Changes in hypothalamic mu-opioid receptor expression following acute olanzapine treatment in female rats: Implications for feeding behavior.

Advances have been made in recent years in using opioid receptor antagonists as an adjunct therapy to psychotropic medication to reduce debilitating weight gain and metabolic adverse effects associated with in particular second generation antipsychotics. However, it is unknown whether second generation antipsychotics produce a change in opioid receptor expression in the brain. The present study investigated early changes in opioid receptor expression in the female rat hypothalamus, a master controller of hunger and metabolic regulation, after acute treatment with olanzapine, a commonly used second generation antipsychotic. Using quantitative spatial in situ hybridization and receptor autoradiography, expression levels of the three opioid receptors; kappa, mu and delta, were determined at mRNA and protein level, respectively, in the five hypothalamic areas: paraventricular nucleus, arcuate nucleus, ventromedial nucleus, dorsomedial nucleus and lateral hypothalamus. After 48 h of olanzapine treatment at clinically relevant plasma concentration weight gain and food intake changes, and increased plasma glucose were observed in female rats. Olanzapine treatment also led to a significant increase in mu opioid receptor availability in the arcuate nucleus, which contains both satiety and hunger controlling neurons. No other areas showed any opioid receptor expressional changes with olanzapine treatment on neither at mRNA nor protein level. Technical difficulties made it impossible to analyze mRNA levels in the lateral hypothalamus and overall binding of delta opioid receptors. Thus, the present study provided insights in to how olanzapine at clinically relevant plasma levels already at an early stage modulated the opioid system in the hypothalamus.

Receptor Transporter Protein 4 (RTP4) in the Hypothalamus Is Involved in the Development of Antinociceptive Tolerance to Morphine.

Receptor transporter protein 4 (RTP4), one of the receptor chaperone proteins, contributes to the maturation and membrane trafficking of opioid receptor heteromers consisting of mu (MOPr) and delta (DOPr) opioid receptors (MOPr-DOPr). Although MOPr-DOPr is known to mediate the development of morphine tolerance, the extent to which RTP4 plays a role in this process has not been elucidated. Given that RTP4 can be upregulated by repeated administration of morphine, especially in the hypothalamus, here we investigated the effect of hypothalamus-selective ablation of RTP4 on the development of antinociceptive tolerance to morphine. In this study, we generated RTP4flox mice and selectively knocked-out RTP4 using local injection of adeno-associated virus expressing Cre recombinase (AAV-Cre) into the hypothalamus. The AAV-Cre injection partially, but significantly, decreased the level of RTP4 expression, and suppressed the development of antinociceptive tolerance to morphine. Next, we examined the mechanism of regulation of RTP4 and found that, in neuronal cells, Rtp4 induction is via Gi and MAPK activation, while, in microglial cells, the induction is via Toll-like receptor 4. Together, these studies highlight the role of MOR activity in regulating RTP4, which, in turn, plays an important role in modulating morphine effects in vivo.

A lesion-mimic mutant of Catharanthus roseus accumulates the opioid agonist, akuammicine.

Catharanthus roseus is a medicinal plant that produces an abundance of monoterpenoid indole alkaloids (MIAs), notably including the anticancer compounds vinblastine and vincristine. While the canonical pathway leading to these drugs has been resolved, the regulatory and catalytic mechanisms controlling many lateral branches of MIA biosynthesis remain largely unknown. Here, we describe an ethyl methanesulfonate (EMS) C. roseus mutant (M2-117523) that accumulates high levels of MIAs. The mutant exhibited stunted growth, partially chlorotic leaves, with deficiencies in chlorophyll biosynthesis, and a lesion-mimic phenotype. The lesions were sporadic and spontaneous, appearing after the first true bifoliate and continuing throughout development. The lesions are also the site of high concentrations of akuammicine, a minor constituent of wild type C. roseus leaves. In addition to akuammicine, the lesions were enriched in 25 other MIAs, resulting, in part, from a higher metabolic flux through the pathway. The unique metabolic shift was associated with significant upregulation of biosynthetic and regulatory genes involved in the MIA pathway, including the transcription factors WRKY1, CrMYC2, and ORCA2, and the biosynthetic genes STR, GO, and Redox1. Following the lesion-mimic mutant (LMM) phenotype, the accumulation of akuammicine is jasmonate (JA)-inducible, suggesting a role in plant defence response. Akuammicine is medicinally significant, as a weak opioid agonist, with a preference for the κ-opioid receptor, and a potential anti-diabetic. Further study of akuammicine biosynthesis and regulation can guide plant and heterologous engineering for medicinal uses.

