Dermal toxicity studies of 1,2-benzisothiazolin-3-one (CAS number: 2634-33-5) in Sprague-Dawley rats.

Biocides are used as preservatives in various household products, and 1,2-benzisothiazolin-3-one (BIT) is one of the popular chemicals. Therefore, BIT is highly likely to be exposed to human skin, necessitating dermal toxicity evaluation. In this study, we aimed to investigate dermal toxicity, eyes and skin irritation, and skin sensitization of BIT. All studies were conducted according to the Organisation for Economic Co-operation and Development testing guidelines. In acute dermal toxicity using rats, no treatment-related responses were observed at the highest dose (2000 mg/kg). A 28-day repeated dermal toxicity study at 1, 4, and 12 mg/kg/day showed transient local skin irritation lesions, including erythema, exfoliation, and crust formation. Based on no systemic effects, the no observed adverse effect level (NOAEL) of BIT of the 28-day repeated dermal toxicity study was determined to be 12 mg/kg/day. Eye and skin irritation tests showed that BIT is a strong irritant and corrosive to the eyes and a mild irritant to the skin. However, BIT showed no skin sensitization reactions in a local lymph node assay. These dermal toxicity studies can provide valuable information for the risk assessment of BIT.

Merkel cells and corpuscles of Stannius as putative targets for polyethylene terephthalate microfibers in sheepshead minnow larvae.

Polyethylene terephthalate (PET) fibers are contaminated in wastewater from various primary sources, such as washing textile waters. PET fibers in the environment can be degraded into microfibers because of weathering processes such as sunlight, physical wear, and heat. Although recent studies reported adverse effects of PET microfibers on aquatic organisms, the lack of information on their toxicity and mode of action hampers the risk assessment of PET microfibers. Therefore, this study aimed to investigate the biological effects of PET microfibers and their underlying mechanisms in early-staged sheepshead minnows (Cyprinodon variegatus). PET microfibers (about 13 µm diameter × 106 µm length) were prepared by cutting PET threads and treated to sheepshead minnow larvae at 10 and 100 mg/L for 10 days. No acute toxicity was found in the minnow, but PET microfibers significantly produced reactive oxygen species and reduced behavioral responses of traveled distance and maximum velocity. The transcriptomic data suggested that Merkel cells (flow sensors) and corpuscles of Stannius (calcium regulator) are putative targets, which were derived from oxidative stress, sensory neuropathy, cognitive impairment, and movement disorders. These findings underscore that although PET microfibers are not directly lethal to sheepshead minnows, they could impact their survival by damaging swimming-related key genes. This study provides new insights into how PET microfibers are toxic to aquatic organisms and disrupt ecosystems beyond survival and pathological changes.

Mitigated toxicity of polystyrene nanoplastics in combination exposure with copper ions by transformation into copper (I) oxide: Inhibits the oxidative potential of nanoplastics.

The combined impact of trace metals and polystyrene (PS) microplastics is extremely concerning for human health because PS microplastics can serve as a vehicle for other contaminants. Herein, we investigated the combined effect of copper ions (Cu2+) on the toxicity of PS nanoplastics in vivo and in vitro. The pristine PS (PPS) and ultraviolet irradiated oxidized PS (OPS) nanoplastics with 50 nm-size were conjugated with Cu2+ (13-27 mg/g) for 4 days to get four types of samples: PPS, OPS, PPS/Cu, and OPS/Cu. The comparative toxic potentials of test samples were evaluated using a mouse pharyngeal aspiration model and relevant human cell lines (A549 and differentiated THP-1 cells). The results showed an antagonistic effect in vivo and in vitro by the presence of Cu ions: PPS > PPS/Cu; OPS > OPS/Cu. Furthermore, the OPS produced significantly increased toxic potentials compared to the corresponding PPS: OPS > PPS; OPS/Cu > PPS/Cu. The antagonistic effect of Cu2+ on the toxicity of PS was due to the transformation of Cu2+ and balanced the surface charge of the nanoplastics, which inhibited the oxidative potential of corresponding nanoplastics. These antagonistic effects may provide a better understanding of the combined effects of metals on the intrinsic toxic potential of microplastics under natural conditions.

