【作者】 王静；

【导师】 倪新强；

【作者基本信息】 广州中医药大学 ， 中医儿科学， 2025， 硕士

【摘要】 目的:1.通过检索国内外相关文献,系统评估中医药治疗ADHD的动物实验研究的设计、实施方法与步骤并归纳作用机制,为规范中医药治疗ADHD动物实验提供理论支撑。2.探究补肾开窍法对ADHD大鼠行为学的改善作用,并进一步研究其对前额叶-纹状体环路突触可塑性以及海马髓鞘再生的影响,为临床运用本法提供真实可靠的实验依据。方法:1.对中国知网（CNKI）、万方数据库、维普中文期刊数据库和Pubmed等数据库进行文献检索,检索时间截止到2024年12月。中文检索主题词为“中医”或“中医药”,“注意力缺陷多动障碍”或“ADHD”或“多动”或“注意缺陷”或“轻微脑功能障碍”,“动物”或“鼠”或“实验”;英文检索主题词为“Chinese medicine”、“Attention deficit hyperactivity disorder”or“ADHD”or“hyperactivity”or“Attention deficit”、“animal”or“rat”or“mouse”or“experiment”。2.（1）将60只SPF级幼龄雄性SHR大鼠（55～65 g）依据旷场实验总运动距离平均分为模型组,哌甲酯组(2 mg·kg^-1·d^-1),补肾开窍方低、中、高剂量组(15.245g·kg^-1·d^-1、30.49 g·kg^-1·d^-1、60.98 g·kg^-1·d^-1),WKY大鼠为正常对照组,连续灌胃给药28 d。记录每组大鼠每日体质量和摄食量,分别于灌胃2、4周时进行旷场实验、高架十字迷宫测试、自发交替实验评估大鼠多动、冲动、注意力不集中等行为。（2）行为学测试后取前额叶、纹状体、海马,对前额叶、纹状体组织进行尼氏染色法观察神经元的形态和数量变化,TUNEL荧光染色法检测神经元凋亡,透射电镜法观察神经元突触超微结构,高尔基染色法观察树突棘密度,免疫荧光法检测PSD95、GAP-43阳性表达情况,Western Blot法检测PSD95、caspase-3、TrκB、GAP-43、BDNF蛋白表达量;对海马组织进行LFB染色法观察髓鞘脱失情况,透射电镜观察海马髓鞘超微结构变化,免疫组化检测CA1、CA3、DG区MBP、PLP阳性表达情况,Western Blot法检测海马MBP、PLP蛋白表达量。结果:1.经文献检索、筛选、去重等过程,共纳入94篇文献,其中中文文献83篇,外文文献11篇。通过分析文献内容,发现近年文献选用模型逐渐标准化,多维度行为学指标评估ADHD症状,机制突破了单一神经递质的局限性,然而仍存在盲法缺失、对照组设置混乱、给药剂量换算不明等不足。2.（1）ADHD大鼠在给药前,给药2、4周时体质量和平均24 h摄食量差异无统计学意义。（2）SHR大鼠显示出高活跃性和多动行为,给药4周后,补肾开窍方高剂量组可降低SHR大鼠在旷场箱内的总运动距离（16.22±4.89 vs 29.88±3.90,P<0.0001）、平均运动速度（5.41±1.63 vs 9.96±1.30,P<0.0001）、直立次数（11.00±3.05 vs26.75±4.14,P<0.0001）、中心区运动距离（1.87±0.72 vs 6.88±1.82,P<0.0001）、中心区停留时间（20.94±6.75 vs 61.32±15.62,P<0.0001）、中心区进入次数（6.08±1.83vs 13.50±2.07,P<0.0001）;降低在高架十字迷宫内的总运动距离（15.80±2.30 vs20.54±2.57,P=0.0003<0.01）、平均运动速度（5.27±0.77 vs 6.85±0.86,P=0.0003<0.01）、探头次数（9.42±3.48 vs 20.75±3.70,P<0.0001）、进入开臂的次数比（32.82±11.73vs 57.19±9.93,P<0.0001）、进入开臂的时间比（13.73±5.36 vs 46.79±8.62,P<0.0001）、闭臂内的直立次数（7.33±2.35 vs 11.58±3.37,P=0.0008<0.01）;降低在Y迷宫内的进臂总次数（17.58±2.43 vs 27.08±4.94,P<0.0001）,增加自发交替率（61.31±8.17 vs44.96±6.17,P<0.0001）。（3）补肾开窍方高剂量组可增加前额叶、纹状体尼氏阳性细胞数量（16.33±2.34vs 7.67±2.16,P<0.0001;28.33±4.13 vs 17.33±3.08,P<0.0001）,提高Neu N阳性细胞率（62.12±5.66 vs 46.61±9.43,P=0.0023<0.01;59.18±7.55 vs 48.04±7.66,P=0.0222<0.05）,降低细胞凋亡率（16.39±6.17 vs 36.70±10.58,P=0.0001<0.01;10.15±7.58 vs33.64±5.08,P=0.0001<0.01）,改善突触结构损伤,增加树突棘密度（0.38±0.03 vs0.23±0.02,P=0.0002<0.01;0.35±0.03 vs 0.22±0.02,P<0.0001）,升高PSD95（0.91±0.06vs 0.54±0.07,P=0.005<0.01;0.83±0.06 vs 0.56±0.06,P=0.009<0.01）、GAP-43（0.86±0.02 vs 0.58±0.04,P<0.0001;0.89±0.09 vs 0.57±0.05,P=0.0016<0.05）蛋白相对荧光强度,提高PSD95（0.95±0.12 vs 0.68±0.01,P=0.0456<0.05;0.94±0.16 vs0.60±0.14,P=0.0498<0.05）、TrκB（0.79±0.03 vs 0.55±0.03,P<0.0001;0.73±0.18 vs0.38±0.04,P=0.0115<0.05）、GAP-43（0.86±0.02 vs 0.58±0.03,P=0.0005<0.01;0.91±0.13 vs 0.45±0.17,P=0.0042<0.01）、BDNF（0.85±0.10 vs 0.55±0.02,P=0.0438<0.05;1.21±0.25 vs 0.56±0.07,P=0.0003<0.01）蛋白表达水平,降低caspase-3（1.06±0.15 vs 1.82±0.11,P<0.0001;1.16±0.35 vs 1.97±0.11,P=0.0191<0.05）蛋白表达水平。