N-methyl-D-aspartate receptors induce M1 polarization of macrophages: Feasibility of targeted imaging in inflammatory response in vivo.

BACKGROUND: N-methyl-D-aspartate receptors (NMDARs) are considered to be involved in several physiological and pathophysiological processes in addition to the progression of neurological disorders. However, how NMDARs are involved in the glycolytic phenotype of M1 macrophage polarization and the possibility of using them as a bio-imaging probe for macrophage-mediated inflammation remain unclear. METHODS: We analyzed cellular responses to NMDAR antagonism and small interfering RNAs using mouse bone marrow-derived macrophages (BMDMs) treated with lipopolysaccharide (LPS). An NMDAR targeting imaging probe, N-TIP, was produced via the introduction of NMDAR antibody and the infrared fluorescent dye FSD Fluor™ 647. N-TIP binding efficiency was tested in intact and LPS-stimulated BMDMs. N-TIP was intravenously administered to mice with carrageenan (CG)- and LPS-induced paw edema, and in vivo fluorescence imaging was conducted. The anti-inflammatory effects of dexamethasone were evaluated using the N-TIP-mediated macrophage imaging technique. RESULTS: NMDARs were overexpressed in LPS-treated macrophages, subsequently inducing M1 macrophage polarization. Mechanistically, NMDAR-mediated Ca2+ accumulation resulted in LPS-stimulated glycolysis via upregulation of PI3K/AKT/mTORC1 signaling. In vivo fluorescence imaging with N-TIP showed LPS- and CG-induced inflamed lesions at 5 h post-inflammation, and the inflamed lesions could be detected until 24 h. Furthermore, our N-TIP-mediated macrophage imaging technique helped successfully visualize the anti-inflammatory effects of dexamethasone in mice with inflammation. CONCLUSION: This study demonstrates that NMDAR-mediated glycolysis plays a critical role in M1 macrophage-related inflammation. Moreover, our results suggest that NMDAR targeting imaging probe may be useful in research on inflammatory response in vivo.

Nephrotoxicity evaluation and proteomic analysis in kidneys of rats exposed to thioacetamide.

Thioacetamide (TAA) was administered orally at 0, 10, and 30 mg/kg body weight (BW) daily to Sprague-Dawley rats aged 6-7 weeks for 28 consecutive days. Nephrotoxicity and proteomics were evaluated in the kidneys of rats exposed to TAA. The BW decreased, however, the relative kidneys weight increased. No significant histopathologic abnormalities were found in the kidneys. The numbers of monocytes and platelets were significantly increased. However, the mean corpuscular volume and hematocrit values were decreased significantly in rats exposed to 30 mg/kg BW TAA. The expression levels of Kim-1 and NGAL were increased 4 to 5-fold in the kidneys, resulting in significant nephrotoxicity. Proteomic analysis was conducted and a total of 5221 proteins spots were resolved. Of these, 3 and 21 protein spots were up- and downregulated, respectively. The validation of seven proteins was performed by Western blot analysis. The expression level of ASAP2 was significantly upregulated, whereas RGS14, MAP7Dl, IL-3Rα, Tmod1, NQO2, and MUP were reduced. Sixteen isoforms of MUP were found by the 2DE immunoblot assay and were significantly downregulated with increasing exposure to TAA. MUP isoforms were compared in the liver, kidneys, and urine of untreated rats and a total of 43 isoforms were found.

Multi-Path and Group-Loss-Based Network for Speech Emotion Recognition in Multi-Domain Datasets.

