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Licorice,Glycyrrhizae radix, is one of the herbal medicines in East Asia that has been commonly used for treating various diseases, including stomach disorders. This study investigated the effect of licorice on arsenite (As)-induced cytotoxicity in H4IIE cells, a rat hepatocyte-derived cell line. Cell viability was significantly diminished in As-treated H4IIE cells in a time and concentration-dependent manner. Furthermore, results from flow cytometric assay and DNA laddering in H4IIE cells showed that As treatment induced apoptotic cell death by activating caspase-3. Licorice (0.1 and 1.0 mg ml−1) treatment significantly inhibited cell death and the activity of caspase-3 in response to As exposure. These results demonstrate that licorice induced a cytoprotective effect against As-induced cell death by inhibition of caspase-3.

To understand antitumor activity of an edible mushroom Paecilomyces japonica , we have investigated its effect on the cell cycle. When Jurkat T cells transfected with vector (JT/Neo) or Bcl-2 gene (JT/Bcl-2) were treated with the ethyl acetate extract (4–6 μg/ml) of P. japonica for 40 h, JT/Neo cells underwent apoptosis with no detectable G 1 -arrest, whereas JT/Bcl-2 cells that failed to induce apoptosis accumulated at the G 1 along with reduction of hyperphosphorylated Rb. The cdk4, cyclin E and A, required for the G 1 -cdks that phosphorylate Rb, were markedly downregulated in JT/Bcl-2 cells, and the G 1 -cdk inhibitor p27 Kip1 was significantly upregulated. GC–MS analysis identified a component structurally similar to diacetoxyscirpenol as the effective ingredient contributing to the G 1 -arrest. These results demonstrate that P. japonica can arrest the cell cycle of JT/Bcl-2 cells at the G 1 by suppressing phosphorylation of Rb through downregulation of the activity of G 1 -cdks, and thus potentiates apoptotic cell death.

The glutamatergic system may play a vital role in regulating neurobehavioral effects of various drugs of abuse. In the present study, we evaluated the effects of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective antagonist of the type 5 metabotropic glutamate receptor (mGluR5) on the acquisition, expression and reinstatement of ethanol conditioned place preference (CPP). In the ethanol acquisition study, mice were conditioned with saline or ethanol (20% v/v, 2g/kg) on alternating days for 8 consecutive days and were given MPEP 10 min before ethanol conditioning. In another experiment, animals were conditioned with 2g/kg ethanol and MPEP was administered 10 min prior to the post-conditioning test. In a reinstatement study, following the extinction phase, animals were pretreated with MPEP 10 min prior to a priming injection of 1.0 g/kg ethanol. The mGluR5 antagonist MPEP significantly reduced the expression and the reinstatement in dose-dependent manner, but not acquisition of ethanol-induced CPP. These results indicate that mGluR5 may be involved in the expression and reinstatement of conditioned rewarding effects of ethanol, but not the acquisition of ethanol, which provide an evidence that mGluR5 blockade might make dissociable contributions during the training (acquisition phase), the performance of behavior (expression phase) and reinstatement.

To develop an oral vaccine against Helicobacter pylori infection, we have expressed the H. pylori cag12 (HP0532) gene, encoding the outer membrane protein Cag12 (31 kDa), in a live delivery vehicle Lactococcus lactis. The cag12 gene was amplified by polymerase chain reaction (PCR) using the genomic DNA of H. pylori K51 isolated from Korean patients. DNA sequence analysis revealed that the cag12 gene of H. pylori K51 has 98.1 and 97.4% identity with individual cag12 genes of the H. pylori 26695 and J99, respectively. The GST-Cag12 fusion protein, produced using the Escherichia coli expression system, was used to raise a rat polyclonal anti-Cag12 antibody. The PCR-amplified cag12 gene of H. pylori K51 was cloned in the E. coli-L. lactis shuttle vector (pMG36e) and transformed into L. lactis. Western blot analysis demonstrated that the Cag12 protein was expressed in the L. lactis transformant, with a maximum level at the log phase without extracellular secretion. The oral administration of the transformant into mice resulted in the generation of the anti-Cag12 antibody in serum in two out of five cases. These results suggest that the recombinant L. lactis, which expresses Cag12, may be applicable as an oral vaccine to induce protective immunity against H. pylori.

To improve the oral bioavailability of poorly water-soluble ibuprofen with poloxamer and menthol, the effects of menthol and poloxamer 188 on the aqueous solubility of ibuprofen were investigated. The dissolution and pharmacokinetic study of ibuprofen delivered by the ibuprofen-loaded preparations composed of poloxamer 188 and menthol were then performed. In the absence of poloxamer, the solubility of ibuprofen increased until the ratio of menthol to ibuprofen increased from 0:10 to 4:6 followed by an abrupt decrease in solubility above the ratio of 4:6, indicating that four parts menthol formed eutectic mixture with six parts ibuprofen. In the presence of poloxamer, the solutions with the same ratio of menthol to ibuprofen showed an abrupt increase in the solubility of ibuprofen. The poloxamer gel with menthol/ibuprofen ratio of 1:9 and higher than 15% poloxamer 188 showed the maximum solubility of ibuprofen, 1.2 mg/mL. The simultaneous addition of menthol and poloxamer 188 significantly improved the dissolution rates of ibuprofen from aqueous solution due to the ibuprofen solubility-improving effect of menthol in the presence of poloxamer. Furthermore, the ibuprofen-loaded preparation with menthol and poloxamer 188 gave significantly higher initial plasma concentrations, Cmax, and AUC of ibuprofen than did the preparation without menthol and poloxamer 188, indicating that the simultaneous addition of menthol and poloxamer 188 could improve the oral bioavailability of ibuprofen in rats. In modern pain management it is always desirable for the ibuprofen-loaded preparation with poloxamer 188 and menthol to show a rapid onset of action with a minimal phase of lag time to feel the decreased pain. From an industry point of view, it is more desirable for a formulation to be fast acting, easy to use, and cost effective. Thus, the ibuprofen-loaded preparation with poloxamer 188 and menthol was a more effective oral dosage form for poorly water-soluble ibuprofen.

