Diagnostic Role of Bile Pigment Components in Biliary Tract Cancer.

Bile pigment, bilirubin, and biliverdin concentrations may change as a results of biliary tract cancer (BTC) altering the mechanisms of radical oxidation and heme breakdown. We explored whether changes in bile pigment components could help distinguish BTC from benign biliary illness by evaluating alterations in patients with BTC. We collected bile fluid from 15 patients with a common bile duct stone (CBD group) and 63 individuals with BTC (BTC group). We examined the bile fluid's bilirubin, biliverdin reductase (BVR), heme oxygenase (HO-1), and bacterial taxonomic abundance. Serum bilirubin levels had no impact on the amounts of bile HO-1, BVR, or bilirubin. In comparison to the control group, the BTC group had considerably higher amounts of HO-1, BVR, and bilirubin in the bile. The areas under the curve for the receiver operating characteristic curve analyses of the BVR and HO-1 were 0.832 (p<0.001) and 0.891 (p<0.001), respectively. Firmicutes was the most prevalent phylum in both CBD and BTC, according to a taxonomic abundance analysis, however the Firmicutes/Bacteroidetes ratio was substantially greater in the BTC group than in the CBD group. The findings of this study showed that, regardless of the existence of obstructive jaundice, biliary carcinogenesis impacts heme degradation and bile pigmentation, and that the bile pigment components HO-1, BVR, and bilirubin in bile fluid have a diagnostic significance in BTC. In tissue biopsies for the diagnosis of BTC, particularly for distinguishing BTC from benign biliary strictures, bile pigment components can be used as additional biomarkers.

Efficient Generation of Dopaminergic Neurons from Mouse Ventral Midbrain Astrocytes.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by tremors, bradykinesia, and rigidity. PD is caused by loss of dopaminergic (DA) neurons in the midbrain substantia nigra (SN) and therefore, replenishment of DA neurons via stem cell-based therapy is a potential treatment option. Astrocytes are the most abundant non-neuronal cells in the central nervous system and are promising candidates for reprogramming into neuronal cells because they share a common origin with neurons. The ability of neural progenitor cells (NPCs) to proliferate and differentiate may overcome the limitations of the reduced viability and function of transplanted cells after cell replacement therapy. Achaete-scute complex homolog-like 1 (Ascl1) is a wellknown neuronal-specific factor that induces various cell types such as human and mouse astrocytes and fibroblasts to differentiate into neurons. Nurr1 is involved in the differentiation and maintenance of DA neurons, and decreased Nurr1 expression is known to be a major risk factor for PD. Previous studies have shown that direct conversion of astrocytes into DA neurons and NPCs can be induced by overexpression of Ascl1 and Nurr1 and additional transcription factors genes such as superoxide dismutase 1 and SRY-box 2. Here, we demonstrate that astrocytes isolated from the ventral midbrain, the origin of SN DA neurons, can be effectively converted into DA neurons and NPCs with enhanced viability. In addition, when these NPCs are inducted to differentiate, they exhibit key characteristics of DA neurons. Thus, direct conversion of midbrain astrocytes is a possible cell therapy strategy to treat neurodegenerative diseases.

Anti-Growth, Anti-Angiogenic, and Pro-Apoptotic Effects by CX-4945, an Inhibitor of Casein Kinase 2, on HuCCT-1 Human Cholangiocarcinoma Cells via Control of Caspase-9/3, DR-4, STAT-3/STAT-5, Mcl-1, eIF-2α, and HIF-1α.

