students who went to bed before 11 p.m. had lower academic performance, while rural male students who went to bed after 2 a.m. had lower academic performance. Later bedtime was associated with increased smartphone usage, skipping breakfast, consuming fast food, and drinking carbonated beverages. Later bedtime was also associated with higher perceived stress levels, particularly among students who went to bed after 2 a.m., higher rates of suicidal ideation, experiencing sadness and despair, as well as the prevalence of clinically significant anxiety disorders. CONCLUSION: These results suggest that middle school students who go to bed too late have higher rates of smoking and alcohol drinking, as well as unhealthy eating habits, stress, suicidal ideation, sadness, and anxiety. Therefore, it is necessary to provide educational and social institutional support to promote adequate sleep for the health of adolescents.

Real-World Treatment Patterns and Clinical Outcomes in Korean Patients With AML Ineligible for First-Line Intensive Chemotherapy: A Subanalysis of the CURRENT Study, a Non-Interventional, Retrospective Chart Review.

BACKGROUND: Although most elderly patients with acute myeloid leukemia (AML) are ineligible for intensive chemotherapy (ICT), treatment options remain limited. CURRENT (UMIN000037786), a real-world, non-interventional, retrospective chart review, evaluated clinical outcomes, clinicopathologic characteristics, and treatment patterns in these patients. We present results from a subanalysis of Korean patients in this study. METHODS: Patients were aged ≥ 18 years with primary or secondary AML ineligible for ICT who initiated first-line systemic therapy or best supportive care (BSC) between 2015 and 2018 across four centers in Korea. Primary endpoint was overall survival (OS) from diagnosis. Secondary endpoints included progression-free survival (PFS), time to treatment failure, and response rates. Data analyses were primarily descriptive, with time-to-event outcomes estimated using the Kaplan-Meier method, and Cox regression used to determine prognostic factors for survival. RESULTS: Among 194 patients enrolled, 84.0% received systemic therapy and 16.0% received BSC. Median age at diagnosis was 74 and 78 years, and Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1 was reported in 73.0% and 48.4% of patients, respectively; poor cytogenetic risk was reported in 30.1% and 16.1% of patients. Median OS was 7.83 vs. 4.50 months, and median PFS was 6.73 vs. 4.50 months in the systemic therapy vs. BSC groups. Prognostic factors affecting OS included secondary AML (hazard ratio, 1.67 [95% confidence interval, 1.13-2.45]), ECOG performance status ≥ 2 (2.41 [1.51-3.83]), poor cytogenetic risk (2.10 [1.36-3.24]), and Charlson comorbidity index ≥ 1 (2.26 [1.43-3.58]). CONCLUSION: Clinical outcomes are poor in Korean patients with AML ineligible for ICT who are prescribed current systemic therapies or BSC. There is a substantial unmet need for novel agents (monotherapy or in combination) to improve clinical outcomes in this patient population.

Effect of sp3/sp2 carbon ratio and hydrodynamic size on the biodistribution kinetics of nanodiamonds in mice via intravenous injection.

BACKGROUND: Nanodiamonds (NDs) have gained a rapidly growing interest in biomedical applications; however, little is known regarding their biokinetics owing to difficulties in measurements and limited synthesis/purification technologies. In this study, we investigated the distribution kinetics of detonation-synthesized NDs in mice via intravenous injection to evaluate the parameters that determine the behavior of the particles. We prepared two distinctive NDs that controlled the sp3/sp2 carbon ratio and particle size by coating them with serum proteins. The four control samples were intravenously injected into mice, and tissue distribution and clearance were evaluated at 30 min and 1, 7, and 28 days post-injection. RESULTS: The sp3/sp2 carbon ratio showed no correlation with the organ distribution of the NDs. However, hydrodynamic size showed an excellent correlation with organ distribution levels: a negative correlation in the liver and positive correlations in the spleen and lungs. Furthermore, the deposition levels of NDs in the lung suggest that particles smaller than 300 nm could avoid lung deposition. Finally, a similar organ distribution pattern was observed in mice injected with carbon black nanoparticles controlled hydrodynamic size. CONCLUSIONS: In conclusion, the tissue distribution of NDs is modulated not by the sp3/sp2 carbon ratio but by the hydrodynamic size, which can provide helpful information for targeting the tissue of NDs. Furthermore, the organ distribution pattern of the NDs may not be specific to NDs but also can apply to other nanoparticles, such as carbon black.

