Corrigendum to' Morphine dependence is attenuated by red ginseng extract and ginsenosides Rh2, Rg3, and compound K' [Journal of Ginseng Research Volume 40, Issue 4, October 2016, Pages 445-452].

[This corrects the article DOI: 10.1016/j.jgr.2016.08.006.].

Concanavalin A Induces Cortical Neuron Apoptosis by Causing ROS Accumulation and Tyrosine Kinase Activation.

The lectin, concanavalin A (Con A), is the most extensively investigated member of the lectin family of plant proteins, but its effects on cortical neurons and astrocytes are poorly understood. In cultured cortical neurons and astrocytes, Con A exhibited dose-dependent neurotoxicity, but this was not observed in astrocytes. Similarly, in the cortical areas of rat brains, intracranial administration of Con A caused neuronal but no astrocyte damage. Methyl-α-D-mannopyranoside, a competitor of Con A, blocked Con A-induced cell death, whereas AMPA/KA receptor antagonists showed partial blocking effects. Furthermore, the mRNA levels of TNF-α, IL-1ß, and IL-6 were elevated in astrocytes and cortical neurons treated with Con A. Intracellular reactive oxygen species (ROS) levels were increased in Con A-treated cortical neurons, and N-acetyl-cysteine (NAC, an antioxidant) and diphenyleneiodonium (DPI, a NADPH oxidase inhibitor) reduced intracellular ROS accumulation. Likewise, AG556 (a TNF-α inhibitor) and AG82 (a tyrosine kinase inhibitor) both reduced Con A-induced intracellular ROS accumulation. Furthermore, Con A-induced tyrosine phosphorylation was decreased by NAC and by AG556. Taken together, Con A-induced apoptosis in cortical neurons occurred as a sequel to Con A binding to neuronal glycoproteins and intracellular ROS accumulation. Interestingly, Con A-induced cellular damage was observed in cortical neurons but not in astrocytes or microglia.

Morphine dependence is attenuated by red ginseng extract and ginsenosides Rh2, Rg3, and compound K.

BACKGROUND: Red ginseng and ginsenosides have shown plethoric effects against various ailments. However, little is known regarding the effect of red ginseng on morphine-induced dependence and tolerance. We therefore investigated the effect of red ginseng extract (RGE) and biotransformed ginsenosides Rh2, Rg3, and compound K on morphine-induced dependence in mice and rats. METHODS: While mice were pretreated with RGE and then morphine was injected intraperitoneally, rats were infused with ginsenosides and morphine intracranially for 7 days. Naloxone-induced morphine withdrawal syndrome was estimated and conditioned place preference test was performed for physical and psychological dependence, respectively. Western blotting was used to measure protein expressions. RESULTS: Whereas RGE inhibited the number of naloxone-precipitated jumps and reduced conditioned place preference score, it restored the level of glutathione in mice. Likewise, ginsenosides Rh2, Rg3, and compound K attenuated morphine-dependent behavioral patterns such as teeth chattering, grooming, wet-dog shake, and escape behavior in rats. Moreover, activated N-methyl-D-aspartate acid receptor subunit 1 and extracellular signal-regulated kinase in the frontal cortex of rats, and cultured cortical neurons from mice were downregulated by ginsenosides Rh2, Rg3, and compound K despite their differential effects. CONCLUSION: RGE and biotransformed ginsenosides could be considered as potential therapeutic agents against morphine-induced dependence.

Modulation of NMDA Receptor Subunits Expression by Concanavalin A.

The processes of N-methyl-D-aspartate (NMDA) receptor subunits expression were examined in cortical neurons and rat brain in order to investigate how the concanavalin A (Con A) modulates neuronal cells. Con A modulated the expression of NMDA receptor subunits in cultured cortical cells. Con A augmented the level of intracellular Ca(2+) by α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA). We determined whether activation of AMPA receptors was involved in the regulation of NMDA receptor expression with Con A by blocking the desensitization of AMPA receptors. The results showed that AMPA receptor antagonists suppressed NMDA receptor subunits expression in Con A-treated cortical neuronal cells. PMA elevated the expression of NMDA receptor subunits, while PKC inhibitor and tyrosine kinases inhibitor suppressed the expression of NMDA receptor subunits. Furthermore, it was shown that NMDA receptor subunits expression was modulated in a region-specific manner after the sustained microinfusion of Con A into the cerebroventricle of the rat brain. Collectively, it could be presumed that the AMPA receptor activation was involved in Con A-induced modulation of NMDA receptor subunits expression.