Speech emotion recognition (SER) is a natural method of recognizing individual emotions in everyday life. To distribute SER models to real-world applications, some key challenges must be overcome, such as the lack of datasets tagged with emotion labels and the weak generalization of the SER model for an unseen target domain. This study proposes a multi-path and group-loss-based network (MPGLN) for SER to support multi-domain adaptation. The proposed model includes a bidirectional long short-term memory-based temporal feature generator and a transferred feature extractor from the pre-trained VGG-like audio classification model (VGGish), and it learns simultaneously based on multiple losses according to the association of emotion labels in the discrete and dimensional models. For the evaluation of the MPGLN SER as applied to multi-cultural domain datasets, the Korean Emotional Speech Database (KESD), including KESDy18 and KESDy19, is constructed, and the English-speaking Interactive Emotional Dyadic Motion Capture database (IEMOCAP) is used. The evaluation of multi-domain adaptation and domain generalization showed 3.7% and 3.5% improvements, respectively, of the F1 score when comparing the performance of MPGLN SER with a baseline SER model that uses a temporal feature generator. We show that the MPGLN SER efficiently supports multi-domain adaptation and reinforces model generalization.

Sensor Data Acquisition and Multimodal Sensor Fusion for Human Activity Recognition Using Deep Learning.

In this paper, we perform a systematic study about the on-body sensor positioning and data acquisition details for Human Activity Recognition (HAR) systems. We build a testbed that consists of eight body-worn Inertial Measurement Units (IMU) sensors and an Android mobile device for activity data collection. We develop a Long Short-Term Memory (LSTM) network framework to support training of a deep learning model on human activity data, which is acquired in both real-world and controlled environments. From the experiment results, we identify that activity data with sampling rate as low as 10 Hz from four sensors at both sides of wrists, right ankle, and waist is sufficient in recognizing Activities of Daily Living (ADLs) including eating and driving activity. We adopt a two-level ensemble model to combine class-probabilities of multiple sensor modalities, and demonstrate that a classifier-level sensor fusion technique can improve the classification performance. By analyzing the accuracy of each sensor on different types of activity, we elaborate custom weights for multimodal sensor fusion that reflect the characteristic of individual activities.

Benexate hydrochloride betadex modulates nitric oxide synthesis and cytokine expression in gastric ulcers.

The present study investigated benexate hydrochloride betadex (BHB)-mediated ulcer healing, and changes to microcirculation modulated through nitric oxide synthase (NOS) and anti-inflammatory activity. A rat model of gastric mucosal injury was established through injection of a 60% acetic acid solution into the stomach. Following ulcer induction, the rats were administered BHB orally for 5 days at doses of 0, 100, 300 or 1,000 mg/kg. The highest dose of BHB was also administered with or without L-NG-nitroarginine methyl ester (L-NAME). The area of gastric ulcers was determined by planimetry, and expression of cyclooxygenases (COX), cytokines and NOS in stomach tissues were measured using western blotting. Compared with the control group, gastric ulcer size was significantly decreased in the 1,000 mg/kg BHB-treated group (P<0.05). Administration of BHB led to a significant increase in endothelial (e)NOS expression (P<0.05). Although acetic acid co-treatment with L-NAME induced more severe mucosal damage, BHB decreased COX expression and tumor necrosis factor-α levels when administered with the nitric oxide inhibitor, L-NAME (P<0.05). BHB exhibited protective effects in a rat model of gastric ulcers, which were associated with a decrease in pro-inflammatory cytokine levels and the activation of eNOS.

Evaluation of toxicological biomarkers in secreted proteins of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and their expressions in the plasma of rats and incineration workers.

