OBJECTIVE:To investigate the mechanism underlying the effect of the Huanglian decoction(黄连汤,HLD)on morphine tolerance(MT),using network pharmacology,and to verify these mechanisms in vitro and in vivo.METHODS:Avail...OBJECTIVE:To investigate the mechanism underlying the effect of the Huanglian decoction(黄连汤,HLD)on morphine tolerance(MT),using network pharmacology,and to verify these mechanisms in vitro and in vivo.METHODS:Available biological data on each drug in the HLD were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform.The target proteins of MT were retrieved from the GeneCards,PharmGkb,Therapeutic Target Database,DrugBank,and Online Mendelian Inheritance in Man databases.Information regarding MT and the drug targets was compared to obtain overlapping elements.This information was imported into the Search Tool for the Retrieval of Interacting Genes/Proteins platform to obtain a protein-protein interaction network diagram.Then,a“component-target”network diagram was constructed using screened drug components and target information,via Cytoscape(Institute for Systems Biology,Seattle,WA,USA).The database for annotation,visualization,and integrated discovery was used for Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathways analyses.Pathway information predicted by network pharmacology was verified using animal studies and cell experiments.RESULTS:Network pharmacology analysis identified 22 active compounds of HLD and revealed that HLD partially ameliorated MT by modulating inflammatory,apoptosis,and nuclear factor kappa B(NF-κB)signaling pathways.Berberine(BBR),one of the main components of HLD,inhibited the development of MT in mice.BBR reduced cell viability while increasing B-cell lymphoma 2(Bcl-2)protein expression and decreasing CD86,NF-κB,Bax,and Caspase-3 protein expression in brain vascular 2(BV2)mcroglia cells treated with morphine.Additionally,BBR contributed to a reduction in pro-inflammatory cytokine release and apoptotic cell number.CONCLUSIONS:BBR,a key component of HLD,effectively suppressed microglial activation and neuroinflammation by regulating the NF-κB and apoptosis signaling pathways,thereby delaying MT.This study offers a novel approach to enhance the clinical analgesic efficacy of morphine.展开更多
Objective：To observe the tolerance and the dependence of endomorphin-1 （EM-1） in rats and the possible mechanisms. Methods：Sixty Sprague-Dawley rats were randomly allocated into saline, acute EM-1-treated and chro...Objective：To observe the tolerance and the dependence of endomorphin-1 （EM-1） in rats and the possible mechanisms. Methods：Sixty Sprague-Dawley rats were randomly allocated into saline, acute EM-1-treated and chronic EM-1-treated groups. The rats were intracerebroventricularly injected with saline, acute EM-1 10 μg/kg 30 rain prior to sacrifice,and chronic EM-1 by daily administration at 8：00 A.M. and 15：00 P.M. from 10 μg/kg on the 1^st day to 50 μg/kg on the 94 day, respectively. In chronic EM-1-treated group, the median antinociceptive dose （AD50） and the catatonic median effective dose （ED50） were determined by the improved Dixon＇s method. Natural withdrawl test was used to assess the dependence of EM-1. Maximal binding capacity （Bmax） and dissociation constant （Kd） of 3H-DAMGO, binding to mu-opioid receptor （MOR） in brain tissue, was measured by Scatchard analysis. Gene expression of MOR was measured by reverse transcription-polymerase chain reaction（RT-PCR）. Results ：Tolerance of the antinociceptic and catatonic effects on the 3rd day （3.1-fold and 1.9-fold ） and the 9th day （28.4-fold and 8.5-fold） were observed in chronic EM-1-treated group （P 〈 0.05）. Jumping times and withdrawal scores of rats were significantly higher in the chronic EM-1-treated group than those in saline group on the 94 day （P 〈 0.05）. Bmax and mRNA expression of MOR in cortex, midbrain and striatum were lower in chronic EM-1-treated group on the 94 day than the other two groups（P 〈 0.05）, but Kd had no significant difference （P 〉 0.05）. AD50,ED50,Bmax ,Kd and gene expression of MOR were recorded. Conclusion： EM-1 possesses the tolerance and the dependence. After a long-term treatment, EM-1 down regulates the binding capacity and mRNA of MOR, which somewhat accounts for the dependence.展开更多
BACKGROUND： Drug addiction involves two main central nervous systems, namely the dopamine and noradrenaline systems. These systems are primarily distributed in five brain regions： the ventral tegmental area, the nuc...BACKGROUND： Drug addiction involves two main central nervous systems, namely the dopamine and noradrenaline systems. These systems are primarily distributed in five brain regions： the ventral tegmental area, the nucleus accumbens, the prefrontal cortex, the hippocampus, and the locus coeruleus. OBJECTIVE： To investigate regional changes of guanine nucleotide binding protein-inhabitant 2 （Gi2） in dopaminergic and noradrenergic neurons in brains of morphine-tolerant and -dependent rats. DESIGN, TIME, AND SETTING： A randomized control study was performed at the Department of Neurobiology in the Second Military Medical University of Chinese PLA （Shanghai, China） between September 2002 and March 2004. MATERIALS： Thirty-six, healthy, male, Sprague-Dawley （SD） rats were used to establish morphine-dependent models. Morphine hydrochloride was a product of Shenyang First Pharmaceutical Factory （China）; naloxone hydrochloride was a product of Beijing Four-Ring Pharmaceutical Factory （China）; and α subunit of Gi2 antibody was offered by Santa Cruz Biotechnology, lnc （USA）. METHODS： Thirty-six SD rats were randomly divided into six groups （n = 6）： （1） acute morphine-dependent group, （2） acute abstinent group, （3） acute control group, （4） chronic morphine-dependent group, （5） chronic abstinent group, and （6） chronic control group. Rats in the acute morphine-dependent and the acute groups were injected with morphine （5 mg/kg）, one injection every two hours, for a total of eight injections. In the acute and chronic morphine-dependent rat models, morphine withdrawal syndrome was precipitated by an injection of naloxone （5 mg/kg）. Rats in the acute control group were given a peritoneal injection of physiological saline at the same administration time as the above two groups. Rats in the chronic morphine-dependent and chronic abstinent groups were injected with morphine three times per day. The administration dose on day 1 was initially 5 mg/kg at 20：00, which increased by 5 mg/kg at 8：00, 12：00, and 20：00 until day 7. On day 13, the dose continuously increased by 10 mg/kg until a chronic morphine-dependent rat model was successfully induced. Afterwards, the rats presented with withdrawal syndromes on naloxone （5 mg/kg） at 8：00 on the same day. Rats in the chronic control group were injected with physiological saline at the same time of the two chronic groups. MAIN OUTCOME MEASURES： The concentration of Gi2 protein in the five brain regions （ventral tegmental area, nucleus accumbens, prefrontal cortex, locus coeruleus, and hippocampus） was detected by immunohistochemistry. RESULTS： In the acute morphine-dependent and acute abstinent groups, Gi2 protein concentration was significantly decreased in the nucleus accumbens, compared to the acute control group （P 〈 0.01）, while no obvious changes were detected in other brain regions. In the chronic morphine-dependent and chronic abstinent groups, Gi2 protein concentration was significantly decreased in the nucleus accumbens, but significantly increased in the locus coeruleus （P 〈 0.01 ） compared to the chronic control group. CONCLUSION： Morphine dependence and tolerance may induce obvious reductions of Gi2 protein levels in the nucleus accumbens of rats. Chronic morphine dependence desensitizes the homologous neurons.展开更多
Nuclear factor kappa B（NF-κB） in the spinal cord is involved in pro-infl ammatory cytokine-mediated pain facilitation. However, the role of NF-κB activation in chronic morphine-induced analgesic tolerance and the ...Nuclear factor kappa B（NF-κB） in the spinal cord is involved in pro-infl ammatory cytokine-mediated pain facilitation. However, the role of NF-κB activation in chronic morphine-induced analgesic tolerance and the underlying mechanisms remain unclear. In the present study, we found that the level of phosphorylated NF-κB p65（p-p65） was increased in the dorsal horn of the lumbar 4–6 segments after intrathecal administration of morphine for 7 consecutive days, and the p-p65 was co-localized with neurons and astrocytes. The expression of TNF-α and IL-1β was also increased in the same area. In addition, pretreatment with pyrrolidinedithiocarbamate（PDTC） or SN50, inhibitors of NF-κB, prevented the development of morphine analgesic tolerance and alleviated morphine withdrawal-induced allodynia and hyperalgesia. The increase in TNF-α and IL-1β expression induced by chronic morphine exposure was also partially blocked by PDTC pretreatment. In another experiment, rats receiving PDTC or SN50 beginning on day 7 of morphine injection showed partial recovery of the anti-nociceptive effects of morphine and attenuation of the withdrawal-induced abnormal pain. Meanwhile, intrathecal pretreatment with lipopolysaccharide from Rhodobacter sphae-roides, an antagonist of toll-like receptor 4（TLR4）, blocked the activation of NF-κB, and prevented the development of morphine tolerance and withdrawal-induced abnormal pain. These data indicated that TLR4-mediated NF-κB activation in the spinal cord is involved in the development and maintenance of morphine analgesic tolerance and withdrawalinduced pain hypersensitivity.展开更多
Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the d...Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance,currently,there is no effective therapy to treat morphine tolerance.In the current study,we aimed to develop a monoclonal antibody(mAb)precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms.We successfully prepared a mAb targeting MOR,named 3A5C7,by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization,and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation.Treatment of two cell lines,HEK293T and SH-SY5Y,with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2(GRK2)/b-arrestin2-dependent mechanism,as demonstrated by immunofluorescence staining,flow cytometry,Western blotting,coimmunoprecipitation,and small interfering ribonucleic acid(siRNA)-based knockdown.This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR.We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid.Western blot,enzyme-linked immunosorbent assays,and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase,the in vitro biomarker of morphine tolerance,via the GRK2/b-arrestin2 pathway.Furthermore,in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice,and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence.Finally,intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/b-arrestin2 pathway.Collectively,our study provided a therapeutic mAb,3A5C7,targeting MOR to treat morphine tolerance,mediated by enhancing morphine-induced MOR endocytosis.The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.展开更多
Objective： Some animal models apply morphine in the drinking water to generate addiction, but related reports are not free of conflicting results. Accordingly, this study aimed to figure out if chronic consumption of...Objective： Some animal models apply morphine in the drinking water to generate addiction, but related reports are not free of conflicting results. Accordingly, this study aimed to figure out if chronic consumption of morphine in the drinking water can induce morphine addiction in Wistar rats. Methods： For 3 weeks, the animals received a daily morphine dose of 35 mg/kg by offering a calculated volume of sugar water （5% sucrose） with morphine （0. l mg/ml） to each rat; animals receiving just sugar water served as controls. Immediately after the treatment phase, the tail immersion test was used to check for morphine tolerance, and all animals were then kept on tap water for one week （withdrawal phase）. Afterwards, all rats were allowed to choose their drinking source by offering two bottles, containing sugar water without and with morphine, simultaneously for two days （preference phase）. Results： While the chronic consumption of morphine led to a reduction in body weight and to morphine tolerance, the morphine-treated Wistar rats did not show any preference for the opiate-containing sugar water. Conclusion： Body weight loss and tolerance do not reveal a condition of drug craving, and current animal models should be re-evaluated regarding their potential to establish morphine addicted animals.展开更多
Objective Our previous study identified Threonine 161 （Thr-161）, located in the second intracellular loop of the 6-opioid receptor （DOR）, as the only consensus phosphorylation Cdte for cyclin-dqpendent kinase 5 （...Objective Our previous study identified Threonine 161 （Thr-161）, located in the second intracellular loop of the 6-opioid receptor （DOR）, as the only consensus phosphorylation Cdte for cyclin-dqpendent kinase 5 （CdkS）. The aim of this study was to assess the function of DOR phosphorylation by Cdk5 in complete Freund＇s adjuvant （CFA）-induced inflammatory pain and morphine tolerance. Methods Dorsal root ganglion （DRG） neurons of rats with CFA-induced in- flammatory pain were acutely dissociated and the biotinylation method was used to explore the membrane localization of phosphorylated DOR at Thr-161 （pThr-161-DOR）, and paw withdrawal latency was measured after intrathecal delivery of drugs or Tat-peptide, using a radiant heat stimulator in rats with CFA-induced inflammatory pain. Results Both the total amount and the surface localization of pThr-161-DOR were significantly enhanced in the ipsilateral DRG following CFA injection. lntrathecal delivery of the engineered Tat fusion-interefering peptide corresponding to the second intracellular loop of DOR （Tat-DOR-2L） increased inflammatory hypersensitivity, and inhibited DOR- but not μ-opioid receptor-mediated spinal analgesia in CFA-treated rats. However, intrathecal delivery of Tat-DOR-2L postponed morphine antinociceptive tolerance in rats with CFA-induced inflammatory pain. Conclusion Phosphorylation of DOR at Thr-161 by Cdk5 attenuates hypersensitivity and potentiates morphine tolerance in rats with CFA-induced inflammatory pain, while disruption of the phosphorylation of DOR at Thr- 161 attenuates morphine tolerance.展开更多
Microglia are important cells involved in the regulation of neuropathic pain(NPP)and morphine tolerance.Information on their plasticity and polarity has been elucidated after determining their physiological structure,...Microglia are important cells involved in the regulation of neuropathic pain(NPP)and morphine tolerance.Information on their plasticity and polarity has been elucidated after determining their physiological structure,but there is still much to learn about the role of this type of cell in NPP and morphine tolerance.Microglia mediate multiple functions in health and disease by controlling damage in the central nervous system(CNS)and endogenous immune responses to disease.Microglial activation can result in altered opioid system activity,and NPP is characterized by resistance to morphine.Here we investigate the regulatory mechanisms of microglia and review the potential of microglial inhibitors for modulating NPP and morphine tolerance.Targeted inhibition of glial activation is a clinically promising approach to the treatment of NPP and the prevention of morphine tolerance.Finally,we suggest directions for future research on microglial inhibitors.展开更多
Objective:The roles of gonadal hormones and nitric oxide(NO) on the analgesic effects of morphine,tolerance to morphine,and their interactions have been widely investigated.In the present study,the effect of L-arginin...Objective:The roles of gonadal hormones and nitric oxide(NO) on the analgesic effects of morphine,tolerance to morphine,and their interactions have been widely investigated.In the present study,the effect of L-arginine(an NO precursor) on morphine tolerance in sham and ovariectomized(OVX) female mice was investigated.Methods:Forty mice were divided into sham and OVX groups.On the first day,a hot plate test((55±0.2) °C;cut-off 30 s) was carried out as a base record 15 min before injection of morphine(10 mg/kg,subcutaneously(s.c.)) and was repeated every 15 min after injection.The sham group was then divided into two subgroups:sham-tolerance-L-arginine(Sham-Tol-LA) and sham-tolerance-saline(Sham-Tol-Sal) which received either L-arginine 50 mg/kg(intraperitoneally(i.p.)) or saline 10 ml/kg(i.p.),respectively,three times in a day for three consecutive days.Morphine tolerance was induced in animals by injecting 30 mg/kg morphine(s.c.) three times/day for three days.This treatment was also used for OVX subgroups.On the fifth day,the hot plate test was repeated.The analgesic effect of morphine was calculated as the maximal percent effect(MPE).The results were compared using repeated measure analysis of variance(ANOVA).Results:There was no significant difference in MPE between the OVX and sham groups.The MPEs in both the Sham-Tol-Sal and OVX-Tol-Sal groups were lower than those in both the sham and OVX groups(P<0.01).The MPE in the OVX-Tol-Sal group was greater than that in the Sham-Tol-Sal group(P<0.01).The MPE in the Sham-Tol-LA group was higher than that in the Sham-Tol-Sal group(P<0.01).However,there was no significant difference between the Sham-Tol-LA and sham groups or between the OVX-Tol-LA and OVX-Tol-Sal groups.Conclusions:The results of the present study showed that repeated administration of morphine causes tolerance to the analgesic effect of morphine.L-Arginine could prevent tolerance to morphine but its effect was different in the presence of ovarian hormones.展开更多
