Isolation, identification, and activity evaluation of diterpenoid alkaloids from Aconitum sinomontanum.

A phytochemical study led to the isolation of 25 diterpenoid alkaloids from Aconitum sinomontanum, of which six were described for the first time. Among them compounds 1-3 are anhydrolycoctonine derivatives, rare rearranged aconitine-type C19-diterpenoid alkaloids. To our best knowledge, less than ten of this type of alkaloids were isolated just from the genus Aconitum. The structures of these unreported compounds were elucidated by extensive analysis of NMR spectroscopic data and X-ray diffraction. The biological activities of compounds 1-3, 5-9, and 12-25 were evaluated. Among the tested compounds, compounds 2 and 17 showed potent inhibitory effect on the capsaicin (selective TRPV1 agonist) mediated activation of transient receptor potential vanilloid 1 (TRPV1) channels expressed in HEK-293 cells with inhibition rate of 31.78% and 30.94% at the concentration of 10 µM. Compounds 1-3, 5-9, 13, and 18-25 exhibited weak cytotoxic activity against human tumor cell lines NCI-H226 and MDA-MB-231 with inhibition rate over 10% at the concentration of 10 µM. Compound 16 showed most inhibitory effect on the expression of Nrf2 (NF-E2-related factor-2)-regulated gene with inhibition rate of 25% at the concentration of 20 µM.

Isoflurane produces antidepressant effects inducing BDNF-TrkB signaling in CUMS mice.

RATIONALE: The volatile anesthetic isoflurane is suggested to produce a rapid and robust antidepressive effect in preliminary clinical trials. Recently, isoflurane was found to activate the tropomyosin receptor kinase B (TrkB) signaling which is the underlying mechanism of the rapid antidepressant ketamine. OBJECTIVE: Our study investigated the effect of isoflurane anesthesia on chronic unpredictable mild stressed (CUMS) model in mice and verified the role of brain-derived neurotrophic factor (BDNF)/TrkB/ the mammalian target of rapamycin (mTOR) signaling in the antidepressant effect of isoflurane. METHODS: We employed the CUMS model of depression to assess the rapid antidepressant effect of isoflurane by the forced swimming test (FST), the sucrose preference test (SPT), and the novelty suppressed feeding test (NSFT). The protein expression of BDNF and TrkB/protein kinase B (PKB or Akt)/mTOR was determined through Western blot. The dendritic spine density in the hippocampus and medial prefrontal cortex (PFC) was measured by the Golgi staining. RESULTS: A brief burst-suppressing isoflurane anesthesia rapidly reversed the behavioral deficits caused by CUMS procedure, normalized the expression of BDNF and further activated the TrkB signaling pathway in CUMS-induced stressed mice in both prefrontal cortex (PFC) and hippocampus (HC). All of those behavioral and proteomic effects were blocked by K252a, a selective receptor inhibitor of TrkB. Isoflurane significantly promoted the formation of dendritic spines in both medial prefrontal cortex (mPFC), CA1, CA3, and DG of the hippocampus. CONCLUSION: Our study indicates that isoflurane exerts a rapid antidepressant-like effect in CUMS depression animal model, and the activation of BDNF/TrkB signaling pathway plays an indispensable role in the biological and behavioral antidepressant effects of isoflurane. A single exposure to isoflurane could repair synaptic damage caused by chronic stimulation.

Blockade of TRPV1 Inhibits Methamphetamine-induced Rewarding Effects.

Methamphetamine (MAP) is the most widely used psychostimulant in the world, but the exact mechanisms underlying MAP addiction are not yet fully understood. Recent studies have identified the distribution of TRPV1 in several brain regions that are related to drug addiction, including nucleus accumbens (NAc) and dorsal striatum (DSt). In the present study, we performed conditioned place preference (CPP) and self-administration tests to examine the effects of capsazepine (CPZ) and SB366791 (SB) on MAP reward. We found that both CPZ and SB significantly inhibited MAP-induced CPP and self-administration; in contrast, TRPV1 knock-out (KO) mice did not develop MAP-induced CPP. Real-time RT-PCR, Western blot and quantitative autoradiographic tests showed up-regulation of TRPV1 mRNA and protein expression in the NAc and/or DSt regions of mice exhibiting MAP-induced CPP. In addition, an in vivo microdialysis experiment showed that CPZ dramatically reduced dopamine (DA) levels in the NAc region of MAP-treated mice. Furthermore, attenuated dopamine transporter (DAT) binding levels in the NAc and DSt regions of MAP-induced CPP mice were reversed by CPZ. Together, these data suggest that TRPV1 plays an important role in MAP reward via the modulation of DA release and DAT density, thereby providing a novel therapeutic target for MAP addiction.