Curcumin induces peripheral antinociception by opioidergic and cannabinoidergic mechanism: Pharmacological evidence.

BACKGROUND: Curcumin is one of the compounds present in plants of the genus Curcuma sp., being very used not only as condiment but also with medicinal purposes. As an analgesic, papers highlight the efficacy of curcumin in the treatment of various types of pain. AIMS: In this study we evaluated the peripheral antinociceptive effect of curcumin and by which mechanisms this effect is induced. MAIN METHODS: The mice paw pressure test was used on animals which had increased pain sensitivity by intraplantar injection of carrageenan. All the drugs were administered in the right hind paw. KEY FINDINGS: Curcumin was administered to the right hind paw animals induced antinociceptive effect. Non -selective antagonist of opioid receptors naloxone reverted the antinociceptive effect induced by curcumin. Selective antagonists for µ, δ and κ opioid receptors clocinnamox, naltrindole and nor- binaltorphimine, respectively, reverted the antinociceptive effect induced by curcumin. Bestatin, enkephalinases inhibitor that degrade peptides opioids, did not change the nociceptive response. Selective antagonists for CB1 and CB2 cannabinoid receptors, AM251 and AM630, respectively, reversed the antinociceptive effect induced by curcumin. The MAFP inhibitor of the enzyme FAAH which breaks down anandamide, JZL184, enzyme inhibitor MAGL which breaks down the 2-AG, as well as the VDM11 anandamide reuptake inhibitor potentiated the antinociceptive effect of curcumin. SIGNIFICANCE: These results suggest that curcumin possibly peripheral antinociception induced by opioid and cannabinoid systems activation and possibly for endocannabinoids and opioids release.

Antinociceptive and Anti-Inflammatory Effects of Saline Extract and Lectin-Rich Fraction from Microgramma vacciniifolia Rhizome in Mice.

Previous studies have characterized a saline extract from Microgramma vacciniifolia rhizome and its lectin (MvRL)-rich fraction with low acute toxicity. In the present study, we evaluated these preparations for acute toxicity (1,000 mg/kg) and antinociceptive and anti-inflammatory activities (100-400 mg/kg for the extract and 25-50 mg/kg for the fraction). No signs of toxicity were observed. Both the extract and fraction increased the latency period for nociception in the hot plate assay, decreased writhing induced by acetic acid, and promoted analgesic effects in phases 1 and 2 of the formalin test. The antinociceptive mechanism was attributed to interactions with opioid receptors and K+ ATPase channels. The extract and fraction decreased carrageenan-induced paw edema in 46.15 % and 77.22 %, respectively, at the highest doses evaluated. Furthermore, the fraction was shown to act on the bradykinin pathway. The ability to decrease leukocyte migration after treatment was also verified in the peritonitis and air pouch models. In exudates collected from air pouches, decreased tumor necrosis factor (TNF)-α and increased interleukin (IL)-10 levels were noted. Both the extract and fraction also effectively inhibited the development of granulomatous tissue. In conclusion, the substances investigated in this study can be used for the development of novel therapeutic options for pain and inflammatory processes.

Electroacupuncture decreases inflammatory pain through a pro-resolving mechanism involving the peripheral annexin A1-formyl peptide receptor 2/ALX-opioid receptor pathway.

The pro-resolving mechanism is a recently described endogenous process that controls inflammation. The present study evaluated components of this mechanism, including annexin 1 (ANXA1) and the formyl peptide receptor 2/ALX (FPR2/ALX) receptor, in the antihyperalgesic effect induced by electroacupuncture (EA) in an animal model of persistent peripheral inflammation. Male Swiss mice underwent intraplantar (i.pl.) injection with complete Freund's adjuvant (CFA). Mechanical hyperalgesia was assessed with von Frey monofilaments. Animals were treated with EA (2-10 Hz, ST36-SP6) or subcutaneous BML-111 injection (FPR2/ALX agonist) for 5 consecutive days. In a separate set of experiments, on the first and fifth days after CFA injection, animals received i.pl. WRW4 (FPR2/ALX antagonist) or naloxone (non-selective opioid receptor antagonist) before EA or BML-111 injection. Paw protein levels of FPR2/ALX and ANXA1 were evaluated on the second day after CFA injection by western blotting technique. EA and BML-111 reduced mechanical hyperalgesia. I.pl. naloxone or WRW4 prevented the antihyperalgesic effect induced by either EA or BML-111. EA increased ANXA1 but did not alter FPR2/ALX receptor levels in the paw. Furthermore, i.pl. pretreatment with WRW4 prevented the increase of ANXA1 levels induced by EA. This work demonstrates that the EA antihyperalgesic effect on inflammatory pain involves the ANXA1/FPR2/ALX pro-resolution pathway. This effect appears to be triggered by the activation of FPR2/ALX receptors and crosstalk communication with the opioid system.