OBJECTIVE: To evaluate the effect of berberine on morphine analgesia, tolerance, and hyperalgesia. METHODS: Morphine-induced acute tolerance model: mice received intraperitoneal berberine at doses of 2.5, 5.0, and 10 ...OBJECTIVE: To evaluate the effect of berberine on morphine analgesia, tolerance, and hyperalgesia. METHODS: Morphine-induced acute tolerance model: mice received intraperitoneal berberine at doses of 2.5, 5.0, and 10 mg/kg;30 min later, subcutaneous morphine 10 mg/kg was injected every hour for nine continuous h. Morphine 10 mg/kg alone was administered at 24 and 48 h. Morphine-induced chronic tolerance model: mice received intraperitoneal berberine 2.5, 5.0, and 10 mg/kg;30 min later, 10 mg/kg morphine was injected subcutaneously for eight consecutive days. On the ninth day, morphine 10 mg/kg was given alone. Morphineinduced established tolerance model: mice were injected subcutaneously with morphine 10 mg/kg once a day for eight consecutive days. Berberine 2.5 mg/kg was administered on day one, four, and seven and morphine 10 mg/kg alone on day nine. The baseline latency(T0) and post-treatment latency(T1) were determined by the hot plate test, and the maximum possible analgesic effect (MPAE) was calculated. Nitric oxide synthase(NOS) activity and nitric oxide(NO) content in the spinal cord were measured by spectrophotometer. Verification of berberine analgesic effect by blocking N-methyl-Daspartate(NMDA) receptor: HT-22 and HEK-293 cells transfected with NMDA plasmid were randomly divided into five groups: control group, NMDA group, berberine low-dose, medium-dose, and high-dose groups(5, 10, 20 μmol/L, respectively). Except for the control group, cells were treated with NMDA(HT-22 cells: 20 mmol/L;HEK-293 cells: 50 μmol/L). After 24 h, cell viability was detected by cell counting kit-8. The molecular mechanism between berberine and the NMDA receptor was studied by molecular docking. RESULTS: Berberine 2.5 and 5.0 mg/kg could prolong the analgesic time of morphine. In acute and chronic morphine tolerance models, berberine could inhibit the decrease of MPAE and baseline latency(P < 0.05). In the established tolerance model, berberine could rapidly reverse the decreased MPAE(P < 0.05). The combination of berberine and morphine on day one could effectively inhibit the morphine-induced increase of NOS activity and NO content in the spinal cord(P < 0.05). Berberine significantly increased the cell viability of NMDA-induced nerve injury in HT-22 and HEK-293 cells(P < 0.05). Molecular docking showed that berberine binds to the receptor pocket of NMDA. CONCLUSIONS: Berberine could effectively enhance and prolong the duration of morphine analgesia and inhibit the development of morphine-induced tolerance and hyperalgesia. Furthermore, berberine has a certain neuroprotective effect, which may be related to the inhibition of NMDA activity.展开更多
Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory p...Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.展开更多
Objective： To investigate the effects of intrathecal administration of ketamine, a non-competitive N-methy-D-aspartate receptor antagonist, combined with morphine on the activation of astrocytes and releases of IL-1...Objective： To investigate the effects of intrathecal administration of ketamine, a non-competitive N-methy-D-aspartate receptor antagonist, combined with morphine on the activation of astrocytes and releases of IL-1β and IL-6 from spinal cord in the rats of morphine tolerance. Methods： Twenty-seven Sprague-Dawley male rats were randomly divided into sham-operated, morphine tolerance, and morphine plus ketamine group. The subarachnoid catheterication of all the rats was prepared by the method of Jianping Yang. Morphine 20μg in 10μl was adminstrated intrathecally to induce spinal morphine tolerance once daily for 5 consecutive days. Morphine and ketamine 250μg in 10μl total volume was given in morphine plus ketamine group. Three groups all received intrathecal morphine 5μg in 10μl for morphine challenge test at 24h after last administration of the morphine. After morphine challenge test, lumbar spinal tissues were taken for measurement of glial fibrillary acidic protein （GFAP） of astrocyte in lumbar spinal horn cord by immunohistochemistry and IL-1β and IL-6 of spinal cord by ELISA. Results： The decrease of %MPE induced by chronic intrathecal morphine was inhibibed by ketamine and hyperalgesia and allodynia induced by morphine-withdrawl were alleviated. The average areas, the average absorbency （A^-）, the integral absorbency （A） of GFAP immuno-reactive cells in the dorsal horn, and IL-1β and IL-6 of spinal cord were significantly larger in morphine tolerance group than in morphine plus ketamine group. Conclusion：Co-administration of ketamine and morphine enhance antinociceptive effect of morphine and prevent the development of morphine tolerance. Ketamine might attenuate the activation of astrocytes and inhibit the release of IL-1β and IL-6 from spinal cord in repeated intrathecal morphine rats.展开更多
The chronic use of morphine and other opioids is associated with opioid-induced hypersensitivity(OIH)and analgesic tolerance.Among the different forms of OIH and tolerance,the opioid receptors and cell types mediating...The chronic use of morphine and other opioids is associated with opioid-induced hypersensitivity(OIH)and analgesic tolerance.Among the different forms of OIH and tolerance,the opioid receptors and cell types mediating opioid-induced mechanical allodynia and anti-allodynic tolerance remain unresolved.Here we demonstrated that the loss of peripheralμ-opioid receptors(MORs)or MOR-expressing neurons attenuated thermal tolerance,but did not affect the expression and maintenance of morphine-induced mechanical allodynia and anti-allodynic tolerance.To confirm this result,we made dorsal root ganglia-dorsal roots-sagittal spinal cord slice preparations and recorded low-threshold Aβ-fiber stimulation-evoked inputs and outputs in superficial dorsal horn neurons.Consistent with the behavioral results,peripheral MOR loss did not prevent the opening of Aβmechanical allodynia pathways in the spinal dorsal horn.Therefore,the peripheral MOR signaling pathway may not be an optimal target for preventing mechanical OIH and analgesic tolerance.Future studies should focus more on central mechanisms.展开更多
Objective: Morphine concentration measured in postmortem tissues may or may not reflect antemortem concentration. We measured levels of morphine in autopsied tissues to determine whether morphine distribution in morph...Objective: Morphine concentration measured in postmortem tissues may or may not reflect antemortem concentration. We measured levels of morphine in autopsied tissues to determine whether morphine distribution in morphine-dependent rats is altered after death. Methods: Solid-phase extraction was used to extract morphine from the samples, and morphine levels were measured at 0-96 h postmortem using gas chromatography. Results: The study of the morphine dependent rats showed a significant (P<0.05) increase of morphine concentration in postmortem cardiac blood, liver tissues and kidneys tissues. A significant increase was also observed at 72 h and 96 h postmortem in the brain, while morphine levels in cardiac tissues only increased at 24 h and 96 h postmortem. These changes were associated with an observed pH rapid decrease: pH of cardiac blood dropped from 7.36±0.15 to 6.86±0.09 (P<0.01), pH of liver tissues from 6.98±0.04 to 6.34±0.03 (P<0.05). Conclusion: The postmortem regional distribution of morphine occurs in dependent rats, but different from the change that occurs in acute poisoning rats. The morphine concentration in cardiac blood and tissues tends to increase during the period of 0-96 h postmortem in dependent rats. Morphine concentration increases with pH rapid decrease. The antemortem internal amount of morphine affects its postmortem regional distribution. It appears that several mechanisms are accountable for postmortem morphine distribution. The understanding of the mechanisms and patterns may eventually lead to better choices of samples which may better represent antemortem drug levels.展开更多
BACKGROUND： Studies have demonstrated that exogenous neurosteroid treatment prevents the development of morphine tolerance and dependence, and attenuates abstinence behavior in mice. However, there are few studies on...BACKGROUND： Studies have demonstrated that exogenous neurosteroid treatment prevents the development of morphine tolerance and dependence, and attenuates abstinence behavior in mice. However, there are few studies on whether the levels of endogenous neurosteroids can be changed by morphine dependence and withdrawal. OBJECTIVE： To investigate the levels of various neurosteroids in rat brain following morphine dependence and withdrawal. To evaluate the expressions of steroidogenic enzyme mRNAs and proteins. To identify the relationship between neurosteroids and morphine dependence at the whole animal behavior, neural biochemistry, and molecular levels. DESIGN, TIME AND SETTING： A randomized, controlled study. Experiments were performed at the Department of Pharmacology of Hebei Medical University and Department of Pharmacology of Beathune International Peace Hospital, China, from June 2004 to October 2007. MATERIALS： Morphine hydrochloride injection （Shenyang First Pharmaceutical Factory, China）, naloxone hydrochloride （Hunan Yiqiao Pharmaceutical Co., China） and a gas chromatography-mass spectrometry system （Agilent, CA, USA） were used in this study. METHODS： Healthy adult Sprague Dawley rats were randomly divided into three groups： a morphine dependence group, morphine withdrawal group and control group （n = 20）. The rats in the morphine dependence and morphine withdrawal groups were given increasing