Toxicological biomarkers of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) were investigated in proteins secreted by HepG2 cells and their expression levels were determined in the plasma of rats exposed to 2,3,7,8-TCDD and in the plasma of incineration workers exposed to dioxins. HepG2 cells were treated with various concentrations of 2,3,7,8-TCDD (0, 0.25, 0.5, 1, 2.5, 5, 10, 25 nM) for 24 or 48 h. MTT and Comet assays were performed to determine cytotoxicities and genotoxicities to select exposure concentrations for the proteomic analysis of proteins secreted by 2,3,7,8-TCDD-treated cells. In the proteomic analysis, dose- and time-dependent toxicological biomarkers were evaluated using two pI ranges (4-7 and 6-9) using a large gel 2-DE system. Fifteen secreted proteins were identified by a nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS and the identities of eight secreted proteins including glyoxalase 1 (GLO 1), homogentisate dioxygenase (HGD), peroxiredoxin 1 (PRX 1), proteasome subunit beta type (PSMB) 5 and 6, UDP-glucose 6-dehydrogenase (UDP-GlcDH), hydroxyacyl-coenzyme A dehydrogenase (HADH) and serotransferrin (STF) were confirmed by western blotting. Of these, PSMB 5 and PRX 1 were also found in the plasma of rats exposed to 2,3,7,8-TCDD, whereas GLO 1, HGD, PSMB 6 and PRX 1 were found in the plasma of incineration workers exposed to dioxins.

Plasma proteomic analysis of patients infected with H1N1 influenza virus.

This study profiled the plasma proteins of patients infected by the 2011 H1N1 influenza virus. Differential protein expression was identified in plasma obtained from noninfected control subjects (n = 15) and H1N1-infected subjects (n = 15). Plasma proteins were separated by a 2DE large gel system and identified by nano-ultra performance LC-MS. Western blot assays were performed to validate proteins. Eight plasma proteins were upregulated and six proteins were downregulated among 3316 plasma proteins in the H1N1-infected group as compared with the control group. Of 14 up- and downregulated proteins, nine plasma proteins were validated by Western blot analysis. Putative protein FAM 157A, leucine-rich alpha 2 glycoprotein, serum amyloid A protein, and dual oxidase 1 showed significant differential expression. The identified plasma proteins could be potential candidates for biomarkers of H1N1 influenza viral infection. Further studies are needed to develop these proteins as diagnostic biomarkers.

An innovative sample-to-answer polymer lab-on-a-chip with on-chip reservoirs for the POCT of thyroid stimulating hormone (TSH).

A new sample-to-answer polymer lab-on-a-chip, which can perform immunoassay with minimum user intervention through on-chip reservoirs for reagents and single-channel assay system, has been designed, developed and successfully characterized as a point-of-care testing (POCT) cartridge for the detection of thyroid stimulating hormone (TSH). Test results were obtained within 30 minutes after a sample was dropped into the POCT cartridge. The analyzed results of TSH showed a linear range of up to 55 µIU mL(-1) with the limit of detection (LOD) of 1.9 µIU mL(-1) at the signal-to-noise ratio (SNR) of 3. The reagents stored in the on-chip reservoirs maintained more than 97% of their initial volume for 120 days of storage time while the detection antibody retained its activity above 98% for 120 days. The sample-to-answer polymer lab-on-a-chip developed in this work using the mass-producible and low-cost polymer is well suited for the point-of-care testing of rapid in vitro diagnostics (IVD) of TSH.

The effect of polaprezinc on gastric mucosal protection in rats with ethanol-induced gastric mucosal damage: comparison study with rebamipide.

AIMS: Polaprezinc (PZ), which consists of l-carnosine and zinc, is widely used to treat gastric ulcers. We compared the effects of PZ with those of rebamipide (RM) on the expression of inflammatory cytokines, antioxidants, growth factors, and heat shock proteins (HSP) in a rat model. MAIN METHODS: Seventy Sprague-Dawley rats were randomly assigned to test groups according to the dose of PZ at 5, 10, or 30 mg/kg or RM at 10, 30, or 100 mg/kg. Next, we obtained ulcer indices from rats with ethanol-induced gastric mucosal damage. Western blot analysis was used to evaluate the expression of various target proteins. KEY FINDINGS: Pathological ulcer indices in the PZ and RM groups were significantly lower than those in the control group. The levels of inflammatory cytokines (interleukin 1ß [IL-1ß], IL-6, IL-8, and tumor necrosis factor α) decreased, whereas the levels of platelet-derived growth factor-B, vascular endothelial growth factor, and nerve growth factor significantly increased after PZ administration. Furthermore, the expression of antioxidants (superoxide dismutase 1 [SOD-1], SOD-2, heme oxygenase-1, glutathione S-transferase, peroxidredoxin-1, and peroxidredoxin-5) was significantly higher in the PZ group, and the levels of HSP 90, 70, 60, 47, 27, and 10 significantly increased with an increase in PZ dose. SIGNIFICANCE: In a rat model of ethanol-induced gastric mucosal damage, PZ administration ameliorated ethanol-induced mucosal injury and showed protective effects on the mucosa by reducing the levels of inflammatory cytokines and increasing the expression of antioxidant enzymes and growth factors. Furthermore, PZ showed cytoprotective effects by increasing the HSP levels.

