[Abstract] Objective The present study was undertaken to investigate the protection of Li's Prescription No.5 against glutamate excitotoxicity to primary cultured cortical neurons. Methods The cerebral cortex of newborn mice was removed for in vitro nerve cell culture and establishment of an in vitro model of glutamate excitotoxicity to nerve cells. Morphological observation and colorimetric MTT assay were carried out to investigate the effect of Li's Prescription No.5 on glutamate excitotoxicity to primary cultured cortical nerve cells. Results The results showed that medicated serum of Li's Prescription No.5 decreased dose-dependently the death rate of the neurons and that decoction (containing volatile oil) of Li's Prescription No.5, although it inhibited the injury of primary cultured cortical nerve cells, did not significantly decrease the death rate of the neurons. Conclusions Findings of the present study suggest that Li's Prescription No.5 antagonizes the neurotoxicity of excitatory amino acid.
[Key words] Sero-pharmacology; Li's Prescription No.5; cultured neurons; glutamate exicitotoxicity


The effect of Li's Prescription No.5 on the promotion of proliferation of PC12 cells and the resistance to their injury has been reported in a precious paper. (1) This paper reports on the effect of Li's Prescription No.5 on glutamate excitotoxicity to primary cultured cortical neurons.
Materials and Methods
Drug to be Tested Medicinal powder of Li's Prescription No.5 was kindly supplied by Prof. Li Zizhong, see the previous paper. (1)
Experimental Animal Mice of Kunming strain, weighing 22±3 g, with a male-female ratio of fifty-fifty, were supplied by the Laboratory Animal Department of China Pharmaceutical University.
Drugs and Reagents Poly-l-lysine, product of Sigma; calf serum, product of Hangzhou Evergreen Bioengineering Materials Research Institute; complete DMEM, product of Gibco, 1 liter of DMEM culture fluid contains 3.7g of NaHCO3, 100U/ml of sodium
penicillin G, 100 μg/ml of streptomycin and 10% calf serum, pH 7.4; trypsinase, product of Sigma, was made into a 0.25% solution with D-Hanks solution and the formed solution was adjusted to pH 7.4, sterilized by filtration and
stored at 4℃ for use; L-glutamate, product of Shanghai Boao Biological Science and Technology Co.; MTT (4,5-dimethyl-2-thiazoly)-2, 5-diphenyl-2-tetrazolium bromide), product of Fluka; nimodipine, product of Tianjin Central Pharmaceutical Factory; and other reagents were all analytical pure preparations on sale.
D-Hanks solution and sugar-free Earle's solution were the same as reported in a previous paper. (1)
Primary Culture of Cortical Nerve Cells of Mouse Brain (2) One-day-old newborn mice were put into 75% alcohol for sterilization, then put in super clean bench and transferred into a beaker containing PBS for a moment's immersion. The mice were then removed and placed in a plate containing D-Hanks solution. The skin of the head was cut with scissors, the cerebral cortex was stripped and quickly put into gelid D-Hanks solution. Meninges and blood vessels were picked out, the cortical tissue was transferred into a small bottle containing small amount of D-Hanks solution and cut into paste with eye scissors. 0.25% trypsinase solution was added to submerge the tissue fragments for digestion at 37℃ for 30 minutes. The brain tissue was made to disperse by repeated blowing and beating. After digestion the DMEM culture fluid containing 10% calf serum was added to terminate digestion. The mixture was allowed to stand at room temperature, the cell suspension in the upper layer was aspirated and transferred into a sterilized graduated centrifuge tube for centrifugation at 1 000 rpm for 10 minutes, the supernatant was discarded, DMEM containing 10% calf serum was added to the precipitate to make up cell suspension, which was inoculated on a plate with 24 wells which had been coated with poly-l-lysine and incubated at 37℃ in an incubator containing 5% CO2. Change of fluid was made 24 hours later and every three days afterwards, and on the 7th day the fluid was changed into culture fluid containing cytarabine (its final concentration was 15 μg/ml) to inhibit the proliferation of such non-nerve cells as glia cells and again changed into normal culture fluid 48 hours later. The culture well in which the cells have close density and consistent state of growth was selected for experiment.
Effect of Li's Prescription No.5 on Nerve Cell Injury Induced by Glutamate (3-5) The preparation of decoction of Li's Prescription No.5 and its medicated serum has been reported in a previous paper. (1) The cells selected were divided into control group (I), glutamate group (Ⅱ), group of glutamate +nimodipine (Ⅲ), group of glutamate+0.1mg/ml of Li's Prescription No.5 (Ⅳ), group of glutamate+0.5mg/ml of Li's Prescription No.5 (Ⅴ), group of glutamate+2.5mg/ml of Li's Prescription No.5 (Ⅵ), drug-free serum control group (Ⅶ), group of glutamate+drug-free serum (Ⅷ), group of glutamate+nimodipine-medicated serum (Ⅸ), group of glutamate+Li's Prescription No.5 (3g/kg)-medicated serum (Ⅹ), group of glutamate+Li's Prescription No.5 (6g/kg)-medicated serum (Ⅺ), and group of glutamate+Li's Prescription No.5 (12g/kg)-medicated serum (Ⅻ).