Overexpression of casein kinase 2 (CK2) has an oncogenic and pro-survival role in many cancers. CX-4945 (Silmitasertib) is a CK2 inhibitor with anti-cancerous and anti-angiogenic effects. Up to date, the anti-cancer effect and mechanism of CX-4945 on human cholangiocarcinoma (CCA) remain unclear. This study investigated whether CX-4945 inhibits growth and induces apoptosis of HuCCT-1 cells, a human CCA cell line. Of note, treatment with CX-4945 at 20 µM markedly reduced survival and induced apoptosis of HuCCT-1 cells, as evidenced by nuclear DNA fragmentation, PARP cleavage, activation of caspase-9/3, and up-regulation of DR-4. Although CX-4945 did not affect the phosphorylation and expression of CK2, it vastly inhibited the phosphorylation of CK2 substrates, supporting the drug's efficacy in inhibiting CK2 and its downstream pathway. Importantly, knockdown of CK2 that partially suppressed the phosphorylation of CK2 substrates resulted in a significant reduction of HuCCT-1 cell survival. In addition, CX-4945 reduced the phosphorylation and expression of STAT-3 and STAT-5 in HuCCT-1 cells, and pharmacological inhibition or respective knockdown of these proteins resulted in significant growth suppression of HuCCT-1 cells. CX-4945 also had abilities to decrease Mcl-1 expression while increasing eIF-2α phosphorylation in HuCCT-1 cells. Furthermore, there was a time-differential negative regulation of HIF-1α expression by CX-4945 in HuCCT-1 cells, and knockdown of HIF-1α caused a significant reduction of the cell survival. In summary, these results demonstrated that CX-4945 has anti-growth, anti-angiogenic, and pro-apoptotic effects on HuCCT-1 cells, which are mediated through control of CK2, caspase-9/3, DR-4, STAT-3/5, Mcl-1, eIF-2α, and HIF-1α.

Circulating microRNAs as biomarkers in bile-derived exosomes of cholangiocarcinoma.

PURPOSE: In this pilot study, using next-generation sequencing and integrated messenger RNA (mRNA) sequencing, we investigated circulating microRNA (miRNA) expression profiling from bile-derived exosomes to identify dysregulated miRNA signatures and oncogenic pathways and determine their effects on targeted mRNAs in cholangiocarcinoma (CCA). Moreover, we explored the possibility that genetic analysis using bile-derived exosomes may replace gene analysis using tissue. METHODS: Bile was collected from a patient with perihilar CCA before curative resection. As a control, bile was collected from a patient with a common bile duct stone. Exosomes were isolated from the bile, and we performed next-generation miRNA sequencing using isolated exosomes. To evaluate miRNA-mRNA interactions, mRNA sequencing was performed using bile fluid in both patients. RESULTS: We identified 22 differentially expressed miRNAs. More than 65% of the predicted mRNA targets of those miRNAs were actually differentially expressed between control and CCA bile samples. In functional pathway analysis, targets of 22 miRNAs were primarily enriched in mitogen-activated protein kinase, platelet derived growth factor, vascular endothelial growth factor, epidermal growth factor receptor, and p53 signaling. In particular, in the functional assessment of miRNA-mRNA interactions, RAS pathways, including downstream pathways (PI3K-AKT-mTOR and RAS-RAF-MEK-ERK), were determined to be enriched. CONCLUSION: Circulating miRNAs in bile-derived exosomes provide new information for the development of miRNA analysis in CCA. These miRNAs may represent the oncogenic characteristics of CCA tissue, enabling them to be used instead of tissue samples for the diagnosis of CCA. Further research investigating circulating miRNAs in bile exosomes may lead to more rational, targeted approaches to treatment.

Liquid Biopsy from Bile-Circulating Tumor DNA in Patients with Biliary Tract Cancer.

Although liquid biopsy of blood is useful for cancer diagnosis and prediction of prognosis, diagnostic and prognostic value of ctDNA in bile fluid for BTCs are not clear yet. To determine whether liquid biopsy for circulating tumor DNA (ctDNA) can replace tissue biopsy when assessing somatic mutations in biliary tract cancers (BTCs). Bile samples were obtained from 42 patients with BTC. Matched formalin-fixed paraffin-embedded (FFPE) samples were obtained from 20 of these patients and matched plasma samples from 16 of them. Droplet digital PCR (ddPCR) was used for detection KRAS somatic mutation. KRAS mutations were identified in the bile ctDNA of 20 of 42 (48%) patients. Patients with mutant KRAS showed significantly worse survival than those with wild-type KRAS (2-year survival rates: 0% vs. 55.5%, respectively; p = 0.018). There was 80.0% mutational concordance between the paired bile ctDNA and FFPE samples, and 42.9% between the plasma and FFPE samples. On transcriptomic sequencing of one set of paired bile and FFPE samples, expression level of KRAS-associated signaling oncogenes in the bile and tissue samples showed a strong positive correlation (r = 0.991, p < 0.001). Liquid biopsy of bile reliably detect mutational variants within the bile ctDNA of BTC patients. These results suggest that bile is an effective biopsy fluid for ctDNA analysis.