Involvement of opioidergic and GABAergic systems in the anti-nociceptive activity of the methanolic extract of Cuscuta Epithymum Murr. in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cuscuta epithymum Murr. (CE) is a parasitic plant used as a traditional medicine to treat various diseases such as muscle and joint pains and headache different parts of the world, Europe in the north, Asia in the east. AIM OF THE STUDY: In this study, we aimed to investigate the anti-nociceptive effect of the methanolic extract of the aerial parts of CE and its probable mechanism(s) in mice. MATERIALS AND METHODS: The anti-nociceptive activity of different doses of CE methanolic extract (2.5, 5, 10, 25, 50 and 100 mg/kg, i.p.) was assessed using tail flick, formalin and writhing tests. Morphine (5 mg/kg, s.c.) was used as positive control drug. The possible mechanisms were evaluated by using naloxone (4 mg/kg, i.p.), ondansetron (4 mg/kg, i.p.), picrotoxin (0.6 mg/kg, i.p.) and MK-801 (0.03 mg/kg, i.p.). RESULTS: GC-MS analysis indicated that one of the main components of CE extract was terpenoid compounds. The CE extract (25-100 mg/kg), like morphine, reduced tail flick latency and nociceptive response in both phases of the formalin test. We also observed that the extract significantly decreased the number of abdominal contractions dose-dependently from 5 to 100 mg/kg. The results of tail flick and the first phase of formalin test proved that unlike ondansetron and MK-801, naloxone and picotroxin were able to reverse the anti-nociceptive effect of CE extract. CONCLUSION: Our observations showed the anti-nociceptive potential of the CE extract, which may be mediated by opioidergic and GABAergic systems.

Label-free cell phenotypic study of opioid receptors and discovery of novel mu opioid ligands from natural products.

ETHNOPHARMACOLOGICAL RELEVANCE: Mu opioid receptor (MOR) is mainly a drug target for analgesia. Opioid-like agonists such as morphine have been clinically used for analgesia but have potential adverse effects. MOR antagonists have been demonstrated to alleviate these side effects. Plants (Carthamus tinctorius L, Cynanchum otophyllum C. K. Schneid., Coffea arabica L., Prinsepia utilis Royle and Lepidium meyenii Walp.) and Ganoderma fungi (Ganoderma hainanense J. D. Zhao, Ganoderma capense (Lloyd) Teng, Ganoderma cochlear (Blume et Nees) Bres., Ganoderma resinaceum Boud and Ganoderma applanatum (Pers.) Pat.) are traditional medicines with beneficial effects on immunoregulation, analgesia and the nervous system, but whether MORs are engaged in their effects remains unknown. AIM OF THE STUDY: This work aimed to identify MOR ligands among compounds isolated from the above-mentioned 10 species, and to investigate selectivity against four opioid receptor subtypes. By analyzing the structure-activity relationship and off-target effects, we could provide a new direction for the future development of MOR drugs. MATERIALS AND METHODS: Four opioid receptor subtype models, including MOR, delta (DOR), kappa (KOR) and nop (NOR), were established with a label-free phenotypic dynamic mass redistribution assay to systematically profile the pharmacological properties of known ligands. Then, 82 natural compounds derived from the 10 species were screened against MOR to identify new ligands. The selectivity of the new ligands was characterized against the four subtypes, and off-target effects were also investigated on eight G protein-coupled receptors (GPCRs). RESULTS: The pharmacological properties of known ligands on transfected HEK293T-MOR, HEK293-DOR, HEK293-KOR and HEK293-NOR cell lines were characterized. Seven compounds purified from Ganoderma cochlear (Blume et Nees) Bres. and Carthamus tinctorius L were MOR antagonists with micromolar potency. Among them, compound 35 showed the strongest antagonistic activity on MOR with an IC50 value of 10.0 ± 3.0 µM. To a certain extent, these seven new antagonists, exhibited antagonistic activity on the other opioid receptor subtypes, and they had almost no effect on other GPCRs, including CB1, CB2, M2 and beta2AR. Additionally, a compound from Lepidium meyenii Walp. displayed MOR agonistic activity. CONCLUSIONS: The established screening models opened new avenues for the discovery and evaluation of opioid receptor ligand selectivity. Together, the novel MOR antagonists and agonists will enrich the inventory of MOR ligands and benefit related therapies.