A high concentration of glucose has been implicated as a causal factor in initiation and progression of diabetic kidney complications, and there is evidence to suggest that hyperglycemia increases the production of free radicals and oxidant stress. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP+-dependent isocitrate dehydrogenase (IDPm) to supply NADPH for antioxidant systems. In this report, we demonstrate that modulation of IDPm activity in HEK293 cells, an embryonic kidney cell line, regulates high glucose-induced apoptosis. When we examined the protective role of IDPm against high glucose-induced apoptosis with HEK293 cells transfected with the cDNA for mouse IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPm expressed in target cells and their susceptibility to apoptosis. The results suggest that IDPm plays an important protective role in apoptosis of HEK293 cells induced by a high concentration of glucose and may contribute to various pathologies associated with the long-term complications of diabetes.

Clinical trials are currently underway to determine the effectiveness of acupuncture in the treatment of drug abuse. However, there are still many unanswered questions about the basic mechanisms of acupuncture. Studies have shown that the GABA B receptor system may play a significant modulatory role in the mesolimbic system in drug abuse, including ethanol. The in vivo microdialysis study was designed to investigate the effect of acupuncture on acute ethanol-induced dopamine release in the nucleus accumbens and the potential role of the GABA B receptor system in acupuncture. Male Sprague–Dawley rats were administered with the highly selective GABA B antagonist SCH 50911 (3 mg/kg, i.p.) 1 h prior to an intraperitoneal injection of ethanol (1 g/kg). Immediately after ethanol treatment, acupuncture was given at bilateral Shenmen (HT7) points for 1 min. Acupuncture at the specific acupoint HT7, but not at control points (PC6 or tail) significantly decreased dopamine release in the nucleus accumbens. Inhibition of dopamine release by acupuncture was completely prevented by SCH 50911. These results suggest that stimulation of specific acupoints inhibits ethanol-induced dopamine release by modulating GABA B activity and imply that acupuncture may be effective in blocking the reinforcing effects of ethanol.

Repeated injections of nicotine can produce behavioral sensitization, as evidenced by an enhanced locomotor response to a subsequent injection of the drug. Behavioral sensitization has been suggested as a model for studying drug addiction. Acupuncture as a therapeutic intervention is widely used for treatment for many functional disorders, such as substance abuse and mental dysfunction. We examined the effect of acupuncture on nicotine-induced behavioral locomotor activity and c-fos expression in the nucleus accumbens and striatum utilizing the immunocytochemical detection of the Fos protein. The rats were given repeated daily nicotine injections (0.4 mg/kg s.c., twice daily for 7 days) followed by one challenging injection on the 4th day after the last daily injection. Acupuncture at zusanli (ST36), but not control, significantly attenuated expected increase in nicotine-induced locomotor activity and Fos-like-immunoreactivity in the nucleus accumebns and striatum to subsequent nicotine challenge. These findings suggest that acupuncture has a therapeutic effect on nicotine addiction, possibly by modulating postsynaptic neuronal activity in the nucleus accumbens and the striatum.

To improve the bioavailability of poorly water-soluble ibuprofen in the rectum with poloxamer and menthol, the effects of menthol and poloxamer 188 on the aqueous solubility of ibuprofen were investigated. The dissolution and pharmacokinetic study of ibuprofen delivered by the poloxamer gels composed of poloxamer 188 and menthol were then performed. In the absence of poloxamer, the solubility of ibuprofen increased until the ratio of menthol to ibuprofen increased from 0:10 to 4:6 followed by an abrupt decrease in solubility above the ratio of 4:6, indicating that four parts menthol formed eutectic mixture with six parts ibuprofen. In the presence of poloxamer, the solutions with the same ratio of menthol to ibuprofen showed abrupt increase in the solubility of ibuprofen. The poloxamer gel with menthol/ibuprofen ratio of 1:9 and higher than 15% poloxamer 188 showed the maximum solubility of ibuprofen, 1.2mg/ml. Menthol improved the dissolution rates of ibuprofen from poloxamer gels. Release mechanism showed that the release rate of ibuprofen from the poloxamer gels without menthol was independent of the time but the drug might be released from the poloxamer gels with menthol by Fickian diffusion. Furthermore, the poloxamer gel with menthol (poloxamer/menthol/ibuprofen (15%/0.25%/2.5%)) gave significantly higher initial plasma concentrations, C(max) and AUC of ibuprofen than did solid suppository, indicating that the drug from poloxamer gel could be more absorbed than that from solid one in rats. Thus, the poloxamer gel with poloxamer 188 and menthol was a more effective rectal dosage form for ibuprofen.

Background: Psychological stressors may cause affective disorders, such as depression and anxiety, by altering expressions of corticotropin releasing factor (CRF), serotonin (5-HT), and tyrosine hydroxylase (TH) in the brain. This study investigated the effects of essential oil from Asarum heterotropoides (EOAH) on depression-like behaviors and brain expressions of CRF, 5-HT, and TH in mice challenged with stress. Methods: Male ICR mice received fragrance inhalation of EOAH (0.25, 0.5, 1.0, and 2.0 g) for 3 h in the special cage capped with a filter paper before start of the forced swimming test (FST) and tail suspension test (TST). The duration of immobility was measured for the determination of depression-like behavior in the FST and TST. The selective serotonin reuptake inhibitor fluoxetine as positive control was administered at a dose of 15 mg/kg (i.p.) 30 min before start of behavioral testing. Immunoreactivities of CRF, 5-HT, and TH in the brain were also measured using separate groups of mice subjected to the FST. Results: EOAH at higher doses (1.0 and 2.0 g) reduced immobility time in the FST and TST. In addition, EOAH at a dose of 1.0 g significantly reduced the expected increases in the expression of CRF positive neurons in the paraventricular nucleus and the expression of TH positive neurons in the locus coeruleus, and the expected decreases of the 5-HT positive neurons in the dorsal raphe nucleus. Conclusion: These results provide strong evidence that EOAH effectively inhibits depression-like behavioral responses, brain CRF and TH expression increases, and brain 5-HT expression decreases in mice challenged with stress.