Usefulness of bile as a biomarker via ferroptosis and cysteine prenylation in cholangiocarcinoma; role of diagnosis and differentiation from benign biliary disease.

BACKGROUND: Cholangiocarcinoma (CCA) is a malignant cancer of the biliary tract with a poor prognosis. Herein, we investigated possible mechanism of extrahepatic CCA (eCCA) by dysregulated iron metabolism and post-translational modifications (PTMs). Further, we evaluated potential biomarkers in the bile fluid for diagnosis of eCCA and differentiation between eCCA and benign biliary disease. METHODS: From August 2018 to April 2019, we obtained bile fluids from 46 patients; 28 patients with eCCA (eCCA group) and 18 patients with common bile duct stone (Control group) via percutaneous transhepatic biliary drainage. We examined the levels of reduced glutathione (GSH), peroxide, ferrous iron [Fe+2], glutathione peroxidase (GPX) and farnesyl transferase/geranylgeranyl transferase type-1 subunit alpha (FNTA) concentration in bile fluids to clarify the mechanism of ferroptosis and prenylation. RESULTS: The remarkable difference of PTMs was that FNTA which means prenylated cysteine as regulator was significantly decreased in eCCA than that of control. In addition, level of GSH, peroxide, GPX and ferrous iron [Fe+2] were significantly depleted in eCCA than control. These results demonstrate that PTM, dysregulated iron metabolism and GPX-regulated ferroptosis with GSH depletion through cysteine modification in bile are possible mechanisms of eCCA. Liquid Chromatography (LC)-Mass Spectrometry (MS) analysis, several oncogenic pathways including MYC target, apoptosis, fatty acid metabolism, P53 and mTORC1 were enriched in eCCA. CONCLUSIONS: In conclusion, redox-dependent modification of cysteine and ferroptosis in bile fluids are possible mechanisms of eCCA. Several protein and oncogenic pathways related to PTM which are seen in eCCA tissues were also enriched in bile fluids. It suggests that bile fluid represents the oncogenic characteristics of eCCA tissues. Therefore, bile fluids have a role of a biomarker for diagnosis in eCCA, especially, differentiation of eCCA from benign biliary stricture.

(-)-Epigallocatechin-3-O-gallate (EGCG) attenuates the hemodynamics stimulated by caffeine through decrease of catecholamines release.

A human study of the effects on hemodynamics of caffeine and epigallocatechin-3-O-gallate (EGCG) was performed. Caffeine tablets (200 mg) were orally administered to healthy males aged between 25 and 35 years 30 min after oral administration of EGCG tablets (100 and 200 mg). The increase in BP induced by caffeine was inhibited when co-administrated with EGCG. We found that caffeine slightly decreased heart rate (HR) in the volunteers. Although EGCG enhanced HR reduction, the effect was not significant. In addition, caffeine increased blood catecholamine levels, but EGCG inhibited the increase in noradrenaline, adrenaline and dopamine levels induced by caffeine. Whether EGCG decreases the elevated HR and systolic perfusion pressure, and ventricular contractility induced by adrenergic agonists in the isolated rat heart was investigated. The modified Krebs-Henseleit solution was perfused through a Langendorff apparatus to the isolated hearts of rats. HR, systolic perfusion pressure, and developed maximal rates of contraction (+dP/dtmax) and relaxation (-dP/dtmax) were increased by epinephrine (EP) and isoproterenol (IP). In contrast, EGCG decreased the elevated HR, systolic perfusion pressure, and left ventricular ±dp/dtmax induced by EP and/or IP. In conclusion, EGCG could attenuate the hemodynamics stimulated by caffeine through decreasing catecholamine release.

