Effects Of Theanine On DA、5-TH And GluR2、PLC-γ1mRNA In Cerebral Ischemia Reperfusion Injury Rats

Object:L-Theanine, also called gamma-glutamylethylamide, is a unique amino acid and a glutamic acid analog, commonly found in tea. Kakuda^[1] found that theanine could combine ionic glutamate receptors （NMDA, AMPA, KA） in central nervous system and thus produce a biological effect, using the radioactive H atoms tracer method. Recently, the protective effects of theanine on nerve damaging have been realized by more and more people^[2], Shen hui^[3] has discussed the effect of theanine on amino acid neurotransmitters in ischemia-reperfusion injury rats,but our study mainly focus on monoamine neurotransmitters and related genes to explore the mechanism of protective effects of theanine on the induced brain injury by ischemia-reperfusion in rats.Method:1. Establish a HPLC method for determination of dopamine and5-hydroxytry-ptamine in rats’brain.Using ODS-SP C₁8chromatography column（4.6mmx250mm,5μm）, the mobile-phase was40mM Sodium acetate-Citric acid buffer solution（containing Sodium acetate40mmol/L, Citric acid30mmol/L, EDTA-2Na0.2mmol/L, Sodium1-octanesulfonate0.4mmol/L, PH3.8）:methanol=70:30. Column temperature was35℃. Flow rate was set at lml/min. Sample volume was20μl. The excitation wavelength was280nm while the emission wavelength was330nm^[4]. Using Standard curve method and Peak area to calculate the content of dopamine and5-hydroxytry-ptamine.2.To observe the protective effects of theanine on cerebral ischemia-reperfusion injury induced by focal cerebal ischemia in rats.A tolal of56male Sprague-Dawley rats in SPF grade（6weeks old,100～120g） were randomly assigned into five groups according to the body weight levels:model group, sham-operation group and three theanine（The） treated groups （theanine-low, middle, high） after adaptively feeding three days. The model group and the theanine groups had12rats respectively while the sham-operation group had8rats. The rats in model group and sham-operation groups were given distilled water,and the rats in theanine groups were given theanine solution （10,30and90mg/kg respectively） intragastrically for fifteen days. Then the cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion （MCAO） in the model and theanine groups, and sham-operation group was used as a fake surgery control. The score of neurological behavior was evaluated at the3rd and24th hours after reperfusion[5]. Rats were sacrificed at24h after reperfusion, The concentrations of dopamine （DA）,,5-hydroxytryptamine （5-HT） and theanine in rats brain following ischemia-reperfu-sion were determined by high performance liquid chromatography with fluorescence detection. At the same time, we determined the levels of mitochondria reactive oxy-gen species（ROS） and activities of catalase （CAT） in the brain. The expressions of GluR2mRNA and PLC-yl mRNA in rat brain were examined by reverse transcription polymerase chain reaction （RT-PCR） technique.Result:1.The determination of dopamine and5-hydroxytryptamine in rats’brain.Under such high performance liquid chromatography condition, monoamine neurotransmitters had good linearity with the concentration ranges of0.005-1.000ug/ml, retention times were appropriate, the chromatographic peaks of dopamine and5-hydroxytryptamine were obvious and separated. In addition, other components of the tissues didn’t disturb the chromatographic peak of them. The regression equation of dopamine and5-hydroxytryptamine were respectively Y=2.33x10⁶X-1793and Y=9.06x10⁶X+58081, the correlation coefficients were0.9998and0.9987respectively. The intraday precisions（RSD,%） of dopamine and5-hydroxytrypt-amine with the concentration of0.1、0.3、0.5ug/ml were lower than7%, while the recoveries were higher than80%. 2. The protective effects of theanine on cerebral ischemia-reperfusion injury induced by focal cerebal ischemia in rats.2.1The score of neurological behavior of rats at3rd and24th hour after reperfusionThe score of neurological behavior of rats in model group（n=8）, theanine-low （n=8）, middle（n=9）, high dose groups（n=10）at the3rd hour were6.000±0.92654.100±0.738,3.444±0.726,2.250±0.886（F=29.696, P<0.01）, and the score at the24th hour in these groups were6.625±0.91,5.000±0.817,3.667±0.707,2.625±0.916（F=34.679, P<0.01）, the rats in sham-operation （n=8） group had no significant home symptoms, but rats in other groups appear nerve injuryed symptom, the scores of theanine treated groups were all lower than model group （P<0.05）2.2The determination of theanine in rats’brainThe content of theanine in different groups of brain tissue depend on the theanine they took in, and the content in model group, theanine-low, middle, high dose groups and sham-operation（S） group were（0.141±0.022）,（0.205±0.051）,（0.375±0.083）,（1.064±0.133）,（0.130±0.034）nmol/g（F=221.950, P<0.01）.2.3The effect of theanine on monoamine neurotransmitters in cerebral ischemia-reperfusion injury rats’brainThe concentrations of dopamine in model group, theanine-low, middle, high dose groups and S group were （10.26±1.12）,（12.48±1.09）,（14.55±0.94）,（15.97±0.92）,（11.98±0.63）μg/g （F=43.755, P<0.01）; The concentrations of5-HT were （1.091±0.160）,（0.818±0.101）,（0.571±0.050）,（0.453±0.111） and （0.863±0.063） μg/g （F=48.681, P<0.01）.The content of dopamine in model group rats’ brain was lower than S group while the content of5-hydroxytryptamine was higher. But dopamine in theanine-low, middle, high dose groups were significantly increased, and5-hydrox-ytryptamine were decreased.2.4Expression of related genes by using reverse transcription polymerase chain reaction （RT-PCR） techniqueThe expressions of GluR2mRNA in model group, theanine-low, middle, high dose groups and S group were0.842±0.020,1.063±0.100,1.170±0.152,1.254±0.131 and1.012±0.056respectively （F=9.232, P<0.01）; The expressions of PLC-yl mRNA in these groups were0.737±0.090,0.887±0.045,0.963±0.025,0.991±0.049and0.867±0.079（F=10.239, P<0.01）.The expression of GluR2mRNA levels in model group was lower than S group and theanine-treated groups. PLC-yl mRNA levels in theanine-low, middle, high dose groups were higher than model group.2.5Determination of levels of reactive oxygen species （ROS） and activities of catalase （CAT） in mitochondria of brain.The levels of ROS in model group, theanine-low, middle, high dose groups and S group were（3.072±0.503）,（1.331±0.268）,（1.295±0.061）,（0.804±0.200）,（2.158±0.218）U-min^-1·mg^-1（F=80.823, P<0.01） and the activities of CAT in these groups were（4.880±1.121）,（8.405±1.356）,（9.535±2.511）,（15.09±4.054）,（21.26±6.054）U/g （F=28.577, P<0.01）.The levels of reactive oxygen species in model group was higher compared with S group but the activities of catalase （CAT） in mitochon-dria of brain decreased.Conclusion:1. With the chromatographic conditions in this study, the chromatographic peaks of dopamine and5-hydroxytryptamine were obvious and separated, and the reten-tion times were appropriate. In addition, other components of the tissues didn’t disturb the chromatographic peaks. The method had good linearity with the concentration ranges of0.005-1.000μg/ml for dopamine and5-hydroxytrypta-mine, the correlation coefficient was0.9998and0.9987.2. Theanine has a protective effect on the induced brain injury by ischemia-reperf-usion in rats, which may be associated with its interaction with monoamine neurotransmitters and up-regulating the expressions of GluR2mRNA and PLC-yl mRNA.