| BackgroundRubiaceae, a Chinese medicine, is commonly used. It is recorded on Pharmacopoeia of the People’s Republic of China(2010)". The authentic rhynchophylla contains:Uncaria rhynchophylla (Miq.)Miq. ex. Havil, Uncaria macrophylla Wall., Uncaria hirsute Havil., Uncaria sinensis (Oliv.) Havil., Uncaria sessilifructus Roxb. J. Medicinal history of Uncaria rhyunchophylla can be traced back to The wei and jin dynasties. It is recorded on " Supplementary Records of Famous Physicians"," Compendium of Materia Medica","Medicinal Theory " and other ancient books. Uncaria rhyunchophylla, cold, acting on the liver and the pericardium, is of clearing-heat and planning-liver, dispelling wind and relievling convulsion. Uncaria rhyunchophylla with other Chinese medicines are used to cure Chronic ulcerated, Convulsions, The mainstay of headache, Dizziness by the doctors of traditional Chinese medicine. At present, Uncaria rhyunchophylla are mainly used to cure primary and secondary hypertension, cerebral neurologic diseases and cerebrovascular diseases, Epilepsy, old age dementia and the prevention and treatment of drug dependence.Rhynchophylline (Rhy) is the highest levels of indoles among Uncaria Alkaloids,20～30%of total alkaloids. Other alkaloids include Isorhynchophylline, Corynoxine, Corynoxine B, Corynoxeine, Isocorynoxeine, Corynantheine. Pharmacological studies shown that Rhy have effects on the cardiovascular and central nervous systems, such as antihypotensive, antiarrhythmic, inhibiting platelet aggregation, protection of brain ischemia hypoxia and NMDA nervous excitement, antiepileptic, anti-anxiety, resistance to drug addiction and relief neuropathy pain. Mechanisms of action are relevant with inhibition of L-calcium channel and potassium channel, antagonism of NMDA receptors (NMDAR) and AMPA receptors (AMPAR), adjusting metabolism of central neurotransmitters such as the glutamic acid, dopamine, noradrenaline and acetylcholine, endorphins, serotonin and others.NMDAR are ionic excitatory amino acid receptors coupled of Ca2+channels. Natural NMDAR are composed of NR1, NR2and NR3. NR2are divided into four subtypes such as NR2A, NR2B, NR2C and NR2D. NR1are the basic functional units. NR2decide the nature of the receptors. NR3in NMDAR mainly exert inhibitory effects. NMDAR are widely distributed in the brain, Spinal Cord and Peripheral tissues. NMDAR antagonists are divided into two major categories of competitive and non-competitive. Competition NMDAR antagonists excite sites of the amino acids, such as AP5, AP7and CPP. Non-competitive NMDAR antagonists affect ion channels or the adjuste sites, such as MK-801, ketamine, PCP, etc. Nonselective NMDAR antagonists have serious adverse reactions, such as psychotomimetic effect and effects on motor function. Selective NR2B antagonists, such as Ifenprodil, Eliprodil, Haloperidol, Styrene acrylic ester ammonia, are of nerve protection, anticonvulsants, analgesia and so on. NMDAR activation can trigger long-term potentiation and long-term depression, which is physiological mechanism of the brain learning, memory, cognition and synaptic plasticity. However, excessive activation of NMDAR also can cause a variety of central nervous system diseases, such as Stroke, Parkinson’s, Alzheimer’s disease, epilepsy, schizophrenia, Lou gehrig’s disease, huntington’s disease and so on.It was reported that Uncaria rhynchophylla is an effective anxiolytic agent. Pharmacodynamics experiments of Rhy showed that Rhy (60mg/kg) significantly increased the time-spent and entries into open arms of the EPM in5minutes, increased the number of visits to enter the light compartment and percentage of time spent in the light compartment. Rhy had anxiolytic-like effect indeedly. However, mechanism was still not clear. Early research found that Rhy inhibited NR2B mRNA and protein expression in the amygdaloid nucleus and nucleus accumbens in rats which induced by Amphetamine in conditioned place preference. And Rhy had no psychological dependence effect. Another research found that Rhy inhibited the NMDA receptors and intracellular calcium overload in primary cortical neurons which induced by Methamphetamine. Rhy might be an NMDA receptor inhibitor. We speculated that anti-anxiety effect of Rhy might be related to NMDA receptor. In view of the above analysis, we studied that Rhy affected NR1and NR2B expression on primary hippocampal neurons.ObjectiveIt was confirmed that Rhy had anti-anxiety effect in pharmacodynamics prediction experiments, but the mechanism was unclear. The pharmacological experiments about drug dependence showed that Rhy might be an NMDA receptor