Low molecular mass chondroitin sulfate suppresses chronic unpredictable mild stress-induced depression-like behavior in mice.

The present study is aimed at testing the antidepressant--like effects and probable mechanisms of action of low molecular mass chondroitin sulfate (LMMCS) on depression induced by chronic unpredictable mild stress (CUMS) in mice. Four weeks of CUMS exposure resulted in depressive-like behavior, expressed by a significant decrease in the locomotor activity and sucrose consumption and increased immobility time in the forced swim test. Further, there was a significant reduction of 5-HT level in the hippocampus region of depressed mice. Treatment of mice for four weeks with LMMCS ameliorated significantly both the behavioral and biochemical changes induced by CUMS. These novel results suggest that LMMCS produces an antidepressant-like effect in mice subjected to CUMS, which might be related, at least in part, to the increase of 5-HT concentration in the hippocampus.

Lactational and postnatal exposure to polychlorinated biphenyls induces sex-specific anxiolytic behavior and cognitive deficit in mice offspring.

The central nervous system is affected by polychlorinated biphenyls (PCBs). Previous studies have indicated that developmental exposure to PCBs impairs behavioral performance and alters cognitive abilities. This study assessed the effects of lactational and postnatal exposure to a commercial PCBs mixture, Aroclor 1254 (A1254), on mice performing several neurobehavioral tasks including the open field test, novel object test, elevated plus maze test, Y-maze test, and tail suspension test. In the open field test, PCBs treatment (6 and 18 mg/kg/day) was associated with increased movement, time duration, and frequency in the central zone in female but not male mice. PCBs-treated female mice (6 and 18 mg/kg/day) also showed decreased novel object recognition, indicating impairment in recognition memory. Finally, we performed autoradiographic receptor binding assays for dopamine (DA) D1 and D2 receptors, dopamine transporter (DAT), and the N-methyl-D-aspartic acid (NMDA) receptor after behavioral tests to examine whether alterations occurred in the dopaminergic and NMDAergic systems of the brain. Our results showed that PCBs treatment did not change D1 and D2 receptors or DAT binding in the dorsal striatum of female mice. However, PCBs treatment significantly decreased NMDA receptor binding in the dorsal striatum, frontal cortex, cingulate cortex, and motor cortex, and CA3 and dentate gyrus (DG) of the hippocampus in female mice. Collectively, our results suggest that long-term PCBs exposure can induce anxiolytic behavior, cognitive deficits, and changes of NMDA receptors.

Modafinil-induced conditioned place preference via dopaminergic system in mice.

Modafinil, a psychostimulant, is used in the treatment of narcolepsy, shift work sleep disorder, and excessive daytime sleepiness associated with obstructive sleep apnea. Preclinical and clinical studies suggest that modafinil may have reinforcing effects. However, a possible rewarding property of modafinil has not been fully investigated. In this study, we assessed the potential rewarding property of modafinil using the conditioned place preference (CPP) paradigm in mice. Using radiolabeled ligands, we observed changes in dopamine, glutamate, and GABA receptor binding in the brains of mice after treatment with modafinil. Modafinil produced significant CPP in mice at an intraperitoneal (i.p.) dose of 125 mg kg⁻¹ and prevented normal body weight gain of mice in a dose-dependent manner. A significant reduction in normal body weight gain was observed when mice were administrated 125 mg kg⁻¹ modafinil. In addition, there were widespread changes in receptor binding in the brains of modafinil-treated mice; Dopamine D1 binding was increased in the caudate putamen, the accumbens, and the substantia nigra, while dopamine D2 binding was decreased in the caudate putamen and the accumbens. Dopamine transporter (DAT) binding was increased in the prefrontal cortex, the caudate putamen, and the nucleus accumbens. No changes were observed in NMDA and GABA(A) receptor binding. These data indicate that modafinil had a significant rewarding property and could be abused as a recreational drug. Dopamine systems may play a key role in the rewarding property of modafinil.