Reactive oxygen species (ROS) have been implicated in the development of behavioral sensitization following repeated cocaine exposure. We hypothesized that increased ROS following cocaine exposure would act as signaling molecules in the mesolimbic dopamine (DA) system, which might play an important role in mediating the reinforcing effects of cocaine. The aim of this study was to evaluate cocaine enhancement of brain metabolic activity and the effects of ROS scavengers on cocaine self-administration behavior, cocaine-induced ROS production in the nucleus accumbens (NAc) and cocaine enhancement of DA release in the NAc. Metabolic neural activity monitored by temperature and oxidative stress were increased in NAc following cocaine exposure. Systemic administration of the ROS scavenger N-tert-butyl-α-phenylnitrone (PBN) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), either pre- or post-treatment, significantly decreased cocaine self-administration without affecting food intake. Infusion of TEMPOL into the NAc inhibited cocaine self-administration. Increased oxidative stress was found mainly on neurons, but not astrocytes, microglia or oligodendrocytes, in NAc of rats self-administering cocaine. TEMPOL significantly attenuated cocaine-induced enhancement of DA release in the NAc, compared to saline controls. TEMPOL had no effect on the enhancement of DA release produced by the DA transporter inhibitor GBR12909. Taken together, these findings suggest that enhancement of ROS production in NAc neurons contributes to the reinforcing effect of cocaine.

In the previous study, acupuncture at HT7 has shown to attenuate the self-administration of morphine at a low dose (0.1mg/kg). In this study, it was further investigated whether acupuncture at HT7 could attenuate the morphine self-administration at a high dose (0.5mg/kg). Male Sprague-Dawley rats weighing 270-300g were used. After surgery of catheterization, animals were trained to self-administer morphine solution (0.5mg/kg) using daily 1h session under fixed ratio 1 schedule for 3 weeks. Animals that had shown stable morphine-taking (establish baseline: variation less than 20% of the mean of three consecutive days) were subjected to the acupuncture treatment. Bicuculline and SCH 50911 were used to investigate the possible relation between the effect of acupuncture and the GABA receptor system. Acupuncture at HT7, but not at control acupoint, LI5, suppressed spontaneous morphine-taking behavior significantly. In addition, the effect of acupuncture was blocked by both GABA receptor antagonists. The results of this study suggest that acupuncture at HT7 suppresses morphine-taking behavior through the mediation of GABA receptor system.

A new cytotoxic guaianolide was isolated from Chrysanthemum boreale Makino. The structure of guaianolide was elucidated as 8-acetoxy-4,10-dihydroxy-2,11(13)-guaiadiene-12,6-olide (1). Compound 1 exhibited cytotoxic activity (IC(50)< or =4 microg/ml) against all five human cancer cell lines tested.

Expression of the active urease of the enterobacterium, Klebsiella aerogenes, requires the presence of the accessory genes (ureD, ureE, ureF, and ureG) in addition to the three structural genes (ureA, ureB, and ureC). These accessory genes are involved in functional assembly of the nickel-metallocenter for the enzyme. Characterization of ureF gene has been hindered, however, since the UreF protein is produced in only minute amount compared to other urease gene products. In order to overexpress the ureF gene, a recombinant pMAL-UreF plasmid was constructed from which the UreF was produced as a fusion with maltose-binding protein. The MBP-UreF fusion protein was purified by using an amylose-affinity column chromatography followed by an anion exchange column chromatography. Polyclonal antibodies raised against the fusion protein were purified and shown to specifically recognize both MBP and UreF peptides. The UreF protein was shown to be unstable when separated from MBP by digestion with factor Xa.

The role of neuropeptide Y (NPY) in the central nucleus of amygdala (CeA) in the preventive effects of acupuncture against ethanol withdrawal-induced anxiety was investigated. Rats were treated with 3g/kg/day of ethanol for 28 days, followed by 3 days of withdrawal. Bilateral acupuncture treatment at HT7 (Shen-Men), PC6 (Nei-Guan) or a non-acupoint was respectively added to the rats during the withdrawal once a day for three days. Enzyme-linked immunosorbent assays and real-time polymerase chain reaction analyses showed there was a significant decrease in NPY protein and mRNA expression in the CeA during ethanol withdrawal, which was reversed by acupuncture at HT7 but neither at PC6 nor at a non-acupoint. Acupuncture at HT7 also greatly inhibited the decrease in cAMP response element-binding protein (CREB) phosphorylation in the CeA. In elevated plus maze tests, a selective NPY Y1 receptor antagonist BIBP 3226 into the CeA before the acupuncture abolished almost completely the anxiolytic effect of acupuncture at HT7. These results suggest that acupuncture at HT7 rescues the depletion of amygdaloid NPY and reverses the decrease in CREB phosphorylation to produce anxiolytic effects during ethanol withdrawal.

To help the clinicians prescribe acupoints easily and effectively, we developed one simple flow chart to select acupoints. This study aimed to evaluate the usefulness of flow chart to select acupoints in dogs. Total 102 dogs showing intervertebral disc disease (IVDD) (n=12), vomiting (n=11), diarrhea (n=2), abdominal pain (n=5), cough (n=66), or epilepsy (n=6) received acupuncture treatment according to the chart, and its outcomes were evaluated as regards clinical symptoms, duration, treatment numbers, and recovery time. Dogs (8/8) with IVDD from grades I to III recovered over periods of 5 days to 6 weeks after 1–12 treatments, while 1/4 dogs with grade IV recovered over 7 weeks after 15 treatments. Vomiting dogs with acute/subacute (n=8) and chronic symptoms (n=3) required about 1 and 7 treatments to recover fully, respectively. All dogs (n=5) with abdominal pain showed fast relief within 24 hours after acupuncture. Two diarrhea cases recovered over 2–9 days after 1-2 treatments. Fifty-four of 66 coughing dogs were recovered by 1-2 treatments. And 5 of 6 epilepsy dogs under a regular acupuncture treatment had no epileptic episode during followup of 12 months. These results suggest that this flow chart can help the clinicians prescribe acupoints effectively.

