Protective Effect Of MK-801 On Manganese Disturbing Glutamate Metabolism And NMDA Receptor Expression

ObjectiveManganese (Mn) is one of essential trace elements found in a variety of biological tissues and constitutes many enzyme active groups or the accessory factor. Simultaneously, it is also certain enzyme activating agent and participates in many biochemical reactions. But excessive manganese enters the body to be able to cause manganism. Mn is possible to penetrate the blood-brain barrier and stores up in the brain organization. Early symptoms are main performance for nerve behavior function change. The long-term exposure Mn main performance for the extra pyramidal system is the damage, has similar symptoms resembling features of Parkinson's disease. Therefore, study on the neurotoxicology of Mn research the general scholar's attention. The mechanisms underlying the neurotoxicity of Mn are still incompletely understood and involve many aspects. The excessive manganese may increase the nerve cell metabolism, break mitochondria, and improve lysosomes activity, occurring self-digestion, causes the nerve cell to degenerative changes and damage. It also activate cytochrome oxidase P-450 to produce free radical. In recent years studied indicated, Mn may disturb excitatory neurotransmitter Glutamate metabolism to induce the indirect excitability neurotoxicity. N-methyl-D-aspartate receptor (NMDAR), an excitatory amino acid neurotransmitter receptor, possesses unique structure function characteristic and exerts essential physiological functions. It not only is the ligand gating acceptor, also is the voltage dependence gating acceptor. Thus in the neuro-physiology, the pathology and the toxicology research the NMDA acceptor has also received the special attention. MK-801 is one kind of powerful NMDA acceptor non-competitive antagonist. Through the blood-brain barrier, it may affect easily in the NMDA acceptor channel internal benzene ring pai-ding position spot carries on the construct adjustment and can effectively hinder the glutamate and postsynaptic membrane receptor combination. MK-801 may block NMDA the acceptor coupling the Ca2+ channel activation decreasing influx of extracellular Ca2+, thus weaken the NMDA receptor function. In order to further understand the relationships between effect of Mn on excitatory neurotransmitter metabolism and the nerve cell damage and protective effect of MK-801 on Mn neurotoxicity, we will test two aspects from in vivo and in vitro to study the following question:①Effect of Mn on Glutamate metabolism.②Effect of Mn on NR1, NR2A and NR2B subunit expression.③Toxic effects of Mn on primary cultured neurons.④Protective effect of MK-801 on Mn-induced nerve damage. It has the extremely vital significance to us further understood the certain mechanism of Mn neurotoxicity and provides the important theoretical and experimental basis for the pathogenesis and reventing and controlling of Mn-induced nerve cell damage.Methods一,In vivo animal experiments(一) Experimental animal and treatment100 Wistar rats obtained from the Laboratory Animal Center of Chinese Medicine University, weighing 170-190 gram. After an acclimation period of a week,100 rats were randomly divided into 5 groups with 20 animals in each group:control group, Mn-treated group (8,40, and 200μmol/kg) and MK-801 pre-treated group. The control group rats were intraperitoneally (i.p.) injected with 0.9%normal saline (group 1). Mn-treated rats were respectively i.p. injected with 8,40,200μmol/kg MnCl2 in sterile deionized water (group 2-4). MK-801 in sterile deionized water was given every two-day. The MK-801 pre-treated rats were subcutaneously (s.c.) injected with 0.3μmol/kg body weight/day, two hour before the i.p. administration with 200μmol MnCl2/kg body weight/day (group 5). The capacity of injection is all 5 ml/kg.4 weeks after administration, the rats were anatomized to get the tissue samples of the striatum.(二) Measuring indexes1. Measurement of Mn concentration in striatumAfter administrated for 4 weeks, each group executes 8 rats. A certain amount striatum were separated and Mn concentration analysis was performed by HITACHI 180-80 atomic absorption spectrophotometer.2. Measurements of Glu and Gln concentrations in striatumAfter administrated for 4 weeks, each group executes 6 rats. A certain amount striatum were separated and Glu and Gln concentrations analysis were performed according to the kits'introduction.3. Measurements of PAG and GS activities in striatumAfter administrated for 4 weeks, each group executes 6 rats. A certain amount striatum were separated and PAG and GS activities analysis were performed according to the methods of Curi and Renis.4. Measurements of NMDAR mRNA and proteins expression in striatumAfter administrated for 4 weeks, each group executes 4 rats. Measurements of NMDAR mRNA and proteins expression were performed by RT-PCR ans Western Blotting analysis and collected total RNA and the total protein according to the conventional method. The changes of intensity of NR1, NR2A and NR2B mRNAs after Mn treatment were normalized using the intensity obtained in the internal control bands (G3PDH). The changes of intensity of NR1, NR2A and NR2B proteins after Mn treatment were normalized using the intensity obtained in the internal control bands (β-actin).5. Apoptosis DetectionEach group 4 rats'striatum were dissected to use for the preparation of dissociated striatum cells, which were analyzed using flow cytometry. After administrated for 4 weeks, each group executes 2 rats. After the rats were perfused with 4% paraformaldehyde, a certain amount striatum were separated and viewed in a JEOL JSM-7300EX transmission electron microscope.