Administration of Phytoceramide Enhances Memory and Upregulates the Expression of pCREB and BDNF in Hippocampus of Mice.

This study was aimed at investigating the possible effects of phytoceramide (Pcer) on learning and memory and their underlying mechanisms. Phytoceramide was orally administered to ICR mice for 7 days. Memory performances were assessed using the passive avoidance test and Y-maze task. The expressions of phosphorylated cAMP response element binding protein (pCREB), brain-derived neurotrophic factor (BDNF) were measured with immunoblot. The incorporation of 5-bromo-2-deoxyuridine (BrdU) in hippocampal regions was investigated by using immunohistochemical methods. Treatment of Pcer enhanced cognitive performances in the passive avoidance test and Y-maze task. Immunoblotting studies revealed that the phosphorylated CREB and BDNF were significantly increased on hippocampus in the Pcer-treated mice. Immunohistochemical studies showed that the number of immunopositive cells to BrdU was significantly increased in the hippocampal dentate gyrus regions after Pcer-treatment for 7 days. These results suggest that Pcer contribute to enhancing memory and BDNF expression and it could be secondary to the elevation of neurogenesis.

Anti-Inflammatory and Neuroprotective Effects of Constituents Isolated from Rhodiola rosea.

To determine the biological activity of Rhodiola rosea, the protein expression of iNOS and proinflammatory cytokines was measured after the activation of murine microglial BV2 cells by LPS under the exposure of constituents of Rhodiola rosea: crude extract, rosin, rosarin, and salidroside (each 1-50 µ g/mL). The LPS-induced expression of iNOS and cytokines in BV2 cells was suppressed by the constituents of Rhodiola rosea in a concentration-dependent manner. Also the expression of the proinflammatory factors iNOS, IL-1 ß , and TNF- α in the kidney and prefrontal cortex of brain in mice was suppressed by the oral administration of Rhodiola rosea crude extract (500 mg/kg). To determine the neuroprotective effect of constituents of Rhodiola rosea, neuronal cells were activated by L-glutamate, and neurotoxicity was analyzed. The L-glutamate-induced neurotoxicity was suppressed by the treatment with rosin but not by rosarin. The level of phosphorylated MAPK, pJNK, and pp38 was increased by L-glutamate treatment but decreased by the treatment with rosin and salidroside. These results indicate that Rhodiola rosea may have therapeutic potential for the treatment of inflammation and neurodegenerative disease.

Enhancement of Anxiety and Modulation of TH and pERK Expressions in Amygdala by Repeated Injections of Corticosterone.

Repeated stress induces corticosterone release. However, it is not clear that stress results in further elevation of corticosterone levels, and the roles of released corticosterone to aggravate stress-related symptoms are also not clear. This study investigated whether neuronal modulation was induced in the amygdala after two kinds of stress, that is, such as electric shock and corticosterone injection. It was found that stress by electric shock decreased the expression of tyrosine hydoroxylase (TH) in the amygdala while the expression of pERK was increased. However, there is no difference in the expressions of TH and pERK in the frontal cortex compared with those of the control group. The level of corticosterone was significantly increased in the serum after stress. To determine the effect of corticosterone on the induction of anxiety and the expression of TH, the rats received corticosterone (20 mg or 40 mg/kg i.p.) for 1 day, 1 week, 2 weeks and 3 weeks, respectively. The spent time in open arms of the EPM (elevated plus maze) test was significantly decreased after 1 week, 2 weeks and 3 weeks. The time spent in open arms of the EPM test after repeated injections of corticosterone was significantly decreased in a dose-dependent manner. The expression of TH in the amygdala was reduced after following repeated corticosterone treatment for 2 weeks and 3 weeks. Collectively, this study suggests that corticosterone has a major role in the induction of anxiety and the modulation of TH expression, at least, in the amygdala.