Early-Life Stress as a Probe to Study the Opioid System in Developing Rodents.

The developmental origins of disease or fetal programming model predict that early (intrauterine and/or postnatal) exposures to external insults of sufficient length and intensity may have enduring or lifelong consequences for physical and psychological health. The method described in this chapter considers an animal model to study the pathophysiological alterations connected to an HPA axis (hypothalamic-pituitary-adrenal) hyperactivity that are induced by an early-life stressful procedure involving the opioid system.

Multifunctional Opioid-Derived Hybrids in Neuropathic Pain: Preclinical Evidence, Ideas and Challenges.

When the first- and second-line therapeutics used to treat neuropathic pain (NP) fail to induce efficient analgesia-which is estimated to relate to more than half of the patients-opioid drugs are prescribed. Still, the pathological changes following the nerve tissue injury, i.a. pronociceptive neuropeptide systems activation, oppose the analgesic effects of opiates, enforcing the use of relatively high therapeutic doses in order to obtain satisfying pain relief. In parallel, the repeated use of opioid agonists is associated with burdensome adverse effects due to compensatory mechanisms that arise thereafter. Rational design of hybrid drugs, in which opioid ligands are combined with other pharmacophores that block the antiopioid action of pronociceptive systems, delivers the opportunity to ameliorate the NP-oriented opioid treatment via addressing neuropathological mechanisms shared both by NP and repeated exposition to opioids. Therewith, the new dually acting drugs, tailored for the specificity of NP, can gain in efficacy under nerve injury conditions and have an improved safety profile as compared to selective opioid agonists. The current review presents the latest ideas on opioid-comprising hybrid drugs designed to treat painful neuropathy, with focus on their biological action, as well as limitations and challenges related to this therapeutic approach.

Antinociceptive activity of Schinus terebinthifolia leaf lectin (SteLL) in sarcoma 180-bearing mice.

ETHNOPHARMACOLOGY RELEVANCE: Schinus terebinthifolia Raddi leaves have been used in folk medicine due to several properties, including antitumor and analgesic effects. The variable efficacy and adverse effects of analgesic drugs have motivated the search for novel antinociceptive agents. It has been reported that the S. terebinthifolia leaf lectin (SteLL) has antitumor activity against sarcoma 180 in mice. AIM OF THE STUDY: This work aimed to evaluate whether SteLL would reduce cancer pain using an orthotopic tumor model. MATERIALS AND METHODS: A sarcoma 180 cell suspension was inoculated into the right hind paws of mice, and the treatments (150 mM NaCl, negative control; 10 mg/kg morphine, positive control; or SteLL at 1 and 2 mg/kg) were administered intraperitoneally 24 h after cell inoculation up to 14 days. Spontaneous nociception, mechanical hyperalgesia, and hot-plate tests were performed. Further, the volume and weight of the tumor-bearing paws were measured. RESULTS: SteLL (2 mg/kg) improved limb use during ambulation. The lectin (1 and 2 mg/kg) also inhibited mechanical hyperalgesia and increased the latency time during the hot-plate test. Naloxone was found to reverse this effect, indicating the involvement of opioid receptors. The tumor-bearing paws of mice treated with SteLL exhibited lower volume and weight. CONCLUSION: SteLL reduced hyperalgesia due to sarcoma 180 in the paws of mice, and this effect can be related to its antitumor action.

Pharmacological and molecular docking assessment of cryptotanshinone as natural-derived analgesic compound.