In this study, we investigated the effects of saikosaponin A (SSA), a major compound of Bupleurum falcatum L., on morphine self-administration behavior. Male Sprague-Dawley rats were trained to self-administer intravenous morphine (0.1mg/kg per injection over 5s) during daily 1-h sessions under a fixed-ratio 1 schedule. Rats were pretreated with SSA (0.25, 0.5, 1.0mg/kg) by intraperitoneal injection 30 min prior to the start of the test session. Results demonstrated that pretreatment with SSA reduced morphine-maintained responding dose-dependently. Additionally, SSA inhibition of morphine-reinforced behavior was blocked by the selective GABA(B) receptor antagonist (2S)(+)-5,5-dimethyl-2-morpholineacetic acid (SCH 50911), but not the selective GABA(A) receptor antagonist bicuculline. Together, these results suggest that SSA may effectively suppress morphine-reinforced behavior by activating GABA(B) receptors.

Our previous studies have shown that acupuncture attenuates morphine self-administration and sensitization behavior as well as withdrawal signs. The present study was designed to investigate the role of acupuncture in the reinstatement of morphine seeking. Male Sprague-Dawley rats weighing 270-300 g were subjected to intravenous catheterization after food training. The animals were trained to self-administer morphine (1.0mg/kg, 3 weeks), followed by extinction (1 week). Extinction conditions were introduced by substituting saline for morphine. The rats were then tested for reinstatement of morphine self-administration by a priming injection of morphine (0.25mg/kg). To see whether acupuncture can reduce morphine reinstatement, acupuncture was performed at SI5 or LI5 for 1 min immediately before a morphine injection. To further test the involvement of gamma aminobutyric acid (GABA) receptors in acupuncture effects, GABA receptor antagonists were injected before acupuncture. In the present results, acupuncture at SI5, but not at control acupoint LI5 attenuated the reinstatement of morphine seeking behavior, which was blocked by the GABA receptor antagonists. It suggests that acupuncture can reduce the reinstatement of morphine seeking, possibly due to the mediation of GABA receptor system.

Sauchinone is a lignan isolated from Saururus chinensis known to suppress nitric oxide (NO) activity. Previous studies demonstrate that NO plays a key role in methamphetamine-induced neurotoxicity. Thus, we hypothesized that sauchinone could have a suppressive effect on the neurotoxicity induced by methamphetamine. Repeated injections of methamphetamine cause degeneration of dopaminergic nerve terminals, whereas sauchinone treatment significantly prevented this degeneration. Sauchinone treatment also inhibited the methamphetamine-induced activation of glia cells and the production of NO via a blockade of inducible NO synthase protein expression. Our results suggest that sauchinone can prevent methamphetamine-induced neurotoxicity through the suppression of NO production. Keywords:: methamphetamine, sauchinone, nitric oxide

Testosterone 5α-reductase inhibitors represent important therapeutic drugs for use against androgen-dependent diseases such as benign prostatic hyperplasia, male pattern baldness, and acne. We have searched for inhibitors of rat prostate testosterone 5α-reductase in the cultured broths of many kinds of soil bacteria, and have found that cultured soybean-casein digest broths of certain bacterial strains have a potent inhibitory effect on the enzyme. We tested 10 selected isoflavonoids, including isoflavones and O-methylated isoflavones, for inhibitory effects on rat prostate testosterone 5α-reductase to determine the important structural elements for inhibition of the enzyme. Genistein, biochanin A, equol, and 3′,4′,7-trihydroxyisoflavone showed considerably higher inhibitory effects whereas daidzein, formononetin, glycitein, prunetin, ipriflavone, and 4′,7-dimethoxyisoflavone showed lower inhibitory effects. The IC 50 values of genistein, biochanin A, equol, 3′,4′,7-trihydroxyisoflavone, and riboflavin, a positive control, for rat prostate testosterone 5α-reductase were 710 μm, 140 μm, 370 μm, 690 μm, and 17 μm, respectively. Daidzein, genistein, biochanin A, formononetin, and equol are already known to be testosterone 5α-reductase inhibitors, but this is the first characterization of 3′,4′,7-trihydroxyisoflavone as an inhibitor of the enzyme.

Repeated morphine administration increases extracellular dopamine levels in the nucleus accumbens, which results in behavioral sensitization that can be suppressed by acupuncture at Shenmen (HT7) points. The present study was conducted to investigate the effects of acupuncture at HT7 on morphine withdrawal syndrome as well as to explore the role of GABA receptors in mediating the effects of HT7 acupuncture. We induced morphine withdrawal by injecting naloxone to rats that self-administer morphine and evaluated the effects of acupuncture and/or GABA receptor antagonists on their withdrawal symptoms. Acupuncture at HT7, but not at the control point LI5, significantly decreased symptoms of morphine withdrawal. HT7 inhibition of the withdrawal syndrome was blocked by pretreatment with either the GABA(A) receptor antagonist bicuculline or the GABA(B) antagonist SCH 50911. These findings suggest that the effects of acupuncture on suppression of morphine withdrawal syndrome are mediated, at least in part, through GABA receptors.