Formulation of Dual-Functional Nonionic Cetomacrogol Creams Incorporated with Bacteriophage and Human Platelet Lysate for Effective Targeting of MDR P. aeruginosa and Enhanced Wound Healing.

Successful development of phage-based therapeutics and their utility predominantly depend on the mode and route of phage administration. Topical and site-directed phage application evokes minimal immune clearance and allows more phage-host adsorption, thereby ensuring higher phage efficacy. However, a notable drawback of conventional topical phage applications is the absence of sustained release. Occlusive emollients guarantee the controlled release of active pharmaceutical ingredients (APIs), thereby facilitating administration, preventing moisture loss, and acting as a skin barrier. In this study, we developed phage and human platelet lysate (h-PL) incorporated cetomacrogol-based creams for combined phage therapy and wound healing. The base material for phage immobilization was formulated by emulsifying paraffin and sterile water with cetomacrogol (emulsifying agent). Specifically, we incorporated a Pseudomonas aeruginosa-infecting lytic phage vB_PaeM_M12PA in the formulation and characterized its genome in this study. Cetomacrogol, a nonionic PEG (polyethylene glycol) based ether, rendered phage stability and allowed initial burst release followed by continuous controlled release of phages from the embedding matrix in the initial 6-8 h. Rheological studies showed that the material has elastic properties with storage moduli (G') values ranging from 109.51 ± 2.10 to 126.02 ± 3.13 kPa, indicating frequency-independent deformation. Platelet lysates in the cream acted as wound healing agents, and in vitro evaluation of cell migration and wound healing capacity of h-PL showed a significant enhancement by the sixth hour compared to untreated groups. The phage-incorporated cream showed sustained phage release in solid media and a significant reduction in bacterial growth in liquid cultures. In vivo wound healing studies in 6-week-old Wistar rats with full-thickness excision wounds and subsequent histopathological studies showed that the formulation enhanced wound healing and tissue restoration efficiency. In conclusion, the study unveils a promising approach for integrated phage therapy and wound healing strategies.

Multimodal pulmonary clearance kinetics of carbon black nanoparticles deposited in the lungs of rats: the role of alveolar macrophages.

BACKGROUND: Alveolar macrophages (AMs) have been predicted to affect the pulmonary clearance of nanomaterials; however, their qualitative and quantitative roles are poorly understood. In this study, carbon black nanoparticles (CBNPs) were instilled into the lungs of Wistar rats at 30, 100, and 300 µg/rat. The concentrations of particles in organs, including the lung, lung-associated lymph nodes (LALN), liver, spleen, and kidney, were evaluated at days 0 (immediately after instillation), 1, 7, 28, 60, and 90 post-instillation. RESULTS: The results indicated a multimodal pulmonary clearance pattern for CBNPs: slow clearance until day 28, fast clearance from days 28 to 60, and slow clearance from days 60 to 90. To determine the mechanism of this unique clearance pattern, CBNPs were instilled into AM-depleted rats using clodronate liposomes (CLO). At 28 days after instillation, the CBNP levels in the lungs treated with CLO showed about 31% higher reduction than in normal rats. In addition, the concentration of CBNPs in LALN treated with CLO significantly increased on day 28, whereas in normal rats, no detectable levels were observed. CONCLUSIONS: This result highlights that the prolonged retention of poorly soluble NPs in the lung until day 28 is mediated by the phagocytosis of AMs, and the fast clearance between days 28-60 is due to the turnover time of AMs, estimated around 1-2 months after birth. Similarly, new generations of AMs mediate the slow phase between days 60 and 90. However, further studies are needed to understand the multimodal clearance mechanism and the modulation of pulmonary clearance of poorly soluble NPs.

The oxidative stress-dependent pulmonary inflammation of inhalable multi-walled carbon nanotube-containing nano-concrete dust and its comparison with conventional concrete dust and DQ12.