Size- and oxidative potential-dependent toxicity of environmentally relevant expanded polystyrene styrofoam microplastics to macrophages.

Expanded polystyrene (EPS), also known as Styrofoam, is a widespread global pollutant, and its lightweight floating property increases its chances of weathering by abrasion and ultraviolet (UV) irradiation, resulting in microplastics. Herein, we investigated the effects of particle size ((1 µm versus 10 µm), UV irradiation (pristine versus UV oxidation), and origin (secondary versus primary) on the toxicity of Styrofoam microplastics. The target cells used in this study were selected based on human exposure-relevant cell lines: differentiated THP-1 cells for macrophages, Caco-2 for enterocytes, HepG2 for hepatocytes, and A549 for alveolar epithelial cells. In the differentiated THP-1 cells, the levels of cytotoxicity and inflammatory cytokines showed size- (1 µm > 10 µm), UV oxidation- (UV > pristine), and origin- (secondary > primary) dependency. Furthermore, the intrinsic oxidative potential of the test particles was positively correlated with cellular oxidative levels and toxicity endpoints, suggesting that the toxicity of Styrofoam microplastics also follows the oxidative stress paradigm. Additionally, all microplastics induced the activation of the pyrin domain-containing protein 3 (NLRP3) inflammasome and the release of interleukin-1ß (IL-1ß). These results imply that weathering process can aggravate the toxicity of Styrofoam microplastics due to the increased oxidative potential and decreased particle size.

Antioxidant effect of ergothioneine on in vitro maturation of porcine oocytes.

BACKGROUND: Ergothioneine (EGT) is a natural amino acid derivative in various animal organs and is a bioactive compound recognized as a food and medicine. OBJECTIVES: This study examined the effects of EGT supplementation during the in vitro maturation (IVM) period on porcine oocyte maturation and subsequent embryonic development competence after in vitro fertilization (IVF). METHODS: Each EGT concentration (0, 10, 50, and 100 µM) was supplemented in the maturation medium during IVM. After IVM, nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels of oocytes were investigated. In addition, the genes related to cumulus function and antioxidant pathways in oocytes or cumulus cells were investigated. Finally, this study examined whether EGT could affect embryonic development after IVF. RESULTS: After IVM, the EGT supplementation group showed significantly higher intracellular GSH levels and significantly lower intracellular ROS levels than the control group. Moreover, the expression levels of hyaluronan synthase 2 and Connexin 43 were significantly higher in the 10 µM EGT group than in the control group. The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and NAD(P)H quinone dehydrogenase 1 (NQO1) were significantly higher in the oocytes of the 10 µM EGT group than in the control group. In the assessment of subsequent embryonic development after IVF, the 10 µM EGT treatment group improved the cleavage and blastocyst rate significantly than the control group. CONCLUSIONS: Supplementation of EGT improved oocyte maturation and embryonic development by reducing oxidative stress in IVM oocytes.

Astrocyte-derived adenosine excites sleep-promoting neurons in the ventrolateral preoptic nucleus: Astrocyte-neuron interactions in the regulation of sleep.

