Identification of Potential Human Ryanodine Receptor 1 Agonists and Molecular Mechanisms of Natural Small-Molecule Phenols as Anxiolytics.

Natural small-molecule phenols (NSMPs) possess certain ubiquitous bioactivities including the anxiolytic effect. Ryanodine receptor 1 (RyR1) may be one of the potentially critical pharmacological targets for studying the anxiolytic activity of NSMPs. However, detailed molecular mechanisms of NSMPs have not been fully clarified. This research was intended to identify potent hRyR1 agonists from NSMPs and investigate whether RyR1 plays a role in their anxiolytic effect. Homology modeling and molecular docking analysis were performed using Accelrys Discovery Studio 2.5. The most appropriate concentrations of NSMPs to activate RyR1 were measured using the MTT assay. Fluorescence analyses of the intracellular calcium levels and western blotting analysis were carried out to validate whether NSMPs could regulate the calcium flux to some extent by activating RyR1. The results demonstrated that xanthotoxol and 5-hydroxy-1,4-naphthalenedione can be screened as hit compounds for potential agonists of hRyR1 to exert the anxiolytic effect. In conclusion, NSMPs might be a kind of pharmacological signal carrier, acting on RyR1 as an agonist and resulting in calcium ion mobilization from intracellular calcium ion store.

Melatonin pretreatment attenuates acute methamphetamine-induced aggression in male ICR mice.

Aggression is one of the symptoms of methamphetamine (MA) use and withdrawal, which can exacerbate MA addiction and relapse. Many studies have demonstrated that poor sleep is significantly associated with aggression. Melatonin has been indicated to be effective in treating sleep disorders induced by MA, and it can also protect neuronal cells against MA-induced neurotoxicity. However, the underlying effects of melatonin on MA-reduced aggression remain unclarified. This study was designed to evaluate the effects of melatonin on acute MA-induced aggressive behavior in male ICR mice and the effects on neurotransmitters related to aggression. Fifty male ICR mice were randomly assigned to control and treatment groups pretreated with MA (3 mg/kg) or melatonin (2.5, 5, 10 mg/kg) plus MA. Aggressive behaviors were observed through isolation-induced aggression in the resident-intruder model. High-performance liquid chromatography combined with electrochemical detection (HPLC-ECD) was used to anatomize the levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA), and the concentrations of serotonin (5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in the hippocampus involved in behavior processing. The results showed that acute MA administration decreased latency to initial attacks and thereby increased the number and total duration of attacks. Furthermore, HVA level as well as 5-HIAA and 5-HT turnover estimated by 5-HIAA/5-HT ratios declined compared to those in the vehicle group. The medium melatonin pretreatment dose (5 mg/kg) could significantly reverse acute MA-induced aggressive behavior in the form of prolonging latency to initial attacks and thereby attenuating the number of attacks and total duration of attacks. HVA and 5-HIAA levels, 5-HT turnover estimated by 5-HIAA/5-HT ratios, and DA turnover estimated by HVA/DA ratios and (DOPAC + HVA)/DA ratios were elevated compared to those in the MA group. These results indicate that the DA and 5-HT systems are involved in the processes of MA-induced aggressive behaviors and that melatonin has the capacity to reverse MA-induced aggressive behaviors.

Anxiolytic effect of a novel 9,10-dihydrophenanthrene, juncuenin H, is associated with metabolic changes in cortical serotonin/dopamine levels in mice.

Two novel phenanthrenoids, juncuenin H (1) and dijuncuenin B (2), together with eight known phenanthrenoids, effusol (3), dehydroeffusol (4), juncusol (5), dehydrojuncusol (6), juncuenin B (7), dehydrojuncuenin B (8), juncuenin A (9), and dehydrojuncuenin A (10), were isolated from the underground parts of Juncus setchuenensis. The structures of the compounds were determined by 1D and 2D NMR and mass spectroscopy. The anxiolytic activities of compounds 1, 6, 9, and 10 were evaluated. In order to explore the mechanisms underlying their anxiolytic activities, the levels of serotonin (5-HT), dopamine (DA), and their metabolites in the cerebral cortex and hippocampus of mice treated with compound 1 were determined by quantitative mass spectrometry. The mice treated with compound 1 had significantly lower levels of 5-HT, 3-methoxytyramine (3-MT), 5-hydroxyindole-3-acetic acid (5-HIAA), homovanillic acid (HVA), and 3, 4-dihydroxyphenylacetic acid (DOPAC) in the cerebral cortex than those of the vehicle control-treated mice. The levels of HVA and 5-HIAA in the hippocampus were also significantly lower in the mice treated with compound 1 than in the control group mice. These results suggest that the metabolic changes, reflected in the levels of DA and/or 5-HT, may contribute to the anxiolytic activity of the phenanthrenoids studied herein.