Suppression of LPS-induced inflammatory responses by inflexanin B in BV2 microglial cells.

Microglia are a type of resident macrophage that functions as an inflammation modulator in the central nervous system. Over-activation of microglia by a range of stimuli disrupts the physiological homeostasis of the brain, and induces inflammatory response and degenerative processes, such as those implicated in neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Therefore, we investigated the possible anti-inflammatory mechanisms of inflexanin B in murine microglial BV2 cells. Lipopolysaccharide (LPS) activated BV2 cells and induced the production of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and cytokines (interleukins-1ß and -6, and tumour necrosis factor α). The LPS-induced production of pro-inflammatory mediators was associated with the enhancement of nuclear factor-kappaB (NF-κB) nuclear translocation and the activation of mitogen-activated protein kinase (MAPK) including ERK1/2 and JNK. Conversely, pretreatment of cells with inflexanin B (10 and 20 µg/mL) significantly reduced the production of pro-inflammatory mediators. This was accompanied with the reduced nuclear translocation of NF-κB and reduced activation of MAPKs. These results suggest that inflexanin B attenuated the LPS-induced inflammatory process by inhibiting the activation of NF-κB and MAPKs.

Methylalpinumisoflavone inhibits lipopolysaccharide-induced inflammation in microglial cells by the NF-kappaB and MAPK signaling pathway.

Neuroinflammation is chronic inflammation within the brain that is attributed to prolonged activation of microglial cells and results in neurodegenerative events, such as neuronal dysfunction and neuronal loss. Therefore, suppression of neuroinflammation would theoretically slow progression of neurodegenerative disease. In this study, we investigated the anti-inflammatory effects of 4'-O-methylalpinumisoflavone (methylalpinumisoflavone), isolated from Cudrania tricuspidata, against LPS-induced microglial activation in BV2 cells. Exposure of BV2 cells to LPS (0.5 µg/mL) significantly increased production of pro-inflammatory mediators, including NO, PGE(2), and pro-inflammatory cytokines. Conversely, pre-treatment with methylalpinumisoflavone (10 and 20 µg/mL) prior to treatment with LPS resulted in a significant decrease of LPS-induced production of pro-inflammatory mediators in a dose-dependent manner. In addition, reduction of pro-inflammatory mediators by treatment with methylalpinumisoflavone prior to treatment with LPS was accompanied by a decrease in translocation of NF-κB p50 and p65 from the cytoplasm to the nucleus and by a decrease in activation of mitogen-activated protein kinases (MAPKs), such as ERK1/2 and JNK. Taken together, these results suggest that methylalpinumisoflavone suppressed LPS-induced microglial activation and production of pro-inflammatory mediators by decreasing NF-κB signaling and by phosphorylation of MAPKs. These results suggest the potential of methylalpinumisoflavone as an anti-inflammatory drug candidate.

The expression of HSPs, anti-oxidants, and cytokines in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome.