（4）补肾开窍方高剂量组可降低海马髓鞘脱失程度,恢复髓鞘结构损伤,提高海马CA1、CA3区MBP（0.45±0.03 vs 0.30±0.02,P<0.0001;0.47±0.02 vs 0.40±0.02,P=0.0011<0.01）、PLP（0.33±0.03 vs 0.26±0.03,P=0.0049<0.01;0.35±0.06 vs0.24±0.01,P=0.0169<0.05）蛋白MOD值,且提高海马MBP（0.93±0.23 vs 0.49±0.10,P=0.0127<0.05）、PLP（0.89±0.15 vs 0.48±0.11,P=0.0024<0.01）蛋白表达水平。结论:1.近年中医药治疗ADHD的实验文献在实验设计、研究方法、研究内容上逐渐规范和完善,逐渐与国际接轨,但在动物模型与中医证型结合、实验结果的准确度和可信度、对照组设置混乱、实验周期不足等方面仍然存在不足。2.（1）补肾开窍方对ADHD大鼠生长发育表征未见影响。（2）补肾开窍方可有效改善ADHD大鼠多动、冲动、注意力不集中的核心症状。（3）补肾开窍方可对ADHD大鼠神经元及突触相关指标产生影响,对前额叶-纹状体环路突触可塑性具有保护作用,且可能与BNDF/TrκB信号通路有关。（4）补肾开窍方可上调MBP、PLP蛋白表达,促进海马髓鞘再生。更多还原

【Abstract】 Objective1.To systematically evaluate the design,methodology,and mechanisms of animal experiments on traditional Chinese medicine（TCM）for attention deficit hyperactivity disorder（ADHD）through a comprehensive literature review,thereby providing theoretical support for standardizing TCM-based ADHD animal studies.2.To investigate the effects of the method of tonifying the kidney and opening the aperture on ADHD behaviors in rats,with emphasis on its regulatory roles in prefrontal-striatal synaptic plasticity and hippocampal remyelination,offering experimental evidence for clinical applications.Methods1.Literature retrieval was conducted across China National Knowledge Infrastructure（CNKI）,Wanfang data,VIP Information,and Pub Med databases up to December,2024,and used the following terms:“Chinese medicine”,“Attention deficit hyperactivity disorder”or“ADHD”or“hyperactivity”or“Attention deficit”、“animal”or“rat”or“mouse”or“experiment”.2.（1）Sixty SPF-grade juvenile male spontaneously hypertensive rats（SHR,55～65 g）were stratified by open-field test performance into model,methylphenidate(2 mg·kg^-1·d^-1),and low-/medium-/high-dose tonifying kidney and opening aperture formula groups(15.245 g·kg^-1·d^-1、30.49 g·kg^-1·d^-1、60.98 g·kg^-1·d^-1).Wistar Kyoto（WKY）rats served as normal controls.The rats were administered by gavage for 28 days.Body weight and food intake were recorded daily.Behavioral assessments,including the open field test,elevated plus maze test,and spontaneous alternation test（Y-maze）,were conducted at 2 and 4 weeks post-gavage to assess hyperactivity,impulsivity,and inattention.（2）Following behavioral assessments,the prefrontal cortex,striatum,and hippocampus were harvested for histopathological and molecular examinations.For the prefrontal cortex and striatum,Nissl staining was performed to observe neuronal morphology and density changes,terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling（TUNEL）fluorescence staining was used to detect neuronal apoptosis,transmission electron microscopy（TEM）was applied