Although ATP and/or adenosine derived from astrocytes are known to regulate sleep, the precise mechanisms underlying the somnogenic effects of ATP and adenosine remain unclear. We selectively expressed channelrhodopsin-2 (ChR2), a light-sensitive ion channel, in astrocytes within the ventrolateral preoptic nucleus (VLPO), which is an essential brain nucleus involved in sleep promotion. We then examined the effects of photostimulation of astrocytic ChR2 on neuronal excitability using whole-cell patch-clamp recordings in two functionally distinct types of VLPO neurons: sleep-promoting GABAergic projection neurons and non-sleep-promoting local GABAergic neurons. Optogenetic stimulation of VLPO astrocytes demonstrated opposite outcomes in the two types of VLPO neurons. It led to the inhibition of non-sleep-promoting neurons and excitation of sleep-promoting neurons. These responses were attenuated by blocking of either adenosine A1 receptors or tissue-nonspecific alkaline phosphatase (TNAP). In contrast, exogenous adenosine decreased the excitability of both VLPO neuron populations. Moreover, TNAP was expressed in galanin-negative VLPO neurons, but not in galanin-positive sleep-promoting projection neurons. Taken together, these results suggest that astrocyte-derived ATP is converted into adenosine by TNAP in non-sleep-promoting neurons. In turn, adenosine decreases the excitability of local GABAergic neurons, thereby increasing the excitability of sleep-promoting GABAergic projection neurons. We propose a novel mechanism involving astrocyte-neuron interactions in sleep regulation, wherein endogenous adenosine derived from astrocytes excites sleep-promoting VLPO neurons, and thus decreases neuronal excitability in arousal-related areas of the brain.

Six-well plate-based colony-forming efficacy assay and Co-Culture application to assess toxicity of metal oxide nanoparticles.

The development of a universal, label-free, and reliable in vitro toxicity testing method for nanoparticles is urgent because most nanoparticles can interfere with toxicity assays. In this regard, the colony-forming efficacy (CFE) assay has been suggested as a suitable in vitro toxicity assay for testing nanoparticles without such interference. Recently, the Organisation for Economic Co-operation and Development (OECD) developed a 60 × 15 mm Petri dish-based CFE assay for testing nanoparticles in MDCK-1 cells. However, further investigations are needed, including testing with other cell types, at a smaller scale for greater efficiency, and the application of the co-culture technique. In this study, we selected TiO2, CuO, CeO2, and SiO2 as test nanoparticles and successfully developed a 6-well plate-based CFE assay using HepG2 and A549 cells and a co-culture assay for combinations of HepG2 cells and THP-1 macrophages or A549 cells and THP-1 monocytes. The results suggest that the 6-wellplate-based CFE assay for HepG2 and A549 cells can be applied to nanoparticles, but the co-culture CFE assay has limitations in that it is not different from the single culture study, and it inhibits colony-formation by A549 cells in the presence of macrophages; this warrant further study.

The reactive oxygen species as pathogenic factors of fragmented microplastics to macrophages.

The presence of microplastics in the various food web raised concerns on human health, but little is known about the target cells and mechanism of toxicity of microplastics. In this study, we evaluated the toxicity of microplastics using relevant cell lines to the oral route of exposure. Approximately 100 µm-sized fragment-type polypropylene (PP) and polystyrene (PS) particles were prepared by sieving after pulverization and further applied the accelerated weathering using ultraviolet and heat. Thus, the panel of microplastics includes fresh PP (f-PP), fresh PS (f-PS), weathered PP (w-PP), and weathered PS (w-PS). The spherical PS with a similar size was used as a reference particle. Treatment of all types of PP and PS did not show any toxic effects to the Caco-2 cells and HepG2 cells. However, the treatment of microplastics to THP-1 macrophages showed significant toxicity in the order of f-PS > f-PP > w-PS > w-PP. The weathering process significantly reduced the reactive oxygen species (ROS) generation potential of both microplastics because the weathered microplastics have an increased affinity to bind serum protein which acts as a ROS scavenger. The intrinsic ROS generation potential of microplastics showed a good correlation with the toxicity endpoints including cytotoxicity and pro-inflammatory cytokines in THP-1 macrophages. In conclusion, the results of this study suggest that the target cell type of microplastics via oral administration can be macrophages and the pathogenic factor to THP-1 macrophages is the intrinsic ROS generation potential of microplastics. Nevertheless, the toxic effect of microplastics tested in this study was much less than that of nano-sized particles.