Modulation of sphingosine 1-phosphate and tyrosine hydroxylase in the stress-induced anxiety.

Stress causes endocrinological changes and leads to induce anxiety. It was determined the anxiety and stress-related endocrinological changes through the observation of the level of glucocorticoid and sphingolipid metabolites in serum after stress. Immobilized stress and electric shock was applied to rats for 7 days. This study investigated the induction of anxiety, changes of TH and pERK expression in cortex and amygdala after stress. Also it was determined the changes of glucocorticoid and anxiety when the rats were given stress after amygdala lesion. The stress-given rats spent a lesser percentage of time significantly in the open arm than the control rats. The elevated level of glucocorticoid after stress was suppressed in amygdala lesion group. The expression of TH in the amygdala was decreased, but the expression of TH was not changed in the cortex after stress. To investigate the changes in sphingolipid metabolites after stress, the levels of sphingosine and the phosphate form of sphingolipid (So-1-P) were analyzed in serum. The level of So-1-P was elevated after stress and anxiety was observed after the So-1-P infusion (100 pmol/10 µl/h, i.c.v., for 7 days). Continuous infusion of So-1-P for 7 days led to the significant decrease of TH expression in the amygdala. In conclusion, the results of this study indicate that the lesion of amygdala suppressed the stress-induced anxiety and elevation of glucocorticoid in serum. It was also observed that expression of TH in amygdala as well as increased levels of glucocorticoid in serum might be responsible biomarker, at least in part, of chronic stress. These results suggest that the elevation of So-1-P might be involved in induction of anxiety during stress by the modulation of dopaminergic system in amygdala.

Design, synthesis, and biological evaluation of 3,4,5-trimethoxyphenyl acrylamides as antinarcotic agents.

The synthesis and biological evaluation of 3,4,5-trimethoxyphenyl acrylamides 1a-f as novel antinarcotic agents are described. The molecules were prepared by the Wittig reaction, followed by a coupling reaction between 3,4,5-trimethoxycinnamic acid (9) and aliphatic amines, which resulted in good yields. When tested for biological activity, compounds 1d-f exhibited strong inhibitory effects on the morphine withdrawal syndrome in mice due to their high binding affinities with serotonergic 5-HT1A receptors.

Alteration of sphingolipid metabolism and pSTAT3 expression by dietary cholesterol in the gallbladder of hamsters.

Cholesterol and sphingolipids are major lipid constituents of the plasma membrane and have been implicated in a number of human diseases, such as atherosclerosis, fatty liver, diabetes mellitus, coronary heart disease, and hypertension. However, the relationship between cholesterol and sphingolipid metabolism has not been investigated. The purpose of this study was to determine whether dietary cholesterol would induce the alteration of sphingolipid metabolism in hamsters. Hypercholesterolemia was induced in hamsters by placing them on an experimental diet containing 0.5% cholesterol plus 0.5% choline chloride for 8 and 12 weeks. The serum profile of the hamsters showed that the administration of cholesterol increased the levels of total cholesterol, LDL cholesterol, and triglycerides as well as the activities of GOT and GPT. The levels of ceramide and sphingosine-1-phosphate (So-1-P) were remarkably elevated by 6-fold, respectively, in the bile juice of cholesterol-fed hamsters. Interestingly, the levels of iNOS and GFAP were increased in the gallbladders of cholesterol-fed hamsters. In addition, the immunostaining of pSTAT3 was increased on the gallbladder epithelium after cholesterol feeding. These results suggest that sphingolipid metabolism may be regulated in the bile juice during cholesterol feeding and may be a potential target for the treatment of hypercholesterolemia-induced diseases.

Synthesis of novel trans-stilbene derivatives and evaluation of their potent antioxidant and neuroprotective effects.

A convenient synthesis and the biological properties of new amides, esters and other derivatives of trans-stilbene are described. The key synthetic strategies involve the Wittig-Horner reaction of a phosphonium salt 9 and an aldehyde 10 to generate (E)- or (Z)-olefins and a coupling reaction of an acid 12 and various amines 13a-n to give trans-stilbene derivatives 15a-n in high yields. A amide derivative 15g showed three times more in vitro free radical-scavenging activity than resveratrol, while another 15d exhibited strong inhibitory activity against lipopolysaccharide (LPS)(a)-induced NO generation. Allylamide analogue 15a showed the most potent neuroprotective activity in glutamate-induced primary cortical neuron cells.