Alleviation of kainic acid-induced brain barrier dysfunction by 4-o-methylhonokiol in in vitro and in vivo models.

This experiment was designed to investigate whether 4-O-methylhonokiol (MH), a principal ingredient of Magnolia (M.) officinalis bark, alleviated acute intraperitoneal (i.p.) kainic acid- (KA-) induced brain blood barrier dysfunction (BBBD) via pathological examination and cytological analyses of the brain tissues of mice. KA (10-30 mg/kg) time- and dose-dependently increased the water content of brain tissues and induced edema and encephalopathy. However, pretreatment with MH (5 and 20 mg/kg, i.p.) significantly reduced the water content of the brain compared to that observed in the KA control group. Furthermore, MH significantly and dose-dependently reversed the remarkable variations in evan's blue dye (EBD) staining and malondialdehyde (MDA) levels that were induced by KA (10 mg/kg, i.p.). MH also decreased the elevated seizure scores that were induced by KA (10 mg/kg, i.p.) in mice in a manner similar to scavengers such as DMTU and trolox. Additionally, MH significantly scavenged intracellular ROS and Ca2+ within hippocampal cells. The tight junction seals mediated by claudin (Cld-5) were also found to be modulated by MH. MH efficiently reduced 1,1-diphenyl-2-picrylhydrazyl (DPPH) (IC50, 52.4 mM) and •OH with an electron spin resonance (ESR) signal rate constant of 4×10(9) M(-1)·S(-1), which is close to the reactivity of the vitamin E analog trolox. Taken together, these results suggest that MH may enhance radical scavenging in lipid and hydrophobic environments, which may be important for the physiological activity of the barrier.

Reducing effect of IL-32α in the development of stroke through blocking of NF-κB, but enhancement of STAT3 pathways.

Neuroinflammation is important for the development of several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and stroke. Since changes of cytokine level are critical for neuroinflammation in the brain, we investigated whether IL-32α overexpression could change neuroinflammation and, thus, affect stroke development. Middle cerebral artery occlusion (MCAO) induced development of ischemia, and ischemic neuronal cell death were reduced in IL-32α-overexpressing transgenic mice (IL-32α mice) brain through the decreased release of neuroinflammatory cytokines (IL-6, IL-1ß, TNF-α) and activation of astrocytes, but enhancement of anti-neuroinflammatory cytokines (IL-10). Reactive oxygen species generation and lipid peroxidation as well as expression of inducible nitric oxide and cyclooxygenase-2 were also reduced in the IL-32α mice brain. Nuclear factor-kappa B (NF-κB), a critical transcriptional factor regulating neuroinflammation, was much lower, but activation of signal transducer and activator of transcription 3 (STAT3), which plays a crucial role in cell survival and proliferation, was much higher in IL-32α-overexpressing mice brain compared to those of wild-type mice brain. These results suggest that IL-32α can prevent cerebral ischemia damage via upregulation of anti-neuroinflammatory cytokine expression and STAT3 activation, but downregulation of neuroinflammatory cytokines and NF-κB activation.

Pachymic Acid Enhances Pentobarbital-Induced Sleeping Behaviors via GABAA-ergic Systems in Mice.

This study was investigated to know whether pachymic acid (PA), one of the predominant triterpenoids in Poria cocos (Hoelen) has the sedative-hypnotic effects, and underlying mechanisms are mediated via γ-aminobutyric acid (GABA)-ergic systems. Oral administration of PA markedly suppressed locomotion activity in mice. This compound also prolonged sleeping time, and reduced sleep latency showing synergic effects with muscimol (0.2 mg/kg) in shortening sleep onset and enhancing sleep time induced by pentobarbital, both at the hypnotic (40 mg/kg) and sub-hypnotic (28 mg/kg) doses. Additionally, PA elevated intracellular chloride levels in hypothalamic primary cultured neuronal cells of rats. Moreover, Western blotting quantitative results showed that PA increased the amount of protein level expression of GAD65/67 over a broader range of doses. PA increased α- and ß-subunits protein levels, but decreased γ-subunit protein levels in GABAA receptors. The present experiment provides evidence for the hypnotic effects as PA enhanced pentobarbital-induced sleeping behaviors via GABAA-ergic mechanisms in rodents. Taken together, it is proposed that PA may be useful for the treatment of sleep disturbed subjects with insomnia.