Medicinal plants from traditional chinese medicine are used increasingly worldwide for their benefits to health and quality of life for the relevant clinical symptoms related to pain. Among them, Salvia miltiorrhiza Bunge is traditionally used in asian countries as antioxidant, anticancer, anti-inflammatory and analgesic agent. In this context, several evidences support the hypothesis that some tanshinones, in particular cryptotanshinone (CRY), extracted from the roots (Danshen) of this plant exhibit analgesic actions. However, it is surprisingly noted that no pharmacological studies have been carried out to explore the possible analgesic action of this compound in terms of modulation of peripheral and/or central pain. Therefore, in the present study, by using peripheral and central pain models of nociception, such as tail flick and hot plate test, the analgesic effect of CRY in mice was evaluated. Successively, by the aim of a computational approach, we have evaluated the interaction mode of this diterpenoid on opioid and cannabinoid system. Finally, CRY was dosed in mice serum by an HPLC method validated according to European Medicines Agency guidelines validation rules. Here, we report that CRY displayed anti-nociceptive activity on both hot plate and tail flick test, with a prominent long-lasting peripheral analgesic effect. These evidences were indirectly confirmed after the daily administration of the tanshinone for 7 and 14 days. In addition, the analgesic effect of CRY was reverted by naloxone and cannabinoid antagonists and amplified by arginine administration. These findings were finally supported by HPLC and docking studies, that revealed a noteworthy presence of CRY on mice serum 1 h after its intraperitoneal administration and a possible interaction of tested compound on µ and k receptors. Taken together, these results provide a new line of evidences showing that CRY can produce analgesia against various phenotypes of nociception with a mechanism that seems to be related to an agonistic activity on opioid system.

Evaluating kratom alkaloids using PHASE.

Kratom is a botanical substance that is marketed and promoted in the US for pharmaceutical opioid indications despite having no US Food and Drug Administration approved uses. Kratom contains over forty alkaloids including two partial agonists at the mu opioid receptor, mitragynine and 7-hydroxymitragynine, that have been subjected to the FDA's scientific and medical evaluation. However, pharmacological and toxicological data for the remaining alkaloids are limited. Therefore, we applied the Public Health Assessment via Structural Evaluation (PHASE) protocol to generate in silico binding profiles for 25 kratom alkaloids to facilitate the risk evaluation of kratom. PHASE demonstrates that kratom alkaloids share structural features with controlled opioids, indicates that several alkaloids bind to the opioid, adrenergic, and serotonin receptors, and suggests that mitragynine and 7-hydroxymitragynine are the strongest binders at the mu opioid receptor. Subsequently, the in silico binding profiles of a subset of the alkaloids were experimentally verified at the opioid, adrenergic, and serotonin receptors using radioligand binding assays. The verified binding profiles demonstrate the ability of PHASE to identify potential safety signals and provide a tool for prioritizing experimental evaluation of high-risk compounds.

Antinociceptive and antineuropathic effects of cuminaldehyde, the major constituent of Cuminum cyminum seeds: Possible mechanisms of action.

ETHNOPHARMACOLOGICAL RELEVANCE: In Iranian traditional medicine, Cuminum cyminum is a unique medicinal herb for pain relief. Cuminaldehyde has been distinguished as the major constituent of C. cyminum seeds; even though, the analgesic effect of cuminaldehyde has not yet been examined. AIM OF THE STUDY: The nobility of this study was to assess cuminaldehyde effect on nociceptive and neuropathic pains; furthermore, evaluation of its possible mechanisms of action. MATERIALS AND METHODS: Hot plate, formalin, and acetic acid-induced writhing tests were used to evaluate nociception in mice. Naloxone (opioid receptors antagonist), L-arginine (nitric oxide (NO) precursor), L-NAME (NO synthase inhibitor), sodium nitroprusside (NO donor), methylene blue (guanylyl cyclase inhibitor), sildenafil (phosphodiesterase inhibitor), and glibenclamide (KATP channel blocker) were used to determine the implication of opioid receptors and L-arginine/NO/cGMP/KATP channel pathway. Allodynia and hyperalgesia were investigated in the CCI (chronic constriction injury) model of neuropathic pain in rats. The ELISA method was used to measure the inflammatory cytokines in serum samples of rats. The entire chemicals were intraperitoneally injected. RESULTS: Cuminaldehyde (100 and 200 mg/kg) significantly decreased the latency to nociceptive response in the hot plate test. The outcome of cuminaldehyde was completely antagonized by naloxone (2 mg/kg). Formalin- and acetic acid-induced nociception was significantly inhibited by cuminaldehyde (12.5-50 mg/kg). The antinociceptive effect of cuminaldehyde was reversed in writhing test by L-arginine (200 mg/kg), sodium nitroprusside (0.25 mg/kg), and sildenafil (0.5 mg/kg); however, L-NAME (30 mg/kg) and methylene blue (20 mg/kg) enhanced the effect of cuminaldehyde. Glibenclamide (10 mg/kg) did not alter the antinociceptive effects of cuminaldehyde. In the CCI-induced neuropathy, cuminaldehyde (25-100 mg/kg) significantly alleviated allodynia and hyperalgesia and decreased the serum levels of TNF-α and IL-1ß. CONCLUSION: It was attained magnificently that cuminaldehyde exerts antinociceptive and antineuropathic effects through the involvement of opioid receptors, L-arginine/NO/cGMP pathway, and anti-inflammatory function.