to examine synaptic ultrastructure,Golgi-Cox staining was conducted to analyze dendritic spine density,immunofluorescence was employed to assess PSD95 and GAP-43 positive expression,and Western blot was utilized to measure protein levels of PSD95,caspase-3,TrκB,GAP-43,and BDNF.For the hippocampus,Luxol fast blue（LFB）staining was carried out to evaluate demyelination,TEM was used to observe myelin ultrastructure alterations,immunohistochemistry（IHC）was performed to detect PLP and MBP positive expression in CA1,CA3,and DG regions,and Western blot was applied to quantify PLP and MBP protein expression levels.Results1.Following literature retrieval,screening,and deduplication processes,a total of 94studies（83 Chinese articles and 11 international publications）were included.Analysis of the included literature revealed that recent studies have demonstrated gradual standardization of models,multi-dimensional behavioral assessments for ADHD symptom evaluation,and mechanisms extending beyond monoaminergic neurotransmitter dysregulation.However,persistent limitations included lack of blinding procedures,inconsistent control group designs,and unclear dose conversion methods in pharmacological interventions.2.（1）There was no statistically significant difference in the body weight and average24-hour food intake of ADHD rats throughout the drug administration period.（2）The SHR rats exhibited heightened activity and hyperactive behaviors.After 4weeks of treatment,the high-dose Bushen Kaiqiao Formula（BSKQ）group significantly reduced the total movement distance（16.22±4.89 vs.29.88±3.90,P<0.0001）,average movement speed（5.41±1.63 vs.9.96±1.30,P<0.0001）,rearing frequency（11.00±3.05 vs.26.75±4.14,P<0.0001）,central zone movement distance（1.87±0.72 vs.6.88±1.82,P<0.0001）,central zone duration（20.94±6.75 vs.61.32±15.62,P<0.0001）,and central zone entries（6.08±1.83 vs.13.50±2.07,P<0.0001）in the open field test.Additionally,the high-dose BSKQ group decreased the total movement distance（15.80±2.30 vs.20.54±2.57,P=0.0003<0.01）,average speed（5.27±0.77 vs.6.85±0.86,P=0.0003<0.01）,head-dipping frequency（9.42±3.48 vs.20.75±3.70,P<0.0001）,open-arm entry ratio（32.82±11.73 vs.57.19±9.93,P<0.0001）,open-arm time ratio（13.73±5.36vs.46.79±8.62,P<0.0001）,and closed-arm rearing frequency（7.33±2.35 vs.11.58±3.37,P=0.0008<0.01）in the elevated plus maze test.In the Y-maze test,the high-dose BSKQ group reduced total arm entries（17.58±2.43 vs.27.08±4.94,P<0.0001）while increasing spontaneous alternation rate（61.31±8.17 vs.44.96±6.17,P<0.0001）.