Combination effect of nanoparticles on the acute pulmonary inflammogenic potential: additive effect and antagonistic effect.

The combination effect of co-exposed different types of nanomaterials is little known although humans are generally exposed to a mixture of nanomaterials from urban ultrafine particles or industrial nanomaterials. Herein, we evaluated the combined effect of nanoparticles (NPs) using three types of NPs in different inflammogenic categories: carbon black (CB), nickel oxide (NiO), and copper oxide (CuO). A single type of NPs or NPs in combination was intratracheally instilled into the lungs of rats and the bronchoalveolar lavage fluid (BALF) was analyzed at 24 h after instillation to evaluate the acute inflammogenic potential. The percentage of neutrophils in BALF was selected as a toxicity endpoint and the potential for reactive oxygen species (ROS) generation, dose-response of the combined effect, sequential treatment of CB and NiO, and uptake of NiO to alveolar macrophages after combined treatment of CB and NiO were evaluated for the mechanism of the combined effect. Co-exposure of CuO and NiO showed an additive effect on the percentage of neutrophils and ROS generation potential, which implies that the physicochemical properties of each NP are not influenced by the other type. While CB exerted an antagonistic effect on the percentage of neutrophils in combined treatment with CuO or NiO. The antagonistic effect of CB was due to the scavenging activity of the ROS generated by the CuO and NiO rather than the competition in cellular uptake to target cells (i.e. alveolar macrophages), which highlight the importance of the combined effect of NPs in the risk assessment.

Astrocytes in the Ventrolateral Preoptic Area Promote Sleep.

Although ventrolateral preoptic (VLPO) nucleus is regarded as a center for sleep promotion, the exact mechanisms underlying the sleep regulation are unknown. Here, we used optogenetic tools to identify the key roles of VLPO astrocytes in sleep promotion. Optogenetic stimulation of VLPO astrocytes increased sleep duration in the active phase in naturally sleep-waking adult male rats (n = 6); it also increased the extracellular ATP concentration (n = 3) and c-Fos expression (n = 3-4) in neurons within the VLPO. In vivo microdialysis analyses revealed an increase in the activity of VLPO astrocytes and ATP levels during sleep states (n = 4). Moreover, metabolic inhibition of VLPO astrocytes reduced ATP levels (n = 4) and diminished sleep duration (n = 4). We further show that tissue-nonspecific alkaline phosphatase (TNAP), an ATP-degrading enzyme, plays a key role in mediating the somnogenic effects of ATP released from astrocytes (n = 5). An appropriate sample size for all experiments was based on statistical power calculations. Our results, taken together, indicate that astrocyte-derived ATP may be hydrolyzed into adenosine by TNAP, which may in turn act on VLPO neurons to promote sleep.SIGNIFICANCE STATEMENT Glia have recently been at the forefront of neuroscience research. Emerging evidence illustrates that astrocytes, the most abundant glial cell type, are the functional determinants for fates of neurons and other glial cells in the central nervous system. In this study, we newly identified the pivotal role of hypothalamic ventrolateral preoptic (VLPO) astrocytes in the sleep regulation, and provide novel insights into the mechanisms underlying the astrocyte-mediated sleep regulation.

The sp3/sp2 carbon ratio as a modulator of in vivo and in vitro toxicity of the chemically purified detonation-synthesized nanodiamond via the reactive oxygen species generation.