The neuroprotective effects of benzylideneacetophenone derivatives on excitotoxicity and inflammation via phosphorylated Janus tyrosine kinase 2/phosphorylated signal transducer and activator of transcription 3 and mitogen-activated protein K pathways.

To search for new neuroprotective compounds, novel benzylideneacetophenone compounds (JCI, (3E)-4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one; JC2, (1E)-1-(4-hydroxy-3-methoxyphenyl)hept-1-en-3-one; JC3, (2E)-3-(4-hydroxy-3-methoxyphenyl)phenylpro-2-en-l-one; JC4, (1E)-1-(4-hydroxy-3-methoxyphenyl)-5-phenylpent-1-en-3-one; JC5, (1E)-3-(4-hydroxy-3-methoxyphenyl)-6-phenylhex-1-en-3-one; JC6, (1E)-1-(4-hydroxy-3-methoxyphenyl]-7-phenylhept-1-en-3-one) were synthesized, and their potential to prevent neurotoxicities were evaluated. All compounds (JC1-JC6) showed considerable effect on free radical scavenging, the inhibition of glutamate-induced neurotoxicity in cortical cells, and the suppression of lipopolysaccharide (LPS)-induced nitric oxide (NO) generation in microglia. (2E)-3-(4-Hydroxy-3-methoxyphenyl)-phenylpro-2-en-1-one (JC3) exhibited the most potent neuroprotective effect in ischemia model using organotypic hippocampal culture and middle cerebral artery occlusion (MCAO). Based on the above-mentioned results, the mechanisms underlying the biological activity of JC3, which exhibited potent antiexcitotoxic and anti-inflammatory effects, were determined using cortical neuronal cells and microglia. Compound JC3 exerted a neuroprotective effect on oxygen-glucose deprivation- and hydrogen peroxide-induced cytotoxicity in cultured cortical cells. In addition, it suppressed the generation of NO, proinflammatory cytokines, and reactive oxygen species in LPS-treated microglial cells. It also suppressed the activation of phosphorylated Janus tyrosine kinase 2/phosphorylated signal transducer and activator of transcription 3 and mitogen-activated protein kinase (MAPK) in activated microglia and in cortex and striatum after 3 days of the MCAO in mice. These results demonstrated that JC3 might affect a set of intracellular signaling cascades, including the Janus tyrosine kinase/signal transducers and activators of transcription and MAPK pathways. This study suggests that benzylideneacetophenone derivative could be useful antineurotoxic agents.

Neuroprotective effect of benzylideneacetophenone derivative on the MPTP model of neurodegeneration in mice.

In this study, we investigated the neuroprotective effect of a benzylideneacetophenone derivative, JC3, in a mouse model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD). C57BL/6 mice were treated with MPTP (30 mg/kg, i.p.) for 5 consecutive days. JC3 (10 mg/kg, i.p.) treatment was initiated 2 h after the first administration of MPTP and then at 24-h intervals for 3 consecutive days. The mice were sacrificed for analyses 7 days after the last MPTP injection. Immunohistochemistry and Western blot were used to determine the expression levels of tyrosine hydroxylase (TH), dopamine transporter (DAT), OX-42 (a marker of microglial activation), and glial fibrillary acid protein (GFAP, a marker of astrocyte activation) in the substantia nigra (SN) and striatum (ST). The results of these experiments demonstrated that JC3 restored the decreased TH-immunoreactivity (IR) and DAT and JC3 attenuated the increase in OX-42, GFAP, and COX-2 on the SN and ST on day 7 post-MPTP injection. These results suggest that JC3 can be a neuroprotective agent in an MPTP-induced model of PD.

Efficient synthesis and neuroprotective effect of substituted 1,3-diphenyl-2-propen-1-ones.