doses of morphine （5, 10, 15, 20, 30, 40 and 50 mg/kg, intraperitoneal） to create morphine dependence. The rats in the morphine withdrawal group were injected with 2 mg/kg naloxone to precipitate withdrawal 1 hour after the last morphine injection. Rats in the control group were treated with an equal volume of saline. MAIN OUTCOME MEASURES： Following morphine dependence and withdrawal, brain levels of the neurosteroids pregnenolone, progesterone and allopregnanolone were analyzed using gas chromatography-mass spectrometry. The mRNA expression of two key steroidogenic enzymes, P450 side-chain cleavage enzyme （P450scc） and 3[B-hydroxysteroid dehydrogenase （313-HSD）, were determined in rat brain regions using reverse transcription-polymerase chain reaction. The distribution and expression of P450scc protein were visualized in brain regions associated with addiction by immunohistochemistry. RESULTS： In brain tissue from the morphine dependence group, the levels of pregnenolone and progesterone were decreased by 62% （P 〈 0.01） and 92% （P 〈 0.01 ） respectively, compared with the control group. In the morphine dependence group, the key steroidogenic enzyme P450scc mRNA was decreased in striatum （P 〈 0.05）, while 3-HSD mRNA was decreased in amygdala （P 〈 0.05）, striatum （P 〈 0.05） and frontal cortex （P 〈 0.05） compared with the control group. Morphine withdrawal induced a significant increase in the neurosteroid levels compared with the control group （P 〈 0.01）. However, there was no significant difference in the expressions of P450scc and 36-HSD mRNAs between the morphine withdrawal and control groups （P 〉 0.05）. CONCLUSION： The neurosteroid levels and expressions of steroidogenic enzymes changed similarly in morphine dependent rats, suggesting that the morphine dependence-induced decrease in neurosteroids might depend on local expression of steroidogenic enzymes in the central nervous system. However, the changes in neurosteroids in morphine withdrawal rats were not in accordance with the changes in the expression of steroidogenic enzymes, suggesting that the effects of morphine withdrawal on brain neurosteroid levels may not depend primarily on the local expression of steroidogenic enzymes in the central nervous system.展开更多
The present study analyzed the effects of Sidiming, a Chinese herbal compound, on withdrawal syndrome, body weight loss, and serum levels of nitric oxide and its synthase in morphine- dependent rats and rhesus monkeys...The present study analyzed the effects of Sidiming, a Chinese herbal compound, on withdrawal syndrome, body weight loss, and serum levels of nitric oxide and its synthase in morphine- dependent rats and rhesus monkeys. These effects were compared with clonidine, an active control drug used for clinical treatment. Results showed that 4 and 8 g/kg Sidiming, respectively, significantly suppressed morphine withdrawal syndrome and reduced body mass loss in morphine-dependent rats. In addition, 2.4 and 4.8 g/kg Sidiming, respectively, significantly attenuated withdrawal syndrome in rhesus monkeys. High-dose Sidiming （8 g/kg in rats and 4.8 g/kg in rhesus monkeys） led to significantly inhibited serum levels of nitric oxide and its synthase in morphine-dependent rats and rhesus monkeys, which were greater than clonidine. These findings suggested that Sidiming treatment attenuated withdrawal syndrome in morphine-dependent rats and rhesus monkeys by inhibiting serum nitric oxide and its synthase.展开更多
Opiate dependence has become one of the most urgent problems of modernsociety. Opiate dependence involves physical and psychical dependence. Although many addicts can bedetoxified, the relapse ratio of 95% in 5 a demo...Opiate dependence has become one of the most urgent problems of modernsociety. Opiate dependence involves physical and psychical dependence. Although many addicts can bedetoxified, the relapse ratio of 95% in 5 a demonstrates that opiate psychical dependence is aproblem more troublesome. It has been reported that acute and chronic administration of L- NNA canmarkedly retard the development of tolerance to physical dependence on morphine, and suppress theabstinence syndromes precipitated by naloxone in opiate dependent rodents, and even reverse theexisting morphine tolerance. However, the effect of L-NNA on the positive reinforcement ofpsychically active substances and its possible mechanism have not yet been reported. In presentstudy, the effect of L- NNA en the psychical dependence induced by opiates was evaluated on thebasis of conditioned place preference.展开更多
Dependence and impairment of learning and memory are two well-established features caused by abused drugs such as opioids. The hippocampus is an important region associated with both drug dependence and learning and m...Dependence and impairment of learning and memory are two well-established features caused by abused drugs such as opioids. The hippocampus is an important region associated with both drug dependence and learning and memory. However, the molecular events in hippocampus following exposure to abused drugs such as opioids are not well understood. Here we examined the effect of chronic morphine treatment on hippocampal protein expression by proteomic analyses. We found that chronic exposure of mice to morphine for 10 days produced robust morphine withdrawal jumping and memory impairment, and also resulted in a significant downregulation of hippocampal protein levels of three metabolic enzymes, including Fe-S protein 1 ofNADH dehydrogenase, dihydrolipoamide acetyltransferase or E2 component of the pyruvate dehydrogenase complex and lactate dehydrogenase 2. Further real-time quantitative PCR analyses confirmed that the levels of the corresponding mRNAs were also remarkably reduced. Consistent with these findings, lower ATP levels and an impaired ability to convert glucose into ATP were also observed in the hippocampus of chronically treated mice. Opioid antagonist naltrexone administrated concomitantly with morphine significantly suppressed morphine withdrawal jumping and reversed the downregulation of these proteins. Acute exposure to morphine also produced robust morphine withdrawal jumping and significant memory impairment, but failed to decrease the expression of these three proteins. Intrahippocampal injection of D-glucose before morphine administration significantly enhanced ATP levels and suppressed morphine withdrawal jumping and memory impairment in acute morphine-treated but not in chronic morphine-treated mice. Intraperitoneal injection of high dose of D-glucose shows a similar effect on morphine-induced withdrawal jumping as the central treatment. Taken together, our results suggest that reduced expression of the three metabolic enzymes in the hippocampus as a result of chronic morphine treatment contributes to the development of drug-induced symptoms such as morphine withdrawal jumping and memory impairment.展开更多
基金Natural Science Foundation-funded Project:Study on the Mechanism of Mechanical Stress Sensing Element Piezo Type Mechanosensitive Ion Channel Component 2 Interacting with Nuclear Receptor Subfamily 4 Group A Member 2 Mediating Traumatic Brain Injury(No.82172190)。
文摘OBJECTIVE:To investigate the mechanism underlying the effect of the Huanglian decoction(黄连汤,HLD)on morphine tolerance(MT),using network pharmacology,and to verify these mechanisms in vitro and in vivo.METHODS:Available biological data on each drug in the HLD were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform.The target proteins of MT were retrieved from the GeneCards,PharmGkb,Therapeutic Target Database,DrugBank,and Online Mendelian Inheritance in Man databases.Information regarding MT and the drug targets was compared to obtain overlapping elements.This information was imported into the Search Tool for the Retrieval of Interacting Genes/Proteins platform to obtain a protein-protein interaction network diagram.Then,a“component-target”network diagram was constructed using screened drug components and target information,via Cytoscape(Institute for Systems Biology,Seattle,WA,USA).The database for annotation,visualization,and integrated discovery was used for Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathways analyses.Pathway information predicted by network pharmacology was verified using animal studies and cell experiments.RESULTS:Network pharmacology analysis identified 22 active compounds of HLD and revealed that HLD partially ameliorated MT by modulating inflammatory,apoptosis,and nuclear factor kappa B(NF-κB)signaling pathways.Berberine(BBR),one of the main components of HLD,inhibited the development of MT in mice.BBR reduced cell viability while increasing B-cell lymphoma 2(Bcl-2)protein expression and decreasing CD86,NF-κB,Bax,and Caspase-3 protein expression in brain vascular 2(BV2)mcroglia cells treated with morphine.Additionally,BBR contributed to a reduction in pro-inflammatory cytokine release and apoptotic cell number.CONCLUSIONS:BBR,a key component of HLD,effectively suppressed microglial activation and neuroinflammation by regulating the NF-κB and apoptosis signaling pathways,thereby delaying MT.This study offers a novel approach to enhance the clinical analgesic efficacy of morphine.