OBJECTIVES: We studied several acute inflammatory materials (AIM) such as various inflammatory cytokines, oxidative stress, and heat shock proteins in ARDS patients by simultaneously measuring from bronchoalveolar lavage fluid (BALF) and plasma. DESIGN AND METHODS: AIM were measured by using plasma and BALF sampling obtained from ARDS group (n=12) and non-ARDS group (n=12). RESULTS: In the BALF, only HSP 47 was significantly increased in ARDS group than non-ARDS group. In plasma, GRP 94, HSP 90, HSP 60, HSP 47, GPx-3, and IL-8 were increased significantly in ARDS group. In short, most of the AIM in BALF or plasma were not significantly different in ARDS group as compared with non-ARDS group. Ninety-day mortality was significantly related to HSP90, HSP 60 and GPx-3 in plasma but not in BALF. CONCLUSION: Alteration of AIM levels in both BALF or plasma of ARDS group was not remarkable compared with the non-ARDS group. Our result suggests the need to reconsider ARDS pathophysiology and therapeutic strategy.

Toxicological biomarkers of 2,3,4,7,8-pentachlorodibenzofuran in proteins secreted by HepG2 cells.

Using a proteomic approach, a study was conducted for determination of the effects of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PCDF) on proteins secreted by HepG2 cells. Briefly, HepG2 cells were exposed to various concentrations of 2,3,4,7,8-PCDF for 24 or 48h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1nM of 2,3,4,7,8-PCDF was the maximum concentration that did not cause cell death. In addition, a dose- and time dependent increase of DNA damage was observed in HepG2 cells exposed to 2,3,4,7,8-PCDF. Therefore, two different concentrations of 2,3,4,7,8-PCDF, 1 and 5nM, were selected for further analysis of proteomic biomarkers using two different pI ranges (4-7 and 6-9) and large two dimensional gel electrophoresis. Results showed identification of 32 proteins ( 29 up- and 3 down-regulated) by nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS. Among these, the identities of pyridoxine-5'-phosphate oxidase, UDP-glucose 6-dehydrogenase, plasminogen activator inhibitor I precursor, plasminogen activator inhibitor-3, proteasome activator complex subunit 1, isoform 1 of 14-3-3 protein sigma, peptidyl-prolyl cis-trans isomerase A, 14-3-3 protein gamma, protein DJ-1, and nucleoside diphosphate kinase A were confirmed by western blot analysis. The differential expression of protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 was further validated in plasma proteins from rats exposed to 2,3,4,7,8-PCDF. These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF.

Ethanol-induced DNA damage and repair-related molecules in human intestinal epithelial Caco-2 cells.

The acute administration of ethanol to intestinal epithelial cells causes increased intestinal permeability and the translocation of endotoxins. The changes caused by ethanol in intestinal cells may be related to oxidative stress and DNA damage. However, DNA damage and repair-related molecules which act against stresses, including ethanol, have not been fully investigated in intestinal cells. Heat shock proteins (Hsps) are involved in the recovery and protection from cell damage and may be associated with DNA repair. Therefore, the aim of our study was to investigate cytotoxicity, DNA damage and the expression of DNA repair-related molecules, antioxidant proteins and Hsps in intestinal cells exposed to ethanol. Human intestinal Caco-2 cells were incubated with 1-8% ethanol for 1 h. Cell viability and DNA damage were determined using the MTT and comet assays, respectively. We measured DNA repair-related molecules, including DNA polymerase ß, apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1), growth arrest and DNA damage 45α (GADD45α) and proliferating cell nuclear antigen (PCNA), in Caco-2 cells using western blot analysis. We also measured glutathione peroxidase-1 (GPx-1), peroxiredoxin-1 (PRX-1), superoxide dismutase-2 (SOD-2), Hsp10, Hsp27, Hsp60, heat shock cognate (Hsc)70, Hsp70 and Hsp90. The viability of the Caco-2 cells exposed to ethanol decreased at concentrations ≥ 7% (P<0.05). The Olive tail moment, indicating DNA damage, increased dose dependently in ≥ 3% ethanol (P<0.05). Among the DNA repair proteins, the expression of PCNA and APE/Ref-1 increased significantly at 1% ethanol. Antioxidant enzymes, including GPx-1, PRX-1 and SOD-2, had an increased expression at 1% ethanol. Hsp10, Hsp27 and Hsp70 expression also increased significantly at 1% ethanol. In conclusion, the expression of DNA repair molecules, antioxidants and Hsps increased in intestinal Caco-2 cells exposed to low concentrations of ethanol. In particular, PCNA, APE/Ref-1, Hsp10, Hsp27 and Hsp70 were sensitive to low ethanol concentrations, indicating that they may be useful in evaluating the DNA repair and cytoprotective effects of the drug against stress in intestinal cells.

