多巴胺对轻微型肝性脑病大鼠脑内谷氨酸摄取能力的影响及其发病机制

Effect of dopamine on intracerebral glutamate uptake ability in rats with minimal hepatic encephalopathy andthe pathogenesis of minimal hepatic encephalopathy

目的研究多巴胺（DA）对神经细胞摄取谷氨酸（Glu）能力的影响，以及DA通过通路对轻微型肝性脑病（MHE）大鼠认知障碍的影响。方法45只SD大鼠随机分为对照组，MHE模型组和DA干预模型组∞=15），其中MHE模型组采用硫代乙酰胺（TAA）腹腔注射、DA干预组采用脑室内注射DA、对照组大鼠腹腔注射等渗盐水，每周2次，共8周；采用脑活体微透析检测MHE大鼠和DA干预大鼠的脑内Glu含量变化，实时定量PCR和免疫印迹分析等检测痕量胺相关受体1（TAARl）、兴奋性氨基酸转运子2（EAAT2）基因mRNA和蛋白质相对表达量；TAARl微小RNA（siRNA）和TAARl质粒侧脑室注射MHE大鼠和DA干预大鼠后检测EAAT2表达情况和细胞外Glu水平变化。计量资料是正态分布且满足方差齐性检验，多组问比较采用单因素方差分析；否则多组间比较采用非参数检验。等级资料多组间比较，采用非参数检验。结果MHE大鼠与对照组相比，DA含量在肝组织[（4．90±0．13）ng／g对比（1．20±0．13）rig／g]、血清[（16．32±1．01）pmol／ml对比（5．50±0．82）pmol／m1]和脑组织[（732．45±78．85）ng／g对比（387．00±23．36）ng／g】均显著升高，P值均〈0．05，差异均有统计学意义。DA干预大鼠在脑室内注射DA后，在40～120min之间细胞外Glu含量显著升高。在MHE大鼠和DA干预大鼠组，TAARl蛋白相对表达量分别为3．72±0．50、4．18±0．43，与对照组的0．96±0．40相比，显著增高，p值均〈0．05，差异均有统计学意义。MHE大鼠和DA干预组大鼠的EAAT2蛋白相对表达水平与对照组相比，显著下降（0．46±0．16对比0．92±0．11，P=0．013；0．51±0．20对比0．92±0．11，p=0．036），差异均有统计学意义。MHE大鼠和DA干预大鼠经TAARlsiRNA干预后脑组织中EAAT2蛋白相对表达量与空载体处理模型组相比上调（0．86±0．142对比0．56±O．060，p=0．028；0．99±0．056对比0．43±0．098，p=0．001），且脑内细胞外的Glu含量在60～120min显著减少。而MHE大鼠和DA干预组大鼠经TAARl质粒干预后与对照组比较，脑组织中EAAT2蛋白相对表达量下调（0．20±0．040对比0．48±0．08；0．24±0．05对比0．54±0．07），P值均〈0．05，差异均有统计学意义；且脑内细胞外的Glu含量在60～100rain显著升高。结论DA通过与脑组织中的TAARl相互作用引起细胞外Glu蓄积，导致MHE大鼠脑组织中TAARl-EAAT2信号通路紊乱，最终损伤其认知功能。

Objective To investigate the effect of dopamine （DA） on the glutamate （Glu） uptake ability of neural cells, as well as its effect on cognitive impairment in rats with minimal hepatic encephalopathy （MHE） via related pathways. Methods A total of 45 Sprague-Dawley rats were randomly divided into control group, MHE model group, and DA intervention model group, with 15 rats in each group. The rats in the MHE model group were given intraperitoneal injection of thioacetamide （TAA）, those in DA intervention model group were given intraventricular injection of DA, and those in the control group were given intraperitoneal injection of physiological saline, with a frequency of twice a week for 8 weeks. Cerebral microdialysis was used to measure the change in the content of Glu in the brain in MHE rats and rats with DA intervention; RT-PCR and Western blotting were used to measure the relative mRNA and protein expression of trace amine-associated receptor 1 （TAAR1） and excitatory amino acid transporter 2 （EAAT2）; the changes in the expression of EAAT2 and extracellular Glu level were measured after intracerebroventricular injection of TAAR1 siRNA and TAAR1 plasmid in MHE rats and rats with DA intervention. One- way analyses of variance for comparison among different groups were performed, categorical data between groups were compared using nonparametric tests. Results Compared with the control group, the MHE model group had significant increases in the content of DA in liver tissue, plasma, and brain tissue （4.90 ± 0.13 ng/g vs 1.20 ± 0.13 ng/g,P 〈 0.05; 16.32 ± 1.01 pmol/ml vs 5.50 ± 0.82 pmol/ml, P 〈 0.05; 732.45 ：i： 78.85 ng/g vs 387.00 ± 23.36 ng/g, P 〈 0.05）. There was a significant increase in the extracellular Glu level within 40-120 minutes after intracerebral injection of DA in the DA intervention model group. Compared with the control group, the MHE model group and the DA intervention model group had a significant increase in the relative protein expression of TAAR1 （3.72 ± 0.50/4.18 ± 0.43 vs 0.96 ± 0.40, bothP 〈 0.05） and a significant reduction in the expression of EAAT2 （0.46 ± 0.16/0.51 ± 0.20 vs 0.92 ± 0.11,P = 0.013 and 0.036）. Compared with the model group treated with empty vector, the MHE model group and the DA intervention model group had a significant increase in the relative protein expression of EAAT2 after TAAR1 siRNA intervention （0.86±0.142 vs 0.56 ± 0.060, P = 0.028; 0.99 ± 0.056 vs 0.43 ± 0.098, P = 0.0010） and a significant reduction in the extracellular Glu level in the brain at 60-120 minutes after injection, while after TAAR1 plasmid intervention, the MHE model group and the DA intervention model group had a significant reduction in the relative protein expression of EAAT2 （0.20 ± 0.040 vs 0.48 ± 0.08, P = 0.006; 0.24 ± 0.05 vs 0.54 ± 0.07, P = 0.004） and a significant increase in the extracellular Glu level in brain at 60-100 minutes after injection. Conclusion DA interacts with TAAR1 in brain tissue to induce extracellular accumulation of Glu, thus leading to the disorder of the TAAR1-EAAT2 signaling pathway in brain tissue and ultimately injuring the cognitive function of MHE rats.