文摘Objective：To observe the tolerance and the dependence of endomorphin-1 （EM-1） in rats and the possible mechanisms. Methods：Sixty Sprague-Dawley rats were randomly allocated into saline, acute EM-1-treated and chronic EM-1-treated groups. The rats were intracerebroventricularly injected with saline, acute EM-1 10 μg/kg 30 rain prior to sacrifice,and chronic EM-1 by daily administration at 8：00 A.M. and 15：00 P.M. from 10 μg/kg on the 1^st day to 50 μg/kg on the 94 day, respectively. In chronic EM-1-treated group, the median antinociceptive dose （AD50） and the catatonic median effective dose （ED50） were determined by the improved Dixon＇s method. Natural withdrawl test was used to assess the dependence of EM-1. Maximal binding capacity （Bmax） and dissociation constant （Kd） of 3H-DAMGO, binding to mu-opioid receptor （MOR） in brain tissue, was measured by Scatchard analysis. Gene expression of MOR was measured by reverse transcription-polymerase chain reaction（RT-PCR）. Results ：Tolerance of the antinociceptic and catatonic effects on the 3rd day （3.1-fold and 1.9-fold ） and the 9th day （28.4-fold and 8.5-fold） were observed in chronic EM-1-treated group （P 〈 0.05）. Jumping times and withdrawal scores of rats were significantly higher in the chronic EM-1-treated group than those in saline group on the 94 day （P 〈 0.05）. Bmax and mRNA expression of MOR in cortex, midbrain and striatum were lower in chronic EM-1-treated group on the 94 day than the other two groups（P 〈 0.05）, but Kd had no significant difference （P 〉 0.05）. AD50,ED50,Bmax ,Kd and gene expression of MOR were recorded. Conclusion： EM-1 possesses the tolerance and the dependence. After a long-term treatment, EM-1 down regulates the binding capacity and mRNA of MOR, which somewhat accounts for the dependence.
文摘BACKGROUND： Drug addiction involves two main central nervous systems, namely the dopamine and noradrenaline systems. These systems are primarily distributed in five brain regions： the ventral tegmental area, the nucleus accumbens, the prefrontal cortex, the hippocampus, and the locus coeruleus. OBJECTIVE： To investigate regional changes of guanine nucleotide binding protein-inhabitant 2 （Gi2） in dopaminergic and noradrenergic neurons in brains of morphine-tolerant and -dependent rats. DESIGN, TIME, AND SETTING： A randomized control study was performed at the Department of Neurobiology in the Second Military Medical University of Chinese PLA （Shanghai, China） between September 2002 and March 2004. MATERIALS： Thirty-six, healthy, male, Sprague-Dawley （SD） rats were used to establish morphine-dependent models. Morphine hydrochloride was a product of Shenyang First Pharmaceutical Factory （China）; naloxone hydrochloride was a product of Beijing Four-Ring Pharmaceutical Factory （China）; and α subunit of Gi2 antibody was offered by Santa Cruz Biotechnology, lnc （USA）. METHODS： Thirty-six SD rats were randomly divided into six groups （n = 6）： （1） acute morphine-dependent group, （2） acute abstinent group, （3） acute control group, （4） chronic morphine-dependent group, （5） chronic abstinent group, and （6） chronic control group. Rats in the acute morphine-dependent and the acute groups were injected with morphine （5 mg/kg）, one injection every two hours, for a total of eight injections. In the acute and chronic morphine-dependent rat models, morphine withdrawal syndrome was precipitated by an injection of naloxone （5 mg/kg）. Rats in the acute control group were given a peritoneal injection of physiological saline at the same administration time as the above two groups. Rats in the chronic morphine-dependent and chronic abstinent groups were injected with morphine three times per day. The administration dose on day 1 was initially 5 mg/kg at 20：00, which increased by 5 mg/kg at 8：00, 12：00, and 20：00 until day 7. On day 13, the dose continuously increased by 10 mg/kg until a chronic morphine-dependent rat model was successfully induced. Afterwards, the rats presented with withdrawal syndromes on naloxone （5 mg/kg） at 8：00 on the same day. Rats in the chronic control group were injected with physiological saline at the same time of the two chronic groups. MAIN OUTCOME MEASURES： The concentration of Gi2 protein in the five brain regions （ventral tegmental area, nucleus accumbens, prefrontal cortex, locus coeruleus, and hippocampus） was detected by immunohistochemistry. RESULTS： In the acute morphine-dependent and acute abstinent groups, Gi2 protein concentration was significantly decreased in the nucleus accumbens, compared to the acute control group （P 〈 0.01）, while no obvious changes were detected in other brain regions. In the chronic morphine-dependent and chronic abstinent groups, Gi2 protein concentration was significantly decreased in the nucleus accumbens, but significantly increased in the locus coeruleus （P 〈 0.01 ） compared to the chronic control group. CONCLUSION： Morphine dependence and tolerance may induce obvious reductions of Gi2 protein levels in the nucleus accumbens of rats. Chronic morphine dependence desensitizes the homologous neurons.
基金supported by grants from the National Natural Science Foundation of China (31171070 and 81171060)
文摘Nuclear factor kappa B（NF-κB） in the spinal cord is involved in pro-infl ammatory cytokine-mediated pain facilitation. However, the role of NF-κB activation in chronic morphine-induced analgesic tolerance and the underlying mechanisms remain unclear. In the present study, we found that the level of phosphorylated NF-κB p65（p-p65） was increased in the dorsal horn of the lumbar 4–6 segments after intrathecal administration of morphine for 7 consecutive days, and the p-p65 was co-localized with neurons and astrocytes. The expression of TNF-α and IL-1β was also increased in the same area. In addition, pretreatment with pyrrolidinedithiocarbamate（PDTC） or SN50, inhibitors of NF-κB, prevented the development of morphine analgesic tolerance and alleviated morphine withdrawal-induced allodynia and hyperalgesia. The increase in TNF-α and IL-1β expression induced by chronic morphine exposure was also partially blocked by PDTC pretreatment. In another experiment, rats receiving PDTC or SN50 beginning on day 7 of morphine injection showed partial recovery of the anti-nociceptive effects of morphine and attenuation of the withdrawal-induced abnormal pain. Meanwhile, intrathecal pretreatment with lipopolysaccharide from Rhodobacter sphae-roides, an antagonist of toll-like receptor 4（TLR4）, blocked the activation of NF-κB, and prevented the development of morphine tolerance and withdrawal-induced abnormal pain. These data indicated that TLR4-mediated NF-κB activation in the spinal cord is involved in the development and maintenance of morphine analgesic tolerance and withdrawalinduced pain hypersensitivity.
