{"title":"A study on the mechanism of Bushen Kaiqiao Formula in modulating microglial activation to alleviate neuroinflammation in ADHD.","authors":"Jiaqi Zhang, Ruxin Sun, Yu Xiong, Yuting Yang, Jing Wang, Kanglin Zhu, Xinqiang Ni, Min Huang","doi":"10.1016/j.bbrep.2025.102211","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Growing evidence suggests that dysregulated microglial activation and neuroinflammation in the prefrontal cortex may underlie the pathophysiology of Attention Deficit Hyperactivity Disorder. Utilizing spontaneously hypertensive rats-a well-validated animal model of ADHD-this study aimed to characterize ADHD-like behavioral phenotypes and prefrontal cortical neuronal development and elucidate the neuroimmunological mechanisms through which the Bushen Kaiqiao Formula (BSKQF) exerts its therapeutic effects by modulating microglial activation states.</p><p><strong>Methods: </strong>Ultra-high-performance liquid chromatography coupled with a tandem mass spectrometer (UHPLC-MS/MS) was used to qualitatively analyze the chemical composition of Bushen Kaiqiao Formula, and network pharmacology was applied to predict its potential therapeutic targets and ADHD-related pathways ADHD-like behaviors, including hyperactivity, impulsivity, spatial memory, and attention, were assessed using the Open Field Test, Elevated Plus Maze, and Y-Maze Test at weeks 2 and 4. Prefrontal cortical neuronal morphology and dendritic spine density were evaluated using Nissl staining and Golgi-Cox staining, respectively. Microglial activation (Iba-1+/iNOS + immunofluorescence), inflammatory cytokine levels (IL-1β and IL-6 by ELISA), and blood-brain barrier integrity (transmission electron microscopy) were examined. In vitro, primary prefrontal cortical neurons and microglia were isolated from neonatal rats and treated with BSKQF-containing serum at different concentrations, with Methylphenidate (MPH) as a control. An oxygen-glucose deprivation/reoxygenation (OGD/R) model was established to mimic neuroinflammatory conditions with inflammatory signaling pathways assessed.</p><p><strong>Results: </strong>UHPLC-MS/MS identified key bioactive components of Bushen Kaiqiao Formula. Network pharmacology analysis suggested that BSKQF may ameliorate ADHD-related behavioral deficits by inhibiting the NF-κB signaling pathway. Behavioral assessments demonstrated dose- and time-dependent effects. BSKQF significantly reduced neuronal loss, promoted synaptic plasticity, reduced the proportion of activated microglia (iNOS+/Iba-1+), and lowered IL-1β and IL-6 levels. Additionally, ultrastructural examination indicated restored blood-brain barrier integrity and reduced perivascular edema. In vitro, BSKQF-containing serum suppressed NF-κB pathway activation (p-NF-κB-p65, p-IκBα) and inflammasome-related proteins (NLRP3, caspase-1).</p><p><strong>Conclusion: </strong>BSKQF effectively alleviates ADHD-like behaviors in SHR rats by inhibiting prefrontal microglial activation and neuroinflammation, reducing neuronal apoptosis, restoring synaptic plasticity, and preserving blood-brain barrier integrity.</p>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":"43 ","pages":"102211"},"PeriodicalIF":2.2000,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12396018/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry and Biophysics Reports","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.bbrep.2025.102211","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/9/1 0:00:00","PubModel":"eCollection","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Background: Growing evidence suggests that dysregulated microglial activation and neuroinflammation in the prefrontal cortex may underlie the pathophysiology of Attention Deficit Hyperactivity Disorder. Utilizing spontaneously hypertensive rats-a well-validated animal model of ADHD-this study aimed to characterize ADHD-like behavioral phenotypes and prefrontal cortical neuronal development and elucidate the neuroimmunological mechanisms through which the Bushen Kaiqiao Formula (BSKQF) exerts its therapeutic effects by modulating microglial activation states.
Methods: Ultra-high-performance liquid chromatography coupled with a tandem mass spectrometer (UHPLC-MS/MS) was used to qualitatively analyze the chemical composition of Bushen Kaiqiao Formula, and network pharmacology was applied to predict its potential therapeutic targets and ADHD-related pathways ADHD-like behaviors, including hyperactivity, impulsivity, spatial memory, and attention, were assessed using the Open Field Test, Elevated Plus Maze, and Y-Maze Test at weeks 2 and 4. Prefrontal cortical neuronal morphology and dendritic spine density were evaluated using Nissl staining and Golgi-Cox staining, respectively. Microglial activation (Iba-1+/iNOS + immunofluorescence), inflammatory cytokine levels (IL-1β and IL-6 by ELISA), and blood-brain barrier integrity (transmission electron microscopy) were examined. In vitro, primary prefrontal cortical neurons and microglia were isolated from neonatal rats and treated with BSKQF-containing serum at different concentrations, with Methylphenidate (MPH) as a control. An oxygen-glucose deprivation/reoxygenation (OGD/R) model was established to mimic neuroinflammatory conditions with inflammatory signaling pathways assessed.