Nanodiamonds have been suggested as biocompatible materials and are suitable for various biomedical applications, but little is known about how to synthesize safer nanodiamonds. Herein, seven different detonation-synthesized nanodiamonds (DNDs) with sequential sp3/sp2 carbon ratios were assembled by controlling the chemical purification parameters and the role of sp3/sp2 carbon ratio on the toxicity of DNDs was investigated. Carbon black and nickel oxide nanoparticles were used as reference particles. The intrinsic reactive oxygen species (ROS) generation potential of DNDs was estimated by a 2'7'-dichlorofluorescein diacetate (DCFH-DA) assay, and these values showed a good negative correlation with the sp3/sp2 carbon ratios, which implies that ROS generation increased as the sp3/sp2 carbon ratio decreased. As a model to investigate inflammogenic potential of DND samples, a rat intratracheal instillation model was used as the lung is very sensitive to nanoparticle exposures. The sp3/sp2 carbon ratios or the estimated values of ROS generation potential showed excellent linear correlations with the number of neutrophils and pro-inflammatory cytokines in bronchoalveolar lavage fluid at 24 h after instillation. Treatment of DND samples to THP-1 derived macrophages also showed that the sp3/sp2 carbon ratios or the estimated values of ROS generation potential were closely related with the toxicity endpoints such as cell viability and pro-inflammatory cytokines. Taken together, these data demonstrate that the sp3/sp2 carbon ratio is the key determinant for the toxicity of DNDs, which can be a useful tool for the safer-by-design approach of DNDs and the safety assessment of carbon nanoparticles.

The early onset and persistent worsening pulmonary alveolar proteinosis in rats by indium oxide nanoparticles.

Workplace inhalation exposure to indium compounds has been reported to produce 'indium lung disease' characterized by pulmonary alveolar proteinosis (PAP), granulomas, and pulmonary fibrosis. However, there is little information about the pulmonary toxicity of nano-sized indium oxide (In2O3), which is widely used in various applications such as liquid crystal displays. In this study, we evaluated the time-course and dose-dependent lung injuries by In2O3 nanoparticles (NPs) after a single intratracheal instillation to rats. In2O3 NPs were instilled to female Wistar rats at 7.5, 30, and 90 cm2/rat and lung injuries were evaluated at day 1, 3, 7, 14, 30, 90, and 180 after a single intratracheal instillation. Treatment of In2O3 NPs induced worsening diverse pathological changes including PAP, persistent neutrophilic inflammation, type II cell hyperplasia, foamy macrophages, and granulomas in a time- and dose-dependent manner. PAP was induced from day 3 and worsened throughout the study. The concentrations of interleukin-1ß, tumor necrosis factor-α, and monocyte chemoattractant protein-1 in bronchoalveolar lavage fluid (BALF) showed dose- and time-dependent increases and the levels of these inflammatory mediators are consistent with the data of inflammatory cells in BALF and progressive lung damages by In2O3 NPs. This study suggests that a single inhalation exposure to In2O3 NPs can produce worsening lung damages such as PAP, chronic active inflammation, infiltration of foamy macrophages, and granulomas. The early onset and persistent PAP even at the very low dose (7.5 cm2/rat) implies that the re-evaluation of occupational recommended exposure limit for In2O3 NPs is urgently needed to protect workers.

Astrocyte-derived lipocalin-2 mediates hippocampal damage and cognitive deficits in experimental models of vascular dementia.

Lipocalin-2 (LCN2) has diverse functions in multiple pathophysiological conditions; however, its pathogenic role in vascular dementia (VaD) is unknown. Here, we investigated the role of LCN2 in VaD using rodent models of global cerebral ischemia and hypoperfusion with cognitive impairment and neuroinflammation. Mice subjected to transient bilateral common carotid artery occlusion (tBCCAo) for 50 min showed neuronal death and gliosis in the hippocampus at 7 days post-tBCCAo. LCN2 expression was observed predominantly in the hippocampal astrocytes, whereas its receptor was mainly detected in neurons, microglia, and astrocytes. Furthermore, Lcn2-deficient mice, compared with wild-type animals, showed significantly weaker CA1 neuronal loss, cognitive decline, white matter damage, blood-brain barrier permeability, glial activation, and proinflammatory cytokine production in the hippocampus after tBCCAo. Lcn2 deficiency also attenuated hippocampal neuronal death and cognitive decline at 30 days after unilateral common carotid artery occlusion (UCCAo). Furthermore, intracerebroventricular (i.c.v) injection of recombinant LCN2 protein elicited CA1-neuronal death and a cognitive deficit. Our studies using cultured glia and hippocampal neurons supported the decisive role of LCN2 in hippocampal neurotoxicity and microglial activation, and the role of the HIF-1α-LCN2-VEGFA axis of astrocytes in vascular injury. Additionally, plasma levels of LCN2 were significantly higher in patients with VaD than in the healthy control subjects. These results indicate that hippocampal damage and cognitive impairment are mediated by LCN2 secreted from reactive astrocytes in VaD.

