| Objective:Pathophysiological mechanism of nerve injury caused by ischemic infarction is in relationship with damage cascade, it mainly includes:excitotoxicity, calcium overload, inflammation and progress cell death (PCD). Cascade begins with excitotoxicity which induced acute necrosis, started PCD, also with the relevant to ischemia inflammatory reaction.L-theanine is a unique composition in the tea, its effection such as depressant, sedative and inhibit the excitability reduced by caffeine have been widely known, recently, the neuroprotective of theanine was attracted more and more attention. This research aims to study the influence and protect mechanism of theanine to rats' central system neurotransmitters, in order to learn the physiological effect of theanine.Method:1. To establish a HPLC-FL method of determining theanine and 4 amino acids in rat serum and brain tissue.Using ODS-SP C18 chromatography column (4.6 mm×250 mm,5μm), the mobile-phase was 0.1 M monopotassium phosphate buffer solution (pH6.0):THF: methanol=63:2:35, the HPLC system with fluorescence detection(FL) was used to examine the theanine in rat serum and brain tissues with pre-column derivatization with o-phthalaldehyde (OPA) in the presence of the internal standard homoserine. Flow rate was set at 0.85 ml/min. The excitation wavelength was 340 nm while the emission wavelength was 455 nm.2. To study the protective effects of theanine on cerebral ischemia-reperfusion injury induced by focal cerebral ischemia in rats.90～120g 64 Sprague-Dawley male rats were randomly assigned into five groups: Model and Shame (SH)control group, low dose of theanine group(Th-L,10 mg/kg bw), middle dose of theanine group(Th-M,30 mg/kg bw),high dose of theanine group(Th-H,90 mg/kg bw),after 15 days intragastrically administered theanine or distilled water to rats, cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion (MCAO) and SH group was used as fake surgery control. The brain index were measured.The score of neurological behavior was evaluated at the 3rd and 24th hours after reperfusion. The rats were put to death 24h after reperfusion.The amino acid contents in rat brain cortex after ischemia-reperfusion were determined. The expressions of BDNF mRNA and Bcl-2 mRNA in hippocampi were examined by using reverse transcription polymerase chain reaction (RT-PCR) technique.Result:1. Result of the HPLC-FL method:The method had good linearity with the concentration ranges of 1～20μmol/L for theanine. The correlation coefficient (r) was 0.9997. The intraday precisions (RSD,%) of theanine with the concentration of 1μmol/L,10μmol/L,20μmol/L were 3.51,1.58,0.75,while the interday precisions(RSD,%) were 4.54,4.25,2.87. The recoveries were between 91.8%-99.1%. The detection limits was 0.02μmol/L. When 200 mg/kg.bw of theanine was intragastrically administered to rats, the serum concentration of theanine reached its highest level during 0.5～1.0 hrs after i.g., while the highest level of brain appeared after 5 hrs.And the other 4 amino acids were separated well in 26 min, had good linearity with the concentration ranges of 1～20μmol/L, correlative coefficience(r)were:Asp:0.9998, Glu:0.9998, Gly:0.9974, GABA:0.9998. The intraday precisions (RSD,%) of 4 amino acids ranged from 0.77 to 7.54,the interday precisions(RSD,%) of 4 amino acids ranged from 2.49 to 16.75.2. Influence of theanine to on cerebral ischemia-reperfusion injury induced by focal cerebral ischemia in rats.2.1 The effect of theanine on the of neurological behavior of rats after 24h cerebral ischemia-reperfusion injury:no significant homer symptoms in SH control group,each cerebral ischemia group after 3&24h,all appear the nerve injuried symptom,while the scores of theanine treated groups all lower than model group (p＜0.05) and the score of Th-H group was lower than others theanine treated groups (p＜0.05), a does-effect relationship can be observated.2.2 The effect of theanine on BrW/BW(g/100g) defict of rats after 24h cerebral ischemia-reperfusion injury:the defict of Model group was much higher than SH group(p＜0.05),reminding cerebral edema.Among the theanine treated groups,with the does increasing,the deficts raising as a does-effect relationship and all significantly higher than the model group (p＜0.05).2.3 The effect of theanine on neurotransmitter concentrations of rats cortex after 24h cerebral ischemia-reperfusion injury:compared with the SH group,the excitatory amino acids concentrations of Asp and Glu in Model rats cortex significantly increased (p＜0.05),and the Th-H group was lower than Th-L group (p＜0.05),but the concentration of Glu(excitatory amino acid) was no significant different between each group(p＞0.05). Concentration of Gly,GABA (inhibitory amino acids) in Th-M,Th-H groups of rat cortex was significant higher than Model group (p＜0.05). Theanine concentration in rats cortex increased with the intragastrically administered does.2.4 The effect of theanine on expressions of BDNF mRNA and Bcl-2 mRNA in rats hippocampi after 24h cerebral ischemia-reperfusion:The expression of BDNF mRNA of hippocampi in the theanine treated groups was higher than Model and SH groups, while the Th-H group was much higher than Th-M,Th-L groups (p＜0.05). The expression of Bcl-2 mRNA of hippocampi in the theanine treated groups was higher than Model group, while the Th-M group was much higher than other groups (p＜0.05).Conclusion:1. This improved high performance liquid chromatography(HPLC) with fluorescence method is sensitive, reproducible and separated and detected theanine in rat serum and brain tissue effectively.2. Theanine has a protective effect on cerebral ischemia-reperfusion injury in rats, which may be associated with its restrain the excitability amino acids of neurotransmitters(glutamate and aspartate, etc.) promote the inhibitory neurotransmitters amio acid (glycine and GABA,etc.) and up-regulation of BDNF mRNA and Bcl-2 mRNA expression. |
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