二,In vitro primary neuronal cultures(一) Primary neuronal culturesCortices were isolated from the brains of neonatal Wister rats and chopped into 2 mm pieces under the light microscope. The cortical chunks were then suspended in trypsin solution. The whole solution was filtered through stainless steel. The cell pellets were resuspended in DMEM containing 10%(v/v) horse serum. The cell suspension was plated at 1×106 cells/1.5ml on six-well dishes pre-coated with poly-L-lysine and incubated at 37℃in humidified 95%air/5%CO2 for 24 h. After 24 h, the culture medium was changed to Phenol Red-free Neurobasal medium (without Phenol Red and estrogen-free) supplemented with B27,100 U/ml penicillin/streptomycin and L-glutamine (0.5 mM). These cells were then treated with cytosine arabinoside on the 3rd day for 48 h to eliminate dividing non-neuronal cells.(二) Identification of neuronsNeuronal morphology was observed with an Olympus IX70 inverted phase-contrast microscope fitted with a digital camera system to capture images. Neuron-specific enolase (NSE) was immunostained by immunocytochemistry.(三) Measuring indexes1. Quantification of viabilityTreatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0-400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells were respectively treated for 6, 12,24,48 h, MTT assay were performed.2. LDH assay in culture solutionTreatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0-400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells were respectively treated for 6, 12,24,48 h, LDH assay were performed.3. TdT-mediated dUTP nick-end labeling (TUNEL) assayTreatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0,25,100, 400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells treated for 12 h, TUNEL assay was examined on paraformaldehyde-fixed cells using the Apop-tagTM in situ apoptosis detection kit.4. Measurements of NMDAR mRNA and proteins expression in neuronsTreatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0,25,100, 400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells treated for 12 h, measurements of NMDAR mRNA and proteins expression were performed by RT-PCR ans Western Blotting analysis and collected total RNA and the total protein according to the conventional method. The changes of intensity of NR1, NR2A and NR2B mRNAs after Mn treatment were normalized using the intensity obtained in the internal control bands (G3PDH). The changes of intensity of NR1, NR2A and NR2B proteins after Mn treatment were normalized using the intensity obtained in the internal control bands (β-actin).5. Measurement of intracellular free calciumTreatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0,25,100, 400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells treated for 12 h, absolute values of [Ca2+]i in neurons were calibrated from the measured fluorescence signals by the use of F-4500 Fluorescence Spectrophotometer.6. Na+-K+-ATPase and Ca2+-ATPase activities assay in neuronsTreatments were performed on the tenth day after plating. Briefly, primary neuronal cells were treated with fresh media with different concentrations of Mn (0,25,100, 400μmol/L) following pre-wash with media. The cells were pre-treated with 10μmol/L MK-801,30 min before treatment with 400μmol/L Mn. After cells treated for 12 h, Na+-K+-ATPase and Ca2+-ATPase activities assay were performed by spectrophotometer at 706 nm.Results一,In vivo animal experiments 1. Mn concentration in striatumThere was a significant increase in Mn concentration in the striatum of Mn-treated rats compared to control rats. In 200μmol/kg Mn-treated group, the Mn concentration significantly increased 4.7-fold in striatum. However, the Mn concentration in MK-801 pre-treated rats was not different compared with 200μmol/kg Mn-treated group.2. Glu and Gln concentrations in striatumWith the increase of administered-MnCl2 dosage, Glu concentration was increased and Gln concentration was decreased. Glu concentration was approximate 2.5-fold increased in 200 umol/kg Mn-treated rats. Gln concentration was decreased in 200μmol/kg Mn-treated rats by 39.4%, relative to saline treated controls. MK-801 pre-treatment did not affect Glu concentration in the striatum. However, MK-801 pre-treatment increased Gln concentration compared with 200μmol/kg Mn-treated group (P＜0.05).3. PAG and GS activities in striatumWith the increase of administered-MnCl2 dosage, GS activity was decreased and PAG activity was increased. A signiffcant (P<0.01) decrease in GS activity was observed in 