Caffeine induces behavioural sensitization and overexpression of cocaine-regulated and amphetamine-regulated transcript peptides in mice.

This study examined whether repeated administration of caffeine would induce behavioural sensitization and overexpression of cocaine-regulated and amphetamine-regulated transcript (CART) peptides in mice. The involvement of dopaminergic receptors and adenosine receptors in caffeine-induced behavioural sensitization and CART overexpression was studied. The relevance of D1R and D2R, and A1R and A(2A)R in the overexpression of CART peptides in mouse striatum was also evaluated. Repeated administration of caffeine induced behavioural sensitization in mice. Significant increases in CART mRNA levels were observed on day 3 and peaked at day 5 of caffeine administration, and then decreased gradually. Higher proportions of CART⁺ cells were observed in the dorsolateral and ventrolateral part of the caudate putamen than in the nucleus accumbens shell and core. The behavioural sensitization induced by caffeine was inhibited by dopaminergic receptor antagonists and adenosine receptor agonists. D1R and D2R, and cyclic AMP (cAMP)/protein kinase A (PKA)/phospho-cAMP response element-binding protein (pCREB) signalling were activated by caffeine, but A1R and A(2A)R were inhibited. Overexpression of caffeine-induced CART peptides and pCREB activity were blocked by N-cyclopentyladenosine (CPA, an A1R agonist) and 4-[2-[[6-amino-9-(N-ethyl-ß-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS 21680, an A(2A)R agonist), but not by R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH 23390, a D1R antagonist) or raclopride (a D2R antagonist). Caffeine-induced overexpression of CART peptides was associated with the inhibition of A1R and A(2A)R, and the activation of cAMP/PKA/pCREB signalling. Moreover, the A(2A)R-D2R heterodimer might be involved in the overexpression of CART peptides induced by caffeine.

Inhibitory effect of a 2,4-bis(4-hydroxyphenyl)-2-butenal diacetate on neuro-inflammatory reactions via inhibition of STAT1 and STAT3 activation in cultured astrocytes and microglial BV-2 cells.

2,4-Bis(p-hydroxyphenyl)-2-butenal (Butenal), a tyrosine-fructose Maillard reaction product has been demonstrated as an effective compound for prevention of neuroinflammatory diseases. However, this compound was vulnerable to environmental factors. Our research has been continuously made to improve druggability of Butenal and identified 2,4-bis(4-hydroxyphenyl)but-2-enal diacetate (HPBD) as an alternative. Herein, to investigate potential anti-neuroinflammatory and anti-amyloidogenic effects of HPBD, we treated HPBD (0.5, 1, and 2 µg/ml) on the lipopolysaccharides (LPS) (1 µg/ml) stimulated astrocytes and microglial BV-2 cell. HPBD inhibited LPS-induced NO and ROS production, and LPS-elevated expression of iNOS, COX2, ß-site APP-cleaving enzyme 1 (BACE1), C99, and Aß1-42 levels as well as attenuation of ß-secretase activities. The activation of nuclear factor-kappaB (NF-κB), signal transducer and activator of transcription1 (STAT1), and STAT3 was concomitantly inhibited by HPBD. Moreover, siRNA targeting STAT3 abolished HPBD-induced inhibitory effects on neuro-inflammation and amyloidogenesis. In addition, pull down assay and docking model showed interaction of HPBD with STAT3. These findings suggest that HPBD may be useful and potentially therapeutic choices for the treatment of neuroinflammatory diseases.

3,4,5-Trimethoxycinnamic acid (TMCA), one of the constituents of Polygalae Radix enhances pentobarbital-induced sleeping behaviors via GABAAergic systems in mice.