Withdrawal from chronic ethanol enhances ventral tegmental area (VTA) GABA neuron excitability and reduces mesolimbic dopamine (DA) neurotransmission, which is suppressed by acupuncture at Shenmen (HT7) points (Zhao et al., 2006). The aim of this study was to evaluate the effects of HT7 acupuncture on VTA GABA neuron excitability, ethanol inhibition of VTA GABA neuron firing rate, and ethanol self-administration. A role for opioid receptors (ORs) in ethanol and acupuncture effects is also explored.Using electrophysiological methods in mature rats, we evaluated the effects of HT7 stimulation and opioid antagonists on VTA GABA neuron firing rate. Using behavioral paradigms in rats, we evaluated the effects of HT7 stimulation and opioid antagonists on ethanol self-administration using a modification of the sucrose-fading procedure.HT7 stimulation produced a biphasic modulation of VTA GABA neuron firing rate characterized by transient enhancement followed by inhibition and subsequent recovery in 5 minutes. HT7 inhibition of VTA GABA neuron firing rate was blocked by systemic administration of the nonselective μ-opioid receptor antagonist naloxone. HT7 stimulation significantly reduced ethanol suppression of VTA GABA neuron firing rate, which was also blocked by naloxone. HT7 acupuncture reduced ethanol self-administration without affecting sucrose consumption. Systemic administration of the δ-opioid receptor (DOR) antagonist naltrindole blocked ethanol suppression of VTA GABA neuron firing rate and significantly reduced ethanol self-administration without affecting sucrose consumption.These findings suggest that DOR-mediated opioid modulation of VTA GABA neurons may mediate acupuncture's role in modulating mesolimbic DA release and suppressing the reinforcing effects of ethanol.

Exposure of Jurkat T cells to mollugin (15–30 μM), purified from the roots of Rubia cordifolia L., caused cytotoxicity and apoptotic DNA fragmentation along with mitochondrial membrane potential disruption, mitochondrial cytochrome c release, phosphorylation of c-Jun N-terminal kinase (JNK), activation of caspase-12, -9, -7, -3, and -8, cleavage of FLIP and Bid, and PARP degradation, without accompanying necrosis. While these mollugin-induced cytotoxicity and apoptotic events including activation of caspase-8 and mitochondria-dependent activation of caspase cascade were completely prevented by overexpression of Bcl-xL, the activation of JNK and caspase-12 was prevented to much lesser extent. Pretreatment of the cells with the pan-caspase inhibitor (z-VAD-fmk), the caspase-9 inhibitor (z-LEHD-fmk), the caspase-3 inhibitor (z-DEVD-fmk) or the caspase-12 inhibitor (z-ATAD-fmk) at the minimal concentration to prevent mollugin-induced apoptosis appeared to completely block the activation of caspase-7 and -8, and PARP degradation, but failed to block the activation of caspase-9 and -3 with allowing a slight enhancement in the level of JNK phosphorylation. Both FADD-positive wild-type Jurkat clone A3 and FADD-deficient Jurkat clone I2.1 exhibited a similar susceptibility to the cytotoxicity of mollugin, excluding involvement of Fas/FasL system in triggering mollugin-induced apoptosis. Normal peripheral T cells were more refractory to the cytotoxicity of mollugin than were Jurkat T cells. These results demonstrated that mollugin-induced cytotoxicity in Jurkat T cells was mainly attributable to apoptosis provoked via endoplasmic reticulum (ER) stress-mediated activation of JNK and caspase-12, and subsequent mitochondria-dependent activation of caspase-9 and -3, leading to activation of caspase-7 and -8, which could be regulated by Bcl-xL.

Ultraviolet (UV) radiation has been shown to generate reactive oxygen species (ROS), such as singlet oxygen, superoxide radicals, hydroxyl radicals and hydrogen peroxide in a variety of cells. These ROS have the potential to damage critical cellular components such as DNA, proteins, and lipids and eventually result in physical and chemical damage to tissues that may lead to cell death. The steamed root of Rehmannia glutinosa (Saeng Jihuang, SJH) is reported to have an antioxidant activity. We investigated the effect of SJH on UV-induced apoptosis in U937 cells. Upon exposure to UV, there was a distinct difference between untreated cells and cells pre-treated with 0.5–2 mg/ml SJH for 12 hours in regard to cellular redox status and morphological change to cells. SJH pre-treated cells showed significant suppression of apoptotic features such as DNA fragmentation, damage to mitochondrial function, and modulation of apoptotic marker proteins upon exposure to UV. This study indicates that SJH may play an important role in regulating the apoptosis induced by UV presumably through scavenging of reactive oxygen species.

Repeated injections of nicotine can produce an increase in locomotor activity and the expression of immediate-early gene, c-fos, in the central dopaminergic areas. Many studies have shown that Coptidis Rhizoma (CR) and its main alkaloid compound, berberine (BER), have a suppressive effect on the central nervous system. We examined the influence of CR or BER on repeated nicotine-induced locomotor activity in rats and the change of c-Fos expression in the brain by using immunohistochemistry. Male Sprague-Dawley rats were given CR and BER before repeated injections of nicotine hydrochloride (0.4 mg kg−1, s.c.) twice daily for 7 days. After 3 days withdrawal, rats received a challenge injection of nicotine. Pretreatment with CR (100 mg kg−1, i.p.) and BER (100 mg kg−1, i.p.) significantly inhibited the nicotine-induced locomotor activity and expression of c-Fos in the striatum and the nucleus accumbens. These results suggest that CR and BER may produce inhibitory effects of nicotine on behavioural sensitization by possibly reducing postsynaptic neuronal activation in the central dopaminergic systems.

Glycyrrhizae radix (licorice) comprises a variety of flavonoids as major constituents including isoliquiritigenin, liquiritin, liquiritigenin, and glycyrrihizin. It has shown various biological activities such as anti-inflammatory, anti-carcinogenic and antihistamic. As very little is known in regard to drug addiction, we carried out a study on the effect of G. radix and its active component, isoliquiritigenin, on acute cocaine-induced extracellular dopamine release in moving rats. Male Sprague-Dawley rats were orally administered with methanolic extracts of G. radix or isoliquiritigenin 1 h prior to an injection of cocaine (20 mg/kg, intraperitoneal (i.p.)). Extracellular dopamine was measured by in vivo microdialysis. Extract of G. radix and isoliquiritigenin inhibited cocaine-induced extracellular dopamine level in the nucleus accumbens by dose-dependent manner. Inhibition of dopamine release by isoliquiritigenin resulted in attenuation of the expression of c-Fos, an immediately early gene induced by cocaine. Effect of isoliquiritigenin was completely prevented by a GABA(B) receptor antagonist. Thus, these results showed that G. radix and isoliquiritigenin inhibit cocaine-induced dopamine release by modulating GABA(B) receptor, suggesting that isoliquiritigenin might be effective in blocking the reinforcing effects of cocaine.