Lavandula angustifolia essential oil inhalation reduces mechanical hyperalgesia in a model of inflammatory and neuropathic pain: The involvement of opioid and cannabinoid receptors.

Lavandula angustifolia (LaEO) essential oil has been widely used by aromatherapy in the treatment of various clinical conditions, with evidence of its analgesic and anti-inflammatory potential. Our results demonstrate that sixty-five substances were identified in LaEO. Among the compounds found, the major ones were linalool (30.61%) and linalyl acetate (20.36%). We found that LaEO inhalation reduces mechanical hyperalgesia in conditions of chronic inflammatory and neuropathic pain. Furthermore, this effect seems to be mediated by peripheral and central opioid and cannabinoid 2 receptors. The findings of the present study suggests that the LaEO inhalation is effective on the chronic pain treatment.

Tibetan medicine Ru-yi-Zhen-bao Pills exhibits anti-migraine effect through mediating PAG anti-nociceptive channel.

ETHNOPHARMACOLOGICAL RELEVANCE: Migraine is a disabling neurovascular disorder, which increases risk of cardiovascular events and is a social burden worldwide. The present first-line anti-migraine medications can cause overwhelming side-effects, of which one includes the onset of cardiovascular disease. As one of the marketed Tibetan drugs, Ru-yi-Zhen-bao Pills (RYZBP) have been clinically used to treat cardiovascular disorders and as anti-migraine medication. However, there is currently no research exploring the anti-migraine actions of RYZBP. AIM OF THE STUDY: The current research was designed to assess the anti-migraine roles of RYZBP and explore the underlying mechanisms in a nitroglycerin (NTG)-induced migraine rat model trial. MATERIALS AND METHODS: 120 rats were randomly divided into the following six groups of 20 rats each: normal control group, model control group, positive control group, and RYZBP high/medium/low-dose groups (Ru-yi-Zhen-bao Pills; TH 1.00 g/kg, TM 0.50 g/kg and TL 0.25 g/kg). All rats were administered intragastrically for 7 consecutive days, which were subcutaneously injected with the NTG (10 mg/kg) after the last gavage (except in the normal control group). 3min after NTG treatment, 30 rats (5 rats from each group) were anesthetized and devoted to electroencephalogram(EEG) testing, which was used to evaluate the analgesic effect of RYZBP. One hour after NTG treatment, the rest of the 90 rats (15 rats from each group) were anesthetized and midbrain tissue sample was dissected. The dissection was then washed with physiological saline and collected. The histopathological changes in the periaqueductal gray(PAG) of 5 tissue samples were determined by aematoxylin-eosin (H&E) staining, as well as an estimation of substance P (SP) and neurokinin 1 receptor (NK1R) expression through immunohistochemically staining(IHC). Another 5 midbrain preparations were carried out to evaluate calcitonin gene-related peptide (CGRP), proenkephalin (PENK), SP, and cholecystokinin (CCK) expressions by real-time quantitative polymerase chain reaction (RT-qPCR). The rest of the 5 brainstem tissues were then used to measure CCK, CGRP, and opioid peptide receptor (DORR) levels by western blotting(WB). RESULTS: In the EEG test, RYZBP (TM 0.50 g / kg) treatment transformed the EEG pain-wave of the NTG-induced migraine model rats in different time period. In the mechanism assay, compared with the model control group, RYZBP pretreatment reduced inflammatory cell infiltration, fibrosis and vacuolation of neuronal cells of PAG tissue seen by HE staining. IHC experiments further showed that RYZBPTM up-regulated SP expression levels and enhanced NK1R levels in the NTG-induced migraine rats (P < 0.05). Therapeutic administration of RYZBP also increased PENK mRNA expression and DORR protein level. Both RT-qPCR and western blotting trials indicated that RYZBP treatment significantly decreased CCK and CGRP expression levels (P < 0.01 or P < 0.05) in the NTG-induced migraine rats. CONCLUSIONS: RYZBP has the potential to be an effective anti-migraine treatment through suppressing the EEG pain-wave, increasing the levels of SP, PENK, DORR and reducing expression of CCK and CGRP. Mediating the PAG anti-nociceptive channel and inhibiting central sensitization were the two potential mechanisms, which offers further evidence for clinical therapy.