基金supported by the National Basic Research Program of China(Grant No.:2015CB553701)the National Science and Technology Major Project,China(Grant No.:2019ZX09732001).
文摘Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance,currently,there is no effective therapy to treat morphine tolerance.In the current study,we aimed to develop a monoclonal antibody(mAb)precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms.We successfully prepared a mAb targeting MOR,named 3A5C7,by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization,and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation.Treatment of two cell lines,HEK293T and SH-SY5Y,with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2(GRK2)/b-arrestin2-dependent mechanism,as demonstrated by immunofluorescence staining,flow cytometry,Western blotting,coimmunoprecipitation,and small interfering ribonucleic acid(siRNA)-based knockdown.This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR.We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid.Western blot,enzyme-linked immunosorbent assays,and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase,the in vitro biomarker of morphine tolerance,via the GRK2/b-arrestin2 pathway.Furthermore,in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice,and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence.Finally,intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/b-arrestin2 pathway.Collectively,our study provided a therapeutic mAb,3A5C7,targeting MOR to treat morphine tolerance,mediated by enhancing morphine-induced MOR endocytosis.The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.
基金Project supported by the National Basic Research Program (973) of China (No. 2003CB515402), and the Science and Technology Council of Zhejiang Province (No. 2005C23G2010166), China
文摘Objective： Some animal models apply morphine in the drinking water to generate addiction, but related reports are not free of conflicting results. Accordingly, this study aimed to figure out if chronic consumption of morphine in the drinking water can induce morphine addiction in Wistar rats. Methods： For 3 weeks, the animals received a daily morphine dose of 35 mg/kg by offering a calculated volume of sugar water （5% sucrose） with morphine （0. l mg/ml） to each rat; animals receiving just sugar water served as controls. Immediately after the treatment phase, the tail immersion test was used to check for morphine tolerance, and all animals were then kept on tap water for one week （withdrawal phase）. Afterwards, all rats were allowed to choose their drinking source by offering two bottles, containing sugar water without and with morphine, simultaneously for two days （preference phase）. Results： While the chronic consumption of morphine led to a reduction in body weight and to morphine tolerance, the morphine-treated Wistar rats did not show any preference for the opiate-containing sugar water. Conclusion： Body weight loss and tolerance do not reveal a condition of drug craving, and current animal models should be re-evaluated regarding their potential to establish morphine addicted animals.
基金supported by grants from the National Natural Science Foundation of China (30830044, 30925015, 30800330, and 81161120497)Beijing Natural Science Foundation (7092061)Specialized Research Fund for Doctoral Program of Higher Education Grants, China (200800011028 and 20060001121)
文摘Objective Our previous study identified Threonine 161 （Thr-161）, located in the second intracellular loop of the 6-opioid receptor （DOR）, as the only consensus phosphorylation Cdte for cyclin-dqpendent kinase 5 （CdkS）. The aim of this study was to assess the function of DOR phosphorylation by Cdk5 in complete Freund＇s adjuvant （CFA）-induced inflammatory pain and morphine tolerance. Methods Dorsal root ganglion （DRG） neurons of rats with CFA-induced in- flammatory pain were acutely dissociated and the biotinylation method was used to explore the membrane localization of phosphorylated DOR at Thr-161 （pThr-161-DOR）, and paw withdrawal latency was measured after intrathecal delivery of drugs or Tat-peptide, using a radiant heat stimulator in rats with CFA-induced inflammatory pain. Results Both the total amount and the surface localization of pThr-161-DOR were significantly enhanced in the ipsilateral DRG following CFA injection. lntrathecal delivery of the engineered Tat fusion-interefering peptide corresponding to the second intracellular loop of DOR （Tat-DOR-2L） increased inflammatory hypersensitivity, and inhibited DOR- but not μ-opioid receptor-mediated spinal analgesia in CFA-treated rats. However, intrathecal delivery of Tat-DOR-2L postponed morphine antinociceptive tolerance in rats with CFA-induced inflammatory pain. Conclusion Phosphorylation of DOR at Thr-161 by Cdk5 attenuates hypersensitivity and potentiates morphine tolerance in rats with CFA-induced inflammatory pain, while disruption of the phosphorylation of DOR at Thr- 161 attenuates morphine tolerance.
基金Project supported by the National Natural Science Foundation of China(No.81660199).
文摘Microglia are important cells involved in the regulation of neuropathic pain(NPP)and morphine tolerance.Information on their plasticity and polarity has been elucidated after determining their physiological structure,but there is still much to learn about the role of this type of cell in NPP and morphine tolerance.Microglia mediate multiple functions in health and disease by controlling damage in the central nervous system(CNS)and endogenous immune responses to disease.Microglial activation can result in altered opioid system activity,and NPP is characterized by resistance to morphine.Here we investigate the regulatory mechanisms of microglia and review the potential of microglial inhibitors for modulating NPP and morphine tolerance.Targeted inhibition of glial activation is a clinically promising approach to the treatment of NPP and the prevention of morphine tolerance.Finally,we suggest directions for future research on microglial inhibitors.
基金Project supported by the Vice Presidency of Research of Mashhad University of Medical Sciences,Iran
文摘Objective:The roles of gonadal hormones and nitric oxide(NO) on the analgesic effects of morphine,tolerance to morphine,and their interactions have been widely investigated.In the present study,the effect of L-arginine(an NO precursor) on morphine tolerance in sham and ovariectomized(OVX) female mice was investigated.Methods:Forty mice were divided into sham and OVX groups.On the first day,a hot plate test((55±0.2) °C;cut-off 30 s) was carried out as a base record 15 min before injection of morphine(10 mg/kg,subcutaneously(s.c.)) and was repeated every 15 min after injection.The sham group was then divided into two subgroups:sham-tolerance-L-arginine(Sham-Tol-LA) and sham-tolerance-saline(Sham-Tol-Sal) which received either L-arginine 50 mg/kg(intraperitoneally(i.p.)) or saline 10 ml/kg(i.p.),respectively,three times in a day for three consecutive days.Morphine tolerance was induced in animals by injecting 30 mg/kg morphine(s.c.) three times/day for three days.This treatment was also used for OVX subgroups.On the fifth day,the hot plate test was repeated.The analgesic effect of morphine was calculated as the maximal percent effect(MPE).The results were compared using repeated measure analysis of variance(ANOVA).Results:There was no significant difference in MPE between the OVX and sham groups.The MPEs in both the Sham-Tol-Sal and OVX-Tol-Sal groups were lower than those in both the sham and OVX groups(P<0.01).The MPE in the OVX-Tol-Sal group was greater than that in the Sham-Tol-Sal group(P<0.01).The MPE in the Sham-Tol-LA group was higher than that in the Sham-Tol-Sal group(P<0.01).However,there was no significant difference between the Sham-Tol-LA and sham groups or between the OVX-Tol-LA and OVX-Tol-Sal groups.Conclusions:The results of the present study showed that repeated administration of morphine causes tolerance to the analgesic effect of morphine.L-Arginine could prevent tolerance to morphine but its effect was different in the presence of ovarian hormones.
