安神定志灵对大鼠脑内突触体多巴胺合成相关因子表达的影响

目的:研究安神定志灵对酪氨酸羟化酶(tyrosine hydroxylase,TH),多巴脱羧酶(DOPA decarboxylase,DDC),蛋白激酶A(protein kinase A,PKA)的调控作用,揭示安神定志灵治疗注意缺陷多动障碍(ADHD)的药效机制。方法:依据随机数字将自发性高血压(SHR)大鼠分为模型组,利他林组,安神定志灵低、中、高剂量(6.75,13.35,26.70 g·kg~(-1))组,每组8只,另设WKY大鼠8只为正常组,分别灌胃4周。Percoll密度梯度离心法制备脑突触体,运用蛋白免疫印迹法(Western blot)检测TH,PKA蛋白表达;运用实时荧光定量聚合酶链式反应(Read-time PCR)技术检测TH,DDC,PKA mRNA表达水平,运用免疫荧光技术检测TH,DDC阳性细胞表达量。结果:与正常组比较,模型组大鼠突触体内TH,DDC,PKA表达量显著降低(P0.01);与模型组比较,利他林组及安神定志灵中剂量组突触体内TH,PKA蛋白及TH,PKA,DDC mRNA表达量及阳性细胞数均有所升高(P0.05);免疫组化阳性细胞计数显示,与正常组比较,模型组及安神定志灵高剂量组TH阳性细胞表达显著降低(P0.01),安神定志灵低剂量组TH阳性细胞表达明显降低(P0.05);模型组及安神定志灵低剂量组中DDC阳性细胞表达显著降低(P0.01);治疗后与模型组比较,利他林组及安神定志灵低、中剂量组TH阳性细胞表达显著上升(P0.01),利他林组及安神定志灵中、高剂量组DDC阳性细胞数上升显著(P0.01)。结论:安神定志灵通过调控TH,DDC,PKA的表达,影响多巴胺的合成速度,安神定志灵控制ADHD核心症状的作用可能与调控多巴胺合成速度有关。

Effect of Anshen Dingzhiling on Synaptosome Dopamine Synthesis Related Factor Expression in Rats

Objective: To study the regulation effect of Anshen Dingzhiling (ADL) on tyrosine hydroxylase (TH), DOPA decarboxylase (DDC), and protein kinase A (PKA), and reveal the effect mechanism of Anshen Dingzhiling in treatment of attention deficit hyperactivity disorder (ADHD). Method: Spontaneously hypertensive (SHR) rats were divided into model group, western medicine group, ADL high, medium and low dose (26.70,13.35,6.75 g·kg^-1) groups based on random number, n=8 in each group. Another 8 WKY rats were set as normal group. The rats in each group received lavage administration for 4 weeks. Brain synaptosome was prepared by using Percoll density gradient centrifugation method. Western blot method was used to detect the protein expression levels of TH and PKA;Real-time polymerase chain reaction (Real-tim PCR) technique was used to detect mRNA expression levels of TH, DDC, PKA; and immunofluorescence technique was used to detect TH, and DDC positive cells expression. Result: As compared with normal group, the synaptic TH, DDC, and PKA expression levels were significantly reduced in model group (P<0.01);as compared with model group, the synaptic TH, PKA protein expression levels, TH, PKA, DDC mRNA expression levels, and number of positive cells were increased in western medicine group and ADL medium dose group (P<0.05). Immunohistochemistry positive cell count results showed that as compared with normal group, TH positive cells expressing level was significantly reduced in model group and ADL high dose group (P<0.01), and TH positive cells expressing level was reduced in ADL low dose group (P<0.05); DDC positive cells expressing level was significantly reduced in model group and ADL low dose group (P<0.01). As compared with model group, TH positive cells expressing level was significantly increased in western medicine group and ADL low dose and medium dose groups (P<0.01); in addition, number of DDC positive cells was increased significantly in the western medicine group and ADL medium and high dose groups (P<0.01). Conclusion: ADL could affect the speed of dopamine synthesis through regulating the expression of TH, DDC, PKA, and the effect of ADL for core symptoms of ADHD may be related to the regulation of dopamine synthesis speed.