These experiments were performed to investigate whether 3,4,5-trimethoxycinnamic acid (TMCA), one of the constituents derived from Polygalae Radix, enhances pentobarbital-induced sleeping behaviors, and to alter sleep architecture through the γ-aminobutyric acid (GABA)ergic systems in mice. TMCA decreased the locomotor activity. TMCA prolonged total sleep time, and reduced sleep latency induced by pentobarbital, similar to muscimol, a GABAA agonist. From the electrocencephalogram recording for 6 h after TMCA administration, the number of sleep/wake cycles were reduced by TMCA. TMCA also increased the total sleep time and non-rapid eye movement (NREM) sleep. In addition, TMCA increased Cl(-) influx in primary cultured cerebellar granule cells of mice. TMCA increased the activation of glutamic acid decarboxylase (GAD) and the expressions of γ-subunit of GABAA receptors in the cerebellar granule cells. However, α- and ß-subunits proteins of GABAA receptors were not increased. Therefore, TMCA would increase pentobarbital induced-sleep and NREM sleep in mice. These results indicate that TMCA may enhance sleep and alter sleep architecture through GABAAergic systyems.

Acceleration of the development of Alzheimer's disease in amyloid beta-infused peroxiredoxin 6 overexpression transgenic mice.

The amyloid beta (Aß) peptide in the brains of patients with Alzheimer's disease (AD) is cytotoxic to neurons and has a central role in the pathogenesis of the disease. Peroxiredoxin 6 (Prdx6) is an antioxidant protein and could act as a cytoprotective protein. However, the role of Prdx6 in neurodegenerative disease has not been studied. Thus, the roles and action mechanisms in the development of AD were examined. Aß1-42-induced memory impairment in Prdx6 transgenic mice was worse than C57BL/6 mice, and the expression of amyloid precursor protein cleavage, C99, ß-site APP-cleaving enzyme 1, inducible nitric oxide synthase, and cyclooxygenase-2 was greatly increased. In addition, the astrocytes and microglia cells of Aß-infused Prdx6 transgenic mice were more activated, and Aß also significantly increased lipid peroxidation and protein carbonyl levels, but decreased glutathione levels. Furthermore, we found that translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus was increased in Aß-infused Prdx6 transgenic mice. These results suggest that the overexpression of Prdx6 could accelerate the development of AD through increased amyloidogenesis through independent PLA2 activation and Nrf2 transcription.

Cordycepin Increases Nonrapid Eye Movement Sleep via Adenosine Receptors in Rats.

Cordycepin (3'-deoxyadenosine) is a naturally occurring adenosine analogue and one of the bioactive constituents isolated from Cordyceps militaris/Cordyceps sinensis, species of the fungal genus Cordyceps. It has traditionally been a prized Chinese folk medicine for the human well-being. Because of similarity of chemical structure of adenosine, cordycepin has been focused on the diverse effects of the central nervous systems (CNSs), like sleep regulation. Therefore, this study was undertaken to know whether cordycepin increases the natural sleep in rats, and its effect is mediated by adenosine receptors (ARs). Sleep was recorded using electroencephalogram (EEG) for 4 hours after oral administration of cordycepin in rats. Sleep architecture and EEG power spectra were analyzed. Cordycepin reduced sleep-wake cycles and increased nonrapid eye movement (NREM) sleep. Interestingly, cordycepin increased θ (theta) waves power density during NREM sleep. In addition, the protein levels of AR subtypes (A1, A2A, and A2B) were increased after the administration of cordycepin, especially in the rat hypothalamus which plays an important role in sleep regulation. Therefore, we suggest that cordycepin increases theta waves power density during NREM sleep via nonspecific AR in rats. In addition, this experiment can provide basic evidence that cordycepin may be helpful for sleep-disturbed subjects.

Red ginseng extract attenuates kainate-induced excitotoxicity by antioxidative effects.