The original culture fluid was aspirated, the cells were gently washed twice with sugar-free Earle's solution, 1ml of sugar-free Earle's solution was added to each well. After 30 minutes' action, nimodipine in the final concentration of 5×10－6 mol/L was added to group Ⅲ; decoction (containing volatile oil) of Li's Prescription No.5 in the final concentration of 0.1mg/ml, 0.5mg/ml and 2.5 mg/ml was added to groups Ⅳ,Ⅴ,Ⅵ, respectively; drug-free serum, nimodipine-medicated serum, and Li's Prescription No.5 (3g/kg, 6g/kg and 12g/kg)-medicated serum was added to groups Ⅷ，Ⅸ，Ⅹ，Ⅺ and Ⅻ, respectively; to make a 5% final serum concentration, after 20 minutes' action, glutamate (Glu) in the final concentration of 500μmol/L was added and the mixture was allowed to stand at room temperature for 30 minutes; in groupⅡonly Glu in the final concentration of 500μmol/L was added; and in groupⅦ drug-free serum in the final concentration of 5% without Glu was added.
The fluid was aspirated, the wells were gently washed twice with D-Hanks solution, 1ml of DMEM culture fluid containing no calf serum was added to each well, and the wells were incubated at 37℃ in an incubator under the condition of 5% CO2 for 24 hours. MTT in the final concentration of 0.5mg/ml was added and the wells were continuously incubated for 4 hours. The culture fluid was aspirated, 200μl of 100% dimethyl sulfoxide was added to each well, and as soon as the blue granules in the wells were completely dissolved, optical density (OD) value was determined at 570 nm with enzyme-linked immunosorbent detector. The calculation of inhibition rate of the decoction (containing volatile oil) and medicated serum of Li's Prescription No.5 for Glu-induced primary cultured cortical nerve cell injury has been reported in a previous paper. (1)
Results
Morphological Observations The primary cultured cortical nerve cell body showed plump appearance, smooth surface and steric projection, and the nerve process formed dense network. The addition of Glu produced excitation, followed by gradual degeneration of injured nerve cells, swelling of cell bodies, occurrence of sedimentation, breaking of nerve process and neuronal disintegration. Pretreatment of cells with medicated serum of Li's Prescription No.5 obviously resisted the morphological change caused by Glu, as manifested by reduced disappearance of cell process structure and reduced formation of cell debris.
Results of MTT Automatic Microdetermination As shown in Fig 1, nimodipine significantly inhibited Glu-mediated nerve injury (p<0.01), the inhibition rate being 86.3%; whereas decoction (containing volatile oil) of Li's Prescription No.5 in the concentrations of 0.1mg/ml, 0.5mg/ml and 2.5mg/ml had no significant inhibition on Glu-mediated nerve injury, the inhibition rate being 3.9%, 13.7% and 17.6%, respectively.
Fig 1 The effect of decoction of Li's Prescription No.5 on glutamate excitotoxicity to primary cultured cortical nerve cells (n=4, ±S; ** p<0.01, compared with the control group; ## p<0.01, compared with the glutamate group).
The effect of medicated serum of Li's Prescription No.5 on glutamate excitotoxicity to primary cultured cortical nerve cells is shown in Fig 2. Nimodipine-medicated serum significantly inhibited Glu-mediated nerve injury (p<0.01), the inhibition rate being 35.7%, medicated serum of 3g/kg of Li's Prescription No.5 had no significant inhibition on Glu-mediated sera of 6g/kg and 12g/kg of Li's Prescription No.5 significantly inhibited Glu-mediated nerve injury (p<0.05 or p<0.01), the inhibition rate being 5.4%, 26.8% and 32.1%, respectively.
Fig 2 The effect of medicated serum of Li's Prescription No.5 on glutamate ecitotoxicity to primary cultured cortical nerve cells (n=4, ±S; ** p<0.01, compared with drug-free serum control group, # p<0.05, ## p<0.01, compared with group of drug-free serum+glutamate.
Discussion
Cell culture has the advantages of short experimental period, easy control of experimental conditions and convenient observation of experimental results and is, therefore, an ideal method for studying
pharmacology of Chinese drugs. However, the direct addition of crude preparations of Chinese drugs to the cell culture system for experiment has many methodological problems, for example, the impurities contained in Chinese drugs, various electrolytes or tannins and the difference in acid-base scale are all factors that affect cell growth, experimental results and conclusions. The sero-pharmacological method for Chinese drugs, which uses serum obtained from animals after their oral administration and absorption of crude preparation of Chinese drugs, can be used to make in vitro experiments without the interference of various affecting factors and is, therefore, more close to the real course of pharmacological action produced by the drugs in in vivo environment, thus improving the degree of confidence of experimental results. In addition, the sero-pharmacological method may well reflet the available compositions which are contained in complex prescription of Chinese drugs and not absorbed by gastrointestinal tract (such as some high-molecular compounds), or inactivated after metabolism, and compositions which have no direct action themselves but produce action after metabolism or have indirect action by stimulation of the second messenger. (6)
Glutamate is one of the main neurotransmitters of excitatory synapse in the brain. Under physiological conditions, it plays, by activating such receptors as NMDA and KA, an important role in learning and memory, transmission of excitatory synapse of neurons, formation of neuronal synapse and architecture of nerve return circuit. In recent years, a large quantity of researches have shown that in the presence of cerebral anoxia and ischemia, brain trauma and epilepsy, glutamate in synaptic cleft increases and that this increase stimulates abnormal excitation of glutamate receptors which causes neuronal degeneration and necrosis, thus severely affecting the functions of nervous system. (7) The results of the present study showed that the decoction (containing volatile oil) of Li's Prescription No.5 had no significant protective effect on glutamate excitotoxicity to primary cultured cortical neurons and that medicated serum of Li's Prescription No.5 dose-dependently inhibited glutamate-mediated nerve injury, the mechanism of which remains to be studied further.
References
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