200μmol/kg Mn-treated group (44.47±6.93μmol/min/g pro). PAG activity was significantly increased by 54.4%in 200μmol/kg Mn-treated rats (41.02±6.60μmol/min/g pro) compared with control animals (P＜0.01). Compared with 200μmol/kg Mn-treated animals, MK-801 pre-treatment blocked GS activity decrease and PAG activity increase.4. NMDAR mRNA and proteins expression in striatumWith the increase of administered-MnCl2 dosage, mRNA and protein levels of NR1, NR2A and NR2B1 were reduced in different extent compared with those of control group. Expression of NR2A mRNA and protein were much more sensitive to Mn than those of NR1 and NR2B. Compared with 200μmol/kg Mn-treated animals, MK-801 pre-treatment increase the levels of NR2A mRNA and protein. However, the levels of NR1 and NR2B mRNA and protein were not found the obvious change.5. Cell apoptosis in striatumTotal apoptosis rate was significantly increased with the increase of administered-MnCl2 dosage using flow cytometry. However, there were significantly reduced apoptotic cells in the MK-801 pre-treated group compared with 200μmol/kg Mn-treated group. Under transmission electron microscopy, in 200μmol/kg Mn-treated group, neurons showed features of apoptosis, characterized by nucleus shrinkage, dense aggregation of chromatin and chromatin margination.二,In vitro primary neuronal cultures1. Viability and LDH assayWith the increase of Mn treatment concentration, viability of neurons and LDH levels in the culture medium displayed the obvious dosage-effect relations and the time-effect relations. According to MTT and LDH analysis resulting, Mn exposures in subsequent experiments were restricted to concentrations of 25,100,400μmol/L Mn for 12 h, which ensure that Mn treatment levels were representative and appropriate concentrations that did not cause severe cytotoxicity and the sufficient concentrations to induce measurable neurologic deficits.2. TUNEL assayWith the increase of Mn treatment concentration, there was a significant increase of apoptotic cell death and Integrated Optical Density (IOD) in a concentration-dependent manner. Compared with 400μmol/L Mn-treated group, MK-801 pre-treatment reduced the cell apoptosis rate and IOD.3. NMDAR mRNA and proteins expression in neuronsWith the increase of Mn treatment concentration, mRNA and protein levels of NR1, NR2A and NR2B were reduced in different extent compared with those of control group. Expression of NR2A mRNA and protein were much more sensitive to Mn than those of NR1 and NR2B. Compared with 400μmol/L Mn-treated group, MK-801 pre-treatment increase the levels of NR1 and NR2A mRNA and protein. However, the levels of NR2B mRNA and protein were not found the obvious change.4. [Ca2+]i in primary cultured neuronsWith the increase of Mn treatment concentration, [Ca2+]; increased evidently. Compared with 400μmol/L Mn-treated group, MK-801 pre-treatment reduced the increase of [Ca2+]i.5. Na+-K+-ATPase and Ca2+-ATPase activities in primary cultured neurons With the increase of Mn treatment concentration, Na+-K+-ATPase and Ca2+-ATPase activities were decreased. Compared with 400μmol/L Mn-treated group, MK-801 pre-treatment raised Na+-K+-ATPase and Ca2+-ATPase activities.Conclusions1. The rat model of manganism was successfully established by intraperitoneally injection with MnCl2 The research discovered Mn level was obviously increased in rat striatum of manganism. Mn may cause the nerve cell to occur apoptosis by transmission electron microscopy and flow cytometry.2. By animal experiments, we discovered Mn could disrupt "Glu-Gln cyclic pathway" in rat striatum, which cause PAG activity increase and GS activity decrease.3. By animal experiments and primary neuronal cultures, we discovered Mn could inhibit expression of NR1, NR2A and NR2B mRNAs and proteins in rat striatum and expression of NR2A mRNA and protein were much more sensitive to Mn than those of NR1 and NR2B. NR2A and NR2B subunits were associated with the different intracellular signal pathways, which might be one of Mn-induced cell apoptosis reasons.4. By primary neuronal cultures, we discovered Mn could cause obviously cytotoxicity in primary cultured neurons. Main performance:the cellular transparence decreased, the cell bodies were shrinkage, the neuraxon of neurons shortened, the network disappeared, and some had already been ruptured and suspended. The viability of neurons was significantly decreased and LDH release significantly increased. Our data demonstrated that significant apoptotic cell death were evident in Mn-treated neurons using apoptotic TUNEL assay.5. Mn can disrupt intracellular calcium homeostasis. Mn treatment increased intracellular [Ca2+]; evidently and inhibited Na+-K+-ATPase and Ca2+-ATPase activities in primary cultured neurons.6. Because MK-801 can effectively hinder the glutamate and postsynaptic membrane receptor combination and not only block NMDA the acceptor coupling the Ca2+channel activation but also L-type calcium channels decreasing influx of extracellular Ca+and antioxidation, MK-801 has certain protective function on Mn-induced neurotoxicity through many kinds of mechanisms.