（3）In the prefrontal cortex and striatum,the high-dose BSKQ group increased the number of Nissl-positive cells in the prefrontal cortex and striatum（16.33±2.34 vs.7.67±2.16,P<0.0001;28.33±4.13 vs.17.33±3.08,P<0.0001）,elevated Neu N-positive cell rate（62.12±5.66 vs.46.61±9.43,P=0.0023<0.01;59.18±7.55 vs.48.04±7.66,P=0.0222<0.05）,and reduced apoptosis rate（16.39±6.17 vs.36.70±10.58,P=0.0001<0.01;10.15±7.58 vs.33.64±5.08,P=0.0001<0.01）;the treatment ameliorated synaptic structural damage,increased dendritic spine density（0.38±0.03 vs.0.23±0.02,P=0.0002<0.01;0.35±0.03 vs.0.22±0.02,P<0.0001）,and upregulated the relative fluorescence intensity of PSD95（0.91±0.06 vs.0.54±0.07,P=0.005<0.01;0.83±0.06 vs.0.56±0.06,P=0.009<0.01）and GAP-43（0.86±0.02 vs.0.58±0.04,P<0.0001;0.89±0.09 vs.0.57±0.05,P=0.0016<0.05）;western blot analysis revealed elevated protein expression levels of PSD95（0.95±0.12 vs.0.68±0.01,P=0.0456<0.05;0.94±0.16 vs.0.60±0.14,P=0.0498<0.05）,TrκB（0.79±0.03 vs.0.55±0.03,P<0.0001;0.73±0.18 vs.0.38±0.04,P=0.0115<0.05）,GAP-43（0.86±0.02 vs.0.58±0.03,P=0.0005<0.01;0.91±0.13vs.0.45±0.17,P=0.0042<0.01）,and BDNF（0.85±0.10 vs.0.55±0.02,P=0.0438<0.05;1.21±0.25 vs.0.56±0.07,P=0.0003<0.01）,while suppressing caspase-3 expression（1.06±0.15 vs.1.82±0.11,P<0.0001;1.16±0.35 vs.1.97±0.11,P=0.0191<0.05）.（4）The high-dose BSKQ group attenuated hippocampal myelin loss,restored myelin structural integrity,and increased the MOD values of MBP（0.45±0.03 vs.0.30±0.02,P<0.0001;0.47±0.02 vs.0.40±0.02,P=0.0011<0.01）and PLP（0.33±0.03 vs.0.26±0.03,P=0.0049<0.01;0.35±0.06 vs.0.24±0.01,P=0.0169<0.05）in the CA1 and CA3 regions.Moreover,it upregulated hippocampal MBP（0.93±0.23 vs.0.49±0.10,P=0.0127<0.05）and PLP（0.89±0.15 vs.0.48±0.11,P=0.0024<0.01）protein expression levels.Conclusion1.Recent experimental studies on TCM for ADHD treatment have shown progressively standardized and improved experimental designs,methodologies,and research content,gradually aligning with international standards.However,deficiencies remain in several aspects including the integration of animal models with TCM syndrome differentiation,accuracy and reliability of experimental results,inconsistent control group settings,and insufficient experimental durations.2.（1）The Bushen Kaiqiao formula showed no observable effects on the growth and development characteristics of ADHD model rats.（2）The Bushen Kaiqiao formula effectively improved core symptoms of ADHD model rats including hyperactivity,impulsivity,and inattention.（3）The Bushen Kaiqiao formula exerted significant effects on neuronal and synaptic-related indicators in ADHD model rats,demonstrating protective effects on synaptic plasticity in the prefrontal cortex-striatum circuit,potentially mediated through the BDNF/TrκB signaling pathway.（4）The Bushen Kaiqiao formula upregulated the expression of MBP and PLP proteins,promoting myelin regeneration in the hippocampus.更多还原