茶氨酸对大鼠脑缺血再灌注后单胺类神经递质及相关基因表达的影响

Effects of theanine on monoamine and related genes in cerebral ischemia-reperfusion injury rats

目的探讨茶氨酸对大鼠脑缺血再灌注后多巴胺（DA）、5-羟色胺（5-HT）的水平及对谷氨酸受体2（GluR2）mRNA、磷脂酶c-11（PLC-11）mRNA表达的影响，讨论其所致神经损伤的保护作用机制。方法将56只6周龄、体重为100—120g的SPF级雄性SD大鼠按体重分层，以随机数字表法分为5组，分别为模型组（12只）、假手术组（8只），以及茶氨酸低（10mg／kg）、中（30mg／kg）、高剂量组（90mg／kg）（每组12只）。茶氨酸剂量组大鼠灌胃每天给予相应剂量的茶氨酸，模型组和假手术组每天灌胃给予等量超纯水。连续灌胃15d后，制备大鼠大脑中动脉栓塞（middlecerebralarteryocclusion，MCAO）模型，于再灌注后第3、24小时进行神经学评分，再灌注后第24小时处死动物，测定脑组织中DA、5-HT和茶氨酸含量，同时测定脑组织线粒体中活性氧（ROS）生成和过氧化氢酶（CAT）活性，并采用RT—PCR法检测脑组织中GluR2mRNA以及PLC-y1mRNA的表达水平。结果模型组、茶氨酸低、中、高剂量组大鼠脑缺血再灌注后第3小时神经学评分分别为（6．000±0．926）、（4．100±0．738）、（3．444±0．726）、（2．250±0．886）分（F=29．70，P〈0．01）；第24／J、时神经学评分分别为（6．625±0．916）、（5．000±0．817）、（3．667±0．707）、（2．625±0．916）分（F=34．68，P〈0．01）。模型组、茶氨酸低、中、高剂量组及假手术组大鼠脑内DA含量分别为（10．26±1．12）、（12．48±1．09）、（14．55±0．94）、（15．97±0．92）、（11．98±0．63）μg／g（F=43．76，P〈0．01）；5-HT含量分另0为（1．091±0．160）、（0．818±0．101）、（0．571±0．050）、（0．453±0．111）、（0．863±0．063）μg（F=48．68，P〈0．01）；大鼠脑线粒体中ROS生成速率分别为（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=80．82，P〈0．01）；CAT活性分别为（4．880±1．121）、（8．405±1．356）、（9．535±2．511）、（15．090±4．054）、（21．260±6．054）U／g（F=28．58，P〈0．01）；大鼠脑内GluR2mRNA相对表达量分别为0．842±0．020、1．063±0．100、1．170±0．152、1．254±0．131、1．012±0．056（F=9．23，P〈0．01）；PLC-y1mRNA相对表达量分别为0．737±0．090、0．887±0．045、0．963±0．025、0．991±0．049、0．867±0．079（F=10．24，P〈0．01）。结论茶氨酸对大鼠脑缺血再灌注损伤具有一定的保护作用，其机制与调节脑内单胺类神经递质含量以及上调GluR2mRNA和PLC-11mRNA表达有关。

Objective To study the effects of theanine on dopamine （DA）, 5-hydroxy tryptamine （5-TH） and glutamate receptor 2 （ GluR2 ） mRNA, phospholipase-y1 （ PLC-u1 ） mRNA in cerebral ischemia-reperfusion injury rats and explore the mechanism of protective effects of theanine on the induced brain injury by ischemia-reperfusion in rats. Methods According to random number table, a total of 56 sprague-dawley rats in SPF grade about six-week old and 100 - 120 grams weighting were divided into five groups according to the body weight levels ： model group （ n = 12 ） , sham-operation group （ n = 8 ） , low theanine group（ 10 mg/kg） , middle theanine group（30 mg/kg） and high theanine group（90 mg/kg）. There were 12 rats in each of the theanine group. The rats in model group and sham-operation groups were given distilled water, and the rats in theanine groups were given corresponding theanine solution intragastrically for fifteen days. Then the cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion （MCAO）. The score of neurological behavior was evaluated at the 3rd and 24th hours after reperfusion. Rats were sacrificed at 24 hours after reperfusion, the concentrations of DA,5-HT and theanine in rats brain following ischemia-reperfusion were determined. At the same time, we determined the levels of reactive oxygen species（ROS） and activities of catalase（CAT） in mitochondria of brain. The expressions of GluR2 mRNA and PLC-y1 mRNA in rat brain were examined by reverse transcription polymerase chain reaction （RT-PCR） technique. Results The score of neurological behavior of rats in model group, theanine-low, middle, high dose groups at the 3rd hour was 6. 000 ±0. 926,4. 100 ± 0. 738,3. 444 ±0. 726 and 2. 250 ± 0. 886 respectively（F = 29. 70,P 〈 0. 01 ）, and the score at the 24th hour in these groups was 6. 625± 0. 916,5. 000 ± 0. 817,3. 667 ± 0. 707 and 2. 625 ± 0. 916 respectively（ F = 34. 68, P 〈 0. 01 ）. The concentration of DA in model group, theanine-low, middle, high dose groups and sham-operation group was （10.26±1.12）, （12.48 ± 1.09）, （14.55 ±0.94）, （15.97 ± 0.92） and （11.98 ± 0.63） p,g/g respectively（ F = 43.76, P 〈 0. 01 ） . The concentration of 5-HT in these groups was （ 1. 091 ±0. 160 ） , （0. 818 ±0. 101 ）, （0. 571 ± 0. 050 ）, （0. 453 ± 0. 111 ） and （0. 863± 0. 063 ） txg/g respectively （ F = 48.68 ,P 〈0. 01 ）. The level of ROS was（3. 072±0. 503）, （ 1. 331 ±0. 268）, （ 1. 295±0. 061 ）, （0. 804 ± 0. 200）and（2. 158 ± 0. 218）U min-1 . mg-1（ F = 80. 82 ,P 〈0. 01 ）respectively and the activities of CAT in these groups were （4. 880 ± 1. 121 ）, （8. 405± 1. 356）, （9. 535 ± 2. 511 ）, （ 15. 090 ± 4. 054） and （21. 260 ± 6. 054）U/g respectively （F = 28.58, P 〈 0. 01 ）. The expressions of GluR2 mRNA were 0. 842 ± 0.020,1.063 ±0.100,1.170 ±0.152,1.254 ±0.131 and 1.012±0.056 respectively （F =9.23, P 〈0. 01 ）. The expressions of PLC-3,1 mRNA in these groups were 0. 737±0. 090,0. 887 ±0. 045,0. 963 ± 0. 025,0. 991± 0. 049 and 0. 867± 0. 079 respectively （F = 10. 24, P 〈 0. 01 ）. Conclusion Theanine has a protective effect on the induced brain injury by ischemia-reperfusion in rats, which might be associated with its interaction with monoamine neurotransmitters and up-regulating the expressions of GluR2 mRNA and PLC-Y1 mRNA.