This study investigated the neuroprotective activity of red ginseng extract (RGE, Panax ginseng, C. A. Meyer) against kainic acid- (KA-) induced excitotoxicity in vitro and in vivo. In hippocampal cells, RGE inhibited KA-induced excitotoxicity in a dose-dependent manner as measured by the MTT assay. To study the possible mechanisms of the RGE-mediated neuroprotective effect against KA-induced cytotoxicity, we examined the levels of intracellular reactive oxygen species (ROS) and [Ca(2+)](i) in cultured hippocampal neurons and found that RGE treatment dose-dependently inhibited intracellular ROS and [Ca(2+)](i) elevation. Oral administration of RGE (30 and 200 mg/kg) in mice decreased the malondialdehyde (MDA) level induced by KA injection (30 mg/kg, i.p.). In addition, similar results were obtained after pretreatment with the radical scavengers Trolox and N, N'-dimethylthiourea (DMTU). Finally, after confirming the protective effect of RGE on hippocampal brain-derived neurotropic factor (BDNF) protein levels, we found that RGE is active compounds mixture in KA-induced hippocampal mossy-fiber function improvement. Furthermore, RGE eliminated 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, and the IC(50) was approximately 10 mg/ml. The reductive activity of RGE, as measured by reaction with hydroxyl radical ((•)OH), was similar to trolox. The second-order rate constant of RGE for (•)OH was 3.5-4.5 × 10(9) M(-1)·S(-1). Therefore, these results indicate that RGE possesses radical reduction activity and alleviates KA-induced excitotoxicity by quenching ROS in hippocampal neurons.

Protection of apigenin against kainate-induced excitotoxicity by anti-oxidative effects.

Apigenin (5,7,4'-trihydroxyflavone) is a principal ingredient of Cirsium japonicum. These experiments were performed to determine whether apigenin has neuroprotective effects against kainic acid (KA)-induced excitotoxicity in vitro and in vivo. Intraperitoneal (i.p.) administration of apigenin (25, 50 mg/kg) decreased the seizure scores induced by KA injection (40 mg/kg, i.p.) in mice. In addition, the convulsion onset time was significantly delayed by apigenin administration. Moreover, we found that apigenin blocked KA-induced seizure-form electroencephalogram (EEG) discharge activity in the brain cortex. In hippocampal cells, apigenin inhibited KA-induced excitotoxicity in a dose-dependent manner as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To study the possible mechanisms underlying the in vitro neuroprotective effects of apigenin against KA-induced cytotoxicity, we also examined the effect of apigenin on intracellular reactive oxygen species (ROS) elevations in cultured hippocampal neurons and found that apigenin treatment dose-dependently inhibited intracellular ROS elevation. The remarkable reduction of glutathione (GSH) levels induced by KA in hippocampal tissues was reversed by apigenin in a dose-dependent manner. In addition, similar results were obtained after pretreatment with free radical scavengers such as trolox and dimethylthiourea (DMTU). Finally, after confirming the protective effect of apigenin in hippocampal CA3 region, we found apigenin is an active compound in KA-induced neuroprotection. These results collectively indicate that apigenin alleviates KA-induced excitotoxicity by quenching ROS as well as inhibiting GSH depletion in hippocampal neurons.

Enhancement of pentobarbital-induced sleep by apigenin through chloride ion channel activation.

This experiment was performed to investigate whether apigenin has hypnotic effects and/or enhances pentobarbital-induced sleep behaviors through the GABAergic systems. Apigenin prolonged sleep time induced by pentobarbital similar to muscimol, a GABA(A) receptors agonist. Apigenin also increased sleep rate and sleep time in the combined administration with pentobarbital at the sub-hypnotic dosage, and showed synergic effects with muscimol in potentiating sleep onset and enhancing sleep time induced by pentobarbital. In addition, both of apigeinin and pentobarbital increased chloride influx in primary cultured cerebellar granule cells. Apigenin increased glutamate decarboxylase (GAD) and had no effect on the expression of GABA(A) receptor α-, ß-, γ-subunits in n hippocampus of mouse brain, showing different expression of subunits from pentobarbital treatment group. In conclusion, it is suggested that apigenin augments pentobarbital-induced sleep behaviors through chloride ion channel activation.