inhibitor. It was studied that Whether Rhy played an anti-anxiety role by adjusting the NMDA receptors in this paper. From the viewpoints of positioning, quantitative and qualitative researches, we studied that how Rhy affected NRland NR2B subunits of NMDA receptors on primary hippocampal neurons in neonatal rats.Methods1. Establish of primary hippocampal neurons in neonatal rats and identifying: Neonatal SD rats (postnatal24h) were disinfected by75%alcohol, sacrificed by decapitation and brain regions hippocampus were dissected bluntly. The tissues were chopped using ophthalmic scissors. The chopped tissues were incubated in0.125%trypsin at37＞for30min in the5%CO2incubator and agitated every5min. DMEM/F12medium with10%Fetal bovine serum was added to stop the typsinization. Then the solution was filtered200mesh sieve and centrifuged5min at1000rpm. Finally, the supernate was removed and the cells inside was resuspended in the plant medium and adjusted planting density of5×105/ml. The cell suspension was added2ml/well and incubated at37＞in5%CO2incubator. After12h, the planting medium was changed for maintaining culture medium, halfly renewed medium every4days. The neurons were observed under the inverted microscope. The neurons were identified by Neuron specificity enolization enzyme immunofluorescence and randomly selected five horizons and count number of positive cells in each field of vision, and finally get the average purity.2. The copositioning of NR1and NR2B on the neurons:the primary hippocampal neurons were plant on precoated small coverslips with5×105/ml. The neurons on d8were dyed by immunofluorescence double labeling staining. The primary antibodies were rabbit anti-rat NR11gG and mouse anti-rat NR2B1gG. The secondary antibodies were FITC-goat anti-rabbit antibody and CY5-goat anti-mouse antibody.The neurons were viewed under the Leica laser confocal microscope and photographed. 3. Neurotoxicity of Rhy and MK-801:According to testing concentration of Rhy and MK-801, the d6neonatal primary hippocampal neurons were divided into the blank group, control group,50μmol/L group,100μmol/L group,200μmol/L group,400μmol/L group,800μmol/L group,1000μmol/L group. The neurons were planted in the pre-coated96-well plates with1×106/ml. Six repeat holes were set in each group.The cells suspension was add into wells at100μl/well, no cells in blank group.1mmol/L Rhy or MK-801concentrated solution were diluted into50μmol/L,100μmol/L,200μmol/L,400μmol/L,800μmol/L,1000μmol/L. Each well of the experimental groups were add100μl mataing medium with drug. Each well of the bank and control groups were add100μl refresh mataing meidium. Then the neurons were incubated48h at37℃in5%CO2incubator. The neurontoxic effects of Rhy and MK-801were measured with MTT cell vability test. The absorbance values at490nm represented the viable neuron numbers. The experiment was repeated3times. The dates were shown in form of x±s. Comparision between differently treated groups was carried out with one-way ANOVA followed by least significant different test, using SPSS13.0software. P<0.05was considered statistically significant.4. Rhy affected mRNA expression of NR1and NR2B:The d6neonatal primary hippocampal neurons were divided into control group, Rhy100μmol/L group, Rhy200μmol/L group, Rhy400μmol/L group, MK-801200μmol/L group.1mmol/L Rhy concentrated solution was diluted into100μmol/L,200μmol/L,400μmol/L.1mmol/L MK-801concentrated solution was diluted into200μmol/L. Each well of the control group was added2ml refresh mataing medium. Each well of the experimental groups was added2ml mataing medium with drug. The neurons were incubated6h and48h at37＞and mRNA expression of NR1and NR2B were detected by the Real-time PCR technology. Three repeat holes were set in each group and the experiment was repeated three times. The dates were shown in form of x±s. Comparision between differently treated groups was carried out with one-way ANOVA followed by least significant different test, using SPSS13.0software. P<0.05were considered statistically significant.5. Rhy affected protein expression of NR1and NR2B:The d6neonatal primary hippocampal neurons were divided into control group, Rhy400μmol/L group, MK-801200μmol/L group.1mmol/L Rhy concentrated solution were diluted into400μmol/L.1mmol/L MK-801concentrated solution were diluted into200μmol/L. Each well of the control group was added2ml refresh mataing medium. Each well of the experimental group was added2ml mataing medium with drug. The neurons were incubated48h at37℃. The neurons were dyed by immunofluorescence double labeling staining. The primary antibodies were rabbit