Nano-concrete, which is an admixture of nanomaterials in concrete recipes, has been investigated to overcome the limitations of existing concrete, such as its stability and strength. However, there is no information on the human health effects of broken-down dust released during the construction and demolition efforts. In this study, we prepared an inhalable fraction of multi-walled carbon nanotube-containing nano-concrete dust and performed comparative toxicity studies with conventional concrete dust and DQ12 using a rat intratracheal instillation model. Although the recipes for concrete and nano-concrete are entirely different, the pulverized dust samples showed similar physicochemical properties, such as 0.46-0.48 µm diameter and chemical composition. Both concrete and nano-concrete dust exhibited similar patterns and magnitudes, representing acute neutrophilic inflammation and chronic active inflammation with lymphocyte infiltration. The toxicity endpoints of the tested particles at both time points showed an excellent correlation with the reactive oxygen species levels released from the alveolar macrophages, highlighting that alveolar macrophages are the primary target cells and that the oxidative stress paradigm is the main toxicity mechanism of the tested particles. In addition, the toxicity potentials of both concrete and nano-concrete dust were more than 10 times lower than that of DQ12.

Disruption of early embryonic development in mice by polymethylmethacrylate nanoplastics in an oxidative stress mechanism.

Polymethylmethacrylate (PMMA) has been used in many products, such as acrylic glass, and is estimated to reach 5.7 million tons of production per year by 2028. Thus, nano-sized PMMA particles in the environment are highly likely due to the weathering process. However, information on the hazards of nanoplastics, including PMMA in mammals, especially reproductive toxicity and action mechanism, is scarce. Herein, we investigated the effect of PMMA nanoplastics on the female reproductive system of mice embryos during pre-implantation. The treated plastic particles in embryos (10, 100, and 1000 µg/mL) were endocytosed into the cytoplasm within 30 min, and the blastocyst development and indices of embryo quality were significantly decreased from at 100 µg/mL. Likewise, the transfer of nanoplastic-treated embryos at 100 µg/mL decreased the morula implantation rate on the oviduct of pseudopregnant mice by 70%, calculated by the pregnant individual, and 31.8% by the number of implanted embryos. The PMMA nanoplastics at 100 µg/mL significantly increased the cellular levels of reactive oxygen species in embryos, which was not related to the intrinsic oxidative potential of nanoplastics. This study highlights that the nanoplastics that enter systemic circulation can affect the early stage of embryos. Thus, suitable action mechanisms can be designed to address nanoplastic occurrence.

Analysis of carbon nanotube levels in organic matter: an inter-laboratory comparison to determine best practice.

Carbon nanotubes (CNTs) are increasingly being used in industrial applications, but their toxicological data in animals and humans are still sparse. To assess the toxicological dose-response of CNTs and to evaluate their pulmonary biopersistence, their quantification in tissues, especially lungs, is crucial. There are currently no reference methods or reference materials for low levels of CNTs in organic matter. Among existing analytical methods, few have been fully and properly validated. To remedy this, we undertook an inter-laboratory comparison on samples of freeze-dried pig lung, ground and doped with CNTs. Eight laboratories were enrolled to analyze 3 types of CNTs at 2 concentration levels each in this organic matrix. Associated with the different analysis techniques used (specific to each laboratory), sample preparation may or may not have involved prior digestion of the matrix, depending on the analysis technique and the material being analyzed. Overall, even challenging, laboratories' ability to quantify CNT levels in organic matter is demonstrated. However, CNT quantification is often overestimated. Trueness analysis identified effective methods, but systematic errors persisted for some. Choosing the assigned value proved complex. Indirect analysis methods, despite added steps, outperform direct methods. The study emphasizes the need for reference materials, enhanced precision, and organized comparisons.

Disruption of phosphofructokinase activity and aerobic glycolysis in human bronchial epithelial cells by atmospheric ultrafine particulate matter.

Exposure to ambient ultrafine particulate matter (UPM) causes respiratory disorders; however, the underlying molecular mechanisms remain unclear. In this study, we synthesized simulated UPM (sUPM) with controlled physicochemical properties using the spark-discharge method. Subsequently, we investigated the biological effects of sUPM using BEAS-2B human bronchial epithelial cells (HBECs) and a mouse intratracheal instillation model. High throughput RNA-sequencing and bioinformatics analyses revealed that dysregulation of the glycolytic metabolism is involved in the inhibited proliferation and survival of HBECs by sUPM treatment. Furthermore, signaling pathway and enzymatic analyses showed that the treatment of BEAS-2B cells with sUPM induces the inactivation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB, also known as AKT), resulting in the downregulation of phosphofructokinase 2 (PFK2) S483 phosphorylation, PFK enzyme activity, and aerobic glycolysis in HBECs in an oxidative stress-independent manner. Additionally, intratracheal instillation of sUPM reduced the phosphorylation of ERK, AKT, and PFK2, decreased proliferation, and increased the apoptosis of bronchial epithelial cells in mice. The findings of this study imply that UPM induces pulmonary toxicity by disrupting aerobic glycolytic metabolism in lung epithelial cells, which can provide novel insights into the toxicity mechanisms of UPM and strategies to prevent their toxic effects.