An efficient synthesis involving a key aldol reaction and biological properties of 1,3-diphenyl-2-propen-1-ones 8- 20 is described. The in vitro activity for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging of 10 and 11 was 2 times higher than that for resveratrol. Compounds 9 and 11 were the strongest in suppression of in vitro nitric oxide (NO) generation and antiexcitotoxicity. Molecular modeling proposes an electron-donating group at the para position of acetophenones that leads to a dramatic increase in the suppression of NO production.

Changes in iNOS, GFAP and NR1 expression in various brain regions and elevation of sphingosine-1-phosphate in serum after immobilized stress.

Several studies have been suggested that long-term exposure to stress has detrimental effects on various brain functions and leads to neurodegenerative changes. However, the precise mechanism by which stress induces brain damage or neurodegenerative change is still a matter of debate. This study investigated the damage of neuronal cells involving in the expression of iNOS, NR1, and GFAP in various brain regions and characterized the change of sphingolipid metabolites as a biomarker of physiological change in serum after 3 weeks of repeated immobilization. In this report, the expression of iNOS, GFAP and NR1 in the brain of rats exposed to chronic immobilization stress was investigated. The expression of iNOS, GFAP and NR1 was elevated in the cortex and hippocampal area after 3 weeks of repeated immobilization. Immunoreactivity for GFAP and vimentin, as a marker of reactive gliosis, was also elevated in the cortex and hippocampus. The level of sphingolipids was measured in order to assess the changes in sphingolipid metabolites in the serum of rats exposed to stress. Interestingly, the level of So-1-P was increased in the plasma of rats subjected to 6-h immobilization stress than repeated immobilization. To further investigate the modulating effect of increased So-1-P in various brain regions, So-1-P was infused into the lateral cerebroventricle at a rate of 100 pmol/10 mul/h for 7 days. The expression of iNOS and NR1 was elevated in the cortex, hippocampus, striatum, and cerebellum after So-1-P infusion into the cerebroventricle, while the level of GFAP was elevated in the hippocampus and striatum. Interestingly, the expression levels of iNOS, GFAP, and NR1 were increased by the direct application of So-1-P to cultured cortical cells. These results suggest that NO production via iNOS expression, the NR1 expression, the activation of astrocytes, and the elevation of So-1-P may cause neurodegenerative changes in rats subjected to chronic immobilization and that the elevation of So-1-P by stress exposure would be one of the stress signal molecules.

Synthesis of 1,3-diphenyl-2-propen-1-one derivatives and evaluation of their biological activities.

A simple synthesis and biological properties of 1,3-diphenyl-2-propen-1-ones 18-22 and 25-26 are described. The key synthetic strategies involve Grignard reaction of aldehyde 2 and oxidation reaction of 8-12 in high yields. The prepared compounds 18-22 and 25-26 were evaluated for free-radical scavenging, suppression of LPS-induced NO generation, and anti-excitotoxicity in vitro. It was found that a couple of compounds, especially 21 and 26, were potent suppressors of NO generation and demonstrated anti-excitotoxicity with the concentration range 10-20 microM in vitro.

Cytochemical alterations in the rat retina by LPS administration.

LPS-induced inflammation and changes in protein phosphorylation and the JAK-STAT pathway accompanying glial activation after LPS treatment, were followed by analyzing secreted proinflammatory cytokine levels. The administration of LPS caused tyrosine phosphorylation of STAT3 in retinae and induced glial fibrillary acidic protein. (GFAP) from the nerve fiber layer to the ganglion cell layer. Our results suggest that the LPS-induced activation of the JAK2/STAT3 signaling pathway may play a key role in the induction of astrogliosis. However, no significant increase in vimentin, OX-42 or inducible nitric oxide synthase (iNOS) expressions were observed after LPS administration. Sphingosine kinase catalyzes the conversion of sphingosine to sphingosine-1-phosphate (So-1-P), a sphingolipid metabolite that plays important roles in angiogenesis, inflammation, and cell growth. In the present study, it was found that sphingolipid metabolite levels were elevated in the serum and retinae of LPS-injected rats. To further investigate the chronic effect of increased So-1-P in the retina, So-1-P was infused intracerebroventricularly (i.c.v.) into rats using an osmotic minipump at 100 pmol/10 microl h(-1) for 7 days, and was found to increase retinal GFAP expression. These observations suggest that LPS induces the activation of retinal astrocytes via JAK2/STAT3 and that LPS affects So-1-P generation. Our findings also suggest that elevated So-1-P in the retina and/or in serum could induce cytochemical alterations in LPS treated or inflamed retinae.