anti-rat NR11gG and mouse anti-rat NR2B1gG. The secondary antibodies were FITC-goat anti-rabbit antibody and CY5-goat anti-mouse antibody. The neurons were viewed under the Leica laser confocal microscope and photographed. Fluorescence intensity was analyzed by IPP5.1software and calculated tha average value of individual neuron.Results1. Neuron morphology observation, identification and purity calculation: After6～8days, the cell bodies were bigger.The nucleuses were triangular, circular or elliptic, pervious to light quality, with halo. The neuron network interweaved and became completely mature, could be used in experiments. After14days, gliocytes increased and neurons became aging slowly. The cells were identified positive neurons by neuron specificity enolization enzyme immunofluorescence. The neurons minimum purity was85.9%, the highest purity was up to97.5%, the average purity was (92.6±4.62)%. Pancreatic enzyme digestion method combined with serum-free culture successfully established the model of primary hippocampal neurons. Morphology and purity of6-8d primary hippocampal neurons could met experimental requirements.2. The copositioning of NRl and NR2B:NRl mainly distributed on neuronal axons and dendrites, cytoplasms near the end of axonals. NR2B maily distributed on the membrane, dendrites and axons membranes, and high density distribution on the node of ranvier and the end of synaptic.3. The OD-C curve of Rhy and MK-801were reverse "S" shape. When≤400∴mol/L, Rhy had no effects on activity of neurons. When400≤Rhy≤1000umol/L, neurontoxicity of Rhy was become more and more strong. When Rhy>1000μmol/L, neurontoxicity of Rhy was most strongest. When MK-801≤200μmol/L, MK-801had no effect on activity of neurons. When200≤MK-801≤800μmol/L, neurontoxicity of MK-801was become more and more strong. When MK-801≥800μmol/L, neurontoxicity of Rhy was most strongest. Neurontoxicity of Rhy was most strongest.The experimental concentrations of Rhy were100μmol/L,200μmol/L,400μmol/L. The highest safe concentration of MK-801was200μmol/L.4. Rhy affected mRNA expression of NRl and NR2B:After6h, MK-801down-rugulated NRl and NR2B mRNA expression. After48h, MK-801up-rugulated NRl and NR2B mRNA expression. After6h, high, medium and low dose groups of Rhy up-regulated NRl mRNA expression, but Rhy had no effect on NR2B mRNA expression. After48h, Rhy up-rugulated NRl mRNA expresstion. But low, medium and high dose groups of Rhy down-regulated NR2B mRNA expression. NRl and NR2B receptors mRNA expression of MK-801droped firstly and then rose. After6or48h, high, medium and low dose groups of Rhy showed a trend of increasing the NRl expression of mRNA, decreasing the NR2B mRNA expression with times fly.5. Rhy affected NRl and NR2B proteins expression:After48h, Rhy up-regreated NRl proteins expression around the nucleus compared with normal group. NRl proteins on the cell membrane were not affected obviously by Rhy. Rhy down-regreated NR2B proteins expression on the Dendrites membrane. MK-801significantly increased the protein dot expression of NRl around the nucleus and NR2B protein expression on dendritic spines.Conclusion1. High purity, stable and reliable neuron models could be set up by the method of trypsin digestion combined with serum-free culture.2. NRl distributed on the cell membrane axons and in the cytoplasm of dendrites and axons. NR2B distributed on nerve cell membrane, the axon membrane, dendritic membrane.there was also high density distribution at the end of the neuron synapses, dendritic spines and Nodes of Ranvier.3. The neurons were protected by Rhy under low concentrations.The maximum safe concentration was400μmol/L and neurotoxicity increased with the increase of concentration. Over1000μmol/L, the cell vitality was to the minimum. MK-801under the low concentration had no effects on neurons. The maximum safe concentration was200mmol/L and neurotoxicity increased with the increase of concentration. Over1000mmol/L, the cell vitality was to the minimum.4. Rhy up-regulated NR1mRNA expression in the concentration dependence and time dependence manners and down-regulated NR2B mRNA expression in time dependence manner. Rhy may be a selective NR2B inhibitor. 5. Rhy up-regulated NR1protein expression around nucleus and down-regulated NR2B protein expression on axons, dendrites, dendritic spines. The number of NR1/NR2B groups decreased at dendritic, dendritic spines and synapticterminal membrane. These showed that Rhy may be a selective noncompetitive NMDA receptor inhibitor. It may be one of the mechanisms of antianxietic effect. |
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