基金Natural Science Foundation of Shandong Province:Mechanism of the Compound Huanglian Decoction Against Morphine Tolerance Based on Network Pharmacology and Microglia M1/M2 Pattern(No.ZR2023MH270)。
文摘OBJECTIVE: To evaluate the effect of berberine on morphine analgesia, tolerance, and hyperalgesia. METHODS: Morphine-induced acute tolerance model: mice received intraperitoneal berberine at doses of 2.5, 5.0, and 10 mg/kg;30 min later, subcutaneous morphine 10 mg/kg was injected every hour for nine continuous h. Morphine 10 mg/kg alone was administered at 24 and 48 h. Morphine-induced chronic tolerance model: mice received intraperitoneal berberine 2.5, 5.0, and 10 mg/kg;30 min later, 10 mg/kg morphine was injected subcutaneously for eight consecutive days. On the ninth day, morphine 10 mg/kg was given alone. Morphineinduced established tolerance model: mice were injected subcutaneously with morphine 10 mg/kg once a day for eight consecutive days. Berberine 2.5 mg/kg was administered on day one, four, and seven and morphine 10 mg/kg alone on day nine. The baseline latency(T0) and post-treatment latency(T1) were determined by the hot plate test, and the maximum possible analgesic effect (MPAE) was calculated. Nitric oxide synthase(NOS) activity and nitric oxide(NO) content in the spinal cord were measured by spectrophotometer. Verification of berberine analgesic effect by blocking N-methyl-Daspartate(NMDA) receptor: HT-22 and HEK-293 cells transfected with NMDA plasmid were randomly divided into five groups: control group, NMDA group, berberine low-dose, medium-dose, and high-dose groups(5, 10, 20 μmol/L, respectively). Except for the control group, cells were treated with NMDA(HT-22 cells: 20 mmol/L;HEK-293 cells: 50 μmol/L). After 24 h, cell viability was detected by cell counting kit-8. The molecular mechanism between berberine and the NMDA receptor was studied by molecular docking. RESULTS: Berberine 2.5 and 5.0 mg/kg could prolong the analgesic time of morphine. In acute and chronic morphine tolerance models, berberine could inhibit the decrease of MPAE and baseline latency(P < 0.05). In the established tolerance model, berberine could rapidly reverse the decreased MPAE(P < 0.05). The combination of berberine and morphine on day one could effectively inhibit the morphine-induced increase of NOS activity and NO content in the spinal cord(P < 0.05). Berberine significantly increased the cell viability of NMDA-induced nerve injury in HT-22 and HEK-293 cells(P < 0.05). Molecular docking showed that berberine binds to the receptor pocket of NMDA. CONCLUSIONS: Berberine could effectively enhance and prolong the duration of morphine analgesia and inhibit the development of morphine-induced tolerance and hyperalgesia. Furthermore, berberine has a certain neuroprotective effect, which may be related to the inhibition of NMDA activity.
基金supported by the National Natural Science Foundation of ChinaNos.81971047 (to WTL) and 82073910 (to XFW)+2 种基金the Natural Science Foundation of Jiangsu Province,No.BK20191253 (to XFW)Key R&D Program (Social Development) Project of Jiangsu Province,No.BE2019 732 (to WTL)Jiangsu Province Hospital (the First Affiliated Hospital of Nanjing Medical University) Clinical Capacity Enhancement Project,No.JSPH-511B2018-8 (to YBP)。
文摘Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.
文摘Objective： To investigate the effects of intrathecal administration of ketamine, a non-competitive N-methy-D-aspartate receptor antagonist, combined with morphine on the activation of astrocytes and releases of IL-1β and IL-6 from spinal cord in the rats of morphine tolerance. Methods： Twenty-seven Sprague-Dawley male rats were randomly divided into sham-operated, morphine tolerance, and morphine plus ketamine group. The subarachnoid catheterication of all the rats was prepared by the method of Jianping Yang. Morphine 20μg in 10μl was adminstrated intrathecally to induce spinal morphine tolerance once daily for 5 consecutive days. Morphine and ketamine 250μg in 10μl total volume was given in morphine plus ketamine group. Three groups all received intrathecal morphine 5μg in 10μl for morphine challenge test at 24h after last administration of the morphine. After morphine challenge test, lumbar spinal tissues were taken for measurement of glial fibrillary acidic protein （GFAP） of astrocyte in lumbar spinal horn cord by immunohistochemistry and IL-1β and IL-6 of spinal cord by ELISA. Results： The decrease of %MPE induced by chronic intrathecal morphine was inhibibed by ketamine and hyperalgesia and allodynia induced by morphine-withdrawl were alleviated. The average areas, the average absorbency （A^-）, the integral absorbency （A） of GFAP immuno-reactive cells in the dorsal horn, and IL-1β and IL-6 of spinal cord were significantly larger in morphine tolerance group than in morphine plus ketamine group. Conclusion：Co-administration of ketamine and morphine enhance antinociceptive effect of morphine and prevent the development of morphine tolerance. Ketamine might attenuate the activation of astrocytes and inhibit the release of IL-1β and IL-6 from spinal cord in repeated intrathecal morphine rats.
基金supported by grants from the Ministry of Science and Technology of China(2021ZD0203302)the National Natural Science Foundation of China(32170996)+4 种基金Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions(2021SHIBS0002)the Guangdong Science and Technology Committee(2019A1515010041,A2021319)the Shenzhen Innovation Committee of Science and Technology(ZDSYS20200811144002008)the Natural Science Foundation of Shenzhen University General Hospital(SUGH2018QD024)the Basic Research Project of Shenzhen Science and Technology Innovation Commission(JCYJ20210324100206017).
文摘The chronic use of morphine and other opioids is associated with opioid-induced hypersensitivity(OIH)and analgesic tolerance.Among the different forms of OIH and tolerance,the opioid receptors and cell types mediating opioid-induced mechanical allodynia and anti-allodynic tolerance remain unresolved.Here we demonstrated that the loss of peripheralμ-opioid receptors(MORs)or MOR-expressing neurons attenuated thermal tolerance,but did not affect the expression and maintenance of morphine-induced mechanical allodynia and anti-allodynic tolerance.To confirm this result,we made dorsal root ganglia-dorsal roots-sagittal spinal cord slice preparations and recorded low-threshold Aβ-fiber stimulation-evoked inputs and outputs in superficial dorsal horn neurons.Consistent with the behavioral results,peripheral MOR loss did not prevent the opening of Aβmechanical allodynia pathways in the spinal dorsal horn.Therefore,the peripheral MOR signaling pathway may not be an optimal target for preventing mechanical OIH and analgesic tolerance.Future studies should focus more on central mechanisms.
文摘Objective: Morphine concentration measured in postmortem tissues may or may not reflect antemortem concentration. We measured levels of morphine in autopsied tissues to determine whether morphine distribution in morphine-dependent rats is altered after death. Methods: Solid-phase extraction was used to extract morphine from the samples, and morphine levels were measured at 0-96 h postmortem using gas chromatography. Results: The study of the morphine dependent rats showed a significant (P<0.05) increase of morphine concentration in postmortem cardiac blood, liver tissues and kidneys tissues. A significant increase was also observed at 72 h and 96 h postmortem in the brain, while morphine levels in cardiac tissues only increased at 24 h and 96 h postmortem. These changes were associated with an observed pH rapid decrease: pH of cardiac blood dropped from 7.36±0.15 to 6.86±0.09 (P<0.01), pH of liver tissues from 6.98±0.04 to 6.34±0.03 (P<0.05). Conclusion: The postmortem regional distribution of morphine occurs in dependent rats, but different from the change that occurs in acute poisoning rats. The morphine concentration in cardiac blood and tissues tends to increase during the period of 0-96 h postmortem in dependent rats. Morphine concentration increases with pH rapid decrease. The antemortem internal amount of morphine affects its postmortem regional distribution. It appears that several mechanisms are accountable for postmortem morphine distribution. The understanding of the mechanisms and patterns may eventually lead to better choices of samples which may better represent antemortem drug levels.