Elevation of sphinganine 1-phosphate as a predictive biomarker for fumonisin exposure and toxicity in mice.

Fumonisins are specific inhibitors of ceramide synthase in sphingolipid metabolism. An alteration in sphingolipid metabolism as a result of fumonisin B1 (FB1) exposure is related to cell death, and sphinganine/sphingosine ratio has been used as an indicator of fumonisin exposure in animals. The objective of this study was to investigate a new biochemical marker for the prediction of fumonisin-induced toxicity. When mice were treated with FB1 (10 mg/kg ip/d) for 5 d, the serum levels of sphingoid bases and their 1-phosphate were markedly elevated. The accumulation of sphingosine 1-phosphate (So-1-P) and sphinganine 1-phosphate (Sa-1-P) in serum following FB1 treatment was more apparent than elevated levels of sphingosine (So) and sphinganine (Sa). Sa-1-P/So-1-P ratio in serum was more elevated than Sa/So ratio following fumonisin B1 treatment, indicating that phosphorylation of sphingoid bases may be a sensitive biomarker for fumonisin exposure. In addition, the tissue levels of Sa and Sa-1-P were also significantly elevated in kidneys, liver, heart, lung and brain. FB1-induced toxicity was confirmed microscopically in both liver and kidneys. Liver lesions consisted of centrilobular hypertrophy and cytoplasmic vacuolization. In addition, hepatic binucleated cells were increased and acidophilic body was observed in FB1-treated mice. Kidney lesions were consistent with tubular nephrosis, and tubules were dilated and contained cell debris in FB1-exposed mice. These results suggested that the elevation of Sa-1-P as well as Sa in serum would be a specific biomarker for predicting FB1 exposure, and elevated tissue levels of Sa-1-P may be related to fumonisin toxicity in animals.

Attenuation of morphine tolerance and withdrawal syndrome by coadministration of nalbuphine.

Morphine has been used widely on the treatment of many types of chronic pain. However the development of tolerance to and dependence on morphine by repeat application is a major problem in pain therapy. The purpose of the present study was to investigate whether combined administration of nalbuphine with morphine affects the development of tolerance to and dependence on morphine. We hypothesize that the use of nalbuphine, kappa-agonist may prove to be useful adjunct therapy to prevent morphine-induced undesirable effects in the management of some forms of chronic pain. Morphine (10 mg/kg) was injected to rats intraperitoneally for 5 day. The variable dose of nalbuphine (0.1, 1.0 and 5.0 mg/kg) was administered (i.p.) in combination with morphine injection. The development of morphine tolerance was assessed by measuring the antinociceptive effect with the Randall-Selitto apparatus. The development of dependence on morphine was determined by the scoring the precipitated withdrawal signs for 30 min after injection of naloxone (10 mg/kg, i.p.). Nalbuphine did not attenuate antinociceptive effect of morphine in rats. Interestingly, combined administration of morphine with nalbuphine (10:1) significantly attenuated the development of dependence on morphine. The elevation of [3H]MK-801 binding in frontal cortex, dentate gyrus, and cerebellum after chronic morphine infusion was suppressed by the coadministration of nalbuphine. In addition, the elevation of NR1 expression by morphine was decreased by the coadministration of nalbuphine in rat cortex. These results suggest that the coadministration of nalbuphine with morphine in chronic pain treatment can be one of therapies to reduce the development of tolerance to and dependence on morphine.

Synthesis and evaluation of biological properties of benzylideneacetophenone derivatives.

A series of yakuchinone B 1f and its analogs 1a-e was synthesized and evaluated for free radical scavenging, suppression of LPS-induced NO generation, cytotoxicity and anti-excitotoxicity in vitro. Compound 1c exhibited potent anti-excitotoxicity, while all compounds 1a-f showed considerable effects of free radical scavenging, suppression of LPS-induced NO generation, and cytotoxicity in microglia.