Prenatal and postnatal exposure to bisphenol a induces anxiolytic behaviors and cognitive deficits in mice.

Bisphenol A (BPA), an environmental endocrine-disrupting chemical, has been extensively evaluated for reproductive toxicity and carcinogenicity. However, little is known about the behavioral and neurochemical effects of BPA exposure. This study examined whether chronic daily exposure to an environmental endocrine-disrupting chemical, bisphenol A [(BPA); 100 microg/kg/day or 500 microg/kg/day, p.o.], from prenatal Day 7 to postnatal Day 36 would lead to changes in anxiety and memory in mice. First, we observed the behavioral alterations of BPA-treated mice using two anxiety-related models, the open field test and elevated plus maze (EPM) test. In the open field test, BPA treatment (100 microg/kg/day) increased movement in the central zone. BPA treatment (500 microg/kg/day) also increased the time spent in the open arms in the EPM test. Second, we measured cognitive ability in the Y-maze test and novel object test. BPA-treated mice showed decreased alternation behavior in the Y-maze at both of doses, indicating working memory impairment. BPA-treated mice (100 microg/kg/day) also showed decreased novel object recognition as expressed by central locomotion and frequency in the central zone, showing recognition memory impairment. Finally, to measure changes in the dopaminergic and NMDAergic systems in the brain, we performed autoradiographic receptor binding assays for dopamine D(1) and D(2) receptors, the NMDA receptor, and the dopamine transporter. BPA treatment increased D(2) receptor binding in the caudate putamen (CPu) but decreased DAT binding. BPA treatment also decreased NMDA receptor binding in the frontal cortex and CA1, CA3, and DG of the hippocampus. Taken together, our results suggest that long-term BPA exposure in mice can induce anxiolytic behaviors, cognitive deficits and changes in the dopaminergic and NMDAergic systems.

Repeated methamphetamine treatment increases expression of TRPV1 mRNA in the frontal cortex but not in the striatum or hippocampus of mice.

The transient receptor potential vanilloid type 1 (TRPV1) is a non-selective ligand-gated cationic channel. The distribution of TRPV1 mRNA in various regions of the brain has been successfully established. Methamphetamine (MAP) is a psychostimulant and a major drug of abuse in many parts of the world. The powerful rewarding properties of MAP are attributed to multiple pharmacological actions, but the mechanistic association between TRPV1 expression and MAP-induced drug addiction has not established. In the present study, we conducted a time-course analysis of TRPV1 mRNA levels in the frontal cortex, striatum, and hippocampus of mouse brain following repeated MAP (2mg/kg, i.p.) treatment. Our results demonstrate that expression of TRPV1 mRNA is significantly increased 1, 2, 6, 24, 48h, and 1 week after the last MAP treatment in the frontal cortex but not in the striatum or hippocampus. These data support a potential role for TRPV1 in the treatment of MAP-induced drug addiction.

7-nitroindazole, nitric oxide synthase inhibitor, attenuates physical dependence on butorphanol in rat.