A high concentration of glucose has been implicated as a causal factor in initiation and progression of diabetic complications and there is evidence to suggest that hyperglycemia increases the production of free radicals and oxidative stress. Therefore, compounds that scavenge reactive oxygen species (ROS) may confer regulatory effects on high glucose-induced apoptosis. Ursolic acid (UA), a pentacyclic triterpene, is reported to have an antioxidant activity. We investigated the effect of UA on high glucose-induced apoptosis in U937 cells. Upon exposure to 35 mM glucose for two days, there was a distinct difference between untreated cells and cells pre-treated with 50 nM UA for 2 h in regard to cellular redox status and oxidative DNA damage to cells. UA pre-treated cells showed significant suppression of apoptotic features such as DNA fragmentation, damage to mitochondrial function and modulation of apoptotic marker proteins upon exposure to high glucose. This study indicates that UA may play an important role in regulating the apoptosis induced by high glucose presumably through scavenging of ROS.

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Therefore, compounds that scavenge reactive oxygen species may regulate heat shock-induced cell death. Recently, it has been shown that the decomposition product of the spin-trapping agent alpha-phenyl-N-t-butylnitrone, N-t-butyl hydroxylamine (NtBHA), mimics alpha-phenyl-N-t-butylnitrone and is much more potent in delaying reactive oxygen species-associated senescence. We investigated the protective role of NtBHA against heat shock-induced apoptosis in U937 cells. Upon exposure to heat shock, there was a distinct difference between the untreated cells and the cells pre-treated with 0.1 mM NtBHA for 2 h in regard to apoptotic parameters, cellular redox status, and mitochondrial function. Upon exposure to heat shock, NtBHA pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax, and down-regulation of Bcl-2 compared to untreated cells. This study indicates that NtBHA may play an important role in regulating the apoptosis induced by heat shock, presumably through scavenging of reactive oxygen species.

Acupuncture as a therapeutic intervention is widely practiced in the treatment of many functional disorders including alcohol abuse. In the present study, the effects of acupuncture on alcohol withdrawal syndrome (AWS) and Fos-like immunoreactivity (FLI) in the striatum and the nucleus accumbens (NAC) of rats were investigated. During 3 days of cessation following chronic administration of ethanol (3 g/kg, i.p. for 3 weeks), rats showed a significant increase in AWS, such as hypermotility, tail rigidity, grooming and tremor, and an increase in FLI in the dopamine terminal areas of the brain. Treatment with acupuncture at zusanli (ST36) or sanyinjiao (SP6) during the withdrawal period inhibited both AWS and FLI of rats undergoing ethanol injection. These results suggest that acupuncture may be useful in the treatment of alcoholism by modulating post-synaptic neural activation in the striatum and NAC.

Behavioral sensitization, as evidenced by the progressive enhanced locomotor response to a subsequent injection of the drug, is the major behavioral outcome produced by repeated injections of nicotine, and a model for studying drug addiction. It is putatively regarded that the alteration of extracellular dopamine release in the nucleus accumbens is closely associated with nicotine-induced behavioral sensitization. The present study was performed to evaluate the effects of the essential oil from Angelica gigas NAKAI (on fragrance inhalation) on repeated nicotine-induced locomotor activity and extracellular dopamine levels in the nucleus accumbens of rats using in vivo microdialysis. Rats were given repeated injections of saline or nicotine (0.4 mg/kg s.c., twice a day for 7 d), followed by one challenge injection on the 4th day after the last daily injection. Systemic challenge with nicotine (0.4 mg/kg s.c.) produced a larger increase in locomotor activity in nicotine-pretreated rats than in saline-pretreated rats. A direct local challenge of 3 mM nicotine via a microdialysis probe also induced a larger increase in dopamine release in nicotine-pretreated rats than in saline-pretreated rats. Most importantly, our results showed that inhalation of the essential oils from Angelica gigas NAKAI significantly decreased both dopamine release in the nucleus accumbens and locomotor activity induced by a nicotine challenge. These results suggest that the essential oils from Angelica gigas NAKAI inhibit nicotine-induced behavioral and neurochemical sensitization, and imply that the essential oil from Angelica gigas NAKAI may be effective in treating nicotine addiction, possibly by modulating dopamine release in the nucleus accumbens.

Many studies have shown that acupuncture can contribute to the biochemical balance in the central nervous system and maintenance or recovery of homeostasis. It is well known that chronic administration of ethanol may produce depletion or sensitization of extracellular dopamine levels in the nucleus accumbens. The present study was designed to investigate the effects of acupuncture on chronic ethanol-induced changes in extracellular dopamine levels in the nucleus accumbens shell (using in vivo microdialysis in unanesthetized rats). Male Sprague-Dawley rats were treated with 3 g/kg/day of ethanol (20%, w/v) or saline by intraperitoneal injection for 21 days. Following 72 h of ethanol withdrawal, acupuncture was applied at bilateral Shenmen (HT7) points for 1 min. Different group of rats using the same paradigm of ethanol treatment were acupunctured at the same points after the systemic ethanol challenge (3 g/kg, i.p.). Acupuncture at the specific acupoint HT7, but not at control points (PC6 or tail) significantly prevented both a decrease of extracellular dopamine levels in the nucleus accumbens during ethanol withdrawal and an increase in accumbal dopamine levels induced by the ethanol challenge. These results provided strong evidence that stimulation of the specific acupoint HT7 helps to normalize the release of dopamine in the mesolimbic system following chronic ethanol treatment.