Nickel oxide nanoparticles can recruit eosinophils in the lungs of rats by the direct release of intracellular eotaxin.

BACKGROUND: Instillation of highly soluble nanoparticles (NPs) into the lungs of rodents can cause acute eosinophilia without any previous sensitizations by the role of dissolved ions. However, whether gradually dissolving NPs can cause the same type of eosinophilia remains to be elucidated. We selected nickel oxide (NiO) as a gradually dissolving NP and evaluated the time course pulmonary inflammation pattern as well as its mechanisms. METHODS: NiO NPs were intratracheally instilled into female Wistar rats at various concentrations (50, 100, and 200 cm(2)/rat) and the lung inflammation was evaluated at various time-points (1, 2, 3, and 4 days). As positive controls, NiCl2 and the ovalbumin-induced allergic airway inflammation model was applied. NiCl2 was instilled at 171.1 µg Ni/rat, which is equivalent nickel concentration of 200 cm(2)/rat of NiO NPs. Cytological analysis and biochemical analysis including lactate dehydrogenase (LDH), total protein, and pro-inflammatory cytokines were measured in bronchoalveolar lavage fluid (BALF). The levels of total immunoglobulin E (IgE) and anaphylatoxins (C3a and C5a) were measured in BALF and serum. The levels of eotaxin were measured in the alveolar macrophages and normal lung tissue before and after addition of cell lysis buffer to evaluate whether the direct lysis of cells can release intracellular eotaxin. RESULTS: NiO NPs produced acute neutrophilic inflammation throughout the study. However, eosinophils were recruited at 3 and 4 days post-instillation of NiO NPs and the magnitude and pattern of inflammation was similar with NiCl2 at 24 h post-instillation. The eosinophil recruitment by NiO NPs was not related with either the levels of total IgE or anaphylatoxins. The lysis of alveolar macrophages and normal lung tissue showed high levels of intracellular eotaxin and the levels of LDH showed positive correlation with the levels of eotaxin. CONCLUSIONS: Instillation of NiO NPs produced neutrophilia at 1 and 2 days after instillation, while the mixed type of neutrophilic and eosinophilic inflammation was produced at 3 and 4 days post-instillation, which was consistent with NiCl2. The mechanism of the eosinophilia involves the direct release of intracellular eotaxin due to the rupture of cells by the accumulated solubilized nickel ions in the phagolysosome.

Indium oxide (In2O3) nanoparticles induce progressive lung injury distinct from lung injuries by copper oxide (CuO) and nickel oxide (NiO) nanoparticles.

Indium is an essential element in the manufacture of liquid crystal displays and other electronic devices, and several forms of indium compounds have been developed, including nanopowders, films, nanowires, and indium metal complexes. Although there are several reports on lung injury caused by indium-containing compounds, the toxicity of nanoscale indium oxide (In2O3) particles has not been reported. Here, we compared lung injury induced by a single exposure to In2O3 nanoparticles (NPs) to that caused by benchmark high-toxicity nickel oxide (NiO) and copper oxide (CuO) NPs. In2O3 NPs at doses of 7.5, 30, and 90 cm(2)/rat (50, 200, and 600 µg/rat) were administered to 6-week-old female Wistar rats via pharyngeal aspiration, and lung inflammation was evaluated 1, 3, 14, and 28 days after treatment. Neutrophilic inflammation was observed on day 1 and worsened until day 28, and severe pulmonary alveolar proteinosis (PAP) was observed on post-aspiration days 14 and 28. In contrast, pharyngeal aspiration of NiO NPs showed severe neutrophilic inflammation on day 1 and lymphocytic inflammation with PAP on day 28. Pharyngeal aspiration of CuO NPs showed severe neutrophilic inflammation on day 1, but symptoms were completely resolved after 14 days and no PAP was observed. The dose of In2O3 NPs that produced progressive neutrophilic inflammation and PAP was much less than the doses of other toxic particles that produced this effect, including crystalline silica and NiO NPs. These results suggest that occupational exposure to In2O3 NPs can cause severe lung injury.