Inhibitory effect of 4-O-methylhonokiol on lipopolysaccharide-induced neuroinflammation, amyloidogenesis and memory impairment via inhibition of nuclear factor-kappaB in vitro and in vivo models.

BACKGROUND: Neuroinflammation is important in the pathogenesis and progression of Alzheimer disease (AD). Previously, we demonstrated that lipopolysaccharide (LPS)-induced neuroinflammation caused memory impairments. In the present study, we investigated the possible preventive effects of 4-O-methylhonokiol, a constituent of Magnolia officinalis, on memory deficiency caused by LPS, along with the underlying mechanisms. METHODS: We investigated whether 4-O-methylhonokiol (0.5 and 1 mg/kg in 0.05% ethanol) prevents memory dysfunction and amyloidogenesis on AD model mice by intraperitoneal LPS (250 µg/kg daily 7 times) injection. In addition, LPS-treated cultured astrocytes and microglial BV-2 cells were investigated for anti-neuroinflammatory and anti-amyloidogenic effect of 4-O-methylhonkiol (0.5, 1 and 2 µM). RESULTS: Oral administration of 4-O-methylhonokiol ameliorated LPS-induced memory impairment in a dose-dependent manner. In addition, 4-O-methylhonokiol prevented the LPS-induced expression of inflammatory proteins; inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as well as activation of astrocytes (expression of glial fibrillary acidic protein; GFAP) in the brain. In in vitro study, we also found that 4-O-methylhonokiol suppressed the expression of iNOS and COX-2 as well as the production of reactive oxygen species, nitric oxide, prostaglandin E2, tumor necrosis factor-α, and interleukin-1ß in the LPS-stimulated cultured astrocytes. 4-O-methylhonokiol also inhibited transcriptional and DNA binding activity of NF-κB via inhibition of IκB degradation as well as p50 and p65 translocation into nucleus of the brain and cultured astrocytes. Consistent with the inhibitory effect on neuroinflammation, 4-O-methylhonokiol inhibited LPS-induced Aß1-42 generation, ß- and γ-secretase activities, and expression of amyloid precursor protein (APP), BACE1 and C99 as well as activation of astrocytes and neuronal cell death in the brain, in cultured astrocytes and in microglial BV-2 cells. CONCLUSION: These results suggest that 4-O-methylhonokiol inhibits LPS-induced amyloidogenesis via anti-inflammatory mechanisms. Thus, 4-O-methylhonokiol can be a useful agent against neuroinflammation-associated development or the progression of AD.

Obovatol improves cognitive functions in animal models for Alzheimer's disease.

Etiology of Alzheimer's disease (AD) is obscure, but neuroinflammation and accumulation of ß-amyloid (Aß) are implicated in pathogenesis of AD. We have shown anti-inflammatory and neurotrophic properties of obovatol, a biphenolic compound isolated from Magnolia obovata. In this study, we examined the effect of obovatol on cognitive deficits in two separate AD models: (i) mice that received intracerebroventricular (i.c.v.) infusion of Aß(1-42) (2.0 µg/mouse) and (ii) Tg2576 mice-expressing mutant human amyloid precursor protein (K670N, M671L). Injection of Aß(1-42) into lateral ventricle caused memory impairments in the Morris water maze and passive avoidance tasks, being associated with neuroinflammation. Aß(1-42) -induced abnormality was significantly attenuated by administration of obovatol. When we analyzed with Tg2576 mice, long-term treatment of obovatol (1 mg/kg/day for 3 months) significantly improved cognitive function. In parallel with the improvement, treatment suppressed astroglial activation, BACE1 expression and NF-κB activity in the transgenic mice. Furthermore, obovatol potently inhibited fibrillation of Aßin vitro in a dose-dependent manner, as determined by Thioflavin T fluorescence and electron microscopic analysis. In conclusion, our data demonstrated that obovatol prevented memory impairments in experimental AD models, which could be attributable to amelioration of neuroinflammation and amyloidogenesis by inhibition of NF-κB signaling pathway and anti-fibrillogenic activity of obovatol.