基金the National Natural Science Foundation of China, No. 30772082the Natural Science Foundation of Hebei Province of China, No. C2005000834
文摘BACKGROUND： Studies have demonstrated that exogenous neurosteroid treatment prevents the development of morphine tolerance and dependence, and attenuates abstinence behavior in mice. However, there are few studies on whether the levels of endogenous neurosteroids can be changed by morphine dependence and withdrawal. OBJECTIVE： To investigate the levels of various neurosteroids in rat brain following morphine dependence and withdrawal. To evaluate the expressions of steroidogenic enzyme mRNAs and proteins. To identify the relationship between neurosteroids and morphine dependence at the whole animal behavior, neural biochemistry, and molecular levels. DESIGN, TIME AND SETTING： A randomized, controlled study. Experiments were performed at the Department of Pharmacology of Hebei Medical University and Department of Pharmacology of Beathune International Peace Hospital, China, from June 2004 to October 2007. MATERIALS： Morphine hydrochloride injection （Shenyang First Pharmaceutical Factory, China）, naloxone hydrochloride （Hunan Yiqiao Pharmaceutical Co., China） and a gas chromatography-mass spectrometry system （Agilent, CA, USA） were used in this study. METHODS： Healthy adult Sprague Dawley rats were randomly divided into three groups： a morphine dependence group, morphine withdrawal group and control group （n = 20）. The rats in the morphine dependence and morphine withdrawal groups were given increasing doses of morphine （5, 10, 15, 20, 30, 40 and 50 mg/kg, intraperitoneal） to create morphine dependence. The rats in the morphine withdrawal group were injected with 2 mg/kg naloxone to precipitate withdrawal 1 hour after the last morphine injection. Rats in the control group were treated with an equal volume of saline. MAIN OUTCOME MEASURES： Following morphine dependence and withdrawal, brain levels of the neurosteroids pregnenolone, progesterone and allopregnanolone were analyzed using gas chromatography-mass spectrometry. The mRNA expression of two key steroidogenic enzymes, P450 side-chain cleavage enzyme （P450scc） and 3[B-hydroxysteroid dehydrogenase （313-HSD）, were determined in rat brain regions using reverse transcription-polymerase chain reaction. The distribution and expression of P450scc protein were visualized in brain regions associated with addiction by immunohistochemistry. RESULTS： In brain tissue from the morphine dependence group, the levels of pregnenolone and progesterone were decreased by 62% （P 〈 0.01） and 92% （P 〈 0.01 ） respectively, compared with the control group. In the morphine dependence group, the key steroidogenic enzyme P450scc mRNA was decreased in striatum （P 〈 0.05）, while 3-HSD mRNA was decreased in amygdala （P 〈 0.05）, striatum （P 〈 0.05） and frontal cortex （P 〈 0.05） compared with the control group. Morphine withdrawal induced a significant increase in the neurosteroid levels compared with the control group （P 〈 0.01）. However, there was no significant difference in the expressions of P450scc and 36-HSD mRNAs between the morphine withdrawal and control groups （P 〉 0.05）. CONCLUSION： The neurosteroid levels and expressions of steroidogenic enzymes changed similarly in morphine dependent rats, suggesting that the morphine dependence-induced decrease in neurosteroids might depend on local expression of steroidogenic enzymes in the central nervous system. However, the changes in neurosteroids in morphine withdrawal rats were not in accordance with the changes in the expression of steroidogenic enzymes, suggesting that the effects of morphine withdrawal on brain neurosteroid levels may not depend primarily on the local expression of steroidogenic enzymes in the central nervous system.
基金the Natural Science Foundation of Guangxi Zhuang Autonomous Region, No.0575066
文摘The present study analyzed the effects of Sidiming, a Chinese herbal compound, on withdrawal syndrome, body weight loss, and serum levels of nitric oxide and its synthase in morphine- dependent rats and rhesus monkeys. These effects were compared with clonidine, an active control drug used for clinical treatment. Results showed that 4 and 8 g/kg Sidiming, respectively, significantly suppressed morphine withdrawal syndrome and reduced body mass loss in morphine-dependent rats. In addition, 2.4 and 4.8 g/kg Sidiming, respectively, significantly attenuated withdrawal syndrome in rhesus monkeys. High-dose Sidiming （8 g/kg in rats and 4.8 g/kg in rhesus monkeys） led to significantly inhibited serum levels of nitric oxide and its synthase in morphine-dependent rats and rhesus monkeys, which were greater than clonidine. These findings suggested that Sidiming treatment attenuated withdrawal syndrome in morphine-dependent rats and rhesus monkeys by inhibiting serum nitric oxide and its synthase.
基金ThisprojectwassupportedbyGrantofthePLALaboratoryforNewDrugEvaluation (No .96-0 2 )
文摘Opiate dependence has become one of the most urgent problems of modernsociety. Opiate dependence involves physical and psychical dependence. Although many addicts can bedetoxified, the relapse ratio of 95% in 5 a demonstrates that opiate psychical dependence is aproblem more troublesome. It has been reported that acute and chronic administration of L- NNA canmarkedly retard the development of tolerance to physical dependence on morphine, and suppress theabstinence syndromes precipitated by naloxone in opiate dependent rodents, and even reverse theexisting morphine tolerance. However, the effect of L-NNA on the positive reinforcement ofpsychically active substances and its possible mechanism have not yet been reported. In presentstudy, the effect of L- NNA en the psychical dependence induced by opiates was evaluated on thebasis of conditioned place preference.
基金Acknowledgments We thank Dr Boja Emily S （NHLBI, NIH, USA） for expert technical assistance in mass spectrometry analyses, and our colleagues Drs Bin Lu, Zhao-Qiu Wu for helpful comments. This work was supported by a National Basic Research Program grant from the Ministry of Science and Technology of China （G2003CB515401）, National Science Fund for Distinguished Young Scholar from the National Natural Science Foundation of China （30425002） and a fund supported by the ＂100 Talents Project＂ of Chinese Academy of Sciences （J-G Liu）.
文摘Dependence and impairment of learning and memory are two well-established features caused by abused drugs such as opioids. The hippocampus is an important region associated with both drug dependence and learning and memory. However, the molecular events in hippocampus following exposure to abused drugs such as opioids are not well understood. Here we examined the effect of chronic morphine treatment on hippocampal protein expression by proteomic analyses. We found that chronic exposure of mice to morphine for 10 days produced robust morphine withdrawal jumping and memory impairment, and also resulted in a significant downregulation of hippocampal protein levels of three metabolic enzymes, including Fe-S protein 1 ofNADH dehydrogenase, dihydrolipoamide acetyltransferase or E2 component of the pyruvate dehydrogenase complex and lactate dehydrogenase 2. Further real-time quantitative PCR analyses confirmed that the levels of the corresponding mRNAs were also remarkably reduced. Consistent with these findings, lower ATP levels and an impaired ability to convert glucose into ATP were also observed in the hippocampus of chronically treated mice. Opioid antagonist naltrexone administrated concomitantly with morphine significantly suppressed morphine withdrawal jumping and reversed the downregulation of these proteins. Acute exposure to morphine also produced robust morphine withdrawal jumping and significant memory impairment, but failed to decrease the expression of these three proteins. Intrahippocampal injection of D-glucose before morphine administration significantly enhanced ATP levels and suppressed morphine withdrawal jumping and memory impairment in acute morphine-treated but not in chronic morphine-treated mice. Intraperitoneal injection of high dose of D-glucose shows a similar effect on morphine-induced withdrawal jumping as the central treatment. Taken together, our results suggest that reduced expression of the three metabolic enzymes in the hippocampus as a result of chronic morphine treatment contributes to the development of drug-induced symptoms such as morphine withdrawal jumping and memory impairment.