Erythropoietin improves memory function with reducing endothelial dysfunction and amyloid-beta burden in Alzheimer's disease models.

Neurovascular degeneration contributes to the pathogenesis of Alzheimer's disease (AD). Because erythropoietin (EPO) promotes endothelial regeneration, we investigated the therapeutic effects of EPO in animal models of AD. In aged Tg2576 mice, EPO receptors (EPORs) were expressed in the cortex and hippocampus. Tg2576 mice were treated with daily injection of EPO (5000 IU/kg/day) for 5 days. At 14 days, EPO improved contextual memory as measured by fear-conditioning test. EPO enhanced endothelial proliferation and the level of synaptophysin expression in the brain. EPO also increased capillary density, and decreased the level of the receptor for advanced glycation endproducts (RAGE) in the brain, while decreasing in the amount of amyloid plaque and amyloid-ß (Aß). In cultured human endothelial cells, EPO enhanced angiogenesis and suppressed the expression of the RAGE. These results show that EPO improves memory and ameliorates endothelial degeneration induced by Aß in AD models. This pre-clinical evidence suggests that EPO may be useful for the treatment of AD.

Proteomic analysis of proteins secreted by HepG2 cells treated with butyl benzyl phthalate.

Proteomic changes in proteins secreted by human hepatocellular carcinomas (HepG2) cells exposed to butyl benzyl phthalate (BBP) were evaluated. HepG2 cells were treated with three different concentrations of BBP (0, 10, or 25 µM) for 24 or 48 h. Following incubation, the cells were subjected to proteomic analysis using two different pI ranges (4-7 and 6-9) and large-size two-dimensional gel electrophoresis. Results showed resolution of a total of 2776 protein spots. Of these, 29, including 19 upregulated and 10 downregulated proteins, were identified by electrospray ionization-mass spectrometry-mass spectrometry (ESI-MS/MS). Among these, the identities of cystatin C, Rho guanine nucleotide dissociation inhibitor, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, heptaglobin-related protein, inter-alpha-trypsin inhibitor heavy chain H2, and electron transfer flavoprotein subunit beta were confirmed by Western blot analysis. These proteins were found to be involved in apoptosis, signaling, tumor progression, energy metabolism, and cell structure and motility. Therefore, these proteins have potential to be employed as biomarkers of BBP exposure and may be useful in understanding mechanisms underlying the adverse effects of BBP.

Neuroprotective effects of constituents of Eragrostis ferruginea against Aß-induced toxicity in PC12 cells.

A new flavonoid, 7-demethylageconyflavone A (1), and five known compounds, tricin (2), ageconyflavone A (3), corylin (4), nectandrin B (5), and 4-ketopinoresinol (6) were isolated from the aerial parts of Eragrostis ferruginea. Their structures were determined using spectroscopic techniques, including 1D- and 2D-NMR. All compounds were tested for the neuroprotective effects against amyloid beta peptide (Abeta) using PC12 cells, a major cause of the pathology of Alzheimer's disease. Tricin (2) was found to have a neuroprotective effect with an ED(50) value of 20.3 microM against Abeta-induced toxicity in PC12 cells. Ageconyflavone A (3), nectandrin B (5) and 4-ketopinoresinol (6) demonstrated moderate neuroprotective effects with ED(50) values of 58.7, 44.1, and 54.8 microM, respectively.