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.

Zinc oxide nanoparticles trigger autophagy-mediated cell death through activating lysosomal TRPML1 in normal kidney cells.

Zinc oxide nanoparticles (ZnO NPs) have been widely used in various materials including sunscreens, cosmetics, over-the-counter topical skin products, and pigments. As traces of the used ZnO NPs have been found in the kidney, it is crucial to uncover their potential risks. The aim of this study is to elucidate detrimental effects of ZnO NPs and the molecular mechanism behind their renal toxicity. Cytotoxic effects were measured by MTT assay after HK2 cells were exposed to ZnO NPs for 24 h and IC50 value was determined. ROS and intracellular Zn2+ levels were detected by flow cytometry, and localization of Zn2+ and lysosome was determined by confocal microscopy. Occurrence of autophagy and detection of autophagic flux were determined by Western blot and confocal microscopy, respectively. We performed unpaired student t test for two groups, and one-way ANOVA with Tukey's post hoc for over three groups. ZnO NPs induced cell death in human renal proximal tubule epithelial cells, HK2. Cytosolic Zn2+ caused autophagy-mediated cell death rather than apoptosis. Cytosolic Zn2+ processed in lysosome was released by TRPML1, and inhibition of TRPML1 significantly decreased autophagic flux and cell death. The findings of this study suggest that ZnO NPs strongly induce autophagy-mediated cell death in human kidney cells. Controlling TRPML1 can be potentially used to prevent the kidney from ZnO NPs-induced toxicity.

Differential particle and ion kinetics of silver nanoparticles in the lungs and biotransformation to insoluble silver sulfide.

The measurement of nanoparticles (NPs) in a biological matrix is essential in various toxicity studies. However, the current knowledge has limitations in differentiating particulate and ionic forms and further identification of their biotransformation. Herein, we evaluate the biotransformation and differential lung clearance kinetics of particulate and ionic forms using PEGylated silver NPs (AgNP-PEGs; 47.51 nm) and PEGylated gold NPs (AuNP-PEGs; 11.76 nm). At 0, 3, and 6 h and 1, 3, 7, and 14 days after a single pharyngeal aspiration in mice at 25 µg/mouse, half of the lung is digested by proteinase K (PK) to separate particulates and ions, and the other half is subjected to the acid digestion method for comparison. The quantitative and qualitative evaluation of lung clearance kinetics suggests that AgNP-PEGs are quickly dissolved and transformed into insoluble silver sulfide (Ag2S), which shows a fast-clearing early phase (0 -6 h; particle T1/2: 4.8 h) and slow-clearing late phase (1 -14 days; particle T1/2: 13.20 days). In contrast, AuNP-PEGs were scarcely cleared or biotransformed in the lungs for 14 days. The lung clearance kinetics of AgNPs and biotransformation shown in this study can be informed by the PK digestion method and cannot be obtained using the acid digestion method.

Mutual regulation between phosphofructokinase 1 platelet isoform and VEGF promotes glioblastoma tumor growth.

Glioblastoma (GBM) is a highly vascular malignant brain tumor that overexpresses vascular endothelial growth factor (VEGF) and phosphofructokinase 1 platelet isoform (PFKP), which catalyzes a rate-limiting reaction in glycolysis. However, whether PFKP and VEGF are reciprocally regulated during GBM tumor growth remains unknown. Here, we show that PFKP can promote EGFR activation-induced VEGF expression in HIF-1α-dependent and -independent manners in GBM cells. Importantly, we demonstrate that EGFR-phosphorylated PFKP Y64 has critical roles in both AKT/SP1-mediated transcriptional expression of HIF-1α and in the AKT-mediated ß-catenin S552 phosphorylation, to fully enhance VEGF transcription, subsequently promoting blood vessel formation and brain tumor growth. Levels of PFKP Y64 phosphorylation in human GBM specimens are positively correlated with HIF-1α expression, ß-catenin S552 phosphorylation, and VEGF expression. Conversely, VEGF upregulates PFKP expression in a PFKP S386 phosphorylation-dependent manner, leading to increased PFK enzyme activity, aerobic glycolysis, and proliferation in GBM cells. These findings highlight a novel mechanism underlying the mutual regulation that occurs between PFKP and VEGF for promoting GBM tumor growth and also suggest that targeting the PFKP/VEGF regulatory loop might show therapeutic potential for treating GBM patients.