Acupuncture as a therapeutic intervention has been used for the treatment of many functional disorders including substance abuse. However, there are still many unanswered question about the basic mechanism underlying acupuncture's effectiveness in the treatment of drug addiction. Repeated injection of psycostimulants or morphine can produce behavioral and neurochemical sensitization and have been used as a model for studying drug addiction. The present study was designed to investigate the effect of acupuncture on repeated morphine-induced changes in extracellular dopamine levels using in vivo microdialysis and repeated morphine-induced behavioral changes. Male Sprague-Dawley rats were treated with saline or increasing doses of morphine (10, 20 and 40 mg/kg, s.c., twice daily for 3 days). Following 15 days of withdrawal, acupuncture was applied at bilateral Shenmen (HT7) points for 1 min after the systemic challenge with morphine HCl (5 mg/kg, s.c.). Results showed that acupuncture at the specific acupoint HT7, but not at control points (TE8 and tail) significantly decreased both dopamine release in the nucleus accumbens and behavioral hyperactivity induced by a systemic morphine challenge. These results suggest that the therapeutic effect of acupuncture on morphine addiction occurs through inhibition of neurochemical and behavioral sensitization to morphine.

This study was performed to determine if Akebia and Aristolochia species (Akebia quinata Decaisne and Aristolochia manshuriensis Kom.) could be identified by genetic analysis of Pyrosequencing method, which is used to assess genetic variation. The Pyrosequencing results of Akebia and Aristolochia species showed different patterns. The Pyrosequencing analysis of A. quinata Decaisne was very different compared with that of A. manshuriensis Kom. Pyrosequencing analyses might be able to identify the Akebia and Aristolochia species.

We have previously shown that electroacupuncture (EA) at Shaohai and Neiguan ( HT 3- PC 6) points significantly attenuated stress-induced peripheral responses, including increases in blood pressure, heart rate and plasma catecholamines. In this study, we examined the central effect of EA on the expression of c-fos, one of the immediate-early genes in the brain of rats subjected to immobilization stress. Immobilization stress (180 minutes) preferentially produced a significant increase in Fos-like immunoreactivity (FLI) in stress-relevant regions including the paraventricular hypothalamic nucleus (PVN), arcuate nucleus (ARN), supraoptic nucleus (SON), suprachiasmatic nucleus (SCN), medial amygdaloid nucleus (AMe), bed nucleus of the stria terminalis (BST), hippocampus, lateral septum (LS), nucleus accumbens, and the locus coeruleus (LC). EA (3 Hz, 0.2 ms rectangular pulses, 20 mA) at HT 3- PC 6 on the heart and pericardium channels for 30 minutes during stress, significantly attenuated stress-induced FLI in the parvocellular PVN, SON, SCN, AMe, LS and the LC. However, EA stimulations at HT 3- PC 6 had no effect on FLI in the magnocelluar PVN, ARN, BST or the hippocampus. EA stimulation at HT 3- PC 6 had a greater inhibitory effect on stress-induced FLI than that at TE 5- LI 11, the triple energizer and large intestine meridian, or non-acupoints. These results demonstrated that EA attenuated stress-induced c-fos expression in brain areas. These results suggest that decreased c-fos expression in hypothalamic and LC neurons, among stress-related areas, may reflect the integrative action of acupuncture in stress response.

Glycyrrhizae radix has been popularly used as one of the oldest and most frequently employed botanicals in herbal medicine in Asian countries, and currently occupies an important place in food products. Cadmium (Cd) induces both apoptotic and non-apoptotic cell death, in which alterations in cellular sulfhydryls participate. In the present study, we determined the effects of G. radix extract (GRE) and its representative active components on cell death induced by Cd and explored the mechanistic basis of cytoprotective effects of G. radix. Incubation of H4IIE cells with GRE inhibited cell death induced by 10 microM Cd. Also, GRE effectively blocked Cd (1 microM)-induced cell death potentiated by buthionine sulfoximine (BSO) without restoration of cellular GSH. GRE prevented both apoptotic and non-apoptotic cell injury induced by Cd (10 microM) or Cd (0.3-1 microM) + BSO. Inhibition of Cd-induced cell injury by pretreatment of cells with GRE suggested that the cytoprotective effect result from alterations in the levels of the protein(s) responsible for cell viability. GRE inhibited mitochondrial Bad translocation by Cd or CD+BSO, and caused restoration of mitochondrial Bcl(xL) and cytochrome c levels. Cd-induced poly(ADP-ribose)polymerase cleavage in control cells or in cells deprived of sulfhydryls was prevented by GRE treatment. Among the major components present in GRE, liquiritigenin, but not liquiritin, isoliquiritigenin or glycyrrhizin, exerted cytoprotective effect. These results demonstrated that GRE blocked Cd-induced cell death by inhibiting the apoptotic processes involving translocation of Bad into mitochondria, decreases in mitochondrial Bcl(xL) and cytochrome c, and poly(ADP-ribose)polymerase cleavage.

Astragali radix, which has tonifying and circulatory effect as well as immune response, is one of the oldest and most frequently used crude drug for oriental medicine in many Asian countries. The present study was conducted to evaluate the effect of aqueous extract of Astragali radix (ARE) on the functions of murine macrophage cell line, RAW 264.7 macrophage cells. In the cell proliferation assay, methotrexate (MTX), an agent of immune suppression, decreased the cell proliferation of RAW 264.7 macrophage cells (IC(50): 100 microg/ml), but the suppression of cell proliferation was significantly protected by ARE treatment in RAW 264.7 macrophage cells. The expressions of cytokine gene by ARE were investigated using reverse transcription polymerase chain reaction (RT-PCR). In RT-PCR, IL-1alpha, IL-1beta and IL-6 mRNA expressions was induced in ARE-treated RAW 264.7 macrophage cells. We also investigated the effect of the nitric oxide (NO) synthesis and inducible nitric oxide synthase (iNOS) mRNA expression by ARE. ARE alone had no effect on NO synthesis and iNOS mRNA expression in RAW 264.7 cells. In the case of lipopolysaccharide (LPS) stimulation, NO production and iNOS mRNA expression were detected in RAW 264.7 cells. However, NO production and iNOS mRNA expression which is induced by LPS decreased after treatment of ARE. These data demonstrate that ARE can reduce the suppression of macrophage cell proliferation induced by MTX, and induce IL-1alpha, IL-1beta and IL-6 mRNA expressions in RAW 264.7 macrophage cells. Also, ARE inhibit NO production in LPS-stimulated RAW 264.7 macrophage cells, and the inhibition of NO production may be associated with the inhibition of iNOS mRNA expression.