Results: UHPLC-MS/MS identified key bioactive components of Bushen Kaiqiao Formula. Network pharmacology analysis suggested that BSKQF may ameliorate ADHD-related behavioral deficits by inhibiting the NF-κB signaling pathway. Behavioral assessments demonstrated dose- and time-dependent effects. BSKQF significantly reduced neuronal loss, promoted synaptic plasticity, reduced the proportion of activated microglia (iNOS+/Iba-1+), and lowered IL-1β and IL-6 levels. Additionally, ultrastructural examination indicated restored blood-brain barrier integrity and reduced perivascular edema. In vitro, BSKQF-containing serum suppressed NF-κB pathway activation (p-NF-κB-p65, p-IκBα) and inflammasome-related proteins (NLRP3, caspase-1).
Conclusion: BSKQF effectively alleviates ADHD-like behaviors in SHR rats by inhibiting prefrontal microglial activation and neuroinflammation, reducing neuronal apoptosis, restoring synaptic plasticity, and preserving blood-brain barrier integrity.
背景：越来越多的证据表明，前额皮质的小胶质细胞激活失调和神经炎症可能是注意缺陷多动障碍的病理生理基础。本研究利用自发性高血压大鼠（一种经过验证的adhd动物模型），研究adhd样行为表型和前额皮质神经元发育特征，阐明补肾开翘方通过调节小胶质细胞激活状态发挥治疗作用的神经免疫学机制。方法：采用超高效液相色谱联用串联质谱（UHPLC-MS/MS）对补神开翘方的化学成分进行定性分析，并应用网络药理学方法预测其潜在的治疗靶点和adhd相关通路，分别在第2周和第4周采用Open Field Test、Elevated Plus Maze、Y-Maze等方法评估adhd样行为，包括多动症、冲动性、空间记忆和注意力。采用尼索染色和高尔基-考克斯染色分别评价大鼠前额叶皮质神经元形态和树突棘密度。检测小胶质细胞活化（Iba-1+/iNOS +免疫荧光）、炎症细胞因子水平（ELISA检测IL-1β和IL-6）和血脑屏障完整性（透射电镜）。体外分离新生大鼠的初级前额皮质神经元和小胶质细胞，用不同浓度含bskqf的血清处理，以哌醋甲酯（MPH）为对照。建立氧-葡萄糖剥夺/再氧化（OGD/R）模型，模拟神经炎症条件，并评估炎症信号通路。结果：UHPLC-MS/MS鉴别出补肾开翘方的主要活性成分。网络药理学分析提示BSKQF可能通过抑制NF-κB信号通路改善adhd相关行为缺陷。行为评估显示了剂量和时间依赖效应。BSKQF显著减少神经元丢失，促进突触可塑性，降低活化小胶质细胞比例（iNOS+/Iba-1+），降低IL-1β和IL-6水平。此外，超微结构检查显示血脑屏障完整性恢复，血管周围水肿减少。在体外，含bskqf的血清抑制NF-κB通路激活（p-NF-κB-p65、p -κB α）和炎性小体相关蛋白（NLRP3、caspase-1）。结论：BSKQF通过抑制前额叶小胶质细胞激活和神经炎症，减少神经元凋亡，恢复突触可塑性，保持血脑屏障完整性，有效缓解SHR大鼠adhd样行为。
期刊介绍：
Open access, online only, peer-reviewed international journal in the Life Sciences, established in 2014 Biochemistry and Biophysics Reports (BB Reports) publishes original research in all aspects of Biochemistry, Biophysics and related areas like Molecular and Cell Biology. BB Reports welcomes solid though more preliminary, descriptive and small scale results if they have the potential to stimulate and/or contribute to future research, leading to new insights or hypothesis. Primary criteria for acceptance is that the work is original, scientifically and technically sound and provides valuable knowledge to life sciences research. We strongly believe all results deserve to be published and documented for the advancement of science. BB Reports specifically appreciates receiving reports on: Negative results, Replication studies, Reanalysis of previous datasets.
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