Butorphanol is a synthetic opioid agonist/antagonist analgesic agent that mainly exerts its effects through kappa-opioid receptors. It has been demonstrated that kappa-opioid receptors preferentially mediate the development of physical dependence upon butorphanol and the associated withdrawal syndrome. However, it is not fully understood whether or not nNOS-containing neurons in the various brain regions play an important role in butorphanol withdrawal. Therefore, this study was conducted to determine whether the selective nNOS inhibitor, 7-NI, modifies the development of butorphanol withdrawal and changes of nNOS expressions in different brain regions in physically butorphanol-dependent rats. The first part of the study focused on withdrawal behaviors in male Sprague-Dawley rats. Physical dependence was induced by a 72-h i.c.v. infusion with butorphanol (26 nmol/mixrol/h) and withdrawal was subsequently precipitated by i.c.v. challenge with naloxone (48 nmol/5 microl/rat) 2 h after termination of the butorphanol infusion. The butorphanol/7-NI coadministration group showed a significant decrease in several signs of withdrawal such as teeth chattering, as compared with the butorphanol-treated group. In the second part of the study, immunohistochemical analysis was performed to determine the expression of nNOS in the various brain regions. In the butorphanol/7-NI coadministration group, the number of cells labeled for nNOS was significantly lower in the various brain regions (including the caudate putamen, nucleus accumbens, and hippocampus) than in the butorphanol group. Therefore, 7-NI decreased in butorphanol-induced physical dependence and nNOS expression. Taken together, these findings suggest that the nNOS system is involved in the development of butorphanol-induced physical dependence, and 7-NI has potential clinical application as a candidate for the treatment of opioid withdrawal syndrome.

Free radical scavenging and hepatoprotective constituents from the leaves of Juglans sinensis.

In the course of searching for hepatoprotective agents from natural products, six compounds were isolated from the MeOH extract of the leaves of Juglans sinensis, as guided by their DPPH free radical scavenging activity. The structures were determined as juglanoside B (1), quercetin 3-O-alpha-L-arabinofuranoside (avicularin, 2), quercetin 3-O-alpha-L-arabinopyranoside (guaijaverin, 3), quercetin 3-O-alpha-L-rhamnopyranoside (quercitrin, 4), (+)-catechin (5) and quercetin 3-O-beta-D-galactopyranoside (hyperin, 6). Compounds 2-6 showed significant DPPH free radical scavenging effects. An evaluation for the hepatoprotective activity of the isolated compounds on drug-induced cytotoxicity was conducted, and compounds 1, 2, and 5 showed protective effects against nitrofurantoin-induced cytotoxicity, and compound 5 also exhibited a moderate protective effect on amiodarone-induced cytotoxicity in Hep G2 cells.

Hepatoprotective constituents of Cudrania tricuspidata.

Phytochemical investigation of the MeOH extract of the root barks of Cudrania tricuspidata Bureau (Moraceae), as guided by hepatoprotective activity in vitro, furnished four isoprenylated xanthones, cudratricusxanthone A (1), cudraxanthone L (2), cudratricusxanthone E (3), and macluraxanthone B (4). All of these compounds showed the significant hepatoprotective effect on tacrine-induced cytotoxicity in human liver-derived Hep G2 cells. Compounds 1, 2, and 4 also exhibited the significant hepatoprotective effect on nitrofurantoin-induced cytotoxicity in human liver-derived Hep G2 cells.

[Inhibitation of tumor angiogenesis, growth and metastasis by blocking VEGF paracrine pathway].

Solid tumors require an adequate vascular supply to grow beyond a certain dimension. It is known that formation of new blood vessels in tumor is mediated by unbalanced expression of angiogenic factors and their inhibitors. Among the former, the vascular endothelial growth factor (VEGF) has been assumed prime candidacy as a major positive physiological effector. To investigate the role of VEGF in angiogenesis associated with development of breast cancer, a sense VEGF and an anti-sense VEGF expression plasmids were constructed, and then were introduced into a human breast carcinoma cell line, MCF-7, expressing middle level of endogenous VEGF. Anti-sense VEGF(121) transfected MCF-7 cells that expressed reduced constitutive levels of VEGF and showed the same growing potential as untransfected MCF-7 cells in vitro, but it showed longer latency, smaller tumor, slower growth and prolonged survival time compared to parental or sense VEGF(165) transfected MCF-7 cells in vivo. Moreover, the tumors derived from anti-sense VEGF(121) transfected MCF-7 cells characterized by minimal vascularization and extensive necrosis. Finally, mice with primary subcutaneous tumors treated with intratumoral administration of anti-sense VEGF, or the plasmid expressing extracellular domain of the Flk-1 VEGF receptor (sFlk-1) followed by electroporation, showed significant tumor suppression. These results suggest that VEGF plays a major angiogenic role in breast cancer and a strategy, which blocks the VEGF paracrine pathway, may provide a means to control tumor growth topically without the risk of systemic antiangiogenesis.