The new diterpene isodojaponin D inhibited LPS-induced microglial activation through NF-kappaB and MAPK signaling pathways.

Neuroinflammation with prolonged microglial activation leads to increased levels of pro-inflammatory mediators and subsequently contributes to neuronal dysfunction and neuronal loss. Therefore, pharmacological suppression of neuroinflammation would theoretically slow the progression of neurodegenerative disease. In this study, we investigated the anti-inflammatory effects and possible mechanisms of isodojaponin D (19-hydroxy-1alpha,6-diacetoxy-6,7-seco-ent-kaur-16-en-15-one-7,20-olide), a new diterpene isolated from Isodon japonicus against lipopolysaccharide(LPS)-induced microglial activation in BV2 cells. Results from RT-PCR and Western blot showed that pretreatment with isodojaponin D (5 and 10 microg/ml) prior to treatment with LPS (1 microg/ml) significantly decreased LPS-induced production of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in a dose-dependent manner. In addition, LPS-induced pro-inflammatory cytokines, including IL-1beta, IL-6, and TNF-alpha, were also decreased by pretreatment with isodojaponin D. This effect was accompanied by a decrease in translocations of Nuclear Factor-KappaB (NF-kappaB) p50 and p65 from the cytoplasm to the nucleus and by a decrease in I kappaB (IkappaB) degradation. In addition, pretreatment with isodojaponin D significantly attenuated LPS-induced mitogen-activated protein kinase (MAPK) activation. Taken together, these results suggest that isodojaponin D suppressed LPS-induced microglial activation and production of pro-inflammatory mediators by inhibition of the NF-kappaB signaling pathway and phosphorylation of MAPKs. These results suggest that isodojaponin D could play a beneficial role in treatment of neurodegenerative disease.

Identification of toxicological biomarkers of di(2-ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis.

The effects of di(2-ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 microM) for 24 or 48 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 microM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose- and time-dependent fashion. Proteomic analysis using two different pI ranges (4-7 and 6-9) and large size 2-DE revealed the presence of 2776 protein spots. A total of 35 (19 up- and 16 down-regulated) proteins were identified as biomarkers of DEHP by ESI-MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up- or down-regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin-1, and haptoglobin-related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.

C-methylflavonoids isolated from Callistemon lanceolatus protect PC12 cells against Abeta-induced toxicity.

Increased beta-amyloid (Abeta) production and its aggregation to the oligomeric state is considered to be a major cause of Alzheimer's disease (AD). Therefore, reducing Abeta-induced neurotoxicity could provide a suitable means of prevention or intervention in the disease course of AD. The neuroprotective effects of isolates from Callistemon lanceolatus DC. (Myrtaceae) against Abeta were evaluated using PC12 cells. To evaluate the effects of Abeta on apoptotic cell death and the effects of Bcl-2 family proteins and caspase-3, TUNEL assays and Western blotting were performed, respectively. Substantial fractionation and purification of the EtOAc-soluble extract of the aerial parts of C. lanceolatus afforded six flavonoids, 4',5-dihydroxy-6,8-dimethyl-7-methoxyflavanone (1), eucalyptin (2), 8-demethyleucalyptin (3), sideroxylin (4), syzalterin (5), and quercetin (6). Compounds 1, 5, and 6 were found to protect PC12 cells effectively against Abeta-induced toxicity. In particular, compound 1 showed the most promising neuroprotective effect with an ED (50) value of 6.7 microM in terms of decreasing Abeta-induced apoptotic cell death, and this was accompanied by a decrease in caspase-3 activation and an increase in Bcl-2/Bax ratio. These results suggest that compound 1 could be developed as a candidate anti-AD agent due to its attenuation of Abeta-induced apoptotic cell death.