Modulation of COX-2, INF-É£, glutamatergic and opioid systems contributes to antinociceptive, anti-inflammatory and anti-hyperalgesic effects of bis(3-amino-2-pyridine) diselenide.

Preclinical assays play a key role in research in research on the neurobiology of pain and the development of novel analgesics. Drugs available for the treatment of inflammatory pain are not fully effective and show adverse effects. Thus, we investigated the antinociceptive, anti-inflammatory and anti-hyperalgesic effects of bis(3-amino-2-pyridine) diselenide (BAPD), a new analgesic drug prototype. BAPD effects were investigated using nociception models induced by chemical (glutamate), immunologic (Freund's Complete Adjuvant - CFA) and thermal stimuli in Swiss mice. Mice were orally (p.o.) treated with BAPD (0.1-50 mg/kg) 30 min prior to the glutamate and hot-plate tests and a time-course (0.5 up to 8 h) of the antinociceptive effect of BAPD (50 mg/kg, p. o.) was evaluated in a CFA model. In the CFA model, BAPD effects on cyclooxygenase-2 (COX-2), tumor necrosis factor (TNFα) and interferon-γ (INF-γ) expression, myeloperoxidase (MPO) activity, oxidative (2,2'-Azino-bis-3-ethylbenzothiazoline 6-sulfonic acid and 2,2-diphe- nyl-1-picrylhydrazyl levels) and histological parameters were evaluated. The safety of the compound (50 and 300 mg/kg, p. o.) was verified for 72 h. BAPD reduced the licking time induced by glutamate and caused an increase in latency response to thermal stimulus. Naloxone reversed the antinociceptive effect of BAPD. Paw edema formation induced by glutamate or CFA injection was reduced by BAPD. Mechanical hyperalgesia induced by CFA was attenuated by BAPD. BAPD did not protect against the increase in MPO activity and decrease of the 2,2'-Azino-bis-3-ethylbenzothiazoline 6-sulfonic acid and 2,2-diphe- nyl-1-picrylhydrazyl levels induced by CFA. BAPD protected against histological alterations and reduction on the levels of gene expression COX-2 and INF-γ in the paw of mice exposed to CFA. BAPD was safe at the doses and time evaluated. BAPD exerts acute antinociceptive, anti-inflammatory and anti-hyperalgesic actions, suggesting that it may represent an alternative in the future development of new therapeutic strategies.

Influence of Silybum Marianum on Morphine Addicted Rats, Biochemical Parameters and Molecular Simulation Studies on µ-Opioid Receptor.

The present study aimed to investigate effects of medicinal plant (Silybum marianum) on the animals with treatment of morphine addiction compared to chemical drug (Naloxane). Also, this study was conducted to evaluate the role of Silybum marianum on factors of serum ALT, AST activities and activity of the antioxidant enzyme:superoxide dismutase (SOD) as well as the extent of lipid peroxidation of Morphine addicted rats.High performance liquid chromatography (HPLC) has been used to measure Morphine in the serum, enzymes functions well as lipid peroxidation of Morphine addicted animals. Results demonstrate treatment with S. Marianum for opium rats at dose 400 mg/kg led to a major reduction in serum morphine compared to Naloxane (400 mg/kg>200 mg/kg>100 mg/kg>Naloxane. The positive effect of dose-dependence on liver enzymes function (ALT and AST) in order of 400 mg/kg>Naloxane>200 mg/kg>100 mg/kg. Furthermore, the findings show that the malondialdehyde levels are increased in opium-treated animals. However, the extracts also demonstrate a significant reduction in the MDA levels compared to the control, 400 mg/kg>200 mg/kg>Naloxane>100 mg/kg. Increase of Silybum marianum extract in rats pretreated significantly elevated the activities of superoxide dismutase (SOD).At last, our data suggest that Silibinin as the main component found in Milk thistle is more selective toward µ-Opioid Receptor than Morphine and Naloxane as a narcotic receptor antagonist.