Biokinetics of fluorophore-conjugated polystyrene microplastics in marine mussels.

Biokinetic information on microplastics in bivalves is required to reduce the human exposure, but little is known about the time-course and size effect on tissue absorption and clearance. The biokinetics of fluorophore-labeled polystyrene microbeads with diameters 10 µm (PL10) and 90 µm (PL90) in Mytilus galloprovincialis marine mussels was investigated in the present study. It was found that both PL10 and PL90 showed a biphasic tissue distribution pattern in digestive and non-digestive tissues, highlighting the significant tissue distribution starting from 48 h post-treatment. The differential size effect on tissue distribution was observed only in the gills, which suggests that PL10 accumulates more than PL90. The depuration kinetics show that particles of both sizes can be cleared in any tissue, but non-digestive tissue requires a longer duration for depuration than digestive tissue. The differential size effect on depuration was observed for both digestive and non-digestive tissues, suggesting that PL10 needed a longer duration for depuration than PL90. More than seven days were needed for depuration of microplastics in mussels, which is an exceptionally longer period compared to conventional depuration of bivalves. The most significant improvement of this study is providing the biokinetics of two different-sized microplastics in mussels and the differential time for purging microplastics from mussels.

Nanodiamond as a Cytokine Sponge in Infectious Diseases.

Cytokine release syndrome (CRS) is a systemic inflammatory response resulting in overexpression of cytokines in serum and tissues, which leads to multiple-organ failure. Due to rapid aggravation of symptoms, timely intervention is paramount; however, current therapies are limited in their capacity to address CRS. Here, we find that the intravenous injection of highly purified detonation-synthesized nanodiamonds (DND) can act as a therapeutic agent for treating CRS by adsorbing inflammatory cytokines. Highly purified DNDs successfully inactivated various key cytokines in plasma from CRS patients with pneumonia, septic shock, and coronavirus disease 2019 pandemic (COVID-19). The intravenous injection of the DND samples in a mouse sepsis model by cecal ligation and puncture significantly improved survival rates and prevented tissue damage by reducing the circulating inflammatory cytokines. The results of this study suggest that the clinical application of highly purified DND can provide survival benefits for CRS patients by adsorbing inflammatory cytokines.

Eugenitol ameliorates memory impairments in 5XFAD mice by reducing Aß plaques and neuroinflammation.

Alzheimer's disease (AD) is caused by various pathological mechanisms; therefore, it is necessary to develop drugs that simultaneously act on multiple targets. In this study, we investigated the effects of eugenitol, which has anti-amyloid ß (Aß) and anti-neuroinflammatory effects, in an AD mouse model. We found that eugenitol potently inhibited Aß plaque and oligomer formation. Moreover, eugenitol dissociated the preformed Aß plaques and reduced Aß-induced nero2a cell death. An in silico docking simulation study showed that eugenitol may interact with Aß1-42 monomers and fibrils. Eugenitol showed radical scavenging effects and potently reduced the release of proinflammatory cytokines from lipopolysaccharide-treated BV2 cells. Systemic administration of eugenitol blocked Aß aggregate-induced memory impairment in the Morris water maze test in a dose-dependent manner. In 5XFAD mice, prolonged administration of eugenitol ameliorated memory and hippocampal long-term potentiation impairment. Moreover, eugenitol significantly reduced Aß deposits and neuroinflammation in the hippocampus of 5XFAD mice. These results suggest that eugenitol, which has anti-Aß aggregation, Aß fibril dissociation, and anti-inflammatory effects, potently modulates AD-like pathologies in 5XFAD mice, and could be a promising candidate for AD therapy.