p38ß MAPK affords cytoprotection against oxidative stress-induced astrocyte apoptosis via induction of αB-crystallin and its anti-apoptotic function.

The activation of p38 mitogen-activated protein kinases (MAPKs) has been implicated in many cellular processes, such as, inflammation, cell death, and survival. In mammals, four distinct genes encode the four known members of p38 MAPKs, p38α, p38ß, p38γ, and p38δ. Despite the fact that p38α and p38ß MAPKs share over 75% homology sequences, they have distinct, perhaps even opposite roles under stress conditions. In our previous report, we showed that p38ß MAPK is induced in activated astrocytes in the penumbra of the postischemic brain, wherein it was co-localized with αB-crystallin and MAPKAPK-2. To investigate the functional significance of p38ß MAPK in astrocytes, a C6 astroglioma cell line stably over-expressing p38ß MAPK was generated. In these cells, hydrogen peroxide-induced apoptosis was reduced to 44.3% of that obtained from normal C6 cells. Interestingly, we found that expression of a small heat shock protein, αB-crystallin, was significantly increased in these cells, but that the expressions of HSP27 and HSP70 were not. Repression of αB-crystallin expression by αB-crystallin siRNA transfection suppressed the protective effect and recovered caspase 3 activity, indicating that αB-crystallin induction plays a crucial role in the protection against H2O2-induced apoptosis observed in p38ß-overexpressing C6 astroglioma cells. We found that the binding between αB-crystallin and partially processed caspase-3 (a p24 intermediate) was significantly increased in p38ß-overexpressing cells, which might result in suppression of caspase 3 activity in these cells. These results indicate that p38ß confers protection against H2O2-induced astrocytes apoptosis by inducing a small heat shock protein, αB-crystallin, which inhibits caspase-3 activation.

alphaB-crystallin suppresses oxidative stress-induced astrocyte apoptosis by inhibiting caspase-3 activation.

alphaB-crystallin is a member of the small heat shock proteins, which is abundantly expressed in various vertebrate tissues including the central nervous system. In our previous report, we showed alphaB-crystallin induction in activated astrocytes in the postischemic brain and in H2O2-treated primary astrocyte cultures. To investigate the functional significance of alphaB-crystallin induction in astrocytes, we generated a stable C6 astroglioma cell line overexpressing alphaB-crystallin. In these cells, hydrogen peroxide-induced apoptosis was reduced by 60% compared to parent cells. Furthermore, the repression of alphaB-crystallin expression by alphaB-crystallin siRNA transfection suppressed this protective effect, indicating that alphaB-crystallin is responsible for the protection against H2O2-induced apoptosis in C6 astroglioma cells. Similar level of aggravation in H2O2-induced apoptosis was observed in primary astrocyte cultures when alphaB-crystallin expression was suppressed by alphaB-crystallin siRNA transfection, confirming the importance of alphaB-crystallin. In addition, the induction of caspase-3 activity after H2O2 treatment was markedly suppressed in alphaB-crystallin-overexpressing cells, and immunoprecipitation proved binding between alphaB-crystallin and partially processed caspase-3 (a p24 intermediate). These results indicate that alphaB-crystallin confers protection against hydrogen peroxide-induced astrocytes apoptosis in part by inhibiting caspase-3 activation.