The present study was conducted to evaluate the immunomodulatory effect of aqueous-extracted Astragali radix (ARE) in methotrexate (MTX)-treated mouse spleen cells. In spleen cell proliferation assay, ARE enhanced mitogenic activity in the dose-response manner. We also investigated the effect of ARE on the reducing of immune suppression caused by MTX in mouse spleen cells. MTX decreased the spleen cell proliferation (IC(50):800 microg/ml). However, ARE significantly reduced the suppression of cell proliferation by MTX in mouse spleen cells. Immunomodulatory effect of ARE were further investigated using reverse transcription polymerase chain reaction (RT-PCR). In RT-PCR, we examined the expressions of various cytokines such as IL-6, IL-1alpha, IL-1beta, IL-12p40, GM-CSF and TNF. Enhancement of IL-1alpha and IL-12p40 mRNA expressions were shown in mouse spleen cells by ARE. In spite of MTX treatment, the expressions of IL-1alpha and IL-12p40 mRNA sustained in spleen cells. These data indicate that (1) ARE has a protective effect of immune suppression, and (2) the immunomodulatory effects of ARE may be, in part, associated with the expressions of IL-1alpha and IL-12p40 mRNA as well as the mitogenic effect on spleen cells.

Previous reports have shown that the methanol extract and the essential oil from Acori graminei Rhizoma (AGR) inhibited excitotoxic neuronal cell death in primary cultured rat cortical cells. In the present study, an active principle was isolated from the methanol extract by biological activity-guided fractionations and identified as asarone. We evaluated neuroprotective actions and action mechanisms of the isolated asarone as well as the alpha- and the beta-asarone obtained commercially. The isolated asarone inhibited the excitotoxicity induced by the exposure of cortical cultures for 15 min to 300 microM NMDA in a concentration-dependent manner, with the IC50 of 56.1 microg/ml. The commercially obtained alpha- and beta-asarone exhibited more potent inhibitions of the NMDA-induced excitotoxicity than the isolated asarone. Their respective IC50 values were 18.2 and 26.5 microg/ml. The excitotoxicity induced by glutamate (Glu) was also inhibited, but with much less potency than the toxicity induced by NMDA. The IC50 values for the alpha-, beta-, and the isolated asarone were 89.7, 121.7, and 279.5 microg/ml, respectively. Based on the receptor-ligand binding studies using a use-dependent NMDA receptor-channel blocker [3H]MK-801, asarone inhibited the specific bindings in a concentration-dependent fashion. These results indicate that asarone, the major essential oil component in AGR, exhibits neuroprotective action against the NMDA- or Glu-induced excitotoxicity through the blockade of NMDA receptor function. The alpha-asarone was found to exhibit more potent inhibition of [3H]MK-801 bindings, which is consistent with its more potent neuroprotective action than the beta- or the isolated asarone.

Thioureas have been employed as potent hydroxyl radical scavengers and also inhibit production of oxygen free radicals. The in vitro oxygen radical scavenging effect by N,N'-substituted thioureas including dimethylthiourea (DMT), diethylthiourea (DET), tetramethylthiourea (TMT) and diphenylthiourea (DPT) was assessed by the conversion of phi x-174 DNA from supercoiled DNA to the open circular form or to fragmented DNA. Addition of the N,N'-substituted thioureas to the incubation mixture significantly prevented a single strand breakage of phi x-174 DNA induced by autooxidation of benzenetriol. These thioureas were also effective in preventing degradation of phi x-174 DNA induced by autooxidation of benzenetriol in the presence of ferrous iron. In view of the in vitro radical scavenging effect by the thioureas and the role of reactive oxygen species in the induction of phase II detoxifying enzymes, expression of microsomal epoxide hydrolase (mEH) and rGSTA2 in response to these agents was investigated in the rat liver. Rats treated with each of the alkylthioureas exhibited marked increases of mEH and rGSTA2 mRNA levels with TMT being the most effective. DPT an arylthiourea, however, was minimally active in increasing the mRNAs. Time-course studies revealed that DMT, DET and TMT increased the mRNA levels to the greatest extent at 24 h after a single dose of treatment. The levels of mEH and rGSTA2 mRNA were elevated in a dose-dependent manner by the alkylthioureas. Immunoblot analysis showed that the alkylthioureas induced mEH and rGSTA2 proteins in the liver (0.6 mmol/kg per day, 3 days), which was consistent with the increases in the mRNA levels. DMT, DET or TMT enhanced CCl4-induced liver toxicity, as monitored by plasma aminotransferase activity, although each of the agents alone caused only slight increase in the alanine aminotransferase activity. In contrast to the effects of the alkylthioureas, DPT protected the liver against the toxicant-induced injury. All of the thioureas prevented decreases in the hepatic glutathione level by CCl4. Expression of cytochrome P450 2E1 and P450 2B1/2, which are implicated with metabolic activation of CCl4, was assessed after treatment with the thioureas. P450 2E1 and P450 2B1/2 were differentially induced by the alkylthioureas with the expression of P450 2E1 being inversely related with that of P450 2B1/2. These results showed that N,N'-substituted alkylthioureas were capable of inducing mEH and rGSTA2 in the liver with elevation of the mRNAs, that induction of mEH and rGSTA2 by these alkylthioureas might be mediated by production of the reactive oxygens derived from metabolic activation of the agents irrespective of their radical scavenging effect and that the agents rather enhanced toxicant-induced liver injury with the induction of P450 2E1 or P450 2B1/2.