Effects of Perilla frutescens var. acuta in amyloid ß toxicity and Alzheimer's disease-like pathology in 5XFAD mice.

Although accumulation of amyloid ß (Aß) plaque is a major hallmark of Alzheimer's disease (AD), various pathologies have been suggested therapeutic targets. Therefore, therapies-targeting multiple pathologies would be required for effective managements of AD. Accordingly, natural products, which has multiple active ingredients, have been receiving a lot of attention. In this study, we tested whether standardized ethanol extract of leaves of Perilla frutescens var. acuta (L.) Britt. (Lamiaceae) (ELPF) could modulate various pathologies in AD using 5XFAD mice. ELPF blocked Aß aggregation and disassembled pre-formed Aß aggregates. ELPF blocked Aß aggregates-induced LTP impairment and ELPF-disassembled Aß aggregates failed to impair hippocampal LTP. Systemic administration of ELPF blocked Aß aggregates-induced memory impairment in a passive avoidance test. ELPF-disassembled Aß aggregates failed to impair passive avoidance memory. Prolonged administration of ELPF ameliorated memory impairments in 5XFAD mice. In the hippocampus of 5XFAD mice, ELPF administration significantly reduced Aß deposits and neuroinflammation. These results demonstrate that ELPF could be a promising therapeutic candidate for AD.

Hydrangea macrophylla and Thunberginol C Attenuate Stress-Induced Anxiety in Mice.

Stress is an important neurological input for successful life. However, chronic stress and stress hormones could be a cause of various neurological disorders including anxiety disorders. Therefore, there have been many efforts to find effective materials for curing stress-induced neurological disorders. In this study, we examined the effect of Hydrangea macrophylla (HM) on corticosterone-induced neurotoxicity, stress-induced anxiety in mice and suggested a possible active ingredient of HM. HM protected cortical neurons against neurotoxicity of corticosterone (CORT), a stress hormone. HM also blocked CORT-induced hippocampal synaptic deficit via regulating Akt signaling. Oral administration of HM improved chronic restraint stress-induced anxiety in Elevated Plus maze test along with reduction of plasma corticosterone and TNF-α levels. Moreover, HM reduced stress-induced neuroinflammation and oxidative stress. Thunberginol C, an active ingredient of HM, also prevented CORT-induced neuronal cell death and restraint stress-induced anxiety. Moreover, thunberginol C reduced plasma TNF-α level and neuroinflammation and oxidative stress. Collectively, HM could be a good candidate for preventing stress-induced neurological disorders and thunberginol C may be an active ingredient of HM for this purpose.

Role of extracellular signal-regulated kinase in rubrofusarin-enhanced cognitive functions and neurite outgrowth.

Memory-enhancing agents have long been required for various reasons such as for obtaining a good score in a test in the young and for retaining memory in the aged. Although many studies have found that several natural products may be good candidates for memory enhancement, there is still a need for better agents. The present study investigated whether rubrofusarin, an active ingredient in Cassiae semen, enhances learning and memory in normal mice. Passive avoidance and Morris water maze tests were performed to determine the memory-enhancing ability of rubrofusarin. To investigate synaptic function, hippocampal long-term potentiation (LTP) was measured. Western blotting was performed to determine protein levels. To investigate neurite outgrowth, DCX immunohistochemistry and cell culture were utilised. Rubrofusarin (1, 3, 10, 30 mg/kg) enhanced memory in passive avoidance and Morris water maze tests. Moreover, rubrofusarin ameliorated scopolamine-induced memory impairment. In the rubrofusarin-treated group, high-frequency stimulation induced higher LTP in the hippocampal Schaffer-collateral pathway compared to the control group. The rubrofusarin-treated group showed a higher number of DCX-positive immature neurons with an increase in the length of dendrites compared to the control group in the hippocampal dentate gyrus region. In vitro experiments showed that rubrofusarin facilitated neurite outgrowth in neuro2a cells through extracellular signal-regulated kinase (ERK). Finally, we found that extracellular signal-regulated kinase (ERK) is required for rubrofusarin-induced enhancement of neurite outgrowth, learning and memory. These results demonstrate that rubrofusarin enhances learning and memory and neurite outgrowth, and these might need activation of ERK pathway.