Dose-Dependent Effect of Hyperbaric Oxygen Treatment on Burn-Induced Neuropathic Pain in Rats.

Hyperbaric oxygen treatment (HBOT) has been used to reduce neuropathic pain. Melatonin and opioid receptors are involved in neuropathic pain, but it is not known if HBOT works through these pathways to achieve its antinociceptive effect. We divided anesthetized rats into two treatment and three sham groups. The two treatment groups received third-degree burns on their right hind paws, one treated in a hyperbaric chamber for a week and the other for two weeks. We evaluated the mechanical paw-withdrawal threshold (MWT) and expression of melatonin receptor 1 (MT1), melatonin receptor 2 (MT2), µ (MOR) and κ (KOR) opioid receptor, brain-derived neurotrophic factor (BDNF), Substance P, and calcitonin gene-related peptide (CGRP) in cuneate nucleus, dorsal horn, and hind paw skin by immunohistochemical, immunofluorescence assays and real-time quantitative polymerase chain reaction (RT-PCR). The group receiving one-week HBOT had increased expressions of MT1, MT2, MOR and KOR and decreased expressions of BDNF, Substance P, and CGRP. Their mechanically measured pain levels returned to normal within a week and lasted three weeks. This anti-allodynia effect lasted twice as long in those treated for two weeks. Our findings suggest that increasing the duration of HBOT can reduce burn-induced mechanical allodynia for an extended period of time in rats. The upregulation of melatonin and opioid receptors observed after one week of HBOT suggests they may be partly involved in attenuation of the mechanical allodynia. Downregulation of BDNF, substance P and CGRP may have also contributed to the overall beneficial effect of HBOT.

Plasma membrane G protein-coupled estrogen receptor 1 (GPER) mediates rapid estradiol facilitation of sexual receptivity through the orphanin-FQ-ORL-1 system in estradiol primed female rats.

In estradiol-primed nonreceptive ovariectomized rats, activation of G protein-coupled estrogen receptor 1 (GPER) in the arcuate nucleus of the hypothalamus (ARH) rapidly facilitates sexual receptivity (lordosis). Estradiol priming activates ARH ß-endorphin (ß-END) neurons that then activate medial preoptic (MPN) µ-opioid receptors (MOP) to inhibit lordosis. ARH infusion of non-esterified 17ß-estradiol (E2) 47.5 h after 17ß-estradiol benzoate (2 µg EB) priming deactivates MPN MOP and rapidly facilitates lordosis within 30 min via activation of GPER. Since it was unclear where GPERs were located in the neuron, we tested the hypothesis that GPER signaling is initiated at the plasma membrane. Membrane impermeable estradiol (17ß-estradiol conjugated to biotin; E-Biotin) infused into the ARH of EB primed rats facilitated lordosis within 30 min, and MPN MOP was deactivated. These actions were blocked by pretreating with GPER antagonist, G-15. Further, we used cell fractionation and western blot techniques to demonstrate that GPER is expressed both in plasma membrane and cytosolic ARH fractions. In previous studies, the orphanin FQ/nociceptin-opioid receptor-like receptor-1 (OFQ/N-ORL-1) system mediated estradiol-only facilitation of lordosis. Therefore, we tested whether the OFQ/N-ORL-1 system mediates E-Biotin-GPER facilitation of lordosis. Pretreatment of UFP-101, an ORL-1 selective antagonist, blocked the facilitation of lordosis and deactivation of MPN MOP by ARH infusion of E-Biotin. Double-label immunohistochemistry revealed that GPER is expressed within approximately 70% of OFQ/N neurons. These data indicate that membrane GPER mediates the E2/E-Biotin facilitation of lordosis by inducing OFQ/N neurotransmission, which inhibits ß-END neurotransmission to reduce MPN MOP activation.