Exposure to female estrous is beneficial for male mice against transient ischemic stroke.

OBJECTIVE: Exposure to female estrous, a natural rewarding experience, alleviates anxiety and depression, and the contribution of this behavior to stroke outcome is unknown. The aim of this study was to evaluate whether exposure to female estrous is beneficial to recovery following transient ischemic stroke in male mice. METHODS: Cerebral ischemia was induced in male ICR mice with thread occlusion of the middle cerebral artery (MCAO) for 30 min followed by reperfusion. MCAO mice were randomly divided into MCAO group and Estrous Female Exposure (EFE) group. The mice in the EFE group were subjected to estrous female mouse interaction from day 1 until the end of the experiment. Mortality was recorded during the investigation. Behavioral functions were assessed by a beam-walking test and corner test from day 1 to day 10 after MCAO. Serum testosterone levels were analyzed with ELISA, and the expression levels of growth-associated protein-43 (GAP-43) and synaptophysin in the cortex of the ischemic hemisphere were determined by western blot on day 7 after MCAO. RESULTS: Exposure to female estrous reduced the mortality induced by cerebral ischemic lesions. The beam-walking test demonstrated that exposure to female estrous significantly improved motor function recovery. The serum testosterone levels and ischemic cortex GAP-43 expression were significantly higher in MCAO male mice exposed to female estrous. CONCLUSION: Exposure to female estrous reduces mortality and improves functional recovery in MCAO male mice. The study provides the first evidence to support the importance of female interaction to male stroke rehabilitation. ABBREVIATIONS: GAP-43: growth-associated protein-43; SYP: Synaptophysin; MCAO: middle cerebral artery occlusion; OVXs: ovariectomies; CCA: common carotid artery; ECA: external carotid artery; EFE: estrous female exposure; TTC: 2,3,5-triphenyltetrazolium chloride; PAGE: polyacrylamide gel electrophoresis; PVDF: polyvinylidene difluoride; ANOVA: analysis of variance; LSD: least significant difference.

Support for Natural Small-Molecule Phenols as Anxiolytics.

Natural small-molecule phenols (NSMPs) share some bioactivities. The anxiolytic activity of NSMPs is attracting attention in the scientific community. This paper provides data supporting the hypothesis that NSMPs are generally anxiolytic. The anxiolytic activities of seven simple phenols, including phloroglucinol, eugenol, protocatechuic aldehyde, vanillin, thymol, ferulic acid, and caffeic acid, were assayed with the elevated plus maze (EPM) test in mice. The oral doses were 5, 10 and 20 mg/kg, except for phloroglucinol for which the doses were 2.5, 5 and 10 mg/kg. All tested phenols had anxiolytic activity in mice. The phenolic hydroxyl group in 4-hydroxycinnamic acid (4-OH CA) was essential for the anxiolytic activity in the EPM test in mice and rats compared to 4-chlorocinnamic acid (4-Cl CA). The in vivo spike recording of rats' hippocampal neurons also showed significant differences between 4-OH CA and 4-Cl CA. Behavioral and neuronal spike recording results converged to indicate the hippocampal CA1 region might be a part of the anxiolytic pathways of 4-OH CA. Therefore, our study provides further experimental data supporting NSMPs sharing anxiolytic activity, which may have general implications for phytotherapy because small phenols occur extensively in herbal medicines.

[Review on anxiolytic effect of natural small-molecule phenols].

Phenolic compounds have multiple bioactivities, such as anti-oxidant, anti-tumor, anti-bacterial, and anti-inflammatory activities. Recent literatures have demonstrated that flavonoids have a significant anti-anxiety effect on the central nervous system. In addition, studies showed that flavonoids acted as pro-drugs, which were transformed into smaller phenols through intestinal microflora. The small phenolic metabolites were crucial for the anxiolytic effects of these flavonoids, indicating that natural small-molecule phenols(NSMP) generally have anxiolytic activities. In this paper, the supporting evidences (before June 2016) from SciFinder database have been summarized. Furthermore, NSMPs were classified according to chemical structures; their anxiolytic effects, mechanisms, and the structure-activity relationships were also discussed.

[Chinese herbal medicine enhances sexual function and c-Fos/nNOS expression in the nucleus accumbens of orchidectomized rats].

RESULTS: There was a decrease in accessory genital organ weight, plasma testosterone, and sexual behavior, as well as a low number of c-Fos-positive cells and a large nNOS-positive cell area in orchidectomized rats. Administration of the herbal medicine increased accessory genital organ weight, testosterone level, mating behavior, and c-Fos-positive cell number, while it decreased the nNOS-positive cell area in orchidectomized rats. CONCLUSION: An increase of plasma testosterone after administration of "kidney-nourishing" herbal medicine might contribute to the elevated sexual function and activity in orchidectomized rats. In addition, a central nervous system mechanism, such as the functional alteration of NAc, might be involved. Abstract OBJECTIVE: To determine whether the central nervous system is involved in the effect of Chinese herbal medicine on sexual function recovery in orchidectomized rats. METHODS: Orchidectomized rats were administered intragastrically with a decoction of "kidney-nourishing" Chinese herbal medicine once per day for 28 days. Accessory genital organ weight, plasma testosterone, and mating behavior were investigated. The expression of c-Fos and neuronal nitric oxide synthase (nNOS) in neuronal cells in the nucleus accumbens (NAc) was analyzed by immunohistochemistry.

Anxiolytic effects of orcinol glucoside and orcinol monohydrate in mice.

CONTEXT: Anxiety is a common psychological disorder, often occurring in combination with depression, but therapeutic drugs with high efficacy and safety are lacking. Orcinol glucoside (OG) was recently found to have an antidepressive action. OBJECTIVE: To study the therapeutic potential of OG and orcinol monohydrate (OM) as anxiolytic agents. MATERIALS AND METHODS: Anxiolytic effects in mice were measured using the elevated plus-maze, hole-board, and open-field tests. Eight groups of mice were included in each test. Thirty minutes before each test, mice in each group received one oral administration of OG (5, 10, or 20 mg/kg), OM (2.5, 5, or 10 mg/kg), the positive control diazepam (1 or 5 mg/kg), or control vehicle. Each mouse underwent only one test. Uptake of orcinol (5 mg/kg) in the brain was qualitatively detected using the HPLC-MS method. RESULTS: OG (5, 10, and 20 mg/kg) and OM (2.5 and 5 mg/kg) increased the time spent in open arms and the number of entries into open arms in the elevated plus-maze test. OG (5 and 10 mg/kg) and OM (2.5 and 5 mg/kg) increased the number of head-dips in the hole-board test. At all tested doses, OG and OM did not significantly affect the locomotion of mice in the open-field test. Orcinol could be detected in the mouse brain homogenates 30 min after oral OM administration, having confirmed that OM is centrally active. DISCUSSION AND CONCLUSION: The results demonstrated that OG and OM are anxiolytic agents without sedative effects, indicating their therapeutic potential for anxiety.

A conjugate vaccine attenuates morphine- and heroin-induced behavior in rats.

BACKGROUND: Currently approved medications for opioid addiction have shown clinical efficacy, but undesired side effects, dependence induced by the medications themselves, and low treatment compliance necessitate the need for novel therapies. METHODS: A novel morphine-keyhole limpet hemocyanin conjugate vaccine was synthesized with 6-glutarylmorphine as the hapten and a lengthened linker of 6 carbon atoms. The titer and specificity of the triggered antibody were assessed by enzyme-linked immunosorbent assay. The effects of the vaccine on the morphine-induced elevation of dopamine levels in the nucleus accumbens were determined by high-performance liquid chromatography. The effects of the vaccine on morphine-induced locomotor sensitization and heroin-primed reinstatement of heroin self-administration were also assessed. RESULTS: After subcutaneous administration in rats, the vaccine triggered a high antibody titer, with comparable specificity for morphine, 6-acetylmorphine, and heroin, but no interaction with dissimilar therapeutic opioid compounds, including buprenorphine, naloxone, and nalorphine, was observed. The vaccine significantly prevented the elevation of dopamine levels in the nucleus accumbens induced by a single morphine challenge. Moreover, the vaccine prevented the expression of morphine-induced locomotor sensitization and heroin-primed reinstatement of heroin seeking, suggesting its potential for preventing relapse. CONCLUSION: These results demonstrate that active immunization with the present vaccine induces a robust morphine/heroin-specific antibody response in rats and attenuates the behavioral effects of morphine and heroin.

Two new anxiolytic phenanthrenes found in the medullae of Juncus effusus.

Six phenanthrenes, 2-methoxy-7-hydroxy-1-methyl-5-vinyl phenanthrene (1), juncusin (2), dehydroeffusol (3), juncusol (4), effusol (5), and dehydroeffusal (6), were isolated from the medullae of Juncus effusus L. Compounds 1 and 2 were identified as being new structures, and both of them showed anxiolytic activity at dosages of 10 and 2.5 mg/kg, respectively.

Effects of dehydroeffusol on spasmogen-induced contractile responses of rat intestinal smooth muscles.

Dehydroeffusol is a naturally occurring phenanthrene isolated from Juncus effusus. In the context of screening new drugs against gastrointestinal spasms, we investigated its effects on isolated rat jejunum in vitro. Dehydroeffusol (30-90 µM) slightly and transiently enhanced contractions in a concentration-dependent manner but significantly inhibited the contractions induced by KCl (100 mM), (±)-Bay-K8644 (5 µM), pilocarpine (90 µM), and histamine (100 µM). These results show that dehydroeffusol may antagonize the spasmogenic activity of various agents, and therefore, could be a promising agent in the treatment of spasms. Its potential spasmolytic mechanism is also discussed.

Involvement of the insular nitric oxide signaling pathway in the expression of morphine-induced conditioned place preference in rats.

Nitric oxide (NO) has been recently reported to play an important role in the rewarding effects of addictive drugs. The regional NO signaling in the brain, however, is not completely clear. Here, we studied the effects of insular NO signaling on the expression of morphine-induced conditioned place preference (CPP). Insular microinjection of the NO inhibitors N-nitro L-arginine methyl ester (L-NAME) and 7-nitroindazole reduced the expression of morphine-induced CPP. The NO donor molsidomine, in contrast, reversed L-NAME-induced reduction of CPP expression. These results suggest that insular NO signaling is involved in the expression of morphine-CPP.

Insular muscarinic signaling regulates anxiety-like behaviors in rats on the elevated plus-maze.

Anxiety is one of the most prevalent neuropsychiatric disorders, and little is known about its pathogenesis. In order to investigate the neural mechanisms of this mental disorder, we used rat behavior in the elevated plus-maze as an animal model of anxiety and the insular cortex (insula) as a brain target. The microinjection of non-selective and selective M1 and M4 muscarinic acetylcholine receptor (mAChR) agonists or antagonists was used to explore whether the insular muscarinic receptor and its subtypes regulate levels of anxiety. The results showed that both non-selective and selective M1 and M4 mAChR agonists increased the time spent on exploring in the open arms, whereas antagonists decreased exploration. Our results indicate that activation of insular mAChRs could produce anxiolytic effects, whereas inhibition of insular mAChRs could increase anxiety. We concluded that the insular muscarinic system plays a role in the modulation of anxiety, and dysfunction of mAChR signaling may be involved in the mechanism of anxiogenesis.

Involvement of insular muscarinic cholinergic receptors in morphine-induced conditioned place preference in rats.

RATIONALE: Drug addiction represents a pathological usurpation of neural processes involved in learning and memory. Retrieval of drug-related memories can result in drug craving and relapse. Recently, the insula was identified as part of the neuronal circuit responsible for the processing of drug memory; however, its precise role remains unclear. OBJECTIVE: To investigate the involvement of insular muscarinic acetylcholine receptors (mAChRs) in the processing of drug memory. METHOD: The morphine-induced conditioned place preference (CPP) was used to assess drug memory. All rats were first trained with morphine to establish the CPP. Sub-groups of these rats were used for contextual cue-induced CPP reinstatement. Other sub-groups of rats underwent extinction of the CPP, and 5 m/kg morphine was used for priming-induced CPP reinstatement. Microinjection of mAChR antagonists or agonists into the insula was performed prior to the CPP tests in order to evaluate their effect on CPP expression. RESULTS: Insular microinjections of the nonselective mAChR antagonist, scopolamine, and the M1 antagonist, pirenzepine, significantly inhibited CPP expression in both contextual cue- and priming-induced CPP reinstatement; the M1 agonist, MCN-A-343, and the M4 antagonist, tropicamide, enhanced CPP expression. The M4 agonist, LY2033298, inhibited CPP expression. The M2 antagonist, methoctramine, and M3 antagonist, 4-DAMP, had no effect on CPP expression. CONCLUSION: Our results demonstrate that insular mAChRs play a role in the processing of drug memory. M1 and M4 mAChRs work paradoxically; M1 activation and M4 inhibition attenuate the expression of drug memory, while M1 inhibition and M4 activation augment the expression of drug memory.

Effects of ginsenosides on opioid-induced hyperalgesia in mice.

Opioid-induced hyperalgesia (OIH) is characterized by nociceptive sensitization caused by the cessation of chronic opioid use. OIH can limit the clinical use of opioid analgesics and complicate withdrawal from opioid addiction. In this study, we investigated the effects of Re, Rg1, and Rb1 ginsenosides, the bioactive components of ginseng, on OIH. OIH was achieved in mice after subcutaneous administration of morphine for 7 consecutive days three times per day. During withdrawal (days 8 and 9), these mice were administered Re, Rg1, or Rb1 intragastrically two times per day. On the test day (day 10), mice were subjected to the thermal sensitivity test and the acetic acid-induced writhing test. Re (300 mg/kg) inhibited OIH in both the thermal sensitivity test and the acetic acid-induced writhing test. However, the Rg1 and Rb1 ginsenosides failed to prevent OIH in either test. Furthermore, Rg1 showed a tendency to aggravate OIH in the acetic acid-induced writhing test. Our data suggested that the ginsenoside Re, but not Rg1 or Rb1, may contribute toward reversal of OIH.

Phenanthrenes from Juncus effusus with anxiolytic and sedative activities.

Eight phenanthrenes, 7-carboxy-2-hydroxy-1-methyl-5-vinyl-phenanthrene (1); 2,7-dihydroxy-1-methyl-5-aldehyde-9,10-dihydrophenanthrene (2); dehydroeffusol (3); dehydrojuncusol (4); 7-carboxy-2-hydroxy-1-methyl-5-vinyl-9,10-dihydrophenanthrene (5); 8-carboxy-2-hydroxy-1-methyl-5-vinyl-9,10-dihydrophenanthrene (6); effusol (7) and juncusol (8), were isolated from the aerial part of Juncus effusus. Compounds 1 and 2 were identified as new constituents. Compounds 7 and 8 showed anxiolytic and sedative activities.

A morphine/heroin vaccine with new hapten design attenuates behavioral effects in rats.

Heroin use has seriously threatened public heath in many countries, but the existing therapies continue to have many limitations. Recently, immunotherapy has shown efficacy in some clinical studies, including vaccines against nicotine and cocaine, but no opioid vaccines have been introduced in clinical studies. The development of a novel opioid antigen designed specifically for the prevention of heroin addiction is necessary. A morphine-keyhole limpet hemocyanin conjugate was prepared and administered subcutaneously in rats. Antibody titers in plasma were measured using an enzyme-linked immunosorbent assay (ELISA). Competitive ELISA was used to assess the selectivity of the antibodies. Dopamine concentrations in the nucleus accumbens in rats after vaccine administration were determined by high-performance liquid chromatography with electrochemical detection. The effects of the vaccine on the heroin-primed restatement of self-administration and locomotor sensitization were evaluated. A novel hapten, 6-glutarylmorphine, was produced, and the vaccine generated a high antibody titer response. This vaccine displayed specificity for both morphine and heroin, but the anti-morphine antibodies could not recognize dissimilar therapeutic opioid compounds, such as buprenorphine, methadone, naloxone, naltrexone, codeine, and nalorphine. The morphine antibody significantly decreased morphine-induced locomotor activity in rats after immunization. Importantly, rats immunized with this vaccine did not exhibit heroin-primed reinstatement of heroin seeking when antibody levels were sufficiently high. The vaccine reduced dopamine levels in the nucleus accumbens after morphine administration, which is consistent with its behavioral effects. These results suggest that immunization with a novel vaccine is an effective means of inducing a morphine-specific antibody response that is able to attenuate the behavioral and psychoactive effects of heroin.

Glycogen synthase kinase 3ß in the nucleus accumbens core is critical for methamphetamine-induced behavioral sensitization.

As a ubiquitous serine/threonine protein kinase, glycogen synthase kinase 3ß (GSK-3ß) has been considered to be important in the synaptic plasticity that underlies dopamine-related behaviors and diseases. We recently found that GSK-3ß activity in the nucleus accumbens (NAc) core is critically involved in cocaine-induced behavioral sensitization. The present study further explored the association between the changes in GSK-3ß activity in the NAc and the chronic administration of methamphetamine. We also examined whether blocking GSK-3ß activity in the NAc could alter the initiation and expression of methamphetamine (1 mg/kg, i.p.)-induced locomotor sensitization in rats using systemic administration of lithium chloride (LiCl, 100 mg/kg, i.p) and brain region-specific administration of the GSK-3ß inhibitor SB216763 (1 ng/side). We found that GSK-3ß activity increased in the NAc core, but not NAc shell, after chronic methamphetamine administration. The initiation and expression of methamphetamine-induced locomotor sensitization was attenuated by systemic administration of LiCl and direct infusion of SB216763 into the NAc core, but not NAc shell. These results indicate that GSK-3ß activity in the NAc core mediates the initiation and expression of methamphetamine-induced locomotor sensitization, suggesting that GSK-3ß may be a potential target for the treatment of psychostimulant addiction.

Anxiolytic and sedative effects of dehydroeffusol from Juncus effusus in mice.

Dehydroeffusol, a phenanthrene isolated from Juncus effusus L., possesses characteristic anxiolytic and sedative properties, as determined by an array of behavioral tests in mice. In the elevated plus-maze test, dehydroeffusol significantly increased the number of entries into the open arms and the time the mice spent in these arms in a dose-dependent manner, with a minimum effective dose of 2.5 mg/kg. Dehydroeffusol also significantly increased the head-dips of mice in the hole-board test in a dose-dependent manner, with a minimum effective dose of 5 mg/kg. Dehydroeffusol reduced mouse locomotion in the open-field test with a minimum effective dose of 5 mg/kg. In the rota-rod test, 1-5 mg/kg dehydroeffusol did not decrease the fall-down time of mice. The above results confirm that dehydroeffusol possesses anxiolytic and sedative properties and does not affect the general movement coordination of mice. This suggests that dehydroeffusol is a novel anxiolytic chemical derived from herbal medicines.

Effects of polyinosinic-polycytidylic acid (Poly I:C) on naloxone-precipitated withdrawal in morphine-dependent mice.

Viral infections are frequently found in opioid addicts, subjecting them to immune challenge. However, the effects of immune challenge on opioid withdrawal are not fully understood. In the present study, mice were intraperitoneally injected with 2mg/kg polyinosinic-polycytidylic acid (Poly I:C, a viral mimetic) for 3 days to induce an immune challenge, followed by subcutaneous injection of morphine 3 times per day for 3 days to induce morphine dependence. Withdrawal was induced by an intraperitoneal injection of 5mg/kg naloxone, an opioid receptor antagonist. The results showed that Poly I:C pretreatment did not alter body weight loss, jumping behavior, or locomotion during naloxone-precipitated withdrawal. In contrast, Poly I:C pretreatment significantly increased immobility time in the tail suspension test. Our findings suggest that Poly I:C-induced immune challenge has no effects on acute physical opioid withdrawal symptoms but facilitates depression-like behavior.