Table9_Long Mu Qing Xin mixture improves behavioral performance in spontaneously hypertensive rats (SHR/NCrl) by upregulating catecholamine neurotransmitters in prefrontal cortex and striatum via DRD1/cAMP/PKA-CREB signaling pathway.XLSX

BackgroundAttention deficit hyperactivity disorder (ADHD), a prevalent neurodevelopmental disorder in children, can be effectively alleviated by the herbal preparation Long Mu Qing Xin Mixture (LMQXM), but its mechanism has not been fully elucidated.
ObjectiveTo scrutinize the potential pharmacological mechanisms by which LMQXM improves behavior in spontaneously hypertensive rats (SHR/NCrl).
MethodsThe SHR/NCrl rats were randomly stratified into the model (SHR) group, the methylphenidate hydrochloride (MPH) group, and groups subjected to varying dosages of LMQXM into the medium dose (MD) group with a clinically effective dose, the low dose (LD) group with 0.5 times the clinically effective dose, and high dose (HD) group with 2 times the clinically effective dose. Furthermore, the WKY/NCrl rats constituted the control group. The evaluation of behavior involved the open field test and the Morris water maze test. HPLC, LC-MS, ELISA, immunohistochemistry, Western blot, and RT-qPCR were utilized to scrutinize the catecholamine neurotransmitter content and the expression of proteins and genes associated with the dopamine receptor D1 (DRD1)/cAMP/protein kinase A (PKA)-cAMP response element-binding (CREB) pathway in prefrontal cortex (PFC) and striatum.
ResultsMPH and LMQXM ameliorated hyperactivity and learning and memory deficits of SHR/NCrl rats. Among them, LMQXM-MD and MPH also upregulated dopamine (DA), norepinephrine (NE), adenylate cyclase (AC) and cAMP levels, and the expression of proteins and genes associated with the DRD1/cAMP/PKA-CREB pathway in PFC and striatum of SHR/NCrl rats. PFC and striatum DA levels were also upregulated in the LMQXM-LD group as well as the striatum DA levels in the LMQXM-HD group, but there were no statistically significant differences in their NE levels compared to the SHR group. LMQXM-LD and LMQXM-HD also upregulated some DRD1/cAMP/PKA-CREB pathway-related proteins and gene expression, but the effects were discernibly disparate in PFC and striatum. Upon comprehensive analysis, LMQXM-MD appeared to be the most effective dose.
ConclusionOur study tentatively suggests that LMQXM may rectify hyperactivity and learning and memory deficits of SHR/NCrl rats by elevating catecholamine neurotransmitters in the PFC and striatum. This effect may be attributed to the potential activation of the DRD1/cAMP/PKA-CREB signaling pathway, which appears to achieve an optimal response at moderate doses.

背景：注意力缺陷多动障碍（Attention Deficit Hyperactivity Disorder, ADHD）是儿童高发的神经发育障碍，龙牡清心合剂（Long Mu Qing Xin Mixture, LMQXM）可有效缓解该病症，但具体作用机制尚未完全阐明。
目的：本研究旨在探讨龙牡清心合剂改善自发性高血压大鼠（Spontaneously Hypertensive Rats, SHR/NCrl）行为症状的潜在药理机制。
方法：将SHR/NCrl大鼠随机分层分为模型（SHR）组、盐酸哌甲酯（Methylphenidate Hydrochloride, MPH）组，以及不同剂量的龙牡清心合剂组：即临床有效剂量的中剂量（MD）组、为临床有效剂量0.5倍的低剂量（LD）组，以及为临床有效剂量2倍的高剂量（HD）组；另以Wistar-Kyoto大鼠（Wistar-Kyoto Rats, WKY/NCrl）作为空白对照组。行为学评价采用旷场实验与莫里斯水迷宫实验。通过高效液相色谱法（High Performance Liquid Chromatography, HPLC）、液相色谱-质谱联用法（Liquid Chromatography-Mass Spectrometry, LC-MS）、酶联免疫吸附试验（Enzyme-Linked Immunosorbent Assay, ELISA）、免疫组化、蛋白质印迹法（Western Blot, Western blot）及实时荧光定量聚合酶链反应（Real-Time Quantitative Polymerase Chain Reaction, RT-qPCR），检测大鼠前额叶皮层（Prefrontal Cortex, PFC）与纹状体（Striatum）中的儿茶酚胺类神经递质含量，以及多巴胺D1受体（Dopamine Receptor D1, DRD1）/环磷酸腺苷（Cyclic Adenosine Monophosphate, cAMP）/蛋白激酶A（Protein Kinase A, PKA）-cAMP反应元件结合蛋白（cAMP Response Element Binding Protein, CREB）通路相关蛋白与基因的表达水平。
结果：盐酸哌甲酯与龙牡清心合剂均可改善SHR/NCrl大鼠的多动症状及学习记忆障碍。其中，龙牡清心合剂中剂量组与盐酸哌甲酯组均可上调SHR/NCrl大鼠前额叶皮层与纹状体中的多巴胺（Dopamine, DA）、去甲肾上腺素（Norepinephrine, NE）、腺苷酸环化酶（Adenylate Cyclase, AC）及cAMP水平，同时上调DRD1/cAMP/PKA-CREB通路相关蛋白与基因的表达。龙牡清心合剂低剂量组的前额叶皮层与纹状体多巴胺水平，以及龙牡清心合剂高剂量组的纹状体多巴胺水平亦有所提升，但二者的去甲肾上腺素水平与模型组相比无统计学显著性差异。龙牡清心合剂低、高剂量组同样可上调部分DRD1/cAMP/PKA-CREB通路相关蛋白与基因的表达，但在前额叶皮层与纹状体中的干预效果存在显著差异。综合分析显示，龙牡清心合剂中剂量组的干预效果最优。
结论：本研究初步表明，龙牡清心合剂可通过提升SHR/NCrl大鼠前额叶皮层与纹状体中的儿茶酚胺类神经递质水平，改善其多动症状与学习记忆障碍。该作用可能与激活DRD1/cAMP/PKA-CREB信号